i ae Ei pay er: i of Universit y Libraries: 0-CHEMICAL JOURNAL ‘ e ep & AMM} lip : me ) | e : ‘pati Ste : = HAY) Bes hey “O Gii\\s» . ; AP Fr Ve Aw : rs = dae eee _ BENJAMIN MOORE, M.A., D.Sc. “EDWARD WHITLEY, M. A.. VOLUME IV 1909 Pte $5.4 ee. g 7 | . COPYRIGHT = | BIO-CHEMICAL DEPARTMENT, JOHNSTON LABORATORIES Bie os UNIVERSITY OF LIVERPOOL « pe ¥ pote tee | | ee prtl at ; r av ‘a’ ~ an } ) De aii CoN” py Oars A \\\\ Lies © fee Nite fis cA Vets |] ie Sed C VE ie ae 4 \ j ape UF a eae 4) i} Ad eS se sialia ees Vf] rei a Agents for America: me G. E, STECHERT & CO. =e 129-133 West Twentietn Street, New York City | « “al ae 7 a hon i it cy i a ee ’ on " ie ial ead = ~~ ‘ ‘ 7 > ue ee es i . f Wea nae —_ am 7 : ‘ rc Si CONTENTS OF VOLUME IV Observations on Certain Marine Organisms of (a) Variations in Reaction to Light, and (}) a Diurnal Periodicity of Phosphorescence. By Benjamin Moore, M.A., D.Sc., Johnston Professor of Bio-Chemistry, University of Liverpool : The Relative Importance of Inorganic Kations, especially those of Sodium and “Calcium in the Causation of Gout and Production of wid ne Bak By William Gordon Little, M.A. (Aber.), M.D. (Edin.) On the Nitrogen-containing Radicle of Lecithin and cetild Phosphates By Hugh MacLean, M.D., Carnegie Research Fellow . Some Observations on the Haemolysis of Blood by Syebslote on iver: osmotic Solutions of Sodium Chloride. By U. N. Brahmachari, M.A., M.D., Lecturer on Medicine at the Campbell Medical School, Calcutta Further Observations on the Action of Muscarin and Plocarpin on a ey By Hugh MacLean, M.D., Carnegie Research Fellow The Occurrence and Distribution of Cholesterol and allied Bodies i in he Nite Kingdom. By Charles a M.A., B.Sc., Lindley Student of the b geivieyd of London Allyl sihininase Sina Aspect o of its Physiological celal By E. Wace Carlier, M.D., F.R.S.E. Choline in Animal Tissues and Fluids. “By W. Webster, M. D., ©. M. Haleais strator of Physiology in the University of Manitoba, Canada The Biuret Reaction and the Cold Nitric Acid Test in the Recognition of Protein. By Karl A. van Norman, M.B. (Toronto) The Properties and Classification of the Oxidising Sasi and heieaies between Enzymic Activity and the Effects of Immune Bodies and Complements. By Benjamin Moore, M.A., D.Sc., Johnston Professor of Biante University of Liverpool, and Edward Whitley, M.A. (Oxon.) ; On the Occurrence of a Mon-amino-diphosphatide Lecithin-like Body i in he Yolk of Egg. By Hugh i 5 M.LD., a Saree Research sb uae of Aberdeen . : Iodo-Eosin as a Test fe Pi ree Alkali j in 0 dried-up Plant aaa. By A. C. Hof, Héchst a, Main On the Growth of the Bacillus Tuberculosis he sale Micte-Onseata in different Percentages of yg 4 om By Benjamin Moore, M.A., D.Sc., Johnston Professor of Bi emistry, University of Liverpool, and R. Stenhouse Williams, M.B., D.P.H., Lecturer on Public Health Bacteriology, University of Liverpool The Electrical Forces of Mitosis and the Origin of Cancer. By A. E. and A. C. Jessup; E. C. C. Baly, F.R.S., Fellow of University College, London; F. W. y, M.D., M.R.C.P. ; and E. Prideaux, M.R.C.S., L.R.C.P. ; The Estimation of Phosphorous in Urine. By G. C. Pata, M.B., B. s. (Melb.), Sharpey Scholar On the Nitrogen-containing Radicle of Lecithin ed Pee Phosphatides. By Hugh Maclean, M.D., Carnegie Research Fellow, University of Aberdeen A Polarimetric Study of the Sucroclastic Enzymes in Beta Vulgaris. By R. A. Robertson, M.A. ; James Oe ean Iving, D.Se., Ph.D.; and Mildred E. Dobson, M.A., B.Se., Carnegie Sch The Output of ic Phosphorus in Urine. ay 3 “Mathison, M. B., B.S. (Melb nae Or : sg On the Relative Haemoglobin Value of the éditent Ery ehisotees tiles he Haemolysis of Blood with Hyposmotic Sodium Chloride Solution, and on the Permeability of the Erythrocytes to Water as a Factor in the production of Haemolysis. By U.N. Brahmachari, M.A., M.D., Ph.D., Lecturer in begeeag —* Medical School, Calcutta PAGE CONTENTS The Isolation of Conium Alkaloids from Animal Tissues, and the Action of - Living Cells and Decomposing Organs on these Alkaloids. M.B., Ch.B. (Aber.), Carnegie Scholar in Pharmacology By Walter J. Dilling, Some Observations upon the Error in the Opsonic Technique. By Ernest E. Glynn, M.A., M.D. (Cantab.), M.R.C.P., Lecturer in Morbid Anatomy and Clinical Pathology, University of Liverpool, Pathologist, Royal Infirmary, Liverpool, and G. Lissant Cox, M.A, M.B., B.C. anne ), Holt Fellow in. Pethelogy, sia of Liverpool . The Relationship of ee of a Dow to the i size of re Animal Treated, aida in regard to the Cause of the Failures to Cure Trypanosomiasis and other Protozoan Diseases in Man and in large Animals. By Benjamin Moore, M.A., D.Sc., gion Professor of Bio-Chemistry, University of Liverpool : Proposals for the Nomenclature of the Lipoids. By ras poesaliien A Comparison of the Methods for the Estimation of Total setae in Urine, By Stanley Ritson, A.K.C. : The Use of Barium Peroxide in the heiadon of Total Sulphur in Urine: Stanley Ritson, A.K.C. A Contribution to the Bio- Chaaety a ean is Chaiiges.{ in ie Solubilities of the Lipoids in presence of one another, and of certain unsaturated organic substances; (+) The Balancing Action of Certain Pairs of Haemolysers i in By PAGE Preventing Haemolysis ; ; (c) The Protective Action of Serum Proteins against | Haemolysers ; (d) The Effects of Oxidising and Reducing Enzymes upon Haemolysis, By Benjamin Moore, M.A., D.Sc., Johnston Professor of Bio~-Chemistry, University of Liverpool ; Frederick P. ae M.D. So . ; and Lancelot purest: as M.D. (Liverpool) : Observations on the Hsemidbyiic mee of Ceftsin Bile ‘Deriestives By Hugh MacLean, M.D., Carnegie Research mm os fame pr of ap sas and Lancenrt Hutchinson, M. D. (Liverpool) The Pharmacology of ge bare Citiebaaehe By J.C. w. Graham, MA, M.D., B.C. (Cantab.) The Physiological Effects of Bclesitid Cieiiscuidil with Relation to ine po . on Glycogen and Sugar Derivatives in the Tissues. ed Charles O. dares M.D. (Liverpool) The Effect of Work on rank ecl. Colic of Muscle. and E. P. Cathcart The Action of Extracts of the Pivcitary Body. By H. H. Dale, M.A., M D. By T. Gish Bind . A Method for the Estimation of the Urea, Allantoin, and Amino Acids in the Urine. By Dorothy E. Lindsay, B.Sc., Carnegie Research Scholar _ , On the Nature of the so-called Fat of Tissues and Organs. By Hugh MacLean, e M.D., Carnegie Fellow, University of Aberdeen, and Owen T..Williams, M.D., | B.Sc. "(Lond .), M.R.C.P., Hon.. Assistant he lacie: Hoepita} for Consumption, 4 Lecturer in Pharmacology, University of Liverpool The Osmotic Pressure of Liquid Foods. By Judah L. ie B. Sc. (Adel) “J The Relationship of Diastatic Efficiency to Average Glycogen Content in the.+~< 52 a = Different Tissues and Organs. Fellow, University of Aberdeen _ tt $554 By — Beieet M.D.,, ee Revearthiy The Osmotic Pressure of the Egg of he Gascon Fowl pe its Changs during Incubation. By W. R. G. Atkins, M.A. (Tri righty College, Dublin) . » 462 ‘dhe ‘ss . SERVATIONS ON CERTAIN MARINE ORGANISMS OF = _ (@) VARIATIONS IN REACTION TO LIGHT, AND (6) A DIURNAL PERIODICITY OF PHOSPHORESCENCE ei sex JAMIN “MOORE, -M.A., D.Sc., Johnston Professor of Bio- me aeeeeese U ey of Liverpool. tag rom the Marine Biological Station, Port Erin, Isle of Man oe fie (Received November 6th, 1908) “he observations recorded in this paper were chiefly conducted upon ) yanisms taken by means of a fine silk tow-net in Port Erin Bay during the spring and summer of 1908.1 In addition certain observations are added upon the reaction to light of young larvae of the plaice _ (Pleuronectes platessa) taken from the Hatchery of the Station. 2 te: ~The experiments on the action of light were made in April, and the ~ attempt in September to investigate the action of light upon the phos- ent organisms then present in the Bay, based on the supposition that organisms which themselves emitted light might possibly show - interesting variations in reaction to incident light from without, led to - the accidental discovery of the diurnal periodicity in the phosphorescence -s of these organisms, which furnishes the subject of the second section of paper. _ The two sets of experiments on the variations in a to light, aay upon the diurnal periodicity in phosphorescence, are really distinct, and will be described in two separate sections. ma A. Vartarioys wy rue Reactions or OrGanisms (Cuierty Nave oF . Batanvs) vo Daytieutr ann Arviricta, Licutr Since the very existence of all living organisms, either directly or directly i is dependent upon the energy of light, and the transformation E this into other types of energy, it is not surprising that reactions to it are amongst the most fundamental and most widely spread ‘throughout the whole world of organized living creatures. Such reactions must have been developed in the very beginning of the dawn when the first living cells commenced to synthesize organic products from the inorganic materials of their environment by the use of the store oe energy from the sunlight. Later on organisms arose which were only Soek., All the sdvesibebhieig eat tn Section B were takén in Port Erin Bay and were surface tow. Prot. Herdman. some of those used in Section A were kindly taken for me outside the Bay by 2 BIO-CHEMICAL JOURNAL dependent upon the light at second-hand, since they were able to consume the synthesized organic products formed by other organisms converting the light energy directly, and so were only indirectly dependent upon the light for their existence. Even for this type of organism, utilizing the light energy indirectly, reactions to light remained essential in the search for food and for other physiological functions, and also there would be an inheritance of relationships to light derived from the earlier ancestry with direct dependence upon light. At a later stage structures or organs arose specially adapted for light reactions, and in those living creatures possessing such organs there probably came a deterioration of the sensitiveness to light of the remaining cells of the body. But in spite of all such decline in direct sensitiveness to light, there must have remained some trace of their old primeval relationships to light. . Experimental evidence of this persistence of relationship to light of all cells exists of two kinds; there is first the deleterious effects of complete withdrawal of light for prolonged periods, and the necessity of sunlight for healthy existence; and, secondly, there is the direct evidence of the effects of application of strong light to animal cells seen in the Finsen effects, and in other forms of radiant energy allied to light. It is, however, in the more lowly organized types of both animal and vegetable organisms that the strongest and most direct reactions to light are observable—apart from the particular case of the reaction of chemical synthesis in the chlorophyll-containing cells of the green parts of the higher plants. Examples of this reactivity are seen in “Te effects of sunlight | upon nearly all types of bacteria; in the sudden outburst of vegetable life in the form of diatoms in the spring of each year as the length of the day increases and the more vertical light reaches and penetrates the water before there is much increase in the temperature of the sea—an outburst upon which the whole life of the sea is as thoroughly dependent as that of the terrestrial world is upon the similar outburst of activity in land plants; and in the most marked movements which occur towards or from the light according to varying circumstances of the minute organisms, either larval or adult, which chiefly constitute the plankton or floating life of the ocean. It is hence clear that the observation of the reactions of living cells to light is of importance both to the student of biology, and to the student of medicine who makes practical applications of the discoveries of biology, using the term in its widest sense. VARIATIONS IN: REACTION TO LIGHT 3 Recent discoveries have proven the value of light treatment as a practical adjunct of medicine, and the study of light effects upon the _ simpler organisms must sooner or later yield a key, both for the rational understanding of such effects, and their extension to further utility. In addition to these utilitarian advantages, the study is one of the most "< oe : a “ : j BIO-CHEMICAL JOURNAL orientation, the conditions under which orientation varies with the condition of the organism and the strength of stimulation, and also the remarkable fact that in higher organisms at any rate there is developed what might be described as resistance to orientation, so that the organisms accumulate either at the proximal or distal point to the light and yet lie in all possible planes of orientation, and, further, that they move about within a certain zone in all possible directions. It is in fact self-evident, and may be taken as axiomatic, that there must have been a certain degree of orientation, or steering, or the organisms would never have been able to move either to or from the light. But this, it is to be observed, is quite different from the organism being turned round when the movement first begins, being definitely held there by the influence of the light in a fixed plane, and then as a result moving towards or away from the light. . The experiments to be recorded later show clearly that there is no such fixed or rigid orientation keeping the organisms in a constant plane, but rather a continually directed control bringing the organism back more or less towards the same direction as it darts about under other varying influences and stimuli, and this on the whole gives steering to the course, so that the animal as a net result moves towards or away from the light. Taking this movement then as a sign of chemical change in the cells of the organism, or certain of those cells, the effects were observed—of exposure of organisms to light of varying intensities, of change in reaction as a result of keeping in light of about constant intensity, of velocity of movement in light of varying intensity, of the effect of light of different colours, and of velocity of movement in such lights, of the effects of converging and diverging light, of the effects of light and shade on organisms in the same vessel, on the association of upward or down- ward movements in level with positive or negative phototaxis, and on | movement in presence of more than one source of light. A very considerable literature exists dealing with heliotropism and phototaxis, but no attempt need be made to quote from this, further than relates to the organisms used for the research, or in incidental relationship to the variations in reaction to light described in the present experiments.! The experiments were made with a free-swimming larval stage of the Barnacle (Nauplii of Balanus), obtained in by far the largest quantity in 1. For a general survey and for literature, reference may be made to Verworn, General Physiology, translation by Lee, 1899; Holt & Lee, American Journal of Physiology, Vol. TV, p. 460, 1901 ; and Loeb, Dynamics of Living Matter, 1906. VARIATIONS IN REACTION TO LIGHT 5 the tow-nettings, mixed with a much smaller number of copepods, and larval spirochaetes The manner in which the organisms congregate at the points of the dish nearest to and farthest from the light was used to pipette them off and separate them from other organisms indifferent to the light, and the positive and negative groups of organisms so obtained were examined separately. Many of the Nauplii were found in both the positive and _ negative groups, but no difference in average size or degree of development _ could be found in the two types to differentiate them, and later experiment _ showed that the same separated group might be artificially varied back- ward and forward between positive and negative according to their previous treatment by light. _ The phototaxis of the Nauplius of Balanus has been examined by , “Loeb, and Loeb and Groom, and Loeb! states that they are positively heliotropic upon leaving the egg, but soon become negatively heliotropic. This I consider is entirely due to over-stimulation by the light, for on keeping for some time in darkness the negative organisms become strongly positive to the same intensity of light in which they were previously negative, and in which part of them left during the same interval have continued negative. The statement of Loeb and Groom that they remain positive in artificial light (gas flame) is confirmed by the results of my experiments, but holds up to a certain intensity of illumination only, for if the light of a small lamp was converged by means of a cylindrical museum jar in which the organisms were contained, the organisms in the strongly illuminated area gradually became negative and passed into the shaded parts or to the distal pole. In later experiments made at Berkeley, U.S.A., Loeb found that tae there behaved differently from those examined in his earlier experiments made at Naples, and showed more complicated reactions. Working with the larvae of Polygordius, and with those of Limulus, Loeb noticed a phenomenon which was also conspicuous throughout the present series of experiments, namely, that the positively phototactic organisms gathered in a group towards the top of the vessel, while the negative organisms at the same time as they gathered away from the light congregated at the lower part of the vessel near the bottom. This I have also invariably observed when a tow-netting is brought into the Station and placed in the diffuse light of a window in a glass jar. ___ The positive organisms are in a compact group nearest to the window, and almost ai the surface of the water; while the negative ones are at the most . 1. Loe. cit, :. = - e DONE Ce Ee a ee 6 BIO-CHEMICAL JOURNAL distal point of the jar from the window and down near, or on, the bottom. The same arrangement holds even in a shallow dish, well illuminated throughout its depth, the positive organisms are up close to the surface, und the negative ones on the bottom of the dish. The arrangement continues when the organisms are lit in the dark room by a candle on the same level as the water—still the positive ones are near the surface and the negative ones near the bottom. I consider that the most probable explanation of this is the constant association, in the natural habitat of the organisms (the sea), of swimming upwards towards the light when positively phototactic, and downwards towards the darker regions of water when negatively phototactic. In addition to the interest of this association on its own account, it seems to me to be valuable as a sign that the light not only affects the | sensitive area on which it acts, but also indirectly affects the whole organism, the chemical changes set up at the sensitive area communicating changes to the whole organism, which stimulate it and cause it to rise or sink in the medium. Loeb, working with Gammarus, found that traces of acid made the organisms more strongly positive, and traces of alkali tended to produce a negative heliotropic effect. I have not been able to obtain similar results with hydrochloric acid, or caustic soda, in Nauplius, although both reagents were pushed to the limits compatible with life, viz., 545 normal. The organisms in the dishes to which either acid or alkali was added seemed to behave exactly like the untreated control. I do not, however, consider this any contradiction of Loeb’s results, since the organisms used were different. Moreover, Loeb’s results are stch as would be expected from the knowledge that alkalies, within the compatible range, excite the activity of living matter, while acids depress it. For, if we regard the light effect as producing an increased chemical activity, then the optimum value of reaction, at which the change would occur from positive to negative as the intensity of the illumination was increased, might be expected to be reached sooner in the case of an organism already made hyperactive by alkali than in the case of an organism where the activity was depressed by added acid. Throughout the whole series of my experiments I have consistently found that the phototaxis is positive with very feeble illumination, and becomes negative as the strength of the light is increased. Further, continued illumination, either by diffuse daylight or by a very bright 1. The limit of acidity or alkalinity compatible with life seems to have nearly the above value for all unprotected minute organisms of either vegetable or animal origin. VARIATIONS IN REACTION TO LIGHT 7 artificial illumination, causes an increasing number of organisms to become negative, and keeping in darkness or in a feeble illumination causes this negativity to pass back to a positive phototaxis. This again is compatible with the view that the effect of light upon the sensitive substances of the organism is always the same whether the effect is shown by a positive or negative phototaxis. The degree of the stimulus determines the reaction of the organism towards it, as shown by the direction of the orientation and consequent movement, but the chemical nature of the stimulus is the same. Below a certain optimum the organism reacts so that the sensitive surface is turned towards the light, that is to say so as to increase the amount of light energy reaching it, and so increase the reaction towards its optimum value for the organism in its condition at the given moment. Above the optimum value of stimulation, the organism conversely reacts so as to turn the sensitive surface into a region of diminished light intensity, and so also to decrease the velocity of the reaction towards its optimum for the organism. This supports the view expressed by Holt and Lee,' that direction of light is only effective in a secondary manner in so far as it alters intensity of light falling upon different parts of the organism, and the orientation is hence primarily a question of intensity of light. The very ingenious experiment of Loeb, showing that an organism which is positively phototactic to direct sunlight will pass from this onward into diffuse sunlight, that is, into a region of lower intensity of illumination, and will not reverse its direction when it finds itself in this region of lower illumination, is quite susceptible of explanation on this view, as well as the result of the experiments given below in this text, upon the movement of negatively phototactic organisms away from the source of illumination in converging light, and still onward past the focus of the light in now diverging light with decreasing intensity. Loeb’s experiment consisted in placing an organism (young cater- pillars of Porthesia chrysorrhaea) in a test-tube the axis of which was horizontal and at right angles to the plane of a window near by, through the upper part of which direct sunlight fell on the more distal portion of the test-tube, while the portion of tube near the window was lit only by diffused daylight. Under such conditions these animals, which react positively, did not halt at the junction of diffuse light and direct sunlight and turn again backwards to the stronger light, but proceeded on in the feebler light toward the incident point right up to the end of the tube, From this experiment, Loeb argues strongly against the anthropo- 1. Loe. cil, 8 BIO-CHEMICAL JOURNAL morphic point of view which would assign any choice to the animal as to whether it sought, or turned from, the light because the light was pleasant to it or the reverse, and urges that the whole process is mechanical or automatic, the animal’s head being turned by the stimulus irresistably towards the light, and the whole movement following inevitably upon this turning. Without assuming any extravagantly anthropomorphic point of view, it may be maintained that the ingenious experiment scarcely supports the interpretation placed upon it, and that the whole matter depends upon the force of the stimulus outweighing the degree of development, of what represents the intelligence of the animal, or, if the expression is more suitable, the development of the nervous system, or, in more general terms still, the co-ordination of the organism. When the animal's body or the sensitive area of it passes from the area of direct sunlight into the less illuminated area of diffuse daylight, in order to turn back into the brighter area of sunlight, the sensitive surface would require for a time to be turned away from even the diffuse light into a region of shadow from its own body, that is to say, it would require for the time to behave as a negatively phototactic animal, and reduce the intensity of illumination of the sensitive area. This supposes a degree of intelligence and of memory for the ‘ pleasanter ’ (or more near the optimum) stimulus which the organism does not possess, and hence it does not turn; but a more highly organized animal would turn, and once more seek the stimulus which suited the organism best. It is such excess of stimulus over organization which makes the moth burn itself in the flame or the bird dash itself to pieces against the lighthouse lantern, and in my opinion this differs in degree of complexity only, but not in kind, from the strength of the irresistible impulse which forces the victim of any drug habit to keep on drugging himself, or leads the unfortunate human being with an incoordinated or improperly balanced nervous system into committing crimes against himself or others. The germs of resistance to stimuli, or rather of reacting so as to alter strength of stimuli, must be present in all living creatures, or life and continuance of the species would speedily become impossible; and it appears to me that denial of this would be nearly as great an error as the view which appears to be held by some opponents of the advance of physiological science, that all organisms and animals are about enna HT sentient to stimulation and to pain. The experiments conducted with organisms under different aaal glasses, described below, in which the relationship of the two halves of the VARIATIONS IN REACTION TO LIGHT 9 dish to the direction of the incident light was identical, also show, from the selection of one-half of the dish by the organisms in preference to the other, that the organisms seek that region where the light activity possesses an optimum for them although there is nothing in the incident - direction of light to lead them to swim under one particular glass as a result of orientation. ‘The same is seen in the experiments of Oltmann! and of Holt and Lee, in which a range of varying intensity of light was arranged by means of a prism placed along the long side of a long glass trough containing organisms. The incident light came in varying intensity perpendicu- larly through the prism, and the organisms were then found to place themselves in certain intermediate positions where the intensity of light suited their optimum, although they had to move to this position practically at right angles to the direction of incidence of the light. _. Experiments were made in the present series of observations upon the velocity with which the organisms moved in light of varying intensity, and also under glasses of varying colour, and it was found that within the limits of the experiment, the velocity of movement was practically constant, thus showing that the chemical reactions set up by the light did not affect the locomotor organs. Description or EXPERIMENTS Experiment I.—A tow-netting was taken in Port Erin Bay, April 2ist, 12-1 p.m. After stirring up in sea-water, it was divided into five portions of 300 ¢.c. each, which were placed in white soup plates and treated as follows :— No. 1.--Control, untreated. N N “ff No. 2,—Added 3 ¢.c, of io HCl, making 7000 solution. ie N . N _ No. 3-Added 6 c.c. of 0 HCl, making 500 Solution. N N No, 4.--Added 3 ¢.c. of 7g NaOH, making [99 solution. y N wie. iN No, 5.-—Added 6 c.c, of i NaOH, making es solution, _ The dishes were left in the diffuse daylight of a north window, and examined after two hours (3 p.m.), the arrangement of the organisms is 1, Quoted by Holt & Lee, loc cit, 10 BLO-CHEMICAL JOURNAL found to be the same in all five plates, showing no change due to acid or alkali, and this persisted throughout the experiment. In each of the dishes there are two prominent groups of organisms, a larger group at the part nearest the window and close to the surface of the water, a smaller, but well-marked group at the diametrical pole farthest from the window and at the bottom of the plate. On shading, for a few minutes, half of one dish with a cardboard, the line of shade of edge of cardboard being at right angles to the plane of the window, in the illuminated half of the plate there is a thick group at the nearest point to the window; in the darkened semicircle, immediately on lifting the card, a smaller group is seen at the point distal to the light, and also there is a diffusely scattered but increased number over all this previously dark half, much greater than in corresponding areas of the illuminated half. Examined again at night (8—9 p.m.) by lamp-light when nearly all the organisms in the plates come to the point nearest the light. Shading as before with shadow parallel to direction of incidence, gives a compact group in the illuminated half near the light, but a great many are in the darkened half which possesses a diffuse group at farthest point from light. Examined again April 23rd, noon (about forty-eight hours from commencement of experiment). ‘Took the control plate of organisms into a south room having direct strong sunlight from an open window. The organisms after a time collect very slightly to sun side, but in the quite open unshaded plate are fairly indifferent, being distributed all over. Now one-half of the dish was shaded by cardboard, the line of shade, as on previous occasions, being arranged parallel to mcidence of the light; at once all the organisms came out into the sunlit half, somewhat more at the point nearest to the sun. On reducing the sunlit part to a very small space, it became crowded with organisms accumulating more densely at the point nearest tosun. This compact group of organisms was pipetted off from the white plate into a black vulcanite half-plate photographic developing dish, containing sea-water, when the organisms, at once almost, accumulate at the part of the dish farthest from the sun. The half of the black dish farthest from the sun, after stirring up, was covered over (that is, with the line of shade.at right angles to plane of incidence), and the organisms all collect, at remotest end of shaded part, away from sun. This peculiar reversal in the black dish is difficult to explain, unless it was due to the absence of reflection. The result could not be repeated in other experiments because the organisms were never again found 1, This is a very exceptional behaviour in sunlight. VARIATIONS IN REACTION TO LIGHT 11 indifferent in sunlight, but always strongly negative, even in the white pistes. : The organisms in this experiment were observed for four days longer ; at the end of the third day they had become very strongly negative in the diffuse daylight of the north window, a reversion, it will be observed, from their original mixed condition with a large preponderance of positive organisms. While in this strongly negative condition they were taken into the dark room and tested with lamplight from a small oil lamp. At once there was a change; three of the five, viz., Nos. 3, 4, and 5, were now altered to positive, while Nos. 1 and 2 were mixed, partially positive and partially negative. The plates were left in the dark room over-night, the only trace of illumination being a very faint ruby light, coming from a small borrowed light through the double thickness of a ruby window and ruby photo- graphic screen. The following morning, as soon as a light was struck in the dark- room, it was seen that all the organisms in all the plates were collected at the points nearest to the faint ruby light. The small oil lamp was lit and the plates arranged round it; all five showed the organisms strongly positive. Taken out immediately from the lamplight to the diffuse daylight of the north window again, the organisms in all five are found to be strongly negative. No interval save the time of shifting the plates out from dark room to window bench elapsed between these two observations with reversed results. Experiment I1.-Tow-netting taken by Professor Herdman, on April 25rd outside the Bay. On standing in diffuse daylight of north window, _ two large groups separate in the glass jar; as usual, one towards light and at top, the other away from light and at bottom of jar. These two groups were separated off by pipetting into two soup plates, one containing the positive group, the other the negative group, and both were found to consist chiefly of Nauplii of Balanus. The negative group was taken first for examination in the dark room. On lighting one candle the organisms swim to the opposite pole; on placing two candles at opposite diameters of the plate, the organisms lie in the middle, half-way between the two lights; with four candles placed around equi-distant, the organisms are clustered compactly at the centre of the plate. The positive organisms similarly examined show a grouping dround the periphery of the plate accentuated opposite each candle. The organisms were left in the dark room overnight and examined in it next morning. On first striking a light, both sets of organisms were ee OO ee : = ; re : Pais R : 12 BLO-CHEMICAL JOURNAL seen clustered at nearest point of each plate to the exceedingly faint ruby light. One candle was lit and placed close to the plate containing the previously negative organisms, these are now nearly all positive to this intensity of light. Next morning (11 a.m.) both sets of organisms, which had remained in the dark room overnight, when tested by candle light were strongly positive. They were at once taken out of the dark-room and placed on the window bench in the north room in fairly strong diffuse daylight (it had been snowing, and the hill across the Bay from the — Station was covered with snow). All the organisms in the originally negative plate were now negative again; those in the originally positive plate were mixed about three-fourths negative and the remainder positive. The two plates were once more carried back to the dark room and tested to candle light. The originally negative plate, which a few minutes before had been conrpletely positive in the dark room to candle light, had now, on account of its short sojourn in the diftuse daylight, turned to partially negative and partially positive in about equal groups. The originally positive group was still all positive to the candle light, although a few minutes previously in the diffuse light of the window about three-fourths of the organisms had been positive. Three points are shown clearly in this experiment. First, that the reaction varies in the same organism at the same time with the intensity of the light, and that feeble illumination gives a positive reaction and strong illumination a negative one. Secondly, with the same intensity of illumination the reaction varies with the previous history and exposure to light of the organism. Exposure to darkness or feeble illumination turns the organism so that it reacts positively, and previous bright illumination changes it so that it reacts negatively to a strength of stimulus to which it before acted positively. 3 | } .. ‘Thirdly, throughout these series of changes the original bias of the particular set of organisms persists, the other effects being superposed in a roughly algebraic summation. Thus the original trends towards positive and negative in the two sets of organisms dawn out again at the end of the experiment. : Experiment L11.—On velocity of movement in light of varying intensity and colour. This experiment on the velocity of movement in light of different intensity and of different colour, was made by observing the time required for the organisms to swim from one end to the other of a flat, black vulcanite dish of rectangular shape. The length of the dish was 17 cm., VARIATIONS IN REACTION TO LIGHT 13 and the organisms were first brought to a compact mass at one side by placing the light to be used at one end and then the time noted for them to swim across and form a similar compact mass at the other end when the source of light was shifted to that end. Then the time was again noted which they require to swim back to their original position; these times are denoted by ‘ Out” and ‘ Back’ the following table. In taking the time ‘ out’ one does not wait for sell organism, but waits till the great majority are in a compact group, this, after a little practice can be done accurately within a quarter of a minute - For different intensities of light, one ordinary paraffin wax candle was used in one case, and four similar candles in the other case. For white light, the dish was simply uncovered, and for coloured lights it was covered completely over with slips of coloured glass, through which the coloured light passed to reach tle organisms. The coloured glasses e which it was possible to obtain were red, green and blue. Regarding the fi total intensity of light passing through the three slips, it appeared to the eye as if the red strip was most obscure, and the green most transparent, the blue being intermediate, but no exact photometric instrument was available. The organisms used were a strongly positive group obtained by pipetting off in diffuse daylight. 1. Illumination intensity = one candle. Redlight ... Time ‘Out’ ... 3 min. 0 sees. Time * Back’ ... 3 min. 0 sees. Blue light ... Time ‘Out’ ... 3 min. 30 sees. Time ‘ Back’ ... 3 min. 20 sees. Green light... Time ‘Out’ ... 3 min. 40 sees. Time ‘ Back’ ... 3 min. 0 sees. 2. Illumination intensity = 4 candles. White light... Time ‘Out’ ... 3 min. 0 sees. Time * Back’ ... 2 min. 50 sees. Red light ... Time ‘Out’ ... 3 min. 0 secs, a Time * Back’ ... 3 min. 0 sees, Experiment IV Selection of position under different coloured glasses with the same direction of incidence. In this experiment, diffuse daylight was used on some occasions and candlelight on others, the long side of the dish being placed parallel to the surface of the window, or next to the candles. Then the two glasses of the two different colours to be compared were placed edge to edge, each covering 4 BIO-CHEMICAL JOURNAL one-half of the dish, the edge where the two slips of glass touched being at right angles to plane of window, so that each half was situated exactly the same as to direction of incidence and intensity of light from the window ; and a similar arrangement was used with the candlelight, the candles being so placed opposite the middle of one of the long sides of the dish that they shed equal light on the two different coloured halves. Before placing the two slips over the dish, the contents were stirred so as to uniformly distribute the organisms, but care was taken that the contents were not rotating when the slips were put over. Also, after the organisms had distributed themselves selectively, and the result had been noted, the two slips were reversed in position, each to each, and the change in distribution observed; the organisms at the time were strongly positive in the candle- light, and strongly negative in the diffuse daylight. First, using four candles in the dark room, and with the red glass on the left-hand half and the blue glass on the right-hand half, in 2 min. 30 secs. from the commencement all the organisms are under the blue glass and next the candles, none under the red glass. The red and blue glasses are now reversed without disturbing candles or organisms, and in a very short time all the organisms have shifted and are once. more under the blue glass in its new situation. Second, similar results obtained with diffuse daylight, except that organisms now swim from the light; with blue and red most of the organisms under blue, a few only under red; with blue and green, two groups form at the two corners distal to the light, the larger of the two groups being under the green. Thus the organisms move with equal velocity under all coloured glasses, but when two colours are offered for selection they accumulate chiefly under one. Further, the direction of movement to pass from one colour to another is across the direction of incidence, and not to or from the light, and the relation to the light of the two halves being the same, it would appear that a preference for a particular colour or wavelength (or the greater or lesser stimulus of different wavelengths), caused the different distribution. If the organisms are carefully watched when they are becoming distributed, it is seen that they do not move directly across from one half to the other, but are moving about apparently freely, an organism every now and then leaving a group and darting off; but there is a certain amount of steering and controlling during these apparently free movements, which ultimately settles them down in their final distribution. . In this type of experiment, observation of the grouped animals shows, as in all the other experiments where the animals are grouped either VARIATIONS IN REACTION TO LIGHT 15 - positively or negatively under the influence of the light, that there is no such thing as fixed and continuous orientation of the minute animals. In every group a great many are moving about in and out amongst one another, and a good many are entering and leaving the group like bees from a hive, but each individual, after a short trip about soon returns to the group. The source of light is, in fact, a strong directive influence, but there is no rigidly fixed orientation, any more than there is in a cluster of midges, or a brood of chickens around their mother. Experiment V.—Movement in converging and in diverging light. In order to obtain converging and diverging light, cylindrical museum jars, about 10-5 centimetres in diameter and 18 centimetres high, were used, which happened to be in stock at the Station. Two such jars were used; the first, filled with clear fresh water, was used only as a cylindrical water lens, and contained none of the organisms; the second jar contained the organisms in sea-water. The first cylinder was placed a variable short distance, up to about one foot, from a small oil lamp with a circular wick, and the second cylinder was placed close up against it, on the other side from the lamp. The lamp and two cylinders were so arranged that the diverging light from the lamp became slightly convergent in passing through the first cylinder, and being still further converged by the second cylinder, it formed a caustic about two-thirds to three-fourths of the way through the second cylinder, and from that onward to the concave surface of the second cylinder the light was diverging. By this arrangement any organism moving along the path of the rays, either towards or away from the light, is forced in one part of its path to travel in converging light, and the remaining part it travels in diverging light. Experiments were carried out both with white light and with coloured lights. The first set of organisms examined were negative; these swam away from the light into light of increasing intensity towards the caustic, and then through this onward in light of decreasing intensity till they reached the glass surface most remote from the light. Positive organisms were next tried, and swam in the exactly reverse direction, first from the most distal part towards the caustic in converging light, and therefore of increasing intensity, and then onward in diverging light, therefore of decreasing intensity, up to the glass surface nearest to the light. At first sight it looks proven from this that intensity of light is of no effect, and the direction of incidence the whole matter, because the 16 BIO-CHEMICAL JOURNAL organisms appear to swim in one direction indifferently, whether the illumination is increasing or decreasing. In reality, however, such a conclusion would be fallacious, for in order that, say, a positive organism should turn when it began to swim in light of gradually decreasing intensity, it would be necessary for it to turn its sentient surface away from the light, and that would plunge it into darkness. 1 The true conclusion is shown by what might be termed secondary — effects seen on carefully watching the above experiment with negative he organisms. These organisms at first accumulate in the narrow band of light at the distal glass surface from the light, where they dart about in sh small curves, keeping close to the glass; but in a few minutes it is found & that a great many of them have accumulated in the two shady margins rs just outside this strongly illuminated band, and on either side of it. The ; probable explanation of this is that for these negative organisms the 2 feebler light outside the band is nearer the optimal stimulus, and when | - they escape from the direct light beam in the course of their “ peregrinations, they find a suitable stimulus in the feebler light. But b when any accident, such as a chance movement stimulated by some other = “4 cause, sends them again into the beam, they are stimulated to turn away ; i from the light, and must again return via the distal glass surface to the | di refuge of the shade again. My This effect is seen still more strikingly when the red glass strip is a interposed on the path of the incident light; then scarcely a single bes organism is seen on the illuminated strip, but two packed masses are seen . ie on each side of it in the shade, and gradually tailing off as the distance « <= from the illuminated strip increases. Similar results are seen with negative organisms if a narrow opaque white strip, such.as a strip of cardboard, be lowered into the jar and held in a vertical position at the caustic. When the light is now placed in position, any organisms in the course of the beam, or swimming into it from the two dark zones at either side of it, turn at once away from the light, and swim along the path of the rays towards the caustic and the card; but they do not accumulate to i any appreciable extent at the card, they swim round its edges and alll accumulate in the narrow feebly-lit space behind it. 4 » oo ge Experiment VI.-With young larvae of the plaice (Pleuronectes ae platessa). or : A number of young plaice larvae, which were five to seven days” ‘lf 3 were taken from the Fish Hatchery attached to the Station, and placed i in ? sea-water in a flat, oblong pie-dish. It was found that they were faintly VARIATIONS IN REACTION TO LIGHT 17 “negatively phototactic in diffuse daylight. Contrary to the case of the Nauplii, this appeared to be increased in lamplight as well as in direct sunlight. When the dish is brought into lamplight in the dark room, it is found that most of the larvae after some time are accumulated in the half of the dish farthest from the lamp, decreasing to a clear space directly e. under the lamp. There is, however, no such tight packing up as in the ease of the Nauplii. ‘ - ‘The interesting point, however, is that there is no evidence whatever _ of orientation in regard to the light; the larvae lie at rest with their nz long axes at all possible angles with the line from the lamplight, some = directly facing it, some straight away from it, others nearly at right angles, and many indiscriminately at all angles. The arrangement is not a chance one, as it looks at first sight, for no matter how often the larvae are disturbed and stirred up, they finally settle with the great majority in oe the distal half, and lying there at rest at all angles to the direction of incidence. On shading one-half of the dish with cardboard, the line of _ shade being parallel to the plane of incidence, the great majority of the larvae are found in the shaded half, more in the distal quadrant, and in all lines of orientation. If cards are arranged so that one quadrant of the dish only is illuminated, that quadrant becomes almost free. ee ee _ Eaperiment VII.—Indifference of phosphorescent organisms to movement in light from without. ry It was thought that organisms which themselves emitted light might " show interesting results in their reactions to light from without, and this 2 led to the work of Section B about to be described; but it was found that _____ the phosphorescent organisms present, probably certain copepoda, were ___ entirely indifferent to incident light, at any rate as far as movement was concerned. Since the organisms could not be made to phosphoresce in the dark room during the day, the procedure was adopted of taking a tow-netting 4 during the day, when the Bay was known, by observations made during the od _ previous night, to contain abundance of phosphorescent organisms. This ___ tow-netting was placed in diffuse daylight, and nearly all the positive organisms were pipetted off into one dish containing sea-water, nearly all _ the negative organisms were similarly pipetted into a separate dish, and finally, a good number of indifferent organisms were pipetted off into a third dish, from the middle of the bottom of the stock jar. The three sets of organisms were then examined for phosphorescence _ after dark, when phosphorescence where organisms were present had 18 BIO-CHEMICAL JOURNAL spontaneously set in and could be further intensified by stirring. It was then found that the positive and negative portions each contained only one or two phosphorescent organisms taken up unavoidably with the others; but the indifferent set contained a large number of phosphorescent organisms. The indifferent set containing the majority of the phosphorescent organisms were also practically indifferent to candle-light. In regard to numbers of organisms in each set, the positive set were by far the most numerous, and the numbers in the indifferent and negative sets were about equal. The experiment was varied in a fresh tow-netting by placing several flat pie-dishes containing the organisms (not separated off on this oceasion as to phototaxis) around the lamp in the photographic room, just after nightfall, until the usual phototactic groups had separated, then extinguishing the lamp, and watching the spontaneous appearance of phosphorescence without disturbing the dishes. There is no spontaneous phosphorescence for a period of about two minutes under such circumstances, then it commences, and it is seen that the phosphorescent organisms are scattered about indiscriminately in each dish, and not arranged in any relationship to where the light had previously been. Sometimes the phosphorescing organisms are moving about rapidly while illuminated, but in the majority of cases they are almost or quite at rest, and it is probable that if there had been any previous movement of a phototactic character while the lamp was lit, the arrangement would not have quite disappeared in the short interval after the light was extinguished before the spontaneous phosphorescence reappeared. The only conclusion from the experiments appears to me to be that these particular phosphorescent organisms are almost or quite indifierestt to incident light. B.—Drvrnat Pertopicrry tv PHosPHORESCENCE The suggestion of the work described in this section arose incidentally, - as above-mentioned, and at the time the experiments were made it was unknown to me that a diurnal periodicity in phosphorescence had previously been observed and described. : A search through the earlier literature, however, revealed a description of its occurrence in Pyrophora by Aubert and R. Dubois,! and in Noctiluca by Massart.2 Henneguy? states that Noctiluca does not 21. Compt. rend, acad., T. XLIX, p. 477, 1884; Compt. rend. soc. d. biol., P 661, 1884. See also papers i in both these Journals by R, Dubois, 1884-6. Bulletin scientifique de la france et de la belgique, T. XXV, p. 72, 1898. Compt. rend. soc. d. biol., XL, p. 707, 1884. re ‘DIURNAL PERIODICITY OF PHOSPHORESCENCE 19 li , h up until it has been kept in the dark for half an hour, and that the ntensity is not at the maximum for another additional half-hour. ae The following passage from Massart describes the variations as in Noctiluca :— _ ~~ +‘ The experiments show that the irritability is dependent on the alternations of day and night, the Noctiluca is hardly excitable on shaking ring the day and shines only during the night. Fact still more curious, the organisms are submitted to the alternations of day and night, a » whether they are maintained in constant illumination or constant _ obscurity, they still remain much more excitable during the night than during the day. It is a veritable phenomenon of memory, everything looks as if the Noctilucae preserved the recollection of the regular succession of the days and nights.’ ______ Massart compares this to the change in position of the leaves of plants . during day and night in the Oxalis and certain Papilionaceae, but adds _ that while the phenomenon lasts only some days in plants, in the ‘ | Bettitncee it lasts until the death of the animal. His experiments at the outside limit, however, lasted for one week “aay, when the organisms died; in the present set of observations the - diurnal alternation of activity was followed with organisms kept in continuous darkness for twelve days, and although the number of living Organisms was decreasing all the period, a few were still left alive and __ phosphorescent at night at the end of the period. Since the fact of this diurnal periodicity is one of the most striking of those alternating habits or functions of the lower invertebrates which bear _ such a curious resemblance to memory in higher vertebrates, and, indeed, have been regarded as a rudimentary memory,! it may be regarded as sufficiently interesting to merit a detailed description. It appears to stand some danger of being forgotten, since it is not mentioned even in the . of the modern text-books, and to the best of my knowledge it has ae " ot been shown to exist in the phosphorescent copepoda, nor demonstrated as persisting for such a long period as in the present experiments. Also its onset at the close of the day and gradual extinction at dawn have not previously been followed with any exactitude. 1. . See F, Darwin, Presidential Address, Brit. Association, Dublin, 1908. 20 BIO-CHEMICAL JOURNAL Diary or ExperIMEN’S Monday, September 21st, 1908 (8-30 p.m.)—Calm night, and sea very phosphorescent. Collected plant (Polysiphonia nigrescens) from the rope of an old mooring buoy. The plant is covered over with phosphorescent organisms which flash most brilliantly. The specimen is preserved in sea-water and examined ashore. It shows most brilliant phosphorescence when rubbed. When a piece is put in fresh tap water in the dark it lights up most brilliantly all over for about three minutes, then gradually the light fades out, and cannot now be evoked by any process of shaking or rubbing. | Tuesday, September 22nd.—The plant was taken into the dark room at 11 a.m. and examined; no phosphorescence could now be evoked by any process, either shaking in air, stirring up in the sea-water, rubbing, or applying fresh water. A tow-netting had just been taken in the Bay (12 noon). This was taken into the dark room at once, but no trace of phosphorescence could be obtained from it, even with most vigorous stirring. In the evening, from 9 to 9-30 p.m., a tow-netting was taken in the Bay, the sea being very phosphorescent wherever touched by the oars. The haul, when taken into the boat, scintillated most brilliantly while being washed into sea-water in a jar. The contents of the jar, taken into the dark room at the Station, are showing spontaneous phosphorescence, and give a vivid show when stirred. Left in the dark room oyer-night. Wednesday, September 23rd—Vixamined the previous night’s tow- netting at 11 a.m.; there is not a trace of phosphorescence to be elicited, even on stirring briskly. Examined at intervals all day in the dark room. There is not a trace of phosphorescence seen till about 6-30 p-m., when sparking first starts on stirring, just as it is growing dusk outside, and at 7 p.m. there is spontaneous phosphorescence. Took also during the day three tow-nettings from a row-boat, each of 15 minutes’ duration, at 12-45 to 1 p.m., 3-45 to 4 p.m., and 5-15 to 5-30 p.m. As each tow-netting was finished, it was taken to the Station, at once emptied into a flat pie-dish, and taken to the dark room to be examined for phosphorescence. On each such occasion the tow-nettin previously there were also examined, as also at other intervals during the _ day. In none of the three was any phosphorescence seen till about 5-40 p.m., when a single organism was seen to spark in the second tow- netting (taken 3-45 to 4 p.m.), but nothing in the first or third. Examined at 6-45, when it is dusk outside, all three are DIURNAL PERIODICITY OF PHOSPHORESCENCE 21 a phosphorescing spontaneously, bright sparks showing up, sometimes three or four at once in each dish. On stirring there is a bright display lighting up each dish. “All three left over-night in dark room. ~ Thursday, September 24th—Examined at 10 a.m., none of the three tow-nettings show any phosphorescence in the dark room. Nos. 1 and 3 were kept in the dark room all day, while No. 2 was kept in the daylight, but taken at intervals to the dark room for examination. No ___ phosphorescence seen in any of the three at any time during the day; but sat night (7 p.m.) all three are sparking spontaneously, showing bright ___ sparks at intervals. The phosphorescence is increased on stirring, so that six to ten phosphorescent spots are visible at once, but the display is not so brilliant as on the previous night, probably owing to deaths. All three left in dark room till Friday morning; the faint ruby light _ from the dark room window is completely shut off by banking it up with eardboard on the outside. This same day, being a bright day with good sunlight, three additional tow-nettings were taken, at 11 to 11-15 a.m., 12-45 to 1 p.m., and 4-45 to 5 p.m., and examined in future along with the other three, 4 being kept in dark room also. Examined as follows in dark room :— . No. 1 observed at 11-30 a.m. efi ... No phosphorescence. a _ Nos. 1 and 2 observed at 1-15 p.m. ... ... No phosphorescence. " Nos. 1, 2 and 8 observed at 5-10 p.m. ... No.1, Nil; No. 2, single - 4 | spark on vigorous a stirring; No. 8, Nil. Nos. 1, 2 and 3 observed at 5-35 p.m. ... Nil; single-spark ; Nil. (Good light outside). Nos. 1, 2 and 3 observed at 6-35 p.m. ... Spontaneous sparking (Almost dark outside). in all three, No. 8 most brilliant. Dish lit up in each case ) on stirring. _ Nos. 1 2 and 3 observed at 6-50 p.m. ... All spontaneously phos- (Quite dark outside). phorescing most brilliantly. Also at 1 p.m. to-day, a further supply of Polysiphonia nigrescens was collected from the old mooring rope, and examined in the dark room. It showed no phosphorescence during the day. On placing in distilled water it gives a feeble sparkling, but incomparably less brilliant than on 22 BIO-CHEMICAL JOURNAL similar treatment at night. After dark, from 6-30 p.m. onwards, the same sample sparkles when stirred, and a piece put in distilled water lights up brilliantly all over for from three to five minutes; then the light dies away, and cannot further be evoked in that piece by any of the Se mentioned, All six of the tow-nettings of yesterday and to-day examined again at 7-15 p.m.; all spontaneously phosphorescing, and showing up me on stirring. Same result when again examined at 8-40 p.m. Friday, September 25th.—Arrived at Biological Station at 4-50 a.m; there is just a trace of dawn in the dull, grey sky. Organisms examined at once in the dark room, where they have all still been kept over-night; all six dishes are flashing spontaneously. On standing quietly by and watching the phosphorescence, the minute organisms are not moving about rapidly in most cases, and one can observe that each active organism is emitting a series of flashes at about the rate of one per minute, and between the flashes there is a dimmer light showing which regularly becomes increased by a flash. The effect on the eye is very similar to that of a revolving light seen at sea at some distance off. There is an almost constant dim light lit up by repeated and fairly regular flashes. Many of the more active organisms are so still that one is able to observe clusters of four or five in nearly constant positions for some minutes, so as to give an impression of constancy of shape to the group for the time resembling a stellar constellation. The effect in the complete darkness of the dark room is very beautiful as the undisturbed organisms spontaneously flash out in the darkness. The organisms were now observed at frequent intervals of about ten minutes, in order to accurately note the decline and disappearance of the phosphorescence. It was observed that the number of organisms flashing out was decreasing all the time. The rate of decrease became very rapid about 5-30 a.m., when the daylight was just beginning to grow rapidly | brighter outside. At 6 a.m. there was only an occasional odd flash in each dish, showing that only a few organisms in each were still active. At 6-15 a.m. only one dish (the third of those collected on Thursday) was still showing an occasional gleam; all the other five dishes had stopped spontaneous phosphorescence. At 6-30 a.m. all spontaneous phosphorescence had disappeared, but a faint display could still be elicited in all six dishes by vigorous stirring. At 7 a.m. no sparking obtainable in any dish, even on most vigorous stirring; same result repeated at 7-30 a.m. DIURNAL PERIODICITY OF PHOSPHORESCENCE 23 ‘The organisms on the Polysiphonia nigrescens behave similarly to the : free organisms in the dishes throughout. It was feared that the organisms ___ would perish if the sea-water were not changed, so Nos. 1 and 2 of the Wednesday tow-nettings were filtered in the dark room through the silk of the tow-netting, and then the net being turned (so that no fresh organisms could be introduced), the organisms were washed into a fresh ; quantity of sea-water poured on to the net. Hence there were in future _ five dishes to observe instead of six, but no alteration in rate of survival - on ‘account of the changing was observed, and, as the other dishes of _ organisms appeared to be doing well, the process of washing into a fresh supply of sea-water, which was exceedingly difficult and awkward in the quite dark room, was abandoned. The organisms were next examined at 1 p.m., when vigorous stirring failed to call forth a single spark in any of the tow-nettings or on the weed. Bi _ The next examination was at 9 p.m., when every one of the dishes ag showed spontaneous phosphorescence. ‘The display in the three Thursday 2g nettings is not so vivid as on the previous night, there being fewer sso ganisms phosphorescing. It is also noticeable that the phosphorescence ig not so vigorous in each individual organism. The flare out is perhaps as great, but the light completely dies out in all cases after each flare, and the period between the flares seems to be lengthened, so that one cannot pick out a particular organism by its flashes and keep track of it. The two dishes from the Wednesday tow-nettings, which are to-night showing for the third time, are not much decreased in vigour from the second night, either in frequency of spontaneous flashing or in vividness on stirring them. Nearly as many phosphorescing organisms appear to be present, and the flashes are about as bright as on the preceding night.! _-—C hese Wednesday organisms have now lit up for the third time, _ having been quite quiescent in the intermediate periods of daylight in the _ outer world. One of the two dishes has been in complete darkness _ throughout the period. From this onward all the sets of organisms are ome _ kept in complete darkness the whole time. __—‘Saturday, September 26th.—The organisms were examined at 11 a.m., _ and again at 1 p.m., when no sparking was occurring, nor could any be ____ evoked by vigorous stirring. The next observation was commenced at 6-07 p.m., when the daylight was commencing to fade outside. The dishes were not stirred, but quietly watched in the complete darkness. When the first spontaneous flash occurred, the dark room was quitted and the time 1. This was observed in nearly all the ts, t drop during the first twent four hours, and then a very slow re Paes in the residue. a great drop ig the first twenty- 24 BIO-CHEMICAL JOURNAL noted; it was 6-13 p.m. Between 6-15 and 6-30, six flashes were counted ; between 6-30 and 6-45, twenty-two flashes; between 6-55 and 7-15 p.m., there were twenty flashes. The display is much less marked than on the previous evenings. On stirring the dishes, three or four organisms can be made to phosphoresce at once in each case. The organisms on the Polysiphonia nigrescens are also phospherssai on stirring. Sunday, September 27th—Kxamined at 10-30 a.m.; no _phos- phorescence, either spontaneous or on stirring, from any of the dishes. Re-examined at 7 p.m., four of the dishes show spontaneous phosphorescence, the rate of sparking being extremely slow. The remaining dish (the third of the Thursday tow-nettings) has undergone putrefaction, and shows no phosphorescence, even on stirring. It is taken from the dark room, and all the organisms in it are seen to be dead. Stirring elicits two to four phosphorescent organisms at the same time in the remaining four dishes. Monday, September 28th.—Examined the four dishes at 2-30 pam. ; no phosphorescence obtainable from any of them. Examined again at 7-30 p.m. ‘Two spontaneous sparks seen in the Wednesday dishes in an interval of about five minutes; no spontaneous phosphorescence seen in - the Thursday dishes. On stirring, about six phosphorescent organisms seen in one of the Wednesday dishes, and three or four in the other; one seen in the first of the Thursday dishes, and three or four in the second. Tuesday, September 29th.—Examined at 3-30 p.m.; no phos- phorescence visible or obtainable. Examined again at 9 p.m., there is spontaneous phosphorescence in both of the Wednesday dishes, and in one there is an organism which remains steadily phosphorescent with a dull glow all the time. On stirring, about six phosphorescent organisms are visible in each of the Wednesday dishes, and the sparking is brilliant. In the Thursday dishes, on stirring, there is less display, only two or three organisms showing up at-once in either. The few organisms are, however, quite active, and a single organism in each case lights up so as to illuminate the contents and sides of the whole dish. Wednesday, September 30th—Examined at 11 a.m.; no _phos- phorescence, spontaneous or otherwise. Examined again at 7-30 p.m., no spontaneous phosphorescence during a period of about 5 minutes, but on stirring there is a good display in all four dishes. This is the eighth night of appearance of phosphorescence in the Wednesday lots, and seventh night for the Thursday organisms. Thursday, October 1st—Examined the four dishes at 11 a.m.; no DIURNAL PERIODICITY OF PHOSPHORESCENCE 26 phorescence, spontaneous or on stirring. Examined again at 7 p.m., a fare is spontaneous phosphorescence at a slow rate in three (two * Wednesday and one Thursday), and in all four on stirring. { Friday, October 2nd.—Examined at 3 p.m.; no phosphorescence, either spontaneous or on stirring. Examined again at 9 p.m.; in one of the Wednesday dishes there is an organism which remains permanently lit up the whole time of observation, about seven minutes. Spontaneous phosphorescence seen in the other Wednesday dish, and in one of the gems dishes. All four give phosphorescence on stirring. This is the second time a continuously phosphorescent organism has : ee observed. It may be a pathological condition of the organism.. : Saturday, October 3rd.—Examined at 4 p.m., no phosphorescence of 2 my kind; did not examine after nightfall this day. i ae - Sunday, October 4th——Examined at 12 noon, no phosphorescence in ny dish, either spontaneously or after vigorous stirring. Examined again at 6-20 p.m., and watched at intervals till 8-30 p.m., but there is no g ‘spontaneous flashing. On stirring, however, there is phosphorescence obtainable in each of the four dishes, one or two organisms only flashing in each case. ‘The experiments were brought to an end at this date. When the dishes are taken to the light it is found that only a small number of a organisms are visible and alive in each, and there is much débris of dead | organisms. _ The diurnal periodicity of the phosphorescence had been observed for _ twelve days and nights in the case of the organisms collected on Wednesday, September 23rd, and for eleven periods in the case of those collected on Thursday, September 24th, without any exception. During ___ this interval, with the exception of one of the Wednesday dishes which had been exposed to light on the first Thursday of the period, all the dishes were kept in continuous darkness, yet at the close of the day the ga always lit up, and lights were extinguished about daylight in . > morning. ____ ‘The four dishes of organisms were now filtered one after the other into RS the small end of the same tow-net, washed out into a little sea-water, and _ fixed with five per cent. formol. a The fixation was carried out in the dark room in order to observe if there was any phosphorescence. About six bright points shone out, two of which persisted brilliantly for about three minutes, and then faded out. a The weed (Polysiphonia nigrescens) was kept in the dark from the Thursday (September 24th) till Wednesday (September 30th), showing 26 BIO-CHEMICAL JOURNAL phosphorescence at night and none during the day. Fearing that it would decompose, it was then placed in ordinary diffuse daylight in a vessel with running sea-water. This treatment increased the amount of phosphorescence enormously, and in a day or two it Was quite as phosphorescent as at first. Taken from the diffuse daylight to the dark room for examination, it was never phosphorescent, but at night it always phosphoresced most brilliantly. It, also, at the end, was fixed in 5 per cent. formol, and in this process lit up about twenty seconds after the application of the formol, and shone vividly for about three minutes before dying out. The examination of the united tow-nettings was difficult on account of the majority of the organisms being dead and in a broken-up condition through the long duration of the experiment, but the following account was kindly given me by Mr. A. Scott, to whom my best thanks are due : — Diatoms.—-Biddulphia mobiliensis, 1,000; Chaetoceros densum, 50; Coseinodiseus radiatus, 50; Trochisea sp., 250, Corrrops.—Calanus helgolandicus, 20; Pseudocalanus elongatus, 680; Temora longicornis, 100; Centropages hamatus, 10; ‘Paracalanus parvus, 100; Isias clavipes, 100; Copepod nauplii, 100; Copepod Juv., 200. . Moxtvsca (larval).__Gasteropods, 150; Lamellibranchs, 500. No Noctilucae were present. It is not probable that ‘the diatoms or molluscan larvae were phosphorescent, so that there is little doubt that the phosphorescence was due to the copepods present, or certain species of these. The following is a statement of the contents of the routine tow- nettings always taken of the plankton of the Bay, for the statistical work - of the Biological Station, on the date (Thursday, September 24th) when the second set of tow-nettings were collected for the observations :— Diatoms.—Biddulphia mobiliensis, 800;° Chaetoceros decipiens, 600; Ch. densum, 440; Coscinodiscus radiatus, 50; ite thamensis, 150; Trochisea sp., 50. DInoFLaGELLatTa, &e.—Ceratium furca, 50; C. fusus, 100; C. tripos, 100; Tintinnopsis sp., 600. DIURNAL PERIODICITY OF PHOSPHORESCENCE 27 _ Corgropa.—Calanus helgolandicus, 50; Pseudocalanus elongatus, 3,180; Temora longicornis, 280; Centropages hamatus, 65; _ Aeartia clausi, 2,200; Oithona similis, 1,750; Paracalanus parvus, 830; Isias clavipes, 160; Copepod nauplii, 3,960; d Copepod juv., 2,180. - Morzusca, &c.—Lamellibranch larvae, 280; Oikopleura, 3,800. Whether this diurnal periodicity has the same physical basis in a rudimentary fashion as memory in higher animals, is still an open question, for it is open to believe that the alternating play of light and darkness upon those cells which produce the phosphorescence may have induced in them a periodicity of activity and rest which still persists after the alternating stimulus is withdrawn. The process may, for example, be __ due to a secretion by certain cells which phosphoresces as each drop is produced, and this process of secretion may have a period of rest during _ the day and activity during the night. The rhythm of this activity may be timed daily under ordinary conditions, and regulated by alternation of ___ light and darkness. During the day there would be storage in the cell, and at night discharge. On the removal of the stimulus of light during the day this state of alternation of rest and action might persist for a long ‘li CoNncLUSIONS 1. The characters of the response of an organism by movement to light are not constant for a given organism, but vary for the same organism at the same time according to the intensity of the light and the ____ previous history of the organism in regard to light. As a general rule, ____ the organism is positive to feeble light and negative to stronger light, and _ for a constant intensity of light at a given moment previous darkness or _ weak stimulation tends to turn organisms positive, and previous exposure to bright light turns them negative. 2. Both the positive and negative behaviour to light may be explained on the basis of one chemical action of light upon the cell _ (akatabolic one). The positive state indicates that the speed of reactions in the cell lies below a certain value, which may be called the optimal value, and the negative state corresponds to a speed of reactions in the cell above the optimal value. In the former case the sentient surfaces are turned into the light to increase velocity of reaction up towards the optimal value; in the latter case the sentient surfaces are turned away 28 BLO-CHEMICAL JOURNAL from the light so as to decrease the velocity of the reactions down towards the optimal value. 3. Asa result of the orientation so caused, there arises movement of the organism towards or away from the source of light, but such orientation is not a fixed orientation, but rather a steering action; the animals as a result do not remain in one fixed plane or direction of movement, but the net result of the movement is that the organisms move to or from the light. When the movement is finished the organisms (plaice) may lie in all possible planes of orientation to the light. 4. Movement towards or away from the light has in some organisms (Nauplii of Balanus) an associated movement upwards or downwards. These two movements would coincide together in natural movements of the organisms under the influence of light alone in the sea. 5. In the case of Nauplii of Balanus, addition of small amounts of acid or alkali was not found to alter the reactions to light. 6. The rate of movement of the organism (Nauplii) is almost the same with different intensities of light and different coloured lights, showing that the locomotor apparatus is not affected by the light, but continues to work at the same rate. 7. The particular organism used (Nauplius of Balanus) moves from red light to blue light, and from blue to green, under such circumstances that the incident light is the same in direction for both coloured regions. This would indicate that with the particular total intensities being used for the experiments, green is a more suitable or optimal stimulus than blue, and blue in turn more optimal than red. 8. Movement in converging and diverging light is described and shown to be explicable on the basis of intensity of light alone, and that direction produces its effects in a secondary manner on account of the light and shade effects of the animal’s own body. 9. The phosphorescent organisms experimented with (certain copepods) were shown to be indifferent, in regard to movement, to light from without. ~ 10. That light from without has another type of influence upon these phosphorescent organisms is shown, however, by the fact that their periods of activity and rest in regard to phosphorescence follow respectively the hours of daylight and darkness. 11. It is shown that this alternating diurnal periodicity can persist for a long period (twelve days) in absence of the accustomed recurring stimulus of the light and darkness of day and night. 12. The phosphorescence of these copepods in captivity is “4 DIURNAL PERIODICITY OF PHOSPHORESCENCE 29 neous, and although increased by mechanical stimulation, it goes on sie even. ee the organisms are undisturbed and quite still. ; the organisms are freshly taken, the character of the »sphorescence is such that a faint light persists, which is increased at als by bright flares or flashes. Ata later period the light disappears ely between the flashes, which have a longer interval between them. ide "probably pathological conditions, after the organisms have been pt confined for a considerable period, there may be lighting up of the gan nisms with a continuous glow. _ The appearance of the spontaneous phosphorescence at nightfall, gpa at dawn, are characterised by the same changes in a reversed order in the two cases. Before the appearance of spontaneous osph 0. nce at night, and after its disappearance in the morning, | eta: of minimal excitability of about half an hour during tiring still calls out phosphorescence. here this the organisms om mnpet refractory. : ; _ Addition of fresh water, or formol, produces, during the period | which the organism is dying, a most vivid phosphorescence, which lasts 1 two to three minutes, and then fades and disappears. ris display is very feeble during a daylight period, compared to what er » after dark when spontaneous phosphorescence is present. ae : ee ——— 30 THE RELATIVE IMPORTANCE OF INORGANIC KATIONS, ESPECIALLY THOSE OF SODIUM AND CALCIUM, IN THE CAUSATION OF GOUT AND PRODUCTION OF GOUTY DEPOSITS' By WILLIAM GORDON LITTLE, M.A. (Aber.), M.D. (Edin.) From the Bio-Chemical Department, University of Liverpool (Received November 14th, 1908) As long ago as 1844, Ure? suggested that calcium salts and sodium salts were deposited in gouty conditions; the sodium as biurate in the synovial membranes and tendons, and the calcium as phosphate in the arterial walls. From the predominance of sodium salts in the fluids of the body it is to be expected that the bulk of any salt should contain that kation either in solution or as a deposit; but the more insoluble any salt-forming anion is, such as that of uric acid, the more important does the presence not of one but of several kations become; for upon such multiplicity of kations does the carrying power of the solvent for the feebly soluble anion depend. The great importance of the relative effects upon one another of these kations, in common solution with uric acid, in dissolving or precipitating the uric acid anion, has not been sufficiently realised, and the solubilities under such conditions have not been sufficiently investigated. The object of the present paper is to supply some further information on this question of solubility and precipitation of uric acid anion as acid salt, from the common solution containing more than one kation. The various salts of calcium and uric acid have been described by Delepine? at length, as well as their occurrence in urine and tissues. He describes (1) an acid salt (biurate) which is comparatively soluble in water, and (2) a basic or neutral salt (normal urate) comparatively insoluble. He then quite justly remarks that this is a reversal of what obtains in the case of alkaline urates, presumably those of sodium and potassium, and calls attention to the evident importance of this in relation to the reaction of the solvent medium. Following Heintz, Delepine and other authors, quote calcium biurate as a highly soluble biurate, being even more soluble than the potassium biurate. 1. Part of the expense of this research has been defrayed by a grant from the British Medical Association. 2. Medical Times, Vol. XI, p. 145, 1844. 3. Journ. of Physiol., Vol. VITI, 1887. INORGANIC KATIONS IN GOUT 31 Thus Neubauer and Vogel give the following figures for solubility of _ different biurates in water in the cold and at boiling point :— ———— Lithium biurate, 1 part dissolves in 370 parts cold, and 39 parts boiling water. Calcium » 1 9s . 603 - 276 Potassium ,, Em 3 oa 790 ~ 75 Sodium a 1 ee mn 1,150 = 112 Magnesium ,, =|, bs 3,750 n 160 The fact that calcium biurate occurs in all tophi, as shown by Ebstein and Sprague,! and usually to the extent of 12 to 15 per cent, alongside of about 57 per cent. of sodium biurate, is sufficient to imdicate that the statements and figures given above as to the solubility of calcium biurate, do not represent the true state of affairs accompanying deposition in the body, which must occur at body temperature, and from media which, me rich in sodium chloride, may behave quite differently as a solvent - from distilled water. It is peculiar that no experiments have been recorded as to the ~ solubility at body temperature, and from salt solutions. Such experi- ments I have undertaken, and have found, first, that caleium biurate must be placed even lower than magnesium biurate at the bottom of the _ seale of solubility of biurates, when the solubility is measured in distilled water kept at body temperature in a thermostat; secondly, that mere traces of a calcium salt added to solutions of sodium biurate causes precipitation ; and thirdly, that the presence of so little as 0-5 per cent. of sodium chloride enormously lowers the solubility of sodium biurate. These facts taken together appear to me to explain why sodium biurate and calcium biurate appear together in tophi, and give a significance to calcium biurate in gout, similar to that shown by O. T. Williams? for the insoluble calcium soaps secreted by the intestinal mucosa in mucous colitis and appendicitis, and by Klotz and others for the _ealeium saponification in arterio-sclerosis, where the insoluble soap aie ‘ sooner or later passes into the form of the likewise insoluble calcium 7 carbonate. The biurates used for our experiments were prepared from Merck's ‘extra pure’ uric acid. In the case of the calcium and magnesium biurates, the composition was controlled by incineration and weighing the calcium oxide and magnesium oxide respectively. The calcium salt yielded 12 per cent. of CaO, theory requiring in the biurate 13°6 per cent., 1. Ergebnisse der Physiologie, Bd. Il, 1903. 2. This Journal, Vol. IT’ p. 395.1907; Vol. TIL p. 391, 1908. 82 BIO-CHEMICAL JOURNAL and in the normal urate 27 per cent. Similarly, the magnesium salt yielded 11-7 per cent. of MgO, the theoretical yield being 11°2 per cent.. for biurate and 21 per cent. for normal urate. It is, therefore, obvious that in each casé We were dealing with the biurate. The results obtained for solubility in distilled water of the four biurates were as follows : — One part of potassium biurate dissolves in 64 parts boiling water and in 550 parts at body temperature » sodium bd = 117 Pa i 1,030 a ” Bt magnesium ,, “4 148 < = 2,440 *» wn 23 calcium * > 666 = x 4,760 * » The last figures are the average of two independent titrations with potassium permanganate, which were carried out most carefully, and the result corroborates the findings of those observers who give calcium biurate a permanent place amongst the constituents of tophi. If it were so soluble as Delepine, and the other authors whom he quotes, would place it, then it could not be present in such gouty deposits. w In the body, however, the various biurates are not dissolved in distilled water, but in a saline solution containing as the chief constituent sodium chloride, along with other inorganic salts in lesser concentrations. Accordingly an attempt was instituted to approach more closely to natural conditions by determining the solubilities of the above biurates in a halt per cent. solution of sodium chloride. Here the most interesting result was obtained that while the solu- bilities of calcium and magnesium biurates were actually somewhat increased in the saline solutions, that of the sodium biurate was reduced almost to zero. This result gives probably the key to two things, first, that in the body the preponderating salt in gouty concretions is sodium biurate, and secondly, on account of the increased solubility of the biurates of the alkiline earths (Ca and Mg) ‘in salines, we see, perhaps, a rational basis for understanding the improving effects of certain saline mineral waters empirically used in gout. In any case, the variation in the figures according to whether the ~ solvent medium for the biurates is water or a dilute sodium chloride solution, gives an indication of the great value which would attach to the study of the solvent action of solutions containing a number of inorganic salts in varying proportion. rai Attention may be specially called to the small amount of soll chloride which produces such a considerable change in the solubilities, illustrating that small variations in relative distribution of the salts of the ; i. sl = i teen ile 5 -* - ie a 2 INORGANIC KATIONS IN GOUT 33 _ plasma, such as might naturally occur from individual to individual, may ' have profound effects on the solubilities of the biurates in the body. 4 __'Phe enormous effects of the sodium chloride on the solubility of the sodium biurate is, in the language of physical chemistry, probably to be referred to the mass action of the common sodium ion of the sodium chloride and sodium biurate, tending to throw out of solution the less soluble of the two salts, viz., the sodium biurate. At least, such an action a has certainly been shown to occur in countless cases in pure solution of 4 salts possessing a common ion in their constitution. Hopkins and Hope! y quote Nernst’s. generalization to the effect that any two salts susceptible of dissociation, which contain an electric ion in common, naturally a diminish each other’s solubility. But the converse of this proposition has ; been found to hold true in certain cases, and salts possessing no electric ion in common may mutually increase each other’s solubility in a fluid. _ The possibility exists, therefore, that the ingestion of a mixed dietary may _ produce such a temporary increase in the proportion of salts other than those of sodium (especially potassium salts) as to increase the solubility of any retained sodium biurate, and so accelerate its excretion. They - eonsider this an important principle in lithiasis. For ease of comparison the results of titration of the saturated solutions of the four different biurates, (a) in distilled water, and (6) in half per cent. sodium chloride solution, in each case at body temperature, n ' are given in the following table. The figures show c.c. of 20 KMn0O,, required to oxidize the urie acid, and to get quantities of uric acid dissolved these must be multiplied by the factor 000375. In the _-_—_—_— second part of the table this has been done and the results re-stated in terms of the amount of fluid in each case required to dissolve one part of uric acid in the form of the respective biurates at body temperature. Amount dissolved by 100c.c. | Amount dissolved by 100 c.c. Biurate taken of distilled water at body of 0-5 per cent sodium temperature chloride solution at body as uric acid temperature * ~ Calcium biurate se 5&7 78 Sodium i ais 25°9 sas 0-6 Magnesium ,, pal 110 ies 13-6 Potassium ,, es 48-4 a 48-0 . Parts of distilled water Parts of 0-5 per cent. sodium Biurate taken required to dissolve one part chloride required to dissolve one part Calcium biurate op 4,760 jes 3,420 Sodium “ a 1,030 i. 44,400 Magnesium ,, Ey 2,440 an 1,960 Potassium —_,, es 550 és 555 1. Journal of Physiology, 1898-99, p. 284. 34 BIO-CHEMICAL JOURNAL Precrerrarine Errecrs or Cancrum Sats on Soprum Brurate Sonvrions Another important relationship is the precipitating effeet of calcium salts on biurate solutions of sodium. It was found that the addition of even small percentages of calcium salts to distilled water had the effect of very much lessening its solvent power on sodium biurate. Kp Experiment I,—Calcium chloride— ; At 87° ©. 100 c.c. distilled water alone in 12 hours took up an equivalent of 25-9 c.c. 5= * KMnO, a 100 c.c. +01% CaCl . ” 3:5.0.0. 2» 2H 100 c.c. * +03 % CaCl, = cs 108 ¢.c. ,, 4 100 ¢.c. 7 + 05 % CaCl, s sf 1-9 ¢.c, Pa So that calcium chloride exerts a decidedly deterrent influence on the solubility of sodium biurate in aqueous solution. y 4 Experiment II.—Calcium sulphate :-— Control in distilled water ... ahi ie is 25-9 .0.35 KMnO, is . + 0-1 % CaSO, ... «190 ee. . re 7 0-2 % CaSO, _.... oaks 15-0 c.c. *. Calcium phosphate also had a decided effect in lowering the solubility, and this has a special interest in being so frequently found in tophi. Experiment I1I1.—Calcium Phosphate.—The following titrations give detailed quantitative results to show the markedly deterrent effect of small percentages of calcium phos on the biurates in aqueous solution. Three solutions containing distilled water (a) with potassium, and (b) sodium bieliabes in excess, and (c) a mixture of the two in excess were placed in the incubator at 37°C. To three other samples of these same solutions } per cent. of calcium phosphate was added, and these were similarly treated. Three titrations were made with the following results per 100 c.c, :— . s (a) Distilled water with KHU in excess after 2 hours at 37° C. had dissolved an equivalent of 63 c.c. SD KMnO, (b) ” ” NaHU ” ” ” 34 ©.c. ” (ec) co a both biurates ,, oe =f 75 c.c. me : Il. (a) Distilled water with both biurates in excess after 72 hours at 37° C. had dissolved an equivalent of 60 c.c. i KMn0, (b) ob eo » pe pe oe 38 c.c. ae (e) *” 9 ” % PA >” 75-5 c.c. ou Til. (a) + 4% CaHPO, in excess after 72 hours at 37° C. had dissolved . an equivalent of ... bas vas .. 4650.0. 6 KMn0, (b) ., - 9 os = 27-5 c.c. > (e) ” ” ” ” ” 54-0 cc, ” INORGANIC KATIONS IN GOUT 35 _ These results derive added importance from the fact that CaHPO, is _ constantly found in tophi, and emphasises the value of recent research in _ demonstrating the excretory function of the intestine for calcium and for _ phosphates. They also go towards giving a rational explanation for the e, empirical treatment by mercurials, salines, etc., which prevailed in the past, and is still recommended, for constipation is a classical symptom in ee eehan). _ Complementary to the general question of solubility comes that of . the formation of deposits and tophi, and the influence of calcium salts in this direction. There is a presumption that its influence is decided, for we have already pointed out that calcium is usually present as a biurate or a phosphate. . Without wishing to exaggerate the value of experiments in vitro, : as applied to body processes, one is tempted to suggest some analogy 4 een an experiment like the following and the conditions prior to an acute attack of gout, for, as Osler states, ‘the formation of tophi must rest upon some physico-chemical basis of precipitation and crystallization.’ B This experiment is intended to demonstrate the powerful precipitating effect of a calcium salt in minute percentages on a solution of sodium - biurate containing amounts varying from 1 in 819 to 1 in 3,275, which Roberts believed to approximate to the amounts possibly occurring in a supersaturated condition of the blood and sera preceding an acute attack. A solution was made of uric acid (Merck’s extra pure) in a ‘2 per cent. _ sodium of bicarbonate of sodium. This was found by direct titration . with 3 KMn0, to require 36 c.c. per 100 c.c. of solution, corresponding to a strength of 1 in 655 of uric acid. Into eight stoppered bottles, the _ following quantities of this solution were poured, 80, 70, 60, 50, 40, 30, _ 20, respectively, and distilled water added to bring each volume up to 100 cc. These represent strengths of biurate of sodium varying from 1 in 819 to 1 in 3,275. To each was added a weighed amount of _ erystallized sulphate of sodium, equivalent to ‘2 per cent. of the anhydrous salt. The bottles were then placed in the incubator at _ 87°C., and left for twenty-four hours. At the end of that time they were still clear. Another 1 per cent. of anhydrous sulphate was added, and as at the end of three hours they were still found to be clear, ‘1 per cent. acid phosphate of soda was added.!. Three hours afterwards they were still clear, and they remained so during the next twenty-four hours. At the end 1. These salts were employed so as to minimize the error in the final titration due to oxidizing of the chloride. 36 BIO-CHEMICAL JOURNAL of that time ‘01 per cent. CaCl, was dropped from a stronger solution, and after sixteen hours they were found to be still clear. At the end of that time another ‘01 per cent. Ca@l, was added. This produced no immediate effect, but after one hour slight floceuli were visible in certain: of the bottles. Ten c.c. of each solution (previously filtered) gave the following results on titration, which show clearly the precipitating effect of CaCl, even in ‘02 per cent. (1) 80 ¢.c. solution + 20 ¢.c. water’+ 0-28{Na,8O. Wa N Milligrammes °% loss of 10 H,O) + 0-2 % eH found theoretical Uric acid + 0-02 CaCl, 2-1 2-9 (2) 70c.c. _,, + 30 c.c. af a4 20 2-4 ia (3) Bec. ,, + 40 c.c. m os % 1-9 2-2 13-6 (4) 50 c.c. a + 50 c.c. > me $s 1-7 18 55 (5) 400.c. __,, + 60 cc, oe *” is 1:3 1-4 yf (6) 30ec. ,, + 70c.c. Ps = - 1-0 1-08 70 (7) Mec. ,, + 80 c.c. ts > ~ 0-7 (0°72) 2-8 Regarding the precipitating effect of sodium chloride in gout, and the use of common salt in the diet, the statements in the literature are somewhat contradictory. Sir W. Roberts! advised abstinence from the use of much culinary salt in the gouty. He believed it could be stored up, especially in the serous fluids, to a concentration sufficient to impede solubility, and presumably by inference cause precipitation. Hofmeister’s work (quoted in Schaefer’s Physiology) tends to corroborate this suggestion by proving the power for adsorption of sodium chloride which colloids of the chondrin and mucin type possess. Roberts himself quotes figures in his published Croonian lectures to show that a maximum sodium content is found in cartilaginous tissues. Mendelssohn? mentions experiments with some of the well-known uric acid solvents. He dissolved piperazin and lysidin in blood serum and ‘ found such solutions to have as great a solvent power for uric acid as their aqueous solutions. If sodium chloride be added precipitation occurs, and in the form of sodium biurate.’ He continues: ‘ The sudden nature of attacks of gout seems to point to sudden formation of some such urate precipitant, and the fact that they generally occur after mistakes in diet, over-feeding, etc., where much salt is consumed seems to indicate sodium chloride to be the cause.’ Dapper and von Noorden, in a monograph recently published on the effect of sodium chloride on metabolism, discuss the effect of sodium chloride waters upon the excretion of uric acid. They find in therapeutic literature the greatest confusion on the point as to whether it increases or diminishes the output of uric acid in gout. They cite quantitative © 1. Croonian Lectures, 1882. 2. Deutsch. med. Wochenach., 1895, p. 283. INORGANIC KATIONS IN GOUT 37 ts from several cases to prove that such saline waters do increase the output of uric acid, arid state that these facts point to the use of saline mineral waters in our treatment of gout as worthy of consideration. Two cases in my own practice have afforded suggestive evidence that sodium chloride has something to do with gouty-joint phenomena. In _ the first of these cases a child of five years developed acute arthritic _ symptoms in the feet and thighs, simulating acute rheumatism, after an overdose of sodium chloride administered as a home cure for intestinal : oo Previously, too, a fairly strong salt solution had been injected per rectum daily for the same purpose. That the sodium chloride had some pathogenic significance was proved by the fact that as soon as its - administration ceased, with no other medication except small doses of _— citrate, the child became rapidly well and has been well ever ae toe joint and then in the other. In this case two samples of 24-hours’ urine (150 c.c.’s in volume) _ were analysed for the quantitative estimation of the bases, (1) one when _ the attack was at its worst, (2) the second when recovery was well _ advanced, at an interval of several days. The exact amount of chlorides was not tabulated. Sodium chloride, however, was found in excess, but _ in regard to the bases, the figures are— a Na,O — K,0O CaO MgO (1) * 2-041 grs. 0-091 grs. =: 0-067 grs. 0-085 grs. | in 150 c.c, wh (2) 1-375 grs. 0-183 grs. 0-122 grs. 0-093 grs. | of urine This, so far as it goes, would suggest that sodium chloride in the acute stage had something to do with arthritic symptoms, and that an we increase of urinary calcium, magnesium, and potassium are at least com- ; patible with recovery from an attack of gout, whilst the reverse is true = Whether sodium chloride can exist in such concentration as to cause _ precipitation or not, it is much more likely that a metal of higher valency than sodium, especially one such as calcium, which can produce a higher insoluble biurate, will cause precipitation. It may be noticed also that a gouty condition is often associated with lead poisoning, as Garrod pointed out in his early researches. In conclusion I have to express my thanks to Dr. Charles E. Harris for valuable assistance in carrying out the solubility experiments. 38 ON THE NITROGEN-CONTAINING RADICLE OF LECITHIN AND OTHER PHOSPHATIDES By HUGH MacLEAN, M.D., Carnegie Research Fellow. Irom the Department of Physiological Chemistry, Institute of Physiology, Berlin (Received November 18th, 1908) Part | Since the investigations of Diaconow and Strecker! it has generally been assumed that lecithin is a compound of fatty acids with glycero- phosphoric acid and a base choline. This assumption is based on the results of elementary analysis combined with the fact that hydrolytic decomposition of the lecithin yields the above-mentioned constituents. From this it is obvious that the total amount of nitrogen present is represented by the nitrogen of the choline radicle, and in this way a knowledge of the total amount of nitrogen yielded by any pure lecithin makes it easy to deduce the amount of choline (C,H,,;NO,) actually present, from a theoretical standpoint? Many experiments have been made in order to obtain the choline content of different lecithins, but in every case the results actually obtained fell far below the theoretical values. Thus Erlandsen? obtained from pure heart lecithin, which had been split up by boiling with barium hydrate, only about 42 per cent. of the theoretical amount, and Heffter,? using lecithin extracted from liver, obtained under similar conditions only 25 per cent. In order, if possible, to elicit the cause of these losses, the following investigation was undertaken; here a comparison of the amounts of choline actually obtained from different lecithins, saponified and manipulated under exactly ‘similar conditions, suggests much as to the real nature and cause of this loss in certain lecithins. Marteriat Usep For the first set of experiments a lecithin sold by the firm of J. D. Riedel, Berlin, under the trade name of ‘lecithol’ was employed. Afterwards, I extracted and purified lecithin from the heart muscle of the ox, as described below. Annal. d, Chem. u. Pharm., 148. L. 2. Zeits. j. Physiol. Chem., Ba. LY, 8. 113. 3. Arch. f. exp., Pathol. 4. Pharm., Ba. XXVIIL, 8. 100. NITROGEN-CONTAINING RADIULE OF PHOSPHATIDES 89 * Leerrnot ’ t first the lecithin salt of cadmium chloride was made use of, the lecithin being dissolved in alcohol, and the solution carefully filtered, and then precipitated by cadmium chloride. This lecithin salt, however, while much more convenient to weigh and handle than lecithin itself, ave rise to much difficulty on attempting to split it up with solution of m hydrate, either in water or alcohol; here it constantly adhered to : : 2s of the flask above the liquid, so that, even by constant shaking, was difficult to ensure complete saponification. For this reason it Si me discarded, and the lecithin itself used. The lecithin was found, however, to contain traces of ammonium compounds, and in order to get vi of these impurities it was treated as follows. Small quantities were radually added to some water in a mortar and ground up until a pletely homogeneous emulsion was formed. This emulsion was ) inte by means of acetone, the lecithin carefully filtered, kneaded a plastic mass with some more acetone, and again emulsified as re. The filtrate gave abundant evidence of the presence of ammonia her n treated with caustic alkali and heated. After the process had been pe ited three times it was found that acetone failed to give a precipitate. is difficulty was overcome by the addition of a few drops of sodium _ chloride solution, when the lecithin separated out quite readily. This ay process of emulsification and precipitation by acetone was carried out five: times, the last two filtrates giving no indication of the presence of ammonia. The lecithin was then thoroughly washed with acetone, and dried in vacuo over sulphuric acid. After drying, it was dissolved in ____ absolute alcohol, and the solution preserved in a well-stoppered dark of bottle. For each experiment 5 c.cm. of this solution was used, the same 1 » each time thoroughly cleaned and dried, being used for This lecithin solution gave the following results on Nitrogen (Kjeldahl’s Method) Tite 6 ca. solution = 10-2 10-0 109 and 10-2 o.°. To Average 10-1 oom. = 14:14 mgrs. N Phosphorus (Neumann's Method) In five experiments 5 c.c. solution = 52-0, 52-5, 51-9, 52:1, 52-3, 0.0, = NaOH Average 52-2 c.om, = 28-89 mgr. P Ratio of P: N = 1: 1-08 _+ 5 -e.c. aleoholic sol. = 14:14 mgrs. N & 0-31073 grm. cholin platinum chloride, In another solution used— 5 cc. = 17-4062 mgr. N = 0-38250 grm. cholin platinum chloride. 40 BIO-CHEMICAL JOURNAL Experiments with * Lecrrso.’ Five c.c. of the above alcoholic solution was taken and added to 100 c.c. of methyl alcohol, saturated with barium hydrate, and boiled in a flask on the water bath for varying periods; the flask, which was fitted with a reflux condenser, was shaken from time to time. After heating for periods of one to ten hours,the mixture was allowed to stand for some time, when a well-marked precipitate separated out and fell to the bottom of the flask, the fluid above remaining fairly clear. This fluid was filtered off and the precipitate returned to the flask; to the flask was added 100 c.c. of ethyl alcohol, and after being well shaken up with the residue was boiled for five minutes. ‘The mixture was then allowed to stand and the clear alcohol filtered off as before. This process was repeated usually about four times, and sometimes oftener, in order to ensure thorough extraction of any choline remaining in the insoluble residue. All these alcoholic extracts were then mixed together and carefully evaporated on the steam bath to about the bulk of the original amount of alcohol (100 c.c.) or sometimes rather less. The barium was then separated by treatment with hydrochloric acid; the barium chloride was filtered off and the fluid evaporated to dryness. In order to avoid losses from bumping, it was found better to perform all these evaporations in flasks. When carried out in this way it was easy to ensure that nothing was lost. The dried residue was then thoroughly extracted with absolute alcohol, evaporated to small bulk and treated with a saturated solution of sublimate in absolute alcohol. This was left to stand till next day when the precipitate was separated by filtration; the filtrate was then evaporated to dryness, and the residue, after being waslied with cold alcohol, added to the first precipitate; the combined precipitates were then dissolved in hot water. This solution was treated with sulphuretted hydrogen and filtered; filtrate was evaporated to dryness and dissolved in a little absolute alcohol. The choline, which was present in the form of — choline chloride, was now precipitated by a solution of platinum chloride in absolute alcohol; after eighteen to twenty-four hours the precipitate was filtered off, washed with cold absolute alcohol, dried and weighed. Sublimate was introduced on the assumption that possibly the presence of impurities might interfere with the precipitation of the choline by platinum chloride, and that those impurities might to a greater extent be got rid of by sublimate. Subsequent results did not bear out this view, and in all my later experiments sublimate was omitted. PROGEN-CONTAINING RADICLE OF PHOSPHATIDES 41 _ An extended series of observations with the above method has been ly published! by the writer, but the following short extract serves ) show the general results obtained. In fifteen experiments the average yercentage of the lecithin nitrogen obtained as choiin nitrogen was only 73 per cent :— CHOLLN E-PLATIN UM-CHLORIDE No. of Percentage actually hours Actual amount found, Theoretical amount, found, in terms of hydrolysed in grms. calculated from N theoretical amount of lecithin, in grms. 1 0-2700 0-38250 70-59 lk _ 02450 0-31073 78°85 “S 0-2941 0-38250 76-89 ant 4 0-2991 0°38250 78-19 2 7 0-2421 0-31073 77-91 ‘ ” 10 0-2390 0-31073 76-91 mde): ae / That the substance obtained was pure choline platinum chloride is evident from the following figures :— 1. 02439 grm. left on ignition 0-0771-grm. Platinum = 31-61 °% 2. 0-1740 grm. * 0-0550 grm. » = 31-61% Calculated for (C;Hj,NOCI), PtCh = 31-64% Pt « In three experiments, carried out exactly as above, only that -_- sublimate was not used, the following results were obtained. Here, also, the alt proved to be pure, giving on ignition a residue of 31:59 per “ CHOLIN E-PLATIN UM-CHLORIDE No. of Percentage actually hours Actual amount found, Theoretical amount, found. in terms of hydrolysed in grms, calculated from N theoretical amount of lecithin, in grms. 2 0-2976 0-38250 77-80 3 0-3003 0-38250 78-51 3 0-3031 ‘ 0-38250 79-24 In all these experiments it is seen that not more than from 77 per = to 79 per cent. of the total nitrogen can be recovered as choline ~ Here it is interesting to note that at the same time as these results were published, a paper appeared by Moruzzi? describing the results of hydrolytic decomposition by sulphuric acid. In his experiments the double salt of cadmium-chloride-lecithin was utilised. The following a figures taken from his paper show practically the same percentage of 1. Zeits. }. physiol. Chemie, Ba. LV, 8. 363, 2, Loe. cit., 8. 352. 42 BIO-CHEMICAL JOURNAL choline platinum chloride (average 77'7 per cent.) as was obtained by the writer with barium hydrate. Amount of CHOLINE-PLATINUM-CHLORIDE mium No, of Percentage petanly Number compound hours Actual amount found, Theoretical amount, found, in terms ¢ used, i hydrolysed in grms. calculated from N theoretical amount grms. of lecithin, in grms. 1 2-84.50 44 0-6642 0-8687 76-5 2 1-4952 4h 03507 O-4574 76-7 — 3 1-8147 4h 04425 05542 79:8 Hypro.tysis in Watery Sotvurion or Bartum Hypratre Some experiments were now made in order to observe what results could be obtained by using a saturated watery solution of barium hydrate instead of an alcoholic fluid. Five c.c. of the lecithol solution was added to 100 ¢.c. of a saturated solution of barium hydrate in water, and boiled with a reflux condenser for 2} hours; during the process the flask was shaken from time to time, especially during the first hour. It was then allowed to stand, and the precipitate filtered off. After thorough washing of the residue, the filtrate was freed from barium by means of CO,; barium carbonate was filtered off, and the filtrate, after the addition of a little hydrochloric acid, evaporated to dryness. Residue was extracted with a little absolute alcohol, and after evaporating to small bulk, was precipitated directly with platinum chloride. Precipitate was then left to stand till next day, filtered, washed, dried and weighed as usual, Here the average result obtained was 77°5 per cent. . In all the above modifications it is seen that the result remains practically the same, so that not more than about 77 per cent. to 79 per cent. of the theoretically calculated choline-platinum-chloride is actually obtainable by experiment from this particular lecithin. The following table indicates the average results of the different methods : — Percentage of Choline-platinum-chloride obtained, calculated on theoretica amount 1. Lecithin hydrolysed with saturated solution of barium hydrate in methyl alcohol, using sub- limate as intermediate precipitant .. = 17-3 % 2. Same as above, without sublimate oe = 78-5 % Saturated watery solution of barium hydrate is 77-5 %, 4. Sulphuric acid (Moruzzi) = 77-7 % Average = 17°75 %, —NITROGEN-CON'TAINING RADICLE OF PHOSPHATIDES 48 Ow rne Causes or ruts Loss or tHe THEORETICALLY CaLcuLarED soma CHOLINE Here we have to deal with a loss of a little over 20 per cent. of the theory, and in order to elicit its cause the following points were considered : — - (1) Is the choline partly destroyed when heated with a saturated —___ solution of barium hydrate ? 3 Since it is known that the free base decomposes on heating into trimethylamine, ethylene oxide, and H,O, and since, as first observed by Heffter, a mixture of lecithin and barium hydrate which has been boiled for some time has a pronounced smell of trimethylamine, it might be thought that this decomposition was the cause of the loss. Gulewitsch,! however, has shown that this destruction of choline is so small as to be * of little practical importance, and some experiments made by the writer entirely supported this statement. Lecithin was boiled with barium hydrate in a flask fitted with a reflux condenser, the latter being connected with a bottle containing io H,SO,. Through this acid the volatile decomposition products of lecithin were led by means of a stream of ammonia-free air, and the amount of acid neutralised estimated by titration. In various experiments lasting from two to eight hours it was found that the amount of 0 H,SO, used was very small, amounting, after long boiling to not more than the equivalent of 1 to 3 per cent. of the total nitrogen of the lecithin used. From this it is obvious that the ____ loss is not accounted for, or at least only to a very small degree, by the 4 decomposition of the choline. q (2) Is the lecithin completely split up, and does the filtrate contain E the total nitrogen of the lecithin used ? That the lecithin is completely split up would appear from the fact _ that boiling for ten hours gives no better result than boiling for two to three hours. As the result of experiment, we may assume that all the lecithin is entirely split wp after two to three hours or even less. With regard to the N-content of the filtrate, it is interesting to note, that in no case was it found to contain all the nitrogen of the saponitied lecithin. An analysis of the residue after boiling also showed that this residue always contained nitrogen in a form insoluble in alcohol, as no amount of washing had any effect in lowering the amount of this residual 1. Zeits. {. physiol, Chemie, Ba, XXIV, 8. 513. 4 BIO-CHEMICAL JOURNAL nitrogen. This residue was always very carefully washed in the following manner; after filtration it was returned to the flask and thoroughly shaken up with about 100 c.c. alcohol; the mixture was then boiled for five to ten minutes, allowed to stand, and again filtered. This process was generally repeated three times, but in some experiments as often as six times; in each case the result was the same. The number of hours during which the mixture was boiled had also no effect on the result. The average amount of insoluble nitrogen found in the residue amounted, as shown by the following table, to 8:5 per cent. of the total nitrogen of the lecithin. Total N of N found in Percentage of Number of hours lecithin used, in residue, in mgrs. total N found in No boiled mgrs. residue i 1 1414 1-19 8-4 2 1 14-14 1-13 8-0 3 1} 14-14 0-98 7-0 4 1 14-14 1-26 8-9 5 3 14-14 1-13 8-0 6 3 14-14 1-19 8-4 7 5 14-14 1-33 4 8 5 14-14 1-26 8-9 9 7 14-14 1-26 8-9 10 7 14-14 1-33 9-4 Average 14-14 1-21 8-5 (3) Does the possible presence of traces of impurities (other decomposition products of lecithin) prevent the complete precipitation of choline by platinum chloride? _ To test this, some pure choline chloride solution (0°1 to 0°2 per cent.) in absolute alcohol was taken and divided into equal parts by means of a — burette. To some of these portions were added glycerophosphoric acid, glycerine and barium chloride; they were precipitated with platinum chloride. The other portions were directly precipitated. A comparison of the results obtained in both cases showed that the presence of these impurities, while tending to lower the percentage of choline-platinum- chloride obtained, did not do so to any marked degree. Considering that only very minute traces of impurities can be present, it is not likely that this factor is of much importance practically, in preventing complete precipitation. (4) Is choline chloride imperfectly precipitated by platinum chloride in alcoholic solution ? Gulewitsch made an experiment bearing on this point in the following manner. He took a 05 per cent. solution of choline chloride in absolute TTROGEN-CONTAINING RADICLE OF PHOSPHATIDES 45 ~aleohol and precipitated it with an alcoholic solution of platinum chloride. After twenty-four hours the precipitate was filtered off and ‘ washed with absolute alcohol. |The filtrate was evaporated to dryness, after being decomposed by sulphuretted hydrogen gas. The residue, which was very small, gave only a slight cloudiness with phosphotungstic acid and with iodine and potassium iodide solution. From this it was -eoncluded that platinum chloride precipitates choline chloride _ quantitatively. Since, however, the quantities obtained in lecithin oe _experiments are necessarily small, it was thought advisable to weigh “some choline-platinum-chloride salt; then dissolve it in H,0, decompose _ __ the solution with sulphuretted hydrogen, evaporate the filtrate to dryness, ____ dissolve out with absolute alcohol and precipitate with platinum chloride. ‘The amount of the latter salt ultimately obtained was compared with the amount used. In all my experiments it was found that the weight of choline salt obtained in this way always fell somewhat short of the : “original amount used. While it is likely that there must be some slight mechanical loss on account of the necessary manipulation, it would seem ____ that the precipitation is not quite complete, and in this way a certain amount of the loss of the theoretical choline of lecithin is explained. In some experiments carried out with erystals that had been several times 3 re-erystallised, and using alcohol that had been treated with barium oxide immediately before use, I obtained on an average from 93 to 97 per cent. of the original salt after the above manipulations. The average percentage, when using alcohol that had not been so treated, _ was somewhat lower. With care this loss is but slight. J The chief losses, therefore, seem due to part of the nitrogen remaining in the residue, together with small losses represented by the necessary : manipulations of the method, combined with the incomplete precipitation of choline chloride by platinum chloride. xg _ When we subtract the nitrogen found in the residue from the total fl nitrogen, we get theoretically the amount present in the filtrate ; . racially, however, owing to the necessary manipulation, it is obvious that the ultimate filtrate, when freed from impurities and ready for precipitation by platinum chloride must contain somewhat less nitrogen. The actual amount of choline platinum chloride found, when reckoned on the nitrogen of the lecithin minus the residue nitrogen, was from 86 to 87 per cent. The relative amount of platinum salt obtained when the filtrate was treated as described on page (55), was from 91 to 92 per cent. of the total filtrate nitrogen. This difference as mentioned, is accounted for by small losses during manipulation, for 46 BIO-CHEMICAL JOURNAL it is obvious that a comparatively swall loss materially influences the percentage. Since, therefore, platinum chloride tends to give incomplete precipita- tion of choline chloride, and since there may be traces of impurities also present tending to hinder precipitation, and when we consider the difficulty | of absolutely exact estimation of the nitrogen in these experiments, it would seem that in this particular lecithin all, or nearly all, the — of the filtrate is present as choline. ; Whether the nitrogen found in the residue is derived from the choline is at present undecided; it will be seen later on that a similar residual nitrogen is found after saponification of pure heart lecithin. Lecrruin From Hearrv Mvuscie In order to compare the choline content of the lecithin of heart muscle with that obtained from Riedel’s ‘lecithol,’ I prepared a pure lecithin in the following manner, much in the same way as Erlandsen has described.!. At the same time the mono-amino-diphosphatide substance Cuorin was separated, as well as another substance behaving somewhat like a phosphatide and similar in physical qualities to the substance isolated by Stern and Thierfelder? from egg yolk, and designated by them ‘ weisse substanz.’ PREPARATION oF Heart Muscie Oxen hearts were procured as soon as possible after the animals had been slaughtered, and the fat and fibrous tissue separated off; the muscular substance was then cut up into small pieces and passed through a mincing machine. This finely divided material was spread out in a thin layer on a glass plate, and dried at 30°C. in a current of air, — generated by a fan arrangement which was worked by a small motor, From time to time this layer was stirred, and the lumpy parts broken up into small pieces, and after twelve to eighteen hours it was generally quite dry. In order to obtain a fine powder suitable for extraction, this fairly friable dried material was broken down with the hand, and finally passed through a coffee mill. In this way a very fine powder was obtained. This was put into a desiccator and preserved in vacuo over H,SO, until quite dry. | 1. Zeits. f. physiol. Chem., Bd. LI, 8. 87. 2. Loe, cit., Bd, LIT, 8. 370. SITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 47 EXTRACTION = About 500 grms. of pints heart substance were taken, and to this ie air in the upper part of the bottle was eiaced Pies CO,, in » coalie to prevent as far as possible any tendency there might be for the lecithin to After thorough shaking it was allowed to stand till next day and : In order to exclude oxygen during filtration, this process ‘ried out under an atmosphere of CO,. A porcelain filter was ref fitted into a glass bottle connected ‘eat a suction pump, and e! at this filter a large glass funnel was placed in an inverted position ; s funnei was connected by a rubber tube to a cylinder containing CO,, ad ag filtration a stream of this gas was allowed to pass so that en was totally excluded. At first the ethereal extract filtered y well, but after some time it became very slow indeed. The filtrate ' Besta to about 150 c.c., and precipitated by excess of acetone. ‘pit that the dissolved substance should separate out as well as »ssible it was found best to connect the dish containing the ether- acetone mixture with an exhaust pump for half an hour or so; the partial evaporation of the liquid generated sufficient cold to give a fairly complete precipitate. The fluid part was then poured off, and the solid substance obtained kneaded together with a fresh portion of acetone. This plastic mass was then dried in vacuo over H,SO,. Bach portion was extracted as above described five times; after this ___ it was found that acetone yielded only traces of a precipitate. All the substance obtained in this way from the different extracts was mixed r and thoroughly dried. This substance, which represents the er soluble part of heart phosphatides, contained, as above mentioned, » ‘lecithin’ as well as other lecithin-like substances‘ Cuorin’ and ite substance’; impurities such as fat and cholesterin were also SepaRATION oF ‘ Wuire Sunsstance,’ Far, Erc. Above mixture of substances was dissolved in ether in a dark : - seapene faapne bottle, and gave a whitish turbid fluid; it was not expected that a clear solution would be obtained, for the first 10 to 20 c.c. fluid that passed during filtration described above was invariably slightly turbid. _ This fluid was now centrifuged, when a great deal of a whitish substance was separated. The clear ethereal solution was evaporated to about W iF, a a * = aa ial mia tart i " ae a — a ' 7 ae ei eee he 48 BLO-CHEMICAL JOURNAL 100 ¢.c., and then precipitated by excess of acetone, and the precipitate treated with a little fresh acetone and kneaded together into a plastic mass as before. This precipitate was mostly composed of dark brown masses, but part was also white and flocculent; these floceulent masses did not fall well, and could not easily be completely separated from the acetone along with the rest of the precipitate. When as much as possible was separated, the ether-acetone fluid still contained some floceulent masses. This mixture was left to stand over night in a closed vessel, being protected from light and air. Next morning it was invariably found that all the white masses were precipitated on the floor of the vessel, and were in all respects similar to the ordinary brownish precipi- tate first obtained, so that the apparent difference seemed to be only a physical one. This portion was then added to the first precipitate and — dried as usual. The dried mass was then dissolved in ether as before, and the process again repeated. This was done until all the white substance was got rid of; at the same time fat and other impurities were separated. As will be seen from the following table it was necessary to centrifuge six times :— No. of Amount of Nature of Arrer CENTRIFUGING times ether used solution centrifuged in ¢.c. Nature of Relative amount of solution ‘white substance’ _ l 250 Very turbid and of Deep yellow- Much ‘ white sub- — whitish appearance brown solution stance ’ From tn deep in tube of diameter 2 250 Fairly turbid but Reddish brown About 4 } amount of much less so than solution white substance in No.1 in No. 1 3 200 Turbid, not markedly Light reddish About }-} amount 80 brown solution found in No. 1 4 200 Slightly turbid i / Trace only 5 160 Slightly turbid ov Fair amount: about three times as much as in No. 4 6 160 Almost clear Very light reddish Slight trace of white solution brown solution substance after long centrifuging Ethereal solution was evaporated each time to about 100 c.c, and precipitated with acetone. After above treatment the substance was thoroughly dried in vacuo over H,SO,. The first ether extracts contained a fair amount of fat, but the last two portions seemed to he quite free from it. SEPARATION OF SUBSTANCE INTO *Lecrruin’ ann * Cvortn ’ A. substance was now obtained from which the ‘white substance ' was Separated, and which was free from fat and cholesterin. This DP iahstince which gave a practically (though not absolutely) clear solution! in ether, contained ‘lecithin ’ which is soluble in alcohol and in ether, and another phosphatide ‘Cuorin’ which is insoluble in alcohol. hese substances were now separated as follows :— xe dried mass was dissolved in 150 ¢.c. ether and to this was added . absolute alcohol ; the mixture was then placed in the ice cupboard ty hours. At once a certain amount of precipitate fell, but much re was evident after some hours, and next day the insoluble part (A) “apparently completely separated ; the fluid was then filtered off, and e a rate, which was of a light reddish colour; was partly evaporated under the pump and then placed in a desiccator in vacuo over , and evaporated to dryness. When quite dry it was re-dissolved absolute alcohol; here a small portion remained undissolved, and this ~ us added to the precipitate first obtained. The alcoholic solution was en evaporated to small bulk, precipitated with acetone and dried as 4 - . It was completely soluble in ether, and appeared on drying as yellowish orange coloured masses (B). _——s«sTn this way the substance was divided into two parts : — : (A) Part insoluble in alcohol = ‘Cuorin’ (impure, containing oer some * white substance ’). (B) Part soluble in ether and in alcohol = ‘ Lecithin ’ proper. Arconot-INsoLtuBLe Portion (A) The part insoluble in alcohol was thoroughly dried in vacuo and then dissolved in 150 c.c. ether. Solution was not clear, so centrifuge was used, and here again a good deal of white substance was separated off. he clear solution was precipitated with about four times its volume of solute alcohol, and left to stand over night under CO, in ice. Next gt alcoholic solution appeared almost colourless. The precipitate tained consisted of a mixture of white flocculent masses and a resinous py material. This precipitate was treated with alcohol and left to in the incubator at 60° ©. for several hours. The white part was | es now dissolved by the warm alcohol, and a dark brown syrup was left. This precipitate was again treated as above and a resinous mass again obtained = ‘Cuorin.’ On cooling the alcoholic solution, the white i. agen masses separated out and were filtered off = Portion A!. ‘On centrifuging part of this solution no precipitate was obtained. 50 BIO-CHEMICAL JOURNAL PURIFICATION OF CUORIN The Cuorin was dissolved in a little ether and gave a fairly but not quite clear solution; CO. was introduced, and after standing in the ice cupboard till morning a perfectly clear solution was obtained; a small amount of sediment was seen on the floor and sides of the glass. Acetone was now added, but the precipitate did not at first fall well, and an emulsion-like fluid was obtained. This was left standing im ice under CO, for sixteen hours; a good precipitate was now obtained as a brownish sticky mass. Acetone-ether mixture was slightly whitish coloured and when centrifuged gave a small precipitate of a brownish substance which was not added to the other portion. After centrifuging, the fluid was absolutely water clear. * The precipitate, after being dried over H,SO,, was treated with warm ethyl acetate freshly distilled, and gave a clear solution ; CO, was now introduced, and fluid was left to stand in ice for some hours, when precipitate settled down as a resinous mass on the floor of the flask. The ethyl acetate was somewhat light yellow coloured, and after separating it from the precipitate, was left to stand in ice till morning; then a little precipitate was found to have formed on the floor of the glass consisting of minute small white balls; this was thrown away. The precipitate was dried and the process repeated: this time the ethyl acetate after standing overnight remained quite clear. The substance was now dried, and a small amount dissolved in ether to test its solubility, when it was found that the solution was not absolutely clear. The whole precipitate was then dissolved in ether and left to stand under CO, till morning; fluid was now quite clear, and a little sediment appeared on the glass. This fluid was then evaporated to dryness and Cuorin obtained which gave quite a clear solution in ether. Sunstance Sorvsie rm Arconor at 60° C. (A, Portion) This, as already mentioned, was obtained when the hot alcoholic solution was allowed to cool; it was precipitated in white flocctilent masses and only a small quantity was present; it was filtered off, and re-dissolved in alcohol at 60°C.; then allowed to stand under CO, in ice, and re-precipitated; it was then filtered, washed with cold absolute alcohol and dried over H,SO, in vacuo. This substance seems to be the same as the ‘white substance’ separated previously by the centrifuge, but this point is at present being investigated. ee NITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 51 Ee The following plan shows roughly the general outline of the process of separation of the diffdrent phosphatides from ethereal extract :— Portion (A,); dissolves at 60° and mo | | falls out on cooling : white sub- stance i cs (Perper =cut s we have separated : () Lecithin ; ae (2) Cuorins (3) ‘ White substance ’ (by centrifuge) ; (4) and part, soluble in alcohol at 60°, which seems to be ‘white substance.’ ; _ Anarysis or Heart Muscie Lecrruin n order *e test the purity of this lecithin the following analysis was ne was estimated according to Neumann’s method, and after Kjeldahl. NITROGEN 1. 0-5232 grm. Substance = 6-9 cc. 7 H, 80.

is a pure lecithin. Savonrrication or Heart Lecrrurn With Methyl Alcohol.—This lecithin was now split up in order to | compare the choline content with that of Riedel’s lecithin. A carefully a weighed quantity (from 03 grm. to 07 grm.) was ground up in a mortar 52 BIO-CHEMICAL JOURNAL with 5 grms. solid barium hydrate. | This was added to 100 ¢.c. methyl alcohol, and the mixture boiled in a flask fitted with a reflux condenser for periods varying generally from two to four hours, but sometimes as long as ten hours. Flask was then allowed to stand for some time when the fluid above appeared quite clear, and a well-marked sediment separated out on the floor of the glass. This clear fluid was carefully poured off and filtered, and from 80 to 100 ¢.c. aleohol added to the flask containing » the residue; this was boiled for five minutes, left to stand and filtered as before. This process was usually repeated three times, and afterwards the residue was poured on to the filter paper, and washed there for some time. On some occasions, instead of pouring the residue on to the filter paper, it was thoroughly ground up in a mortar with some alcohol, and the boiling process repeated as above; thus the residue was sometimes boiled in all six times. The total filtrates were mixed and evaporated to about 100 c.c., and the barium precipitated by concentrated HCl; the mixture was carefully heated, allowed to cool and filtered from barium salts. The filtrate was evaporated to dryness and dissolved in water; this was filtered and filtrate again evaporated to dryness. Residue was now dissolved in a little absolute alcohol, and after being evaporated to small bulk was precipitated with platinum chloride and allowed to stand over night; precipitate was then washed, dried and weighed as usual. Several slight modifications of the above method were from time to time introduced, but they did not in any way alter the results, and the above seemed, on the whole, the best. Here the amount of choline-platinum-chloride obtained was only, on an average, about 41 per cent. of the theoretical amount when the substance was boiled for two to four hours. In some experiments in which boiling was carried out for five to ten hours the results were somewhat lower, the average being between 37 per cent. and 38 percent. In no case was I able to obtain a higher percentage than 42°2. The following figures, _ quoted from a series, show generally the results obtained : — Amount of No. of CHOLIN E-PLATINUM-CHLORIDE Number lecethin hours | used, in grms. boiled Found, ingrms. Calculated from Percentage of ‘lecithin N, in grms. theoretical amount found 1 0-5231 2 0-0879 * 0-2127 413 2 0-4875 2 0-0792 0-1982 40-0 3 0-5916 3 0-1015 02405 42-2 “4 0-4757 3 0-08 10 0°1934 42-0 Average = 414 A mixture of above platinum salts yielded on ignition 31:9 per cent. Pt. NITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 58 Restpve arrer Savontrication witn Ba(OH), or This : residue, “after being thoroughly washed as above described, was now examined for nitrogen. As in ‘lecithol’ it was found that this | psidue pararisbly contained a certain amount of alcohol insoluble eke the number of times that the substance was washed “6 not seem to have any appreciable effect on the nitrogen content. That this residual nitrogen was not present as unchanged lecithin is obvious from the fact that the lecithin would have dissolved in the alcohol ; also e number of hours during which the lecithin was boiled made little e1 in the result. The nitrogen was calculated by Kjeldahl’s , a blind experiment being made with the chemicals in each case. No. of times No. of c.c.’s No. of mgrs. No. of mgrs. of — Percentage sg ashed a : . . Pilean ached: sae ee me aeet | tn roche boiled for 5 mins. residue _ (average 1-85 % each time nitrogen content) 3 0-44 0-616 9-68 6-4 4 0:39 0-546 901 6-0 3 0-45 0-630 10-94 5:8 1 035 0-490 8-8 5-6 2 0-49 0-686 — 1L-50 5-9 5 0-57 0-798 15°37 5-2 3 0-39 0-546 14-09 3-9 3 0-36 0-504 9-64 5-2 3 0-40 0-560 12-05 46 3 0-42 0-588 12-15 48 > ‘ Average = 5-34 % he ‘Thus it is seen that something over 5 per cent. of the nitrogen of ithin is found in the residue after boiling; this, however, while rather © ult to. explain, does not account, to any appreciable extent, for the ts 1 Loss i in choline-platinum-chloride. NirroGeN ov FILTRATE ‘The determination of the nitrogen in the filtrate was, of course, of 4 great importance ; if the filtrate did not contain much more nitrogen than Was represented by the choline-platinum-chloride found, it was likely that _ the loss was due to volatilisation during boiling. The nitrogen of the ‘= filtrate plus that of the residue when subtracted from the amount actually _ present in lecithin represents the amount lost through decomposition and _ volatilisation during boiling. On an average it was found that the 54 BIO-CHEMICAL JOURNAL iiltrate contained about 80 per cent. of the total nitrogen of the lecithin used. Thus it will be noticed that the loss due to the formation of volatile products is greater than that in * lecithol.’ Nrrrocen Founp No. of —- — TotalN Difference No. grms. No. of In In Total of between of tota lecithin hours residue, filtrate, found, lecithin foundand N in used boiled mgrs. mgrs. mgrs. used calculated, filtrate in mgrs. 1 0-4393 2 0-58 6-58 7-16 8-12 0-96 8L-0. 2 0-6721 3 0-76 9-81 10-57 12-43 1-86 79-7 3 O-3114 3 0-54 4-69 5-23 5-76 0-53 81-4 4 0-4219 4 0-49 6-02 6-51 78 1-29 770 Average = 79-78% Since the filtrate contains about 80 per cent. of the total nitrogen of the lecithin, and since the average amount of choline-platinum-chloride found corresponds only to about 40 per cent. of the theoretical amount, it is obvious that the total amount of nitrogen recovered as the double platinum salt of choline is only about 50 per cent. of the whole. In order to test this directly without the intervention of filtration and other mechanical manipulations by which slight losses might be incurred, the following relative experiments were undertaken. It will be seen that ~ the result is absolutely in accordance with the above, the average’ percentage found being 50°5 of the total N. Lecithin was boiled as before for some hours with Ba(OH), and the filtrate evaporated to about 60 c.c.; it was then cooled, and excess of acetone added, in order to make sure that no unchanged lecithin was present. A very slight flocculent precipitate formed, which was filtered - off, after standing for some time in the ice cupboard. The acetone was then evaporated off and HCl added; BaCl, was filtered off and the filtrate evaporated to dryness. Residue was dissolved in H,O, filtered, and filtrate again evaporated to dryness. Residue was now dissolved in absolute alcohol and filtered; it was then re-evaporated to dryness, and re-dissolved in absolute alcohol, and ‘evaporated to 5 c.c. or so; to this solution, which was perfectly clear, about 53 c.c. absolute alcohol was added, and, after thorough shaking, divided by a burette into two parts of 26 c.c. each. One part was run directly into a Kjeldahl flask and the nitrogen estimated; the other was run into a small beaker glass, evaporated to about 2 c.c., precipitated with platinum chloride, and left to stand for twenty-four hours. In this way experiments 1 to 5 in the following table were carried out; experiments 6 and 7 were also done in the same -NITROGEN-CONTAINING RADICLE OF PHOSPHATIDES 55 vay only acetone was not used. In experiments 8 and 9 the filtrate was - careally evaporated to dryness without the addition of HCl; the residue _ was then dissolved in absolute alcohol and to this was added a little HCI; it was then treated as above. This plan was adopted in order to get rid of traces of glycerophosphoric acid which might possibly interfere with y precipitation. The results, however, showed, as already found, that this acid does not materially interfere with the separation of the choline as the i platinum salt. 25 c.c. for NrrroGEN 25 0.0. FOR CHOLINE-PLATINUM- CHLORIDE ay | 4 Nitrogen found, in Choline-platinum- Amount of Choline- Percentage of th mgrs. chloride, caleu- platinum-chloride _— total N found, as lated from N actually found, in _— platinum salt of arent found, in grms. grms. Choline mis 8 5-60 012300 0-0619 50-3 | ae 7-70 0-1692 0-0864 51-1 m8 588 0-1292 0-0648 50-2 4 8-596 0-1889 0.0983 52-0 5 714 0-1569 0-0786 50-1 pe 5-04 0-1108 0-0566 51.1 uy 574 0-1261 0-0624 49-5 8 3-78 0-0830 "00408 49-2 9 2-80 0-0615 0-0314 51-0 Average = 50-5 % Analysis of the above salts gave the following percentages of Pt. :— Mixture of Nos. 1, 2 and 3 31-79 % Platinum or * 4 and 5 31-93 % » ” a ‘6 and 7 31-79 % Ps As a result of the above experiments we may assunie that, of the ‘nitrogen contained in the filtrate of this lecithin after saponification with Ba (OH),, not more than about 50 per cent. can be recovered in the form of the double platinum salt of choline. As a control, two experiments with Riedel’s ‘ lecithol’ were carried out as above described, when 92°3 per cent. and 91-2 per cent. of the filtrate nitrogen was recovered as choline platinum chloride; these results agree well with those mentioned under ‘ lecithol,’ being, however, a little higher. It is worthy of note that in almost every case the platinum content of the choline salt is slightly above the theoretical amount; possibly it is not quite pure, but contains traces of some other substance. The high content of platinum, } however, proves that no part of the lecithin can be present as lecithin- ee platinum-chloride, otherwise the platinum content should be much lower— in other words the lecithin must be split up fairly completely. CO 56 BIO-CHEMICAL JOURNAL SpLirrinGc up or LecrrHin 1N WATERY SOLUTION Small quantities of the lecithin were thoroughly ground up in a mortar with barium hydrate, and then boiled for varying periods with enough water to give a saturated barium solution. The flask, which was fitted with a reflux condenser, was at first constantly shaken in order to overcome the marked tendency of the substance to adhere to the sides of the glass above the level of the fluid. The subsequent treatment was similar to that described under lecithol, the choline being obtained as the platinum salt. The following results were obtained by these means; it will be seen that they are practically the same as those obtained with methyl alcohol, the amount of choline-platinum-chloride actually found representing only about 40 per cent. of the total nitrogen of the lecithin used, CHOLIN E-PLATINUM-CHLORIDE Number of Amount of Percentage of No. hours boiled substance, Found, in Calculated from _ theoretical in grms. grms. WN of lecithin,ingrms. amount 1 2 0-4227 0-0634 0-1718 37-0 2 4 0-5244 0-0856 0-2132 40-1 3 5 0-7678 0-1330 03121 42-61 4 7 0-6820 0-1085 0-2773 39-13 5 10 0-4132 0-0676 0-1679 40-26 Average = 39-82 % Resipur The residue found after boiling was now examined for nitrogen in the same way as the aleoholic part; here, as before, a certain amount of nitrogen was always found. When compared with the nitrogen of the methyl alcohol residue there was a slight increase, but it was by no means . marked; here it may be mentioned that an absolutely exact quantitative determination of nitrogen when dealing with these small quantities is exceedingly difficult, and this may occasionally account for small differences. In making two parallel blind experiments with chemicals alone, it is often noticed that despite the most careful precautions, the results are not absolutely the same; in general the difference is so exceedingly slight as not to interfere with the result, but in the above experiments very minute differences interfere to an extent that changes the result to a slight degree. EN-CONTAINING RADICLE OF PHOSPHATIDES 57 Bi In these residues the average amount of the total nitrogen found vas 848 per cent. as shown in the table. PEE acdes nin Resipue : ped iper opin Total nitrogen found, Percentage a co) vidaian leet 986 “ae 70 0-798 8-23 a 631 O48 7 1 8-17 nn 0672 $-29 oi 7-62 0-686 +0 Average =a 8-48 % - Fivrrare ae fedt ti, % htt Owing to lack: of wiatenial only one experiment was lena in aie to test what percentage of the total nitrogen was present in the filtrate; after peilias for three hours the result gave 79°9 per cent. of the total rogen : this agrees with that found in the alcoholic filtrate. 3 Two experiments were made as described on page (54), in which the concentrated filtrates were divided into two exact parts, the nitrogen f one ee directly estimated by Kjeldahl’s method, and the choline . of the other by platinum chloride. Here, as with methyl ) “ a thes over 50 per: cent. of the total nitrogen was recovered as the ‘inum salt. 25 ¢.c. for NITROGEN 25 0c. FOR CHOLINE-PLATINUM- ‘ . CHLORIDE N found, in Choline-platinum- Amount of Choline- Percen mgrs. chloride, caleu- platinum-chloride total N as bes lated from N actually found, in salt found, in grms. grms, 2-50 0-057 , 0-0295 5L-75 3-22 0-0707 0-0358 50-6 Average = 51-18%, the above experiments it is seen that in this pure lecithin not more ‘than about 42 per cent. of the total nitrogen can be recovered as the double salt of platinum when the substance is boiled with alcohol or water saturated with barium hydrate : also that not more than about an average _ of 50 per cent. of the nitrogen in the filtrate can be recovered as choline. _ A comparison of results obtained by means of similar experiments from *lecithol * shows that over 77 per cent. of the total nitrogen is recoverable as choline, while a little over 90 per cent. of the filtrate nitrogen is [ ee ee 1 ee 58 BIO-CHEMICAL JOURNAL represented by choline actually obtained as the platinum salt. The following indicates the general relationship as found by a few experiments : — Percentace or Tota N OBTAINED AS PERCENTAGE OF FILTRATE N OBTAINED CHOLIN E-PLATINU M-CHLORIDE AS CHOLINE-PLATINUM-CHLORIDE No. Lecithol Heart-muscle Lecithol Heart-muscle Lecithin Lecithin l 78°85 41-3 92-3 50-3 2 78-19 42-2 91-2 5l-1 3 77-91 39-1 —— 52-1 4 76-91 42-0 — 49-5 The result of all these experiments indicates that it is probable that heart muscle lecithin differs in constitution from certain other lecithins with regard to the manner of combination of its nitrogen; that all the nitrogen present is not represented by the choline radical, and that this lecithin contains another nitrogen-containing complex. Investigations bearing on the nature of this nitrogen are at present being carried out. CuorINn Cuorin obtained as above described was also examined with regard to its nitrogen-containing radical. This seems much more difficult to hydrolyse than lecithin, but in common with Erlandsen, the writer, after performing several experiments, is of opinion that the base of Cuorin is not choline. The results of certain experiments with this and other substances I hope to give in a later article. OME OBSERVATIONS ON THE HAEMOLYSIS OF BLOOD _ BY HYPOSMOTIC AND HYPEROSMOTIC SOLUTIONS OF SODIUM CHLORIDE By U. N. BRAHMACHARI, M.A., M.D., Lecturer on Medicine at the a Cam pbell Medical School and First Physician, Campbell Hospital, Calcutta. (Received November 28th, 1908)* In the Lancet for April 2nd, 1904, Sir A. E. Wright and Kilner, in 4 Wactibing a new method of testing the blood and the urine, state that ‘complete haemolysis takes place when a dark coloration is observed in a mixture of one volume of suspension of erythrocytes with one volume of a progressive dilution of a deci-normal sodium chloride solution in a capillary tube. Later on, Wright and Ross? point out that instead of making a preparation of the suspension of the red corpuscles, all that is required is to take a measured volume of the blood and to mix with it two __ - volumes of the progressive dilution of the deci-normal sodium chloride fet” solution, and then to observe when the dark coloration takes place. " It will be seen from the above, that it is assumed, firstly, that it is possible to bring about complete haemolysis by mixing one volume of blood with two volumes of a sufficiently dilute solution of sodium chloride, and secondly, that the point of complete haemolysis can be determined by letting light fall obliquely upon capillary tubes containing the mixture, it being supposed to be arrived at when there is a dark coloration of the ___ blood, and there is no bright appearance to be seen in it. In this way, Wright and Ross conclude that the average European blood haemolyses eS completely with two parts of a5 sodium chloride solution. IT am unable to agree with the observations of Wright, Kilner and - Ross, that complete haemolysis can be brought about in the above way. By treating normal blood with two volumes of 50 sodium chloride solution 3 ‘as well : as with two volumes of distilled water, q rates succeeded in demon- strating that in none of these is complete haemolysis obtained. 1 consider that the most accurate conception of complete haemolysis is that the blood supposed to be completely haemolysed should be perfectly transparent, or if it is not perfectly transparent, it should give, on centri- fugalisation, a sediment which, when thoroughly washed with an inactive fluid should not be red. Further, it should not show the presence of 1. Calcutta postmark, November 12th, 1908. 2. Lancet, October 21st, 1908, 60 BLO-CHEMICAL JOURNAL haemoglobin-containing erythrocytes, which can be stained with proper stains. By an inactive fluid is meant a fluid which has no action on the erythrocytes, and cannot, therefore, dissolve the haemoglobin contained in them, but can dissolve any free haemogiobin. To determine whether the sediment is red or not, the supernatant fluid is to be pipetted off and the sediment treated with a solution of — sodium chloride which cannot cause any more haemolysis in the blood under consideration. The mixture is then centrifugalised again and the sediment separated and treated in the same way as before, and if after a sufficient number of washings, it is found that the supernatant fluid at the top is colourless and the sediment is red, then it is evident that complete haemolysis has not taken place; the sediment may further be tested for the presence of haemoglobin-containing corpuscles, and stained with a proper stain to show the presence of stained erythrocytes. If, ou the other hand, the sediment is colourless, then evidently complete haemolysis has taken place. ! Under ordinary circumstances, an =) sodium chloride solution will serve the purpose of the inactive fluid mentioned above. We shall, however, see, later on, that this solution cannot always be used for the above purpose, as, for instance, when the blood has been previously treated with a saturated sodium chloride solution. We began our investigations by testing different specimens of blood from the healthy students of the Campbell Medical School, Caleutta. The blood of a large number of students was examined in the above way, n : i 100 sodium chloride solution, generally the latter. In none of these cases did I observe complete haemolysis conforming to the definition given above. In other words, I always obtained a red sediment after the blood was treated in the above way. At the same time the dark coloration described by Wright n n and others was generally obtained with two volumes of —— a to a the diluting fluid being either distilled water or sodium chloride solution. That the red sediment obtained in the above experiments contains undissolved erythrocytes can be shown in the following way :—- (1) The sediment though insoluble in 0 sodium chloride solution is dissolved after being repeatedly washed with mn 100 case may be. sodium chloride solution or distilled water as the HAEMOLYSIS OF BLOOD 61 (2) The sediment shows the presence of haemoglobin-containing oe, _ erythrocytes under the microscope. = ~ (3) The sediment, when stained with a proper stain, shows the presence of stained erythrocytes. In some of my cases I put the mixture of blood with distilled water, a well as the mixture with sodium chloride solution, for nearly . » hours in corked tubes, and it was found that complete haemolysis a fat taken place even after this period, the temperature of the room ping 29°C. during the day. ‘Phe question now arises as to how many parts of distilled water or _" sodium chloride solution can completely haemolyse one part of = human blood. I have made dilutions of blood several times with one, a two, and up to nine parts of distilled water, as well as +00 sodium - chloride solution, and have obtained the red sediment in all of them after = repeated washing of the sediment with a sodium chloride solution. _ The sediment also showed the presence of haemoglobin-containing erythrocytes, which took easily eosin stain. In one case I diluted a specimen of blood with 40 parts of distilled water, and kept the mixture for twelve hours in a small tube, and could detect the presence of haemo- globin-containing erythrocytes, easily taking the eosin stain. The corpuscles containing haemoglobin, and which are found in the ee cerment described above will, in future, be called sediment corpuscles. “4 The sediment corpuscles can be fixed in pure methyl alcohol or aus alcohol, and stained with a dilute solution of eosin in water, by _ immersing the slides from twelve to fourteen hours in the solution, or may ‘: be stained by mixing the sediment with a dilute solution of eosin in —— “0 gaan chloride solution. a. 2 append here a plate showing the sediment corpuscles after being : fixed and stained in the above manner, they having been obtained by a mixing human blood with 2 vols. of 0 ’ sodium chloride solution, and the mixture left undisturbed for one hour at the temperature of the room (29°C.). ee 62 BIO-CHEMICAL JOURNAL A similar phenomenon is seen when blood is treated with nine volumes of ia sodium chloride solution, except that the sediment corpuscles are much fewer in number and the destructive changes noticed in them are more marked. The laking of blood by hyposmotic sodium chloride solutions has been supposed to be due partly to osmosis and partly to the specific sensibility of the cortical layer of the erythrocytes or the membranes holding the haemoglobin within the corpuscles. I consider that at least a third factor is present upon which the above phenomenon is to some extent dependent. If we examine the sediment corpuscles, it is easily seen that a large number of them have undergone marked changes in shape, size and in the amount of contained haemoglobin. Some of them are Sediment corpuscles obtained after treating one part of human blood with two parts of 165 sodium chloride solution. (4 oil immersion. No. 3 eye piece.) Depth of shading shows amount of haemoglobin. Drawn by B, L. Doss, Caleutta. resistant in the sense that they have not at al! discharged their haemo- globin. But there are others which show marked diminution in the amount of haemoglobin. Some show marked changes in the distribu- tion of their contained haemoglobin, as compared with the normal (see plate). Evidently in these the cortical layer of the envelope has been ruptured, leading to partial escape of the haemoglobin. What is it, therefore, that prevents the remaining portion of the haemoglobin from being completely discharged? The most probable assumption is that the process is to some extent allied to Mass Action that takes place in - chemical reactions. In other words, there probably exists a union allied to chemical combination between the haemoglobin of the erythrocytes and other portions of their structure, perhaps including the salts. This combination is probably broken up when water enters their structure, e.g., when they are treated with hyposmotic sodium chloride solutions, but the amount of decomposition will depend upon the relative masses of the interacting compounds. HAEMOLYSIS OF BLOOD 638 Resistance or tur Eryrurocyres to HAEMOLYSIS UNDER ABNorMAL CoNDITIONS If one volume of normal blood is mixed with two volumes = sodium chloride solution, slight haemolysis is not infrequently observed, while with —_ it is often distinct or sometimes even marked. In certain n forms of anaemia, 3 causes no haemolysis, while go causes very slight or no haemolysis. In other words, in some forms of anaemia, the blood resists haemolysis more than normal blood. Captain McCay by estimating the haemosozic value of serum in certain forms of anaemia, also arrives at the same conclusion. He thinks that this might be due to __ the presence of something of the nature of an antihaemolysin.! Curicat Data or A Case or ANAEMIA Havinc Hieu Resistine Power to HAEMOLYsIs Patient (aet. 25) was admitted into my wards on September 16th, 1908. Condition on admission: patient anaemic, slight oedema of the extremities, no albument in the urine, stools contain ova of ankylostomata. On September 22nd, 1908, he had red cells—2,700,000, haemoglobin—-20 per cent. One volume of blood plus two volumes of oy sodium n chloride—no haemolysis; one volume of blood plus two volumes of sy sodium chloride—very slight haemolysis. The patient has been treated with thymol since admission, but. has been worse since coming into hospital. He is now markedly oedematous, is more anaemic, and his condition is considered hopeless. On November 10th, 1908, his erythro- cytes showed more resistance to haemolysis, two volumes of 5 plus one volume of blood not showing the slightest amount of haemolysis. To determine whether this resisting power was due to anything present in the serum, I washed the erythrocytes several times with a deci-normal sodium chloride solution, till the supernatant fluid obtained on centri- fugalisation was found to be perfectly free from the slightest trace of of albumen. One volume of the suspension of the erythrocytes was treated with two volumes of oe sodium chloride solution, the resulting mixture 1, MeCay, Bio-Chemical Journal, Vol. T11, 1908, p, 97. 64 BLO-CHEMICAL JOURNAL did not show any haemolysis at all. Salinity was 0585 per cent., and alkalinity, estimated in the way pointed out by Moore and Wilson,} was 0095 H,SO,. Red cells—1,770,000; haemoglobin—-13 per cent. It will thus be seen that the resistance to haemolysis was not due to anything present in the plasma, as the same resistance was observed when the serum was replaced by a deci-normal sodium chloride solution. There was a marked diminution in the alkalinity of the blood, which can not, however, account for the resistance of the erythrocytes to haemolysis. BeHAviour or THE Eryrurocytres or MAN AND THE RABBIT TOWARDS SATURATED SOLUTION OF SopruM CHLORIDE When one volume of human blood is mixed with fifteen volumes of a saturated solution of sodium chloride in distilled water the mixture at once becomes turbid. This turbidity is quickly followed by a marked ~ solution of the erythrocytes, and the mixture at the same time becomes clear to a great extent. In the rabbit’s blood no such clearing up of the mixture takes place in a short time, and the fluid remains turbid for a longer time. If the rabbit's blood, after it has been treated in the above way, be centrifugalised within ten minutes, it is found that the super- natant fluid is faintly red, showing that only a slight haemolysis has taken place by this time, contrary to what is found in the case of human blood in which the supernatant fluid is found to be markedly red. _ If, however, the sediment of the rabbit's blood obtained above, is mixed with the super- natant fluid at its top, it possesses the remarkable property of dissolving to some extent, showing as it were that some haemoglobin was squeezed out of the erythrocytes during the process of centrifugalisation. The n same sediment, when treated with O° sodium chloride solution, dissolves to a much greater extent. It is thus evident that the undissolved erythrocytes are markedly altered in their constitution after the treat- ment of the blood with saturated sodium chloride solution. Examined , under the microscope they are found to be much contracted, but most of them retain their globular shape and do not look crenated or wrinkled. The most probable explanation of this haemolysis appears to me to be a marked change in the outer walls of the erythrocytes brought about by the sodium chloride of the saturated sodium chloride solution; probably a sort of combination takes place between the sodium chloride and the outer layer of the erythrocytes which finally leads to its destruction. When the blood is mixed with the saturated sodium chloride solution, no 1, Moore and Wilson, Bio-Chemical Journal, Vol. 1, 1906, p. 297. ae eS eee ee eee a F ie i ~~ ae HAEMOLYSIS OF BLOOD 65 | bt water comes out of the erythrocytes by the process of osmosis and ey accordingly contract; when the sediment from the above is treated _ with = sodium chloride solution water re-enters their structure, and, as a result of this, they try to expand and regain their original size. But either they burst before or as soon as they recover their original size, or it may be that the water dissolves the compound of sodium chloride and the outer wall of the erythrocytes and consequently a marked haemolysis results. ‘The initial turbidity, mentioned above, is probably due to the production of the compound, which is probably very easily decomposed. It is evident that osmosis alone cannot explain the haemolysis of blood by saturated sodium chloride solution. The remarkable phenomenon of haemoglobin coming out of the corpuscles during centrifugalisation is probably explained by assuming that the damaged walls of the erythrocytes allow haemoglobin to pass out of them 2 by a process allied to filtration under very high pressure. As soon as the unstable compound of the sodium chloride with the outer layer of the erythrocytes is decomposed, the latter behave like small spheres of sponges ig containing dissolved colouring matter. 4 4 66 FURTHER OBSERVATIONS ON THE ACTION OF MUSCARIN AND PILOCARPIN ON THE HEART By HUGH MacLEAN, M.D., Carnegie Fellow, formerly Lecturer on Chemical Physiology, in the University of Aberdeen. aw From the Physiological Laboratory, University of Aberdeen (Received December 7th, 1908) In a former paper! I described the parallelism which obtains between vagus inhibition and the effects of muscarin and pilocarpin on the hearts of certain vertebrates. This parallelism has two aspects :— T—Paratier Distrrevtion or Errecrs oN THE Parts oF THE HEART — All the evidence afforded by my experiments goes to show that in the vertebrate heart (adult) the action of muscarin and _ pilocarpin reproduces in a remarkable way the effects of stimulation of the inhibitory nervous apparatus, the incidence of the effects on the different portions of the heart being similar in the two cases. Only the parts of any particular type of heart that are supplied with inhibitory nerves are acted on in the characteristic fashion by a suitable dose of muscarin or pilocarpin— causing an arrest which, like that induced by vagus stimulation, is set aside by atropin.2 Thus the ventricle of the eel and tortoise are not acted upon, while that of the frog and newt are, the latter very strongly. It is not simply that the parts endowed with the highest power of spontaneous rhythm, are more readily acted upon by the drugs*; the case — of the newt’s ventricle, with little or no spontaneous rhythmic power, affords important evidence in this connection. TI—Parattet VartaTion IN EFFrEecTIVENESS OF VaGus INHIBITION AND THE Two Drvueas Such variation may be due to different causes, such as :— (1) Seasonal changes associated with the breeding season. Inanition may play some part in frogs and other animals that have been in captivity for considerable periods, but similar changes were observed in recently caught eels. 1, This Journal, Vol. III, p. 1. 2. The drugs were tried both by dropping on the heart and by intravascular injection- methods which, as is known, may not in the case of some drugs yield identical results. 8. See Gaskell’s criticism (Journal of Physiology, Vol, VIII, p. 408) of Kobert’s results (Arch. f. erp. Pathol. u. Pharmakol., Ba. XX, s. 92). ACTION OF MUSCARIN AND PILOCARPIN 67 - (2) Overdosing with the drugs so that immunity becomes established.! (3) The ‘exhaustion’ following prolonged stimulation of the inhibitory nerves. The activity of the inhibitory nervous mechanism was tested by : — (a) Reflex excitation of the vagus. (b) Faradisation of the nerve in the usual way. (ce) Faradisation of the sinus in the eel and newt, and of the sino-auricular junction in the frog (‘posterior white crescent’); weak and moderate currents were employed, such as when applied in normal heart give the characteristic inhibitory effects readily abolished by atropin. In the course of my work I was quite aware (in 1905) of the fact that when the usual stimulation of the vagus nerve or the sino-auricular junction in the amphibian heart failed to inhibit—-whether from absence of inhibitory power depending on seasonal changes, overdosing with musearin or pilocarpin, exhaustion of the inhibitory apparatus after repeated and prolonged stimulation, &e.—it was still possible to arrest the heart beat for very long periods by running up the secondary coil of the induction machine with the electrodes applied in a certain manner to the sino-auricular junction. I did not, however, attach any importance to these results—obtained by the use of such strong currents and differing in yarious ways from the phenomena of ordinary vagus inhibition—as indications of the condition of the inhibitory nervous. apparatus. That _ faradisation with certain strengths of current can stop the heart in certain conditions when pilocarpin is ineffective, has very lately been noticed independently by McQueen.? _ Some more recent observations I have made entirely confirm the opinion I then formed in regard to this form of arrest being due to local effects on the cardiac tissue produced by the powerful current and quite different in their nature from true vagus inhibition. The arrest is easily got in the amphibian heart (frog, newt, salamander) when a sufficient strength of current is employed, more especially when the electrodes ; are made to embrace the sino-auricular junction. Application in the | usual way (1-2 mm. apart) to the ‘ posterior crescent’ may also produce 4 the phenomenon, but not with so much facility, as a rule. During the - application of the current there is commonly an acceleration of the heart % beat, and then, when the current is stopped, a standstill of auricles and 1. See Marshall, Journal of Physiology, Vol. XXX1, p. 129. 2. This Journal, Vol. ILI, p, 402 (Preliminary communication). — es oe - - ae a a Al ee + ies. 2 ic ° - Ny 2 2", : Bhai tie : re. 7 P ‘ a s ae ot ae “A att aii r es ed ages, ou oe 68 BLO-CHEMICAL JOURNAL ventricle of very variable duration—from a few seconds up to eight or ten minutes or more. Sometimes the period of standstill begins while the current is still applied. The sinus commonly goes on beating regularly during the whole period, but its beats fail to be propagated to the auricles and ventricle, being blocked at the place where the current was applied. Sometimes the sinus stops also. A single stimulus applied to the ventricle during the period of standstill gives a single reversed beat of ventricle and auricles, the contraction failing to pass the blocked area to the sinus. Again, when recommencement takes place, a condition of partial blocking at the faradised area often remains evident for some time, only every second or third beat passing (at first) from sinus to auricle and ventricle. When the current is kept applied for some little time a naked-eye change becomes evident in the faradised area—a distinct whitening or opacity near the electrodes and between them ; a feature noted long ago by Wesley Mills as a result of the application of very strong currents. The narrow isthmus of the sino-auricular connection where these blocking effects are readily induced is, of course, of known structural and physiological peculiarity and complexity. When the heart recommences beating after a period of standstill, the beats are of good strength, as are also beats artificially excited by direct stimulation of the ventricle during the arrest of the normal rhythm. This is, of course, what might be expected in a ventricle which only stopped on account of blocking; there is no sign of the marked depression of contraction force and excitability such as may be induced by vagus inhibition. When the action recommences there is no increase in force beyond what is shown by the first beat—except such as one may see in the frog- heart as a result of a prolonged period of quiescence (staircase), e.g., resulting from Stannius ligature. Re-application of the current during standstill may excite beats during its continuance; this obviously arises from spreading of current to the auricle. There are various other minor - features in the behaviour of the heart under the influence of strong currents, applied in various ways, which hardly seem to call for detailed description—results depending on escape of current to other parts of the organ, electrolytic and thermal effects, &c. Blocking can, of course, be induced in this region, as elsewhere, by other and simpler means which do not involve the same complications. The same heart may be brought to a standstill répildtedly by applying the current at intervals. There is not the same susceptibility to ‘ fatigue ’ that is seen in the case of true inhibitory nerve excitation. Again, a ACTION OF MUSCARIN AND PILOCARPIN 69 weak ill-nourished heart seems to be more easily stopped than a vigorous - one—in contrast to what has been often noticed in regard to vagus The arrest occurs almost immediately or after some seconds, or, in the case of somewhat less powerful currents, after more prolonged and repeated application. The exact strength of current sufficient to produce these effects varies according to circumstances—conditions affecting nt density in the faradised tissue, size and position of electrodes, 988 or emptiness of the organ, the duration of the application of the ent, the state of the heart, ete. | With one Daniell cell in the primary circuit and an ordinary Be is induction machine, the secondary coil at 7-8 em. usually suffices ; the current is perceptible on the tongue at about 24-25 cm., and causes cular contraction when applied to a frog’s sciatic nerve at 45-50 cm. Such a current is strongly felt on the dorsum of the dry hand. With the small Harvard inductorium the secondary coil has to be moved up to about 4 or 5cm.; the current is perceptible on the tongue with secondary coil at full distance and inclined to 45°; and stimulates the sciatic nerve with the secondary coil at its full distance and inclined to 85° or 86°. Under certain _ conditions as to the mode of applying the electrodes, duration and density of current, state of heart, etc., currents weaker than these may produce essentially similar results. The currents employed to produce the cardiac arrest are quite sufficient, when applied to the frog’s sciatic nerve, or skeletal muscle, to _ cause the depression or abolition of excitability, well known as the Wedensky effect. They are obviously quite unsuitable for testing the condition of the inhibitory nervous mechanism of the heart. It is clear that an incautious strengthening of the faradic current beyond certain a may lead to confusion between two different forms of cardiac rest :— (1) Ordinary inhibition due to excitation of the inhibitory ne nervous apparatus, and (2) A stoppage depending on the direct effect of the current on the faradised tissue causing depression, blocking, etc. This phenomenon may be termed pseudo-inhibition. It is by no means impossible that such confusion may have sometimes oceurred in the past; it is often difficult in consulting the writings on the subject to be quite sure as to the exact strength of current employed, the duration of its passage, and the precise mode of applying the electrodes ; a brief application of current is, ceteris paribus, less likely to give the 70 BIO-CHEMICAL JOURNAL second-mentioned form of arrest. Burdon-Sanderson, in his F Practical ixercises,’ long ago directed the faradisation to be for a second or less, the points of the electrodes being not more than a couple of millimetres apart, and Porter, in his recent * Introduction to Physiology ’ (1906) specifies faradisation ‘ for a moment.’ ‘Lhe effect of atropin on the arrest indicates the type to which it belongs. > Fic. 1. Faradisation of heart immune to pilocarpin; pseudo inhibition. Figs. 1 and 2 show tracings of the ventricle of a frog’s heart. Fig. 1 is from a heart which was found to be immune to pilocarpin. Faradisa- tion of the white crescent in the usual way with the secondary coil at 10-12 cm. (primary cell 09 volt) gave no sign of inhibition. The secondary coil was then moved up to 6°5 em. and standstill resulted, as seen in the tracing—beginning when the current was stopped after being applied for about fifteen seconds. Fig. 2 shows a similar standstill in the same heart after a large dose of atropin. The sinus action continues in each case, though much more visible in the tracing in Fig. 1. The time tracing shows seconds in eat h case, Fic. 2. Faradisation of the same heart after atropine ; pseudo-inhibition. The parallelism between the effects of muscarin and pilocarpin and those of electrical stimulation of the inhibitory nerves is not affected by the results described above—obviously due as the latter are to the what holds = tion of the sinus in the eel and newt or of the sino-auricular junction in a a a a ad a < ih ah Bias a Thai 7 — a ee, oe il ‘ ve " ' s —_ i iy ve = oP. i ret : : eat ee ee, . BA cin 7 = pe “i ‘ * i igs ak > fice ga! 2 ¢ ? ei the ‘ 5 L os : es y ’ . aN ACTION OF MUSCARIN AND PILOCARPIN 71 x effects of the excessively strong current on the cardiac tissue "(causing blocking, etc.) and not affected by atropin—in contrast with — regard to the inhibitory influence excited by faradisa- the frog when done in the ordinary way with moderate currents, an experiment long familiar to physiologists. It is known that atropin also abolishes the local inhibitory effects produced by weak faradic currents in the auricle of the tortoise and eel, a __ and in the auricle and ventricle of the newt. Evidence has been adduced by Gaskell" in the case of the tortoise’s auricle, and by MacWilliam? in that of the eel, showing that these local inhibitory effects are due to excitation of inhibitory fibres in the auricular wall and not to direct effects on the muscle. The exact place of attack by atropin (whether on “nerve endings’ or muscle proper) is immaterial in the present connection ; the essential point is that the influence of inhibitory fibres (preganglionic or postganglionic) is cut out by atropin. Even strong currents are then unable to cause the usual inhibitory arrest in a state of relaxation which is, in ordinary circumstances, easily and strikingly obtained in the eel’s auricle and newt’s ventricle. In the light of the evidence available from various sources regarding the action of muscarin and pilocarpin, it is clear that their effects are closely bound up with the functional efficiency of the inhibitory nerves, and their special distribution in the various parts of the vertebrate heart. The effects of the drugs are, with most probability, to be ascribed to a * stimulating ’ influence (by chemical interaction, no doubt) on some part of what, for the sake of brevity, has been termed ‘ nerve endings’ of the vagus (postganglionic) fibres, i.e., on some part of the linkage between the nerve fibre and the fundamental contractile mechanism of the muscular fibre (myoreural junction, ete.). I have to thank Professor MacWilliam for the facilities he has kindly afforded me for carrying out my experiments in his laboratory. 1. Sehifer’s Tert-book of Physiology, Vol. TI, p. 208. 2. Journal of Physiology, Vol. VI, p. 228. 72 THE OCCURRENCE AND DISTRIBUTION OF CHOLESTEROL | AND ALLIED BODIES IN THE ANIMAL KINGDOM — By CHARLES DOREE, M.A., B.Sc., Lindley Student of the University of London. , From the Physiological Laboratory, University of London (Received January Ist, 1909) A large number of observations, accumulated in the course of the past fifty years, have shown that cholesterol is a constant constituent, so far as they have been examined, of all animal tissues. The investigations recorded relate chiefly to man and a few of the more common mammals and birds, in all of the organs of which, and in most of their fluids and secretions, cholesterol] has been found. It is evident that the constant presence of such a substance indicates its great importance from a vital standpoint, and necessitates its recognition as a primary constituent of all protoplasm. ‘This conclusion has recently been fully emphasised by various writers, who have called into question the dictum of Pfliiger ‘Nur das Eiweiss ist lebendig,’ and have assigned to the lipoid class of bodies, of which cholesterol is one of the most important members, functions second only in importance to those of the proteins themselves. What these functions may be in the case of cholesterol we do not know. They would, one must suppose, be quite different from those of the proteins, for whereas these bodies are, from a chemical point of view, unstable, being capable of rapid transformation into innumerable other substances, more or less complex as required by the life of the organism, cholesterol is characterised chemically by a remarkable stability. That it is constantly associated in the cell with lecithin has long been known ; that lecithin can assist the action of various poisons which act by producing haemolysis, and that cholesterol, on the other hand, functions . us an antitoxin in this respect, are facts which have recently come to light, and in them, possibly, may be found a clue to the imporfant question of the part played by cholesterol in the life of the organism. It is a remarkable fact that while cholesterol has been isolated uniformly from the tissues of mammals and birds, whenever the lipoid bodies contained in them have been investigated, no other substance similar to it had, until quite recently, been discovered in animal proto- plasm.'| From the vegetable kingdom, on the other hand, a large 1. With the exception of the isocholesterol of Schiilze, which will be discussed in the sequel. Pt ra. ae ¥. 7 “ae an a rag Oe oF DISTRIBUTION OF CHOLESTEROL 73 Diaibex of bodies, isomeric with, and no doubt closely related to, cholesterol, have been obtained—the phytosterols. Although recent _ observation shows that the number of these is not so great as previously supposed, owing to the fact that many of those described under different names are mixtures of phytosterol itself, which occurs in a pure form in wheat germ,! with other allied bodies, still, not only do many different vegetable cholesterols exist, but, frequently, different forms occur im the same plant, each associated with one or other of the plant _ structures. With cholesterol these various phytosterols make up the cholesterol group. Except in the case of cholesterol itself; we have _ little or no knowledge of the chemical nature of any of these bodies, but, : for the present purpose we may consider the cholesterol group as consisting of :— ae (a) A number of isomeric secondary alcohols C,,H,,0, which are all unsaturated, and possess one well-defined double fab: in the molecule. l= These are all laevorotatory and give the colour tests of Salkowski and Liebermann. d : (b) A number of bodies, almost entirely of vegetable origin, which are undoubtedly similar to those of the preceding class, but which differ from them in their general properties, and, probably, are not isomeric with cholesterol. Their relationships to cholesterol and to one another are entirely unknown. (ec) The natural derived product coprosterol, with which, for reasons given below, may be classed isocholesterol. If, then, cholesterol is a body which is one of the primary constituents ____—s of animal protoplasm, we should expect to find it not only in the highly ; ji ‘organised animals, but throughout the series from Chordata to Protozoa— _ or if cholesterol itself were not present its place should be filled by other _ and closely related forms. In the latter case it might be found that each of the great classes of the animal kingdom was characterised by the presence of a different member of the cholesterol group. On the other hand, if cholesterol is not of primary importance to all forms of life, it is not impossible that animals might be found into the composition of whose protoplasm it did not enter. In regard to the lipoids contained in the lower animals our information is very scanty. In the older literature we find occasional references to cholesterol as a constituent of one or other of these, but no precise identification of the body was, as a rule, made. Quite recently the work of Henze? on the Sponge, Suberites domuneula, 1. Burian, Monatshe/te, XVTIT, 553. 2. Zeit. physiol. Chem., 1908, LV, 427. OES 2. ee y i — a * a co) ‘ 74 BIO-CHLEMICAL JOURNAL and that of Menozzi and Moreschi! on the pupae of Bombyx mori, have shown that in the invertebrata there exist cholesterols which differ from the common cholesterol of the vertebrata. The present writer, on the other hand, in a preliminary investigation,? found that in two species of sea anemone (Coelenterata), cholesterol itself was present, and with a view to throwing more light on the distribution of cholesterol in the animal series, and to solve, if possible, some of the questions referred to above, it was decided to examine the cholesterol bodies contained in a number of animals typical, so far as possible, of each of the great sub- kingdoms of Animalia. An account of these experiments will be given in the following pages. tate CLASSIFICATION AND T'ypES SELECTED The types selected for experiment may be classified as follows :—* CHORDATA— MammMaria Lepus euniculus, the rabbit. Reprinia Tropidonotus natric, the grass snake. Pisces Scomber scombrus, the mackerel. MOLLUSCA- Gastrrovopa Buccinum undatum, the whelk. ARTHROPODA— Crustacea Carcinus menas, the crab. INSECTA Blatta orientalis, the cockroach. ANNULATA— Cua@roropa Launbricus terrestris, the earth worm. ECHINODERMATA~— ASTEROIDEA Asterias rubens, the starfish. COELENTERATA— Actinozoa —- Tealia crassicornis, | ake i sea anemones. Actinia equina, j PORIFERA Cliona celata, E phydatia fluviatilis, | Spongdla lacustris, eae wise Bete Atti della R, Accad> dei Lincei [V] XVII, 95. 2. Proc. physiol. Soc., XXXVII, July, 1908. 3. Based upon the system given by Parker and Haswell, T'ext Book of Zoology, Vol. 1, Macmillan, 1897. DISTRIBUTION OF CHOLESTEROL 15 Meruop or ExPreriMENT The animals were, if necessary, killed with chloroform, and then ‘ground up in a mortar with coarse sand, plaster of Paris being added from time to time to dry up the crushed material. If the tissues were very tough, as in the case, for instance, of the starfish, they were first passed - through a mincing machine, all juices expressed being collected in plaster ____ of Paris. The mass so obtained was left till it had become perfectly dry and hard, after which it was coarsely powdered, and then extracted in a large Soxhlet apparatus with ether, for periods which varied between seven and fifteen days. The ether solution so obtained was at once saponified with a large excess of alcoholic solution of sodium ethylate, according to the method of Kossel and Obermiiller. To ensure complete saponification the liquid was allowed to stand at least twelve hours, after which the precipitated soaps were filtered off and well washed with ether. The filtrate was then shaken several times with water to remove traces of soap, the ethereal solution dried over calcium chloride and the ether distilled off. ‘The crude unsaponifiable residue so obtained is generally described as cholesterol, and weighed as such. But in the case of animal extracts, it always contains brownish oily, or resinous substances, which may constitute as much as three-quarters of the total residue. For the purpose of the present work it was necessary to isolate the cholesterol in a pure state so that its identity could be established beyond question. If the quantity of residue was sufficient it was dissolved in absolute alcohol, filtered, the hot alcohol diluted to about 90 per cent. strength and the liquid allowed to crystallise. A microscopic examination at this point gave valuable indications of the presence of cholesterol, pure or in an admixed state. After the separation of crops of crystalline matter, the filtrates, evaporated to dryness (or if very small the original unsaponi- fiable material), were treated by the following method, in which the cholesterol is isolated in the form of cholesterol benzoate. ‘The dried residue was dissolved in pyridine (20 ¢.c. to each gram of substance), and un excess of benzoy! chloride dissolved in pyridine added and the mixture allowed to stand overnight. In this way the cholesterol is converted quantitatively to benzoate.! The pyridine solution was then poured into water, which precipitated the organic matter, and the whole allowed to stand, if necessary, until the colloidal solution, which at first formed, cougulated. This usually happened in a few hours, but very occasionally 1. Dorée and Gardner, Proe. Roy. Soe., Series B, 1908, LXXX, 228. , 7 oe eS —_—T- Ss Oe Rel ie a ode ay .2°* a a= ‘i 76 BIO-CHEMICAL JOURNAL salting out was necessary. After washing and drying the precipitate it was boiled out with absolute alcohol, the liquid allowed to cool, and the benzoate filtered off. Cholesterol benzoate is very insoluble in alcohol in the cold-according to a determination made for the author, 100 e.c. of commercial absolute alcohol dissolve only 0°12 gram at 20° C.—so that the unchanged oily matters can be completely separated from the benzoate, and if the quantity of alcohol used is measured, a correetion can be applied to the quantity of benzoate obtained. The benzoate crystallises well from ethyl acetate in large rectangular plates. The formation of a benzoate by this method serves in itself for the ? recognition of cholesterol, since, so far as the author's investigations at present carry, vegetable cholesterols do not benzoylate, or if so, imperfectly under the conditions described. But the characteristic properties of cholesterol benzoate enable it to be identified with certainty. It dissolves with difficulty in alcohol, from which it crystallises in square plates; it melts at 145°C. to a turbid liquid, which clears suddenly at 178°C., and this on cooling, shows a brilliant display of colours, of which a light blue at the higher temperature followed by a deep violet at the lower are very characteristic. Some other benzoates of members of this group are now known to give colours on solidifying. One being the sitosterol of Burian,! the colour phenomena of which are described by Ritter,? and the others the new sponge cholesterols described in the present paper. The colour phenomena of these, however, are quite different from those shown by cholesterol benzoate.* A second important method for the isolation and identification of cholesterol consists in its conversion to a dibromide by the method of Windaus.* In this process one gram of the substance dissolved in 10 c.c. of ether is mixed with a solution of 0°5 gram of bromine dissolved in 5 c.e. of glacial acetic acid. The mixture is allowed to stand at 0° C., when a crystalline precipitate of cholesterol dibromide forms, which is filtered off and washed successively with acetic acid, 50 per cent. acetic acid and ° water, after which it is pure. By this means an almost complete separation of cholesterol from, at any rate, vegetable cholesterols is possible. Whereas 100c¢.c. of the ether glacial acetic acid mixture dissolve only 0°6 gram of the dibromide at 20° C., phytosterol dibromide 1. Loc. cit.; from wheat germ. This body should now be called phytosterol. 2. Zeit. physiol. Chem., XXXIV, 431. 3. The colours may be observed very clearly if the benzoate is melted in a thin layer be —— oe a: plates. As the cooling is comparatively slow the order of the colours can be . exactly notec 4. Ber., 1906, XX XIX, 518; Chemiker Zeit., 1906, XXX, 1011. | “ —— — Te se He op Sa ee ee Se! DISTRIBUTION OF CHOLESTEROL 77 is very soluble. This reaction, until recently, was characteristic for cholesterol, but it has lately been found that the bombicesterol of Menozzi and Moreschi, and the sponge cholesterols described below also give the reaction in the same way as cholesterol. Since all these dibromides give the same analytical figures, the melting points become important as a means of identification. | Windaus gave the melting point of cholesterol dibromide, prepared and purified by his method as 123°C. Bondzynski and Humnicki,! who prepared it by the addition of a solution of bromine in light petroleum to a solution of cholesterol in the same solvent, found 109°C. as the melting point. Menozzi and Moreschi again,? in an examination of the cholesterol from hen’s eggs (which proved identical in all respects with the cholesterol from gall stones), found that the dibromide prepared according to Windaus’ instructions, but subsequently erystallised from alcohol, melted at 111°C. The figure obtained for the bromine content of this body agreed closely with that required for cholesterol dibromide, so that the substance was not altered in constitution by treatment with alcohol. The present writer, on the other hand, has found difficulty in obtaining it from alcohol in a crystalline form, but in order to decide the question of the melting point a sample of cholesterol was prepared from human gall stones in the usual way and converted to the dibromide by Windaus’ method, the instructions as to washing, etc., being exactly followed. The pure white substance was, without further purification, dried in vacuo, and found to melt at 123° C., decomposing at a few degrees higher. Windaus’ statement is thus perfectly correct. Clionasterol dibromide, prepared and washed in an exactly similar way, melted sharply at 114°C., and decomposed rapidly between 116° and 120°C. The melting point of bombicesterol dibromide, similarly made, is stated to be 111° C. Systematic Examination or Types SELECTED In the protoplasm of vertebrate animals cholesterol is universally present. In man it is especially abundant in the brain (2°5 per cent.) and nervous tissue (1 per cent.), while in the fat it is found to the extent of 0°35 per cent. and in dry muscle 0°23 per cent. It is also present in bile, 0°07 per cent., in blood, 0-09 per cent., and in milk, 0°082 per cent. In herbivorous animals, whose food contains no cholesterol, the figures, so far as they are available, follow very much in the same order. The “1. Zeit. physiol. Chem., XXII, 396. 2, Atti della R. Acead. dei Lineei [V] XVI, 91. 78 BIOCHEMICAL JOURNAL fact that the cholesterol obtained from various animal sources is one and the same substance, has been demonstrated by Menozzi,! who made a careful comparison of the physical and chemical properties of the cholesterol isolated from cow's milk, from horse brain, and from hen’s eggs. Recently, also, Diels and Linn,? on account of an apparent slight variation in the chemical behaviour of cholesterol obtained from egg-yelk, made a comparison of this specimen with others obtained from gall stones and from brain, respectively, and showed that when properly purified the melting point and rotation of the cholesterol from each of these sources was substantially the same. eine Cholesterol is the only body of its kind so far found in connection with the higher vertebrate animals, with the exception of the so-called iso-cholesterol, to which reference has already been made. This has only been obtained from the wool-fat of the sheep.’ It differs markedly from cholesterol in its properties, and seeing that it is a product excreted by the skin, may, perhaps, be classed with coprosterol, which is a derivative of cholesterol, normally excreted in the faeces by men,* and by carnivorous animals when fed on a raw brain diet.5 The relation of coprosterol to cholesterol is at present unknown, but coprosterol, unlike cholesterol, is a saturated compound, and probably contains two hydrogen atoms more in the molecule. It is not, however a simple reduction | product of cholesterol, and, most probably, has a somewhat different carbon skeleton. But the physical properties of coprosterol stand in marked agreement with those of iso-cholesterol. Alone among the known members of the cholesterol group, iso-cholesterol does not give the characteristic colour-reaction with sulphuric acid and_ chloroform (Salkowski’s test); coprosterol gives it in a modified way. Both these bodies too, are, unlike all the others, dextro-rotatory.. An alteration in rotatory power from negative to positive has been observed generally to be brought about by the saturation or modification of the side chain of cholesterol, which contains the double bond. The dextro-rotatory power . of coprosterol is, no doubt, due to such a modification, but whether that of iso-cholesterol can be ascribed to a similar change is less probable, since, according to Darmstidter and Lifschiitz,® it is, like cholesterol, unsaturated, readily absorbing bromine in chloroform solution, — Tso- Atti della R. Accad, dei Lincei (V) XVII, 91. Ber. 1908, XLI, 260. Schiilze, Ber. V, 1075; VI, 251; XII, 249. Zeit. physiol. Chem., XXII, 396. Proc. Roy. Soc., B. LXXX, 228. Ber., 1898, XXX, 97, 1122. ee -~ eo DISTRIBUTION OF CHOLESTEROL 79 cholesterol is still further distinguished from cholesterol and its isomers by the melting point and crystalline form of its acetate and benzoate. While the benzoates of the latter all melt at about 145° C. and crystallise from alcohol in rectangular plates, the benzoate of iso-cholesterol melts at 191°C. and crystallises from alcohol in needles. These facts are collected together in the following table :—~ ACETATE BENZOATE Crystal form pn YW tay ey See a q (dilute M.p. {ajo Mp. Crystal M.p. Crystal alcohol) (ether) form form Oblong 147° —31° 114° Plates 145° ~—s— Plates 2 Frocks Tsocholestero! ide ks 137° +60° Below100° Amorphous 191° Needles 100° +24° 88° Needles 122° Plates __ The presence of cholesterol in the blood and eggs of birds has been frequently observed. In the case of reptilia its occurrence has not, apparently been recorded, but it is well known as a constituent of oils obtained from various species of fish. Dog-fish oil, for example, is said to contain four to five per cent. of cholesterol. In order to investigate this point, and to ascertain whether any other member of the cholesterol group was present in the tissues of reptiles and fishes, an experiment was carried out with a typical representative of each of these classes. For purposes of comparison the cholesterol contained in the whole body of a small mammal (rabbit) was estimated by a similar process. CHORDATA. Mamaria. Lepus euniculus, the rabbit—A rabbit weighing 28 kilos. was killed, and the blood, which weighed 75 grams, was collected separately, mixed with sand and plaster of Paris, and, when dry, ground to powder; the rest of the animal, including the fur, was then passed several times through a sausage machine, the minced material being ground up with coarse sand, and dried with plaster of Paris. The total mass obtained was extracted for twenty days with ether, and the solution saponified with sodium ethylate and washed as already described. The total unsaponifiable matter weighed 6:0 grams. It was at once dissolved in pyridine and treated with an excess of benzoyl chloride. The product which was obtained on precipitation with water was boiled out with absolute alcohol, in which it was very difficultly soluble, and after re-crystallisation from ethyl acetate appeared in the typical crystalline form of cholesterol benzoate. In all 4112 grams of benzoate were obtained, which melted correctly and showed the characteristic colour play. This corresponds to 3239 grams of cholesterol, or 0117 per cent. A microscopic examination of the residues soluble in alcohol Ng ES es ee ieee! = (i ae eae ’ an —— 80 BIO-CHEMICAL JOURNAL (consisting of brown oily matter which slowly became resinous) euvetield no signs of any other crystalline matter. Rerrmia. Tropidonotus natriz, the grass snake.—Four grass sniadals weighing 246 grams, were killed, passed through the mincing machine and ground up with sand and plaster of Paris. The dry mass was extracted for eight days with ether, and the pale yellow extract saponi- fied with sodium ethylate. A large quantity of a pale brown soap separated, which was filtered off and washed. From the filtrate, after the usual treatment, 0°44 gram of unsaponifiable matter was obtained, which was usually free from colour. It was dissolved in alcohol and- crops of crystals weighing 0°12 gram were separated. These, after re-crystallisation, melted sharply at 145° to 146°C., and under the microscope showed the characteristic crystal form of cholesterol. On benzoylation of the residues in pyridine solution 0°09 gram of benzoate was isolated. This was very difficultly soluble in alcohol, from which it crystallised in square plates. It melted at 144° to 145°C. to a turbid liquid, which cleared at 178° C., and on cooling showed the colour play of cholesterol benzoate. The total yield of cholesterol was thus 0°21 gram, or 0-08 per cent. No other similar body was observed in the residues. Pisces. Scomber scombrus, the mackerel.—Five mackerel, weighing 1,452 grams, were ground up in a mortar with coarse sand mixed with plaster of Paris, and allowed to dry. The mass was then reduced to a coarse powder and extracted in a Soxhlet apparatus for eight days. After washing in the manner described above, the ether solution was pale yellow, and on evaporation left 1:07 grams of brownish crystalline matter. This was dissolved in 90 per cent. alcohol, and under the microscope showed perfectly formed, typical, cholesterol crystals, no sign of any other crystal or mixed form being observed. A small crop, weighing (08 gram, was isolated, re-crystallized from acetone, and found to melt at 145° to 146°C. The whole filtrate from this was benzoylated in - pyridine solution and yielded 0°37 gram of a benzoate, which, after re-crystallisation from acetic ether, appeared in the form of shining rectangular plates. These melted at 145°C. to a turbid liquid, which became clear at 180°C., and showed the colours characteristic of cholesterol benzoate. The alcoholic filtrate from the crude benzoate weighed 0:43 gram, and consisted of a brown resin which, on long standing, only showed minute traces of crystalline matter. The cholesterol obtained was thus about 0°42 gram, or 0°03 per cent., and no other similar body was observed. 7 = DISTRIBUTION OF CHOLESTEROL 81 MOLLUSCA. Gastrropopa. Buceinum undatum, the whelk.—A number of whelks, weighing 1,179 grams after removal of the shells, were minced up and treated with sand and plaster of Paris, the mass extracted for six days with ether, and the extract saponified as described above. The soaps were considerable in quantity and dark brown in colour; the filtrate was also dark brown, and on evaporation left 4°5 grams of beautifully crystalline unsaponifiable matter (0°38 per cent.). This was dissolved in dilute alcohol, and a microscopic examination showed the presence of typical cholesterol crystals, the same crystalline forms being observed down to complete dryness. Three crops of white erystals, weighing 0°75, 0°25 and 0°09 gram were separated and found to melt at 142° to 143°C. The whole 1:09 grams put together and re-crystallised from acetone, melted at 144° to 145° C., and appeared as pure cholesterol. The whole of the residues from these were dissolved in pyridine and treated with benzoyl chloride in the usual way. By this" means 0°37 gram of cholesterol benzoate was obtained, which, after re-crystallisation from ethyl acetate, melted at 145°C. to a turbid liquid, which became clear at 185° C., and on cooling showed the characteristic colour play. A sample of the pure cholesterol dissolved in ether was then treated with a solution of bromine in glacial acetic acid. In a few minutes the solution set almost solid. The precipitated dibromide, after washing and drying in the usual way, melted at 118° to 119°C. with decomposition, in which respect it agreed fairly well with cholesterol dibromide. The total yield of cholesterol was thus approximately 1-46 grams, or (124 per cent. The residue left after separation of the benzoate was a clear, brown oil, which, after standing for six months, contained no trace of crystalline matter, and showed no signs of solidification. Crruatopopa.— Henze! has recently shown that the hepato-pancreas of Octopus vulgaris contains fats and ‘ not inconsiderable quantities’ of cholesterol. ARTHROPODA. Crustacea. Carcinus menas, the edible crab.— A whole crab, weighing 538 grams, was ground up with sand, etc., and extracted for eight days with ether. On saponification of the extract a considerable quantity of a stiff reddish coloured soap was obtained. The filtrate from this, after washing, was pale yellow, and left 1°0 gram, or 19 per cent., of erude residue. This, dissolved in 90 per cent. alcohol, left a small quantity of brown insoluble substance. A microscopic 1, Zeit, physiol. Chem., 1908, LV, 438. 82 BIO-CHEMICAL JOURNAL examination of the solution showed abundance of typical cholesterol crystals, and from it 0°25 gram of nearly pure cholesterol was ultimately separated. After re-crystallisation from acetone, it was pure white, and melted sharply at 146° to 147°C. — Its identity with cholesterol was confirmed by the preparation of the following derivatives :— (a) The dibromide: 0°12 gram of substance was dissolved in 2 ¢.c. of ether and mixed with 0°6 c.c. of Windaus’ solution of bromine in glacial acetic acid. The mixture set almost immediately to a mass of crystals, which were filtered off and washed. After drying in vacuo, these melted at 120°C., turning brown and decomposing at 124° to 126°C. The filtrate on treatment with water deposited a white solid, which contained bromine, and melted at 100° C., decomposing at 120°C. (b) The benzoate: This was prepared by heating the dried substance with benzoyl chloride to 165° ©. for five minutes. The residue was boiled out with alcohol, and the insoluble white crystalline residue purified by crystallisation from ethyl acetate, after which it melted at 145°C. to a turbid liquid, became clear at 178° C., and on cooling showed the colour play characteristic of cholesterol benzoate. | The cholesterol isolated was, therefore, about 0°25 gram, or 0048 per cent. Insecta. Bombyx mori, the silkworm moth.—Owing to the fact that an oil has been obtained commercially from the pupae of this moth, several investigations have been made with a view to decide whether the unsaponifiable residue of the ‘ chrysalis oil’ contains cholesterol—as do other animal oils—or a phytosterol. Lewkowitsch,! who first examined the question, came to the conclusion that cholesterol was present in the oil, since, by the use of Bémer’s acetate method, he was able to isolate from it an acetate whose melting point, after successive crystallisations, finally stood at 114°C. M.Tsujimoto,? however, who has recently made a further investigation, obtained 1°63 per cent. of unsaponifiable residue from the oil, from which, after repeated purification, he prepared a substance of M.p. 143° C., which in crystalline form and the meltin point of the acetate (125° C.) corresponded rather with phytosterol. Realising that the question of the presence or absence of cholesterol could be definitely decided by the application of the methods described in the introduction to this paper, a quantity of the pupae was obtained from France with a view to the extraction of the cholesterol body from 1. Zeit. fur Nahr. u. Genussmittel, 1907, XIII, 552. 2. Journ. of the Coll. of Engineering, Tokyo, Japan, 1908, TV, 63. DISTRIBUTION OF CHOLESTEROL 83 _ them on a large scale, when a paper by Menozzi and Moreschi! appeared which rendered the proposed investigation unnecessary. These authors showed that chrysalis oil contains 10 per cent. of its weight of unsaponi- fiable matter, and that in this there are probably at least four different substances, two of which are paraffin hydrocarbons. One of these hydro- carbons has apparently the formula C,,H,,, and a melting point of 62°. The main constituent, however, is a new isomer of cholesterol, to which the name of bombicesterol has been given, and which, in all its properties, bears an extraordinarily close resemblance to cholesterol itself. The melting point and rotation of the two bodies, and of their benzoates _ correspond exactly with one another, and the melting points of the ____ dibromides (111° C.) are stated to be the same. The crystalline form -___ of bombicesterol differs somewhat from that of cholesterol, but the most important difference between the two lies in the melting points of the formiates and acetates. Bombicesterol formiate melts at 101°C. (as compared with 96°C.), and the acetate at 129°C. (as compared with 114°C.). The benzoate is said to show on melting the phenomenon of liquid crystals, but apparently this is not accompanied by a play of colours, as in the case of cholesterol benzoate. The authors further mention that from the crude acetate of bombi- cesterol they obtained, by the method of Windaus and Hauth, two dibromides. One of these remained in the ether acetic acid solution, and was thrown out on the addition of water. On reduction it yielded bombicesterol acetate, melting at 129°C. The other dibromide erystallised out at once in the ether acetic acid solution, and on reduction gave an acetate of melting point 114°C., which is the same as that of cholesterol acetate. The point is apparently still under investigation, but it is obvious that the possibility of the presence of cholesterol in the pupae of Bombyx mori is not wholly excluded. In order to discover whether the larvae of Bombyx mori contained cholesterol, a quantity of the worms, weighing 102 grams, were kept without food for three hours and then killed. On grinding them up with sand a strong leafy smell was observed. The dried mass was extracted for five days with ether, and the deep green extract saponified. The soap was moss green in colour, this being, no doubt, due to the presence of chlorophyll. The green filtrate from the soap, also, on washing, became pale yellow, all*the green colour going into solution in water. The unsaponifiable residue weighed 01 gram (or 01 per cent). With this 1. Atti della R. Acead. dei Lincei, (V| XVI, 95. ‘ Sipeor ae id al B4 BIO-CHEMICAL JOURNAL small quantity little more than a microscopic examination was possible. The residue was dissolved in 90 per cent. aleohol, and a drop of the solution was allowed to crystallise on a slide. At first, long, very narrow plates, not quite rectangular, were seen, which, although quite different from those of cholesterol, answered to the description of the crystalline form of bombicesterol (lamine allungate ed acuminate) given by Menozzi and — Moreschi. The later crystals were in the form of long, narrow hexagons, — agreeing in this respect with those of the vegetable cholesterols. They probably consisted of the phytosterol of the mulberry leaves on which the worms were fed. The residue was crystallised from methyl alcohol,! in which it was very insoluble, and the small crops of white crystalline matter obtained — melted at 125° C., but not sharply. The eggs of Bombyx mori were examined in 1885 by Tichomiroff.? The eggs, which are laid in the summer, develop up to a certain point, pass the winter in this state, and continue development in the spring. Tichomiroff, using Hoppe-Seyler’s methods, obtained the following percentage figures: (A) for eggs which had reached the winter stage; (B) for eggs on the point of hatching :— Fat Lecithin Cholesterol A dew i 8-08 1-04 0-40 B say oe 4-421 1-76 0-35 As, however, no examination was made of the ‘ cholesterol’ obtained, its identity with either cholesterol or bombicesterol must remain uncertain. Blatta orientalis, the cockroach.—Cockroaches, weighing 194 grams, were killed and dried in the steam oven, after which the weight was - 65°5 grams. The solid matter was ground to powder with a little sand, and extracted with ether for fourteen days. The brownish ether extract on saponification gave a brownish coloured soap in considerable amount, which was very solid when dry. The unsaponifiable residue, which was — very liquid at 100° C., weighed 0°5 gram. It was treated with absolute alcohol in which a part readily dissolved, leaving an insoluble portion, which, on warming the liquid to about 40° C., melted, forming heavy oil drops. By decanting off the alcohol from these and repeating the process several times, a separation of the insoluble and the soluble parts was — effected. A. The part insoluble in aleohol weighed about 0°2 gram. Tt was 1. The methyl alcohol used here and elsewhere was purified, but not absolute. 2. *Chemische Studien iiber die Entwicklung der Insecteneier,’ Zeit. vhysiol. Chem., 1X, 525. DISTRIBUTION OF CHOLESTEROL 85 tely soluble i in petroleum ether, and Sip so in benzene. From i eca-arsralline On heating daw ly in a wide

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(Fig. 4.) With the vagus nerve things are different, it loses its efficiency upon the heart as the strength of the dose increases, failing to reduce the blood pressure, though it can still impede the heart beat to some extent; in this the action of the isothiocyanate closely resembles that of allyl sulphide. (Fig. 5.) | - On skeletal muscles the drug acts powerfully, producing spasm, the extensor groups being more affected than the flexor. Its action in small doses on cardiac muscles is slight and of short duration; beginning early, it passes off in about 150 seconds and amounts to no more than a slight decrease in the rate of the heart beats, with lengthening of the systole. With the frog’s heart phenomena occasionally seen with allyl sulphide es are well marked with the isothiocyanate, more especially so with winter frogs. In all cases pithed and decerebrated frogs were used, and the records obtained by placing the frog on a Pembry myograph, exposing the heart, inserting a hook in the apex of the ventricle, from which a thread passed to the lever of the apparatus. The drug, either pure or diluted with olive oil, was passed directly into an auricle by means of a hypodermic syringe. A dose of 1 minim (0°06 c.c.) of the pure drug stops the heart at once, but a dose of half that quantity only takes effect after thirty seconds, the ventricle muscle passing into delirium before finally ceasing to beat. The auricles, however, continued to beat synchronously, though jerkily, for a considerable time longer. ‘ One minim (0°06 c.c.) of a 33 per cent. solution produces some diminution both in amplitude and rate, followed suddenly some sixty seconds later by a marked change in speed with increase of amplitude, which gradually declines as the speed again increases for a while This is followed by marked slowing just before the sudden arrest of ventricle comes on, some 280 seconds after the injection. The auricles beat normally throughout, and continue so doing for another seventy seconds at least, after which their speed very gradually diminishes. Though arrested, the ventricle is not permanently stopped but resumes beating again with occasional stops, that become shorter as time wears on, until, by the end of fifieen minutes, the whole heart so far recovers as to beat quite rhythmically though slowly and with diminished force. /RNAL IO-CHEMICAL JOl : ; Ll4 “41 WlOJ} O9lj OSOYY PUB Buryoo]q SuULMOYS Spotoed oq} JO suOTyeINp GAIZV[OI OY} OS]V puv ‘UMOYS A[IVETO SI YOOTG JaveY Jo UOMeUIOUaYd oN, *dOIgN[OS OyBUNAOOTYVOSTI [ATV c OZ v jo °0°0 90-0 = UWITUTUT T Jo ofOLINe ouO OFUT UOTZoeLUI Aq JveY S,3013 oY} UOdN peonpord yooye oy] ee a ee es ee ee wee | Janne) ro Bul MOU"US 11! ISOTHIOCYANATE ALLYL pues ‘apo og U1 1v ‘gIQqUa B JO UTOA es aaa lal lel iil Mii bbibild bibiditidbebd bile dibiddiddddbbbbadhdddadddddbadihddiadbaaiaidaddhdaasiadadhaainaaderecenrmorrn me eee . TONTTTNTT GENETTTTY wrETeTreY wrererres ore Peete eee ioddn ony syooy Aq p TORII [ATTe It OFBUBASOIY OSI 2 ¥,0% = O10M SAO] jo WoTyNYlO 116 BIO-CHEMICAL JOURNAL With this dose the results naturally vary somewhat with the size and vigour of the frog used; with small weak frogs the heart is permanently arrested, the auricles always outlasting the ventricle for a considerable time; with large and vigorous frogs partial or complete recovery in the rhythmic sequence is customary; the speed, however, seems always permanently diminished. I have obtained the best results with an injection of 1 minim (0°06 c.c.) of a 20 per cent. solution into the auricle, as may be seen from the tracing here given, in which the production of heart block, with gradual recovery therefrom, is better illustrated than is possible by mere description. (Fig. 6.) When frogs, taken fresh from the fields in summer, are treated as above, heart block rarely supervenes, the heart either stopping suddenly as a whole with large doses, or becoming gradually slower and feebler with smaller ones, until it finally ceases to beat altogether. The effect of the drug upon the mammalian heart may be studied in the rabbit, provided artificial respiration is had recourse to, because the large doses required are very much more than sufficient to paralyse the respiratory mechanism. In all cases the ventricles are more profoundly affected than the auricles; with a dose of 7} minims (0°45 c.c.) of a 20 per cent. solution injected into the jugular vein the amplitude of the heart beat is first lessened, this is followed by diminished speed with some increase-in amplitude, but the heart is finally paralysed in about twenty minutes. (Fig. 7.) Only in one case was some blocking noticed, towards the end of the experiment and after the animal had received several consecutive and increasingly larger doses of the drug. In this case three auricular beats corresponded with one of the ventricles. With these large doses the auricles occasionally fail a fow seconds before the ventricles, but this is not the general rule. ConcLusION Allyl sulphide and allyl isothiocyanate act in a similar manner upon the organism, the latter drug being the more powerful; both paralyse the respiratory and vaso-motor centres, both produce muscular spasms and affect the heart beat, and both lower the body temperature, as most | essential oils do. Neither can be recommended for internal adminis- tration, despite the fact that they are commonly taken in minute quantity with food. a a y= . $ 117 - CHOLINE IN ANIMAL TISSUES AND FLUIDS By W. WEBSTER, M.D., C.M., Demonstrator of Physiology in the University of Manitoba, Winnipeg, Canada. From the Physiological Laboratory, University of Manitoba Communicated by Professor Swale Vincent (Received February 4th, 1909) IntTRopuctToRY AND HistroricaL _ Although the morphological changes accompanying nerve degeneration have been very minutely studied and certain definite results obtained, the study of the corresponding chemical changes has not, so far, thrown any light on the metabolism of the nervous tissues. Gulewitsch! was the first to isolate free choline, one of the substances entering into the constitution of nerve tissue, from extracts of ox-brain. Vincent and Cramer? isolated very small amounts of a similar substance by a process less likely to produce decomposition. These observations constitute the only direct chemical evidence of metabolic processes in nervous tissues. But it must be pointed out that the possibility of post-mortem changes, or of changes occurring during the manipulations involved in the isolation, cannot be altogether excluded. This latter objection seems, however, to lose its weight in view of the observations of Gumprecht,? who found by a micro-chemical test that a substance giving the reactions of choline is present in the normal cerebro-spinal fluid of healthy animals and of patients suffering from diseases other than nervous. Another fact pointing to the existence of metabolic processes in nervous tissue has been brought forward by Waller,4 who suggested that the increase in electrical response after repeated excitation of nerve is due to carbonic acid having been evolved in consequence of the activity of the nerve, The evidence put forward by Halliburton for the presence of choline 1. Zeitachr. {. physiol. Chem., XXTV, 8. 513, 1898. * 2. Journ. of Physiol., Vol. XXX, p. 149, 1904. 3. Verhandl. d. Congress /. innere Med. Wiesbaden, s. 326, 1900. 4. Centralbl. {. Physiol., XII, s. 745, 1899.' 5. Journ. of Physiol., Vol. XXVI, 1900-1, and numerous other publications. 118 BIO-CHEMICAL JOURNAL in saline extracts of nervous tissue can not, in our opinion, be accepted. He believes that the effect on the blood pressure of nervous tissue extracts, observed by himself and by Osborne and Vincent,® is due to the presence of choline, and that by means of this ‘ Physiological test ’ the presence of choline in animal extracts and fluids can be detected. This was disputed by Osborne and Vincent,’ Vincent and Sheen,* and Vincent and Cramer,® who pointed out the difference in behaviour between brain extracts and choline after the administration of atropine, and demonstrated that the depressor effects are due to substances other than choline. The chemical evidence submitted by Halliburton, which is based on the appearance of crystals of a double salt of platinum chloride in brain extracts, has been shown to be unsound. Vincent and Cramer, and independently Mansfeld,'° showed that these crystals consist almost entirely of inorganic salts, from which it is impossible to free the extracts even after repeated extraction with absolute alcohol. These results were confirmed by French and Allen;!! and Bayliss '? in his investigations on adsorption, showed that this phenomenon is due to the adsorption of inorganic electrolytes by organic colloids. The chemical evidence as to changes in pathological conditions of the nervous system is of a more definite character. Mott and Barrat!* were the first to show that degeneration in the spinal cord leads to a diminution of the phosphorus contents. Noll!4 found that the protagon diminishes after nerve section, and Halliburton!5 also showed that a diminution of phosphorus occurs under these conditions. These results led Mott and Halliburton!® to seek for choline in the blood in conditions of nerve degeneration. In a series of experiments on cats the sciatic nerves were divided, and the physiological and chemical tests for choline were applied to the blood of these animals. The results showed a striking parallelism between the effect on the blood pressure and 6. Journ. of Physiol., Vol. XXV, 1900. 7. Loe. cit. 8. Journ. of Physiol., XXIX, p. 242, 1903. 9. Loc. cit. 10. Zeitschr. f. physiol. Chem., Bd. XLII, 1904. ll. Proc. Physiol. Soc., p. 29, 1903; Journ. of Physiol., p. 30. 12. Bio-Chemical Journ., Vol. I, p. 209, 1906. 13. Proc. Physiol. Soc., p. 3; Journ. of Physiol., Vol. XXIV, 1899. 14. Zeitschr. {. physiol. Chem., XXVII, p. 370, 1899. 15. Croonian Lectures, 1901. i ae Eee. Physiol. Soc., Feb., 1898; Journ. of Physiol., Vols. XXI, XXII, and XXIV, eb., 3 CHOLINE IN ANIMAL TISSUES AND FLUIDS 119 the number of crystals obtained. These results constitute the only experimental basis for the so-called ‘ Choline hypothesis,’ that degenerative processes in nervous tissues can be detected by the appearance of choline in the blood. Although it is now generally admitted that the tests employed in these experiments are fallacious, the question has not, up to the present, been re-investigated in experiments in which more reliable tests for choline have been employed. Such tests are recommended by Gumprecht,’’ French and Allen,'® Donath,!® and Rosenheim,?® but have only been applied to the cerebro-spinal fluid in the case of patients suffering from nervous diseases. With the exception of Gumprecht’s observations, no controls have been made upon patients suffering from other kinds of diseases, although the facts given above as to the presence of a substance giving the reactions for choline in normal cerebro-spinal fluid would appear to have rendered some such control imperative. The attempt to find choline in the blood in cases of nerve degeneration has now been almost completely abandoned. The results of clinical investigation by various observers are very conflicting, and are explained by each author as being due to the inferiority of the tests employed by the others. _ EXPERIMENTAL In view of the conflicting nature of the evidence before us, it seemed to me very desirable to test the choline hypothesis by means of a series of experimental lesions in animals. My observations have so far been restricted to testing for choline in the blood of those animals where choline is normally not present. The fact that choline is found in normal cerebro- spinal fluid would necessitate a quantitative estimation of choline in this fluid under various conditions, a proceeding which appears to present very considerable difficulties. In my experiments I have employed seven dogs and one cat. In six dogs and in the cat both sciatic nerves were divided and a long piece resected; the blood was taken a variable number of days after the operation. The results are put together in the following table :— 17. Loe. cit. " 18. Loe. eit, 19. Loe, cit, 20. Journ. of Physiol., Vol. XXXIII, p. 220, 1905-6, pol tiane: Normal ee Effect of extract 110 c.c. of € of extract Ae eos 9 reheat 7 1 ay tot q * 4 Pie hink fi ; aad . | ee — ee CHOLINE IN ANIMAL TISSUES AND FLUIDS 121 cf It may be mentioned that because dogs were employed instead of cats 7 in these experiments the precaution was taken to compensate for their larger size by injecting an extract from a large quantity of blood four to five times as much as the quantity employed by Halliburton.?? In order to produce even more extensive degeneration, in one dog a portion of the dorsal spinal cord about an inch in length was excised. After eight days the animal was killed, and its blood subjected to the physiological test. The effect produced upon the blood pressure was identical with that produced by an extract of an equal quantity of normal blood. It will be seen that there is no progressive increase in the depressor effect such as was described by Halliburton. In fact, in no case did the extract of pathological blood give a fall greater than that obtained by the extract from a normal animal. Further, the effects were precisely the same after the administration of atropine as before. This result completely confirms the observations of Vincent and Cramer, who arrived at the conclusion that the substance present in the _ pathological blood which gave a fall of blood pressure in Halliburton’s experiments was not choline, nor indeed anything arising from morbid processes, but some substance common to normal blood and to all animal tissues. CHEMICAL I have applied the chemical test as recommended by Rosenheim to the blood of animals operated upon as described above. Using a pure choline solution, the formation of dark brown crystals can be easily verified. They are very few in number, and small when a solution of choline of 1-200000 is used. I have, however, been unable to find any similar crystals in the blood of any of the animals experimented upon. An abundant crop of the platinum-chloride crystals can be obtained from both normal and pathological blood if the old test originally recommended by Halliburton is used. This, again, is confirmatory of the result obtained by Vincent and Cramer, and renders very doubtful the present claim of Halliburton that ‘the obtaining of a large crop of crystals, whether they be those of the choline salt or a mixture of the potassium and choline salts, is diagnostic of an extensive breakdown in nervous tissues.’23 .22. Such a precaution is really not necessary, because, together with the increase in size, there is an increase in the weight of the sciatic nerves, and, ecaalnar F of the amount of nervous tissue degenerating after section of the nerve. 23. ‘Oliver Sharpey Lectures,’ Brit, Med, Jowrn., May 4th, 1907. 122 BIO-CHEMICAL JOURNAL The addition of iodine solution to these crystals does not cause the formation of choline pericodide. Some brown crystals are formed, irregular in shape and varying in size, but none are oblong—the characteristic shape of the periodide crystals—neither do any of them resolve themselves into ‘oily droplets’ on drying of the solution, but retain their form days or weeks after drying has occurred. I have been greatly disappointed to find that none of the statements which constitute the basis of the choline hypothesis can be verified. The observations upon which the hypothesis rests are the outcome of investi- gations which have not been sufficiently controlled by the examination of normal tissues and fluids, since all the effects alleged to be characteristic of pathological conditions can be obtained in the normal state. That my results are not due to faulty technique can be proved by the following calculation :— . Taking the total weight of the central nervous system as 1,400 grammes, it can be calculated on the basis of our present knowledge of the chemistry of nervous tissue that about 8°5 grammes of choline are contained therein. This follows from the observations of Coriat,24 who found that 10 grammes of moist brain tissue yield 0°547 gramme of choline, as against the theoretical amount of 0°0584 gramme of choline calculated from Koch’s figures. It follows then that 1 gramme of moist brain substance contains 0:006 gramme of choline. The average volume of total blood is 4,700 c.c. After a sudden destruction of 1 gramme of brain substance the blood of a patient would contain 0°006 gramme of choline. This corresponds to a dilution of 1 in 800,000, therefore 20 c.c., the volume recommended and generally employed by Mott and Halliburton, would contain 0:000025 gramme of choline = one-fortieth of a milligramme. This only holds good if 1 gramme of brain is destroyed suddenly, as choline is oxidised in the organism. Asa rule, degenerative processes destroying 1 gramme of brain substance would be spread over a considerable time, so that the amount of . choline at any particular moment would be much smaller than is given — above. Now, none of the tests hitherto employed are sufficiently delicate to indicate choline in such degrees of dilution.25 Any statement hitherto made to the effect that choline has been demonstrated in the blood may therefore be taken as showing either that the test by which it has been 24. American Journ. of Physiol., Vol. XII, p. 353, 1905. 25. Some very delicate tests have been devised by Gumprecht and by Reid Hunt, but these tests have as yet not been applied to the investigation of this q CHOLINE IN ANIMAL TISSUES AND FLUIDS 128 recognised is fallacious, or if that can be excluded, that the choline which | is present is not derived from the degeneration of nervous tissues. It is interesting to compare these figures with the amount of choline found i in the blood by Halliburton and by Donath. The former observer _ found in the blood of cats he experimented upon choline up to the amount of 0°0052 to 0-0078 per cent. at a time when the degeneration was stated by him to be at its height. Taking the volume of the blood of a cat at 150 e.c., these figures would correspond to a sudden destruction of more than 1 gramme of nervous tissue at the time when the animal, a cat, was killed. As choline is stated to be abundant from the eighth to the thirteenth day after section of the nerves, the amount of nervous tissue destroyed in order to account for the amount of choline alleged to be present would be enormous. With regard to the cerebro-spinal fluid, the physiological effect on _ the blood pressure produced by injections of 10 c.c. of cerebro-spinal fluid from cases of general paralysis is compared with the effect of injections of 1 to 5 c.c. of a 0:2 per cent. solution of pure choline. The amount of choline injected in the latter case is 0:002 to 0°01 gramme, and is, according to ~ Halliburton,2¢ comparable to the amount of the base presumably present 7 _ in pathological cerebro-spinal fluid. We have seen that (if caleulated for | the total cerebro-spinal fluid = 100 ¢.c.) such an amount could be produced only by the sudden destruction of about 3 to 15 grammes of nervous tissue. These considerations completely justify the statement with which Vincent and Cramer concluded their paper, and to which Halliburton has taken exception. Indeed, it appears, a priori, improbable that the substance in these pathological specimens of blood should be choline : derived from nervous tissue. That such comparatively slight destruction ___ of nervous elements as takes place even in extensive disease should supply ___—_—_—s sufficient choline to the blood to give the physiological test seems scarcely ———s goneeivable, especially when we remember that choline is not a very : powerful depressor substance. Donath?’ determined the amount of choline in the cerebro-spinal fluid of patients. Taking the volume of this fluid at the minimal value of 100 ¢.c., his results indicate the presence of from 20 to 40 milligrammes of oe choline. This would correspond to a sudden destruction of 3 to 7 grammes of nerve tissue, and that in cases of epilepsy, both Jacksonian and of idiopathic, and of neurasthenia. 26. Croonian Lectures, p. 51, 1901, 27. Deutache Zeitachrift fiir Nervenheitkunde, Vol, XXVU, p. 71, 1904, 124 BIO-CHEMICAL JOURNAL Quite recently Halliburton, although admitting that the platino- chloride crystals obtained from blood are due partly to the presence of potassium, makes the claim?* that the obtaining of a large crop of erystals, ‘whether they be of the choline salt or a mixture of the potassium and choline salts, is diagnostic of extensive breakdown in nervous tissues. The contrast between such cases and the insignificant yield from normal blood is most striking. This is quite intelligible, when we take into account the very high percentage of potassium that nervous tissues contain.’ If the presence of potassium in the blood leads to the formation of these crystals, it may be asked why the considerable amount of potassium which is known to be present in normal serum should give an insignificant yield of crystals, while an increase which must necessarily be very limited so long as the kidneys are performing their work, should produce an abundant crop. Asa matter of fact, I have never been able to find such a difference, having obtained as abundant a crop of crystals from normal blood as from the blood of animals with recent nerve lesions. The idea that degenerative processes in the nervous system should lead to such an increase in the amount of potassium in the blood that it ean be recognised qualitatively by the yield of the platino chloride crystals is, as in the case of the choline hypothesis, based on a complete misconception of the quantitative relationships. Taking the minimal values given?? for the amount of potassium in the central nervous tissues as 17 per mille, it follows that 1 gramme of brain contains 0°0017 gramme K,0; taking the minimal value of K,O content of blood as 0°2 per mille and the total volume again as 4,700 c.c., we find that in man a sudden destruction of 1 gramme of nervous tissue would add 00017 gramme K,O to 09 gramme K,O in the total blood. This is a difference smaller than that which appears in Abderhalden’s* very careful analyses of samples of blood from two different animals of the same species. In 20 c.c. of blood the amount of potassium would be increased by 0°000007 gramme, or less than 1/100th of a milligramme. In smaller animals, e.g., in cats, the proportion between the astioaill of potassium normally present in the blood.and the amount which may be set free at any given moment by degenerative processes in nervous tissue remains of course essentially the same, since with a diminished volume of blood we have also a diminished amount of nervous tissue. 28. ‘Oliver Sharpey Lectures,’ Brit. Med. Journ., May 5th, 1907. 29. See, for instance, Hammarsten, Text Book of Physiological Chemistry, p. 414, 1904, 30. Zeitachrijft. |. physiolog. Chem., XXV, p. 65, 1898. CHOLINE IN ANIMAL TISSUES AND FLUIDS 125 ay It is clear that even the most extensive degeneration which can possibly oeeur is quite incapable of yielding sufficient choline and potassium to account for the striking contrast in the yield of crystals to which Halliburton refers. If the preliminary statement by the same author is correct, that actual estimations of the potassium content of the blood show an increase in some cases of acute degenerative diseases, it is te ually clear that this increase must be due to other factors than the ration of potassium by the degenerating nervous tissue. _ The smaller volume of cerebro-spinal fluid makes this a more likely place wherein to find the products of degeneration of nervous tissue. But from the data given above it follows that the destruction of nervous tissue would have to be very considerable, 1 gramme at least, before one would be able to detect choline in the cerebro-spinal fluid by means of ~_ Rosenheim’s modification of the iodine test, which is stated to indicate ae choline in a dilution of 1 : 20000. Kaufmann,*! who collected a litre of cerebro-spinal fluid from various cases of nervous diseases’ and who was ius in a position to investigate the subject by exact cheinical methods G instead of relying upon micro-chemical tests, was unable to isolate choline from the cerebro-spinal fluid. The fact that Kaufmann found another organic base to be present which, although having many reactions in . common with choline, proved on more detailed chemical examination to S be altogether different from choline, detracts from the value of observations .; based upon micro-chemical reactions for the detection of a substance ‘7 which, if it is present at all, is present only in fractions of a milligramme. Those who first advanced the choline hypothesis were under the __ impression that the amount of choline liberated in diseases could be measured by multiples of milligrammes; and the relatively great quantities of choline liberated, which their experiments appeared to _ indicate, was one of the strongest arguments in favour of their hypothesis. We know now that the amounts which can possibly appear as the result of degenerative processes in nervous tissue are exceedingly minute, and the question arises whether such small amounts may not be derived from other tissues. Lecithin is a constituent common to all cells. A destruction of a great number of white or red blood corpuscles may just as well lead to choline being set free. Thus the leucocytosis occurring in the cerebro- spinal fluid in some diseases, or the degeneration of a mass of red blood corpuscles after a haemorrhage may be a source of choline. 31. Neurologisches Contralblatt, Vol. XXVII, p. 260 1908 ti =” A ae ee ee Se a a ee ee ee eee 126 BIO-CHEMICAL JOURNAL SuMMARY 1. No choline can be detected in normal blood, provided that the decomposition of the lecithin be prevented in the methods employed. Nor can choline be detected in blood of animals after extensive lesions of the central or peripheral nervous system. : 2. The maximal quantity of choline which would be set free in oe | processes of degeneration is too small to be detected in the blood by any of the methods hitherto employed. The same holds good for potassium. 3. The physiological and chemical tests given by pathological blood, and alleged to be characteristic of choline, are exhibited in exactly the same manner and to the same degree by normal blood. 4. The micro-chemical reactions recommended for the detection of choline are given irregularly both by normal and pathological cerebro- spinal fluid. The presence in the cerebro-spinal fluid of a substance giving the micro-chemical reactions of choline cannot, therefore, be considered as indicative of degenerative changes in the nervous system; and in view of Kaufmann’s results it may even be doubted whether any of the micro- chemical tests at present in use are specific for choline. Whether such changes increase the amount of choline in the cerebro-spinal fiuid by fractions of a milligramme, and whether the same effect may not be produced by the disintegration of tissue other than nervous, will have to be determined by further experimental investigation. 127 + | ; THE BIURET REACTION AND THE COLD NITRIC ACID "TEST IN THE RECOGNITION OF PROTEIN By KARL Hl. VAN NORMAN, M.B. (Loronto). Communicated by Francis M. Goodbody, M.D., M.R.C.P. From the Pathological Chemistry Department, University College, London. (Received February 9th, 1909) In all books on physiological chemistry and clinical diagnosis one is recommended to place great reliance on the biuret reaction. In the course of some investigations on the comparative reliability of various tests for serum albumen I found that I obtained very contradictory results with this test, and therefore I think it would be of interest to give the results which I obtained in an exhaustive series of tests. The usual method given in books for carrying out this test is: that one adds to the albumen solution some soda solution and then, drop by drop, a very dilute solution of copper sulphate, a blue precipitate appearing which, on shaking, dissolves with a pink tinge, finally changing to a reddish violet." But on doing this I found that one could not be certain of obtaining the reaction in a urine which by other tests, such as cold nitric acid, acetic acid and heat, and potassium ferrocyanide and acetic acid, gave clear indications of the presence of albumen. It was therefore necessary to make up a solution containing a definite quantity of serum albumen and to repeat the tests. On doing this I found that it was impossible to make certain of obtaining constant results unless one used solutions of soda and copper sulphate of definite strength, for the addition of the soda solution in varying amounts made a great difference in the result of the test. It is generally recognised that it is necessary to add a dilute solution of copper sulphate, and I found that, although with a high percentage of albumen a 2} per cent. solution of copper sulphate may be used, yet for all practical purposes the solution of copper sulphate should not be stronger than 1 per cent. Another point on which the various text-books, which I have had an epportunity of consulting, lay little stress is on that of heating, and 1. Some text books advise adding the copper sulphate solution before the soda solution. 128 BLO-CHEMICAL JOURNAL although one can obtain a more or less definite result in the cold, I found that the reaction is much more marked on heating. In fact, in a very weak solution of albumen the violet is not perceptible until the solution is boiled, Pe In consequence of these difficulties I decided to work with solutions of albumen, soda and copper sulphate of definite strength, and the original — solutions which I made up were as follows :— I. Albumen solution. Distilled water containing 0°2 per cent. of — albumen, If. A solution containing sodium hydrate 10 grammes and distilled water to 100 c.c. III. A solution containing re-crystallised copper sulphate 5 grammes — and distilled water to 100 c.c. Solution I.—Beginning with the original solution of 0°2 per cent. of albumen, I made an exhaustive series of dilutions with distilled water, these diluted solutions varying in strength from 0°05 per cent. to 0°00033 per cent. of albumen. A number of these dilutions are referred to in Tables I and II. Solution IJ.—As a result of many tests in which I used varying perceniages of sodium hydrate, in albumen solutions of different strengths, — I found that a 10 per cent. solution of sodium hydrate gave the most constant results. The quantity of this soda solution used in each bettie: final result—is given in Tables I and II. Solution I1I.-The 5 per cent. solution of copper cciphdtom was too | strong, for even in the original solution of albumen (Solution I) a brown coloration with a white flocculent precipitate was obtained on the addition of one drop of this copper solution with soda. I therefore diluted to a 2} per cent. solution, but, after three or four estimations, I found this in turn too strong, and it was necessary to dilute to a 1 per cent. solution. Table I shows results on using a 2} per cent. solution. Table II 9 ” 1 oe) oe) For adding the copper sulphate and soda solutions drop i drop, I used an ordinary glass pipette, that is, ordinary glass tubing, drawn toa small calibre at one end. It was necessary to do a great many tests before being able to determine the best method of performing the test. I found as follows :- — (a) 10 c.c. of the albumen solution is the best amount to use. (6) In all the albumen solutions of from 0°004 per cent. to the limit | oe oe eS bi ig ak late THE BLIURET REACTION 129 sodium trydrate (10 per cent.) gave the best results. - In the albumen solutions stronger than 0°004 per cent. two to ten drops of copper sulphate (1 per cent.) can be added, according to the strength of the solution of albumen. In the solutions from 0°05 per cent. to 0°012 per cent. of albumen I used 2} per cent. copper sulphate solution. (See Tables I and II.) ; : _ (e) The copper sulphate should be added first, then the soda solution. ‘The contents of the test tube should then be mixed by inverting twice. (d) After adding the copper and soda the solution should be heated to boiling, when the violet is intensified. mw All text-books I have consulted mention that the colour obtained 4 _ in the biuret reaction is pink or red, changing to violet. I was unable to obtain this play of colours, as all my positive tests gave the violet colour at once. I found that the stronger the solution of albumen the more marked was the violet colour—gradually diminishing with dilution—and, also, that in strong solutions of albumen the violet is intensified by the _ addition of copper sulphate solution drop by drop, boiling between drops. In the very dilute solutions of albumen there is some difficulty in _ recognising the violet colour. I found that the colour was best seen as follows :--- 1. In the meniscus. 2. On looking down the test tube—against white. Re 3. On comparing the albumen solution with a test tube containing an equal quantity of distilled water. The two tubes should be held side Ta _ by side and compared : — (a) On looking down both—against white. (6) In the meniscus—- The best light is that which strikes from above. Both tubes should be slanted towards the operator at about 35°. One should hold the tubes above the head and look up through the meniscus from below, against a dark background. sataph Taste I . Albumen CuS0, Solution NaOH Solutioi per cent. 24 per cent. 10 per cent, Result Remarks S080 3 dro 5 dro Violet Reaction well marked 0-016 my on a se ahett ta ” ws oo fs Reac 0-012 1 drop ee . ee 130 BIO-CHEMICAL JOURNAL In the next dilution—solution of 0°008 per cent. of albumen—one drop of copper sulphate solution (2) per cent.) and five drops of sodium hydrate solution (10 per cent.) were added, and the solution at once turned brown. On boiling, the brown colour deepened with the formation of a whip flocculent precipitate. The result might be shown thus :— Albumen CuSO, Solution NaOH Solution pect Remarks per cent. 2) per cent. 10 per cent. 0-008 1 drop 5 drops No violet Solution at once turns brown In all the succeeding tests I used al per cent. solution of copper sulphate. Tanie IL Albumen CuSO, Solution NaOH Solution Result Bisiarks per cent. 1 per cent. 10 per cent. i 0-0080 2 drops 5 drops Violet Colour deepened on addition of one more drop CuSO, solution. 0-0040 1 drop - ir ; 0-0026 ” ’ *s 0-0020 oy ” 0-0016 bs “F aS 00013 ” bb] ? 0-O011 a sg su 0-0010 ” ” ” 0-00088 9 ” ” Violet faint before boiling. 0-00080 ” ” : 0-00072 o ‘9 ” | 0-00066 ” ” a Violet very faint before boiling. — 0-00061 ” ” ” llea comparison, 0-00056 re a ” distilled water as gern oy is just perceptible before boiling. 0-00053 ” ” ” 0-00050 ” ” ” 0-00047 a mn ‘i Violet imperceptible | before boiling a on comparison with dis- water. 0-00044 ” ” ” 0-00042 ”? ” ” 0-00040 af - Violet imperceptible be before pie ied voles, cal joo pean tilled water, jus after boiling when compared with distilled water. 0-00038 Er $d Z On standing for two minutes solu- tion turns brown. 0-00036 0 ” - On standing for one minute solu-— tion turns brown. 0-00034 ” ” » On standing for twenty seconds , solution turns brown. 0-00033 a a No violet Solution at once turns brown, with the formation of a white floceu- lent precipitate. From the results which I obtained in a watery solution of albumen, I decided to make further tests, using as my original solution urine and albumen. TI therefore took a urine free from albumen and added albumen to make a 2 per cent. solution. 4 ee a ee Oa yh az. i ee a THE BIURET REACTION 131 , _ As in the previous tests, I made a great many dilutions with distilled ed out investigations as before. The more or less abridged Its are seen in Table ITI. In these reactions I used sodium hydrate (10 per cent.) and copper Walt Ll. ‘ BD toe ‘ lrop sed copper sulphate solution was used, but in 0°04 per cent. and all he succeeding solutions to the limit of delicacy I found that one drop of ‘copper sulphate solution gave the best results. In all cases five drops of sodium hydrate gave most constant results. On account of the added colour of the urine, I found it more difficult to recognise the violet colour in this than in the first series of tests. Tansie UL - CuSO; Beanies NaOH Solution Result Remarks 1 per cent. 10 per cent. Five drops Five drops Violet Colour deepened to maximum on addition of ten more drops of Cu80,—drop by drop—boiling between drops. me as -s Colour deepened to maximum on addition of five more drops of CuSO,—drop by drop—boiling between drops. One drop Five drops Violet Reaction good. ” ” ” i x Violet faint before boiling. ‘i 2 Violet very faint before ee si Z ns Violet perceptible before boiling only on comparison with dis- tilled water. a ” - ” a tbe i $iggr: a gy ible before boil- ter boiling violet just percept on comparison with tilled water, ” ” ” But changing almost at once to ; brown, with the formation of a white flocculent precipitate. albumen, and in a urine containing albumen diluted with water, I next tried to obtain the limit of delicacy in a urine containing albumen when urine free from albumen was used for dilution. ye Having determined the delicacy of the test in a watery solution of — 132 BLO-CHEMICAL JOURNAL According to Hammarsten,' an excess of creatinin reduces copper sulphate, and Kellas and Wethered? have shown that an excess of uric acid and urates, as well as creatinin, causes a reduction of copper sulphate. The first urine which I used for diluting contained an excess of uric acid, and in these solutions I was unable to obtain the violet colour except in the stronger solutions of albumen—0°2 per cent. and stronger. : I then did further tests with solutions made by diluting the albuminous urine with urines containing an excess of urates and creatinin, but was unable to obtain positive results except in the stronger solutions of albumen—0O'2 per cent. and stronger. While working on the biuret reaction it occurred to me that it would be of interest to determine the delicacy of the cold nitric acid test for albumen, as this test is so well known and so commonly used. The limit of delicacy of this test given in text-books is 0°002 per cent., but I was able to obtain positive results in very much weaker solutions, as will be seen in Table IV. To obtain satisfactory results in doing the test the following points should be noted :— (a) 12 c.c. of albumen solution should be used. (b) 2 c.c. of nitric acid should be used. (c) A pipette should be used for adding the nitric acid to the albumen solution. This pipette is made as follows:—One end of an ordinary piece of glass tubing, 5 mm. diameter, is drawn to a calibre which allows the nitric acid to escape from the pipette by the drop and not in a continuous stream, when the finger is removed from the large end of the pipette. The pipette should be not less than 28 em. in length. Technique of the Test-—-Take 12 c.c. of albumen solution in a test tube. Draw up 2 c.c. of nitric acid in the pipette, place the finger over the end and carefully lower the pipette into the albumen solution until the point of the pipette is on the bottom of the test tube. Remove the finger from the pipette and allow the nitric acid to run in. When no more nitric acid runs in place the finger over the end of the pipette and carefully withdraw it. Care must be taken not to shake the test tube during the test. By this method the line of demarcation between the albumen solution and the nitric acid is narrow and very distinct, thus facilitating the recognition of the albumen ring as it forms. 1. Physiologischen Chemie. 2. The Lancet, October, 1906. ee . wae “Ccles ane 38 * “sf . . "a ee. h a THE BIURET REACTION 138 *% My first series of tests was done with diluted solutions of my original albu ation, that is, distilled water containing 02 per cent. of bumen. The Beeslic in a number of these solutions are given in One finds in text-books the statement that, for the cold nitric acid r albumen to be positive, the albumen ring should occur within minutes, but on referring to Table IV it will be seen that in very lut ‘solutions of albumen the ring did not occur until as long as fifteen utes, and reached its maximum density in one hour after adding the Taste IV Vere Result-- Time 2 c.c Positive In 20 seconds soy bi ST] maliawte bed ” ” 1 29 ” ” ” 1} minutes bid ” ” 2 b 2 ” bb 7? 2 ” ” ” ” 23 ” ” ” s”? 7 ” 2 tite ” ” bd 4 Phd ” ” ” 4) ” ” bb ” 5 °° ” ” ” 5 ” ” ” ” 5S ” ” ” bed 6 ” ” ” ” 6) ” ” ” ” 7 bed ” ” ” a ” . : 9 is Ring most marked in 15 minutes ” ” ” 10 ” ” ” 20 ” ” ” ” ll ” °° ’ 25 ” ” ” » 12 ” ” ” 30 ” ” ” ” 13 ” ” ” 35 ” ” ” ” a ” ” ” 40 ” X ae ll + aaah oe oe ” ” ” 15 ” ” ” 1 hour ” _ aw ” ” ” 1 hour. Very faint a For my next series of tests I used urine containing 2 per cent. of _ albumen—made by taking a urine free from albumen and adding albumen to make 2 per cent. Taking this as my original solution I made a. large number of dilutions, using for dilution a urine free from albumen. The results in a number of these solutions are given in Table VY. ¥ _. <1 Q « a 134 BIO-CHEMICAL JOURNAL In doing the cold nitrie acid test in albuminous urine containing an excess of uric acid, after adding the nitric acid a cloudiness appears in a few minutes, gradually increasing in density all through the urine from the top of the solution down to the nitric acid ring. To differentiate between this cloudiness, due to uric acid, and the albumen ring, the urine should be heated gently just above the nitric acid ring. The cloudiness due to uric acid disappears and the albumen ring remains. There will then be seen in such cases, in order from below, the nitric acid ring, the white albumen ring, a clear space, and above this more or less cloudiness. It must be remembered in this connection that albumose may be _ present also. If so, on gently heating just above the nitric acid ring, the — depth of the white precipitate will diminish, since the albumose is dissolved and the albumen is left. By this means one can obtain a rough idea as to the quantity of albumose present. In Table V it will be seen that in very weak solutions of albumen the albumen ring did not enn until ten minutes after the addition of the nitrie acid. TABLE V Albumen Nitric Result Time per cent. Acid 0-04 2 c.c. Positive In 1 minute 0-0040 ” ra + 1} minutes 0-0020 ” ” s¢ 2 ss 00013 ys a8 a | a 0-0010 a ” » 3 ” 0-00080 oe - eer | Pr 0-00060 an s9 pe ” 0-00057 . ° 99 43 9 0-00050 me o ae »» 0-00044 ” ” 9? 5} ”? 0-00040 Ld 7” s¢ 6 ” 0-00036 os ‘ 64 ” 0-00033 ; . 7 0-00030 * “a 0-00028 , * 8 0-00026 ” ? ’ 8} 0-00025 : 9 ; 0-00023 = of 0-00022 ae ae 9 Faint 0-00021 i a ae a 0-00020 * * » 10 9% Very faint CONCLUSIONS A. Biuret Reaction.—Limit of Delicacy. I. Ina watery solution of albumen is 0°0004 per cent. or four parts of albumen in 1,000,000 parts of distilled water. THE BIURET REACTION 135 io” II. In albuminous urine diluted with distilled water is 0°001 per am ‘cent, or one part of albumen in 100,000 parts of urine and distilled water. Ill. In albuminous urine diluted with urine free from albumen. The biuret reaction is very difficult to obtain in concentrated urines containing an excess of uric acid, urates or creatinin. In such cases it is only in the stronger solutions—0'2 per cent.—that the reaction is good. In the weaker solutions of albumen the violet colour may be obtained, but almost at once the colour changes to brown. In most cases no violet colour is obtained, the solution at once turning brown with the formation of a white flocculent precipitate. B. Cold Nitric Acid Test.Limit of Delicacy. ____-L.__ Ina watery solution of albumen is 0°00006 per cent. or six parts of albumen in 10,000,000 parts of distilled water. a II. In albuminous urine diluted with urine free from albumen is a ca per cent. or two parts of albumen in 1,000,000 parts of urine. ee tiny best thanks are due to Professor Harley and Dr. Goodbody for much kind assistance and advice. 136 THE PROPERTIES AND CLASSIFICATION OF THE OXIDIZING ENZYMES, AND ANALOGIES BETWEEN ENZYMIC ACTIVITY AND THE EFFECTS OF IMMUNE BODIES AND COMPLEMENTS yee By BENJAMIN MOORE, M.A., D.Sc., Johnston Professor of Bio- chemistry, University of Liverpool, and EDWARD WHITLEY, M.A. (Owon.). Lea (Received March 15th, 1909) - po The presence in plant and animal juices of bodies possessing the properties of ferments which act as oxidizing agents for unstable bodies, such as the guaiaconic acid of guaiacum resin, was first demonstrated by Schénbein! in 1856. . The action of these bodies was studied in greater detail by Bertrand? in a series of papers beginning in 1894. Bertrand first showed that the formation of Japanese lacquer was due to the oxidation of a substance present in the plant juice of certain species of Rhus by a ferment which he termed /accase. The browning and blackening of the cut surfaces of fruits and other parts of vegetable tissues is also due to the action of oxidizing ferments. From the juice of the potato an oxidizing ferment termed tyrosinase was obtained, which acted vigorously upon the isolated tyrosin, yielding a dark brown substance, richer in oxygen than the tyrosin from which it was formed. These browning or blackening ferments are distinct in nature from the oxidizing ferments present in most fresh plant juices, and also in pus, milk, blood, and extracts of some animal organs, which possess the property of turning tincture of guaiacum blue, of oxidizing and thereby rendering coloured certain phenolic bodies, or of setting free iodine from a solution of hydriodic acid. Bertrand showed that this latter class of oxidizing ferments was present in many different types of plant, and he expressed the view that they were universally present in all plants. It is, however, certain that they are not present in all plant juices, although they may be present in other parts of the same plant. Our own experiments recorded below show, for example, that the class of oxidizing 1. Zeitasch. f. Biol., Bd. TI, 8. 325. 2. Compt. rend., p. 1215, 1894. Bi r. ; - Pe PROPERTIES OF OXIDIZING ENZYMES 137 - ferments is absent in the fresh juice of the fruit of the lime, lemon, and orange, but present in the crushed seeds of lemon and orange. Other experiments have demonstrated to us that their distribution in different parts of a root or fruit varies widely, and that they are present in greatest quantity in the region containing the greatest abundance of respiratory vessels, Thus a section across a carrot stains almost at once in the protoxylem, and the staining radiates out from this along the path of vessels, while internally to the protoxylem there is very little staining (see Expt. XIX). It was found by many observers that the guaiacum test for these oxidizing ferments often failed, especially in fluids of animal origin and when freshly made tincture of guaiacum was used for testing. Further, it was found that in such cases the blueing could still be obtained, provided either hydrogen peroxide were added or some organic form of peroxide, containing oxygen linked in the peroxide form. A similar oxidation is seen in the well-known test for traces of blood in urine or other fluids in which tincture of guaiacum and ozonic ether (a ae form of peroxide) are added to the suspected fluid. This test also demonstrates that bodies other than oxidizing ferments occurring in living tissues are capable of giving the guaiacum blueing. Many inorganic and organic bodies also give it, such as sulphurous and nitrous acids, ferrous salts, and potassium permanganate. The substances causing this reaction may be either oxidizing or reducing bodies, so long as they can act as oxygen carriers. In this connection it may also be pointed out, as has been done by Bach,’ that all the oxidations shown to occur with this class of oxidizing enzymes, viz., oxidation of hydriodic acid, of the aromatic amines and phenols, and of the guaiaconic acid of the guaiacum resin, possess the _ eommon factor of a moveable or dynamic hydrogen atom in the molecule. Between this hydrogen atom and the atom of the oxygen in the peroxide form there is already a strong tendency to reaction, and the catalyst simply increases this tendency. On account of the too great readiness with which the guaiacum underwent the oxidation change giving the characteristic blueing, and also on aceount of the fact that blood, and other oxidizing bodies not ferments, readily yielded the guaiacum test, other more definite tests were sought out. A considerable number of tests have been described in which phenols or amido-phenols are oxidized to bodies which possess various 3. Berichte d. deut, Chem, Gesellach., Vol. XL, p. 230," 1907. SS Le ee ee a 138 BIO-CHEMICAL JOURNAL colours. These colour tests for oxidizing enzymes are all very similar, and when one of them has been obtained, or not obtained, in any given case, a similar result is usually obtained with all the others, probably because the potential of chemical energy required for oxidation lies at about the same level for all of them.* The results of carrying out a number of these colour tests will be — given later, but one important difference which we have found may be stated here in general terms, since, in our opinion, it leads to certain important conclusions regarding the classification and mode of action of this class of oxidizing enzymes different from those generally accepted at the present time. This result is that none of the colour tests, with the exception of the guaiacum test, are appreciably increased in velocity, or catalysed, by the solution of the oxidizing ferment alone, except when the fresh juice is taken immediately after its preparation (see Expt. VII), and are only so catalysed when hydrogen peroxide or some other form of peroxide is added at the same time. The reasons why the guaiacum test is often positive when the others are negative are (1) the presence of organic peroxide in the guaiacum itself, and (2) that the oxidation occurs much more readily than with the other colour tests with the oxy- and amido-phenols, and such like bodies, and so the naturally occurring organic peroxide of the juice or of the reagent is sufficient to give an oxidation. Whereas the more stable amino-acids only break up with appreciable velocity in presence of the more readily decomposed, and hence more powerful, free hydrogen peroxide, which has been artifically added. No attempt will be made in this paper to give a full account of the very wealthy literature of the oxidizing ferments,® sincé accounts in English have recently appeared in ‘The Nature of Enzyme Action,’ by Bayliss,® and the ‘ Intracellular Ferments’* of Vernon;‘ but some outline of the classification introduced by Bach and Chodat, and of the basis for that classification, must be given in order that our own views and experiments may be more easily followed. It may be added that this classification has been almost unanimously adopted by subsequent writers. 4. Minor differences in reactivity of the different colour reagents are given in the experi- mental part of the paper. 5.” Citations of the literature are also to be found in the comprehensive review by Bach. and Chodat, Biochemischen Centralblatt, 1903; in ‘* Uber tierische Peroxydasen,’ Ernst von Czyhlarz u. Otto von Fiirth, Beitrage z. Chem. Physiol. u. Path., Zeitsch. {. Biochemie, Ba. X, 8. 358, 1907; Bach, Berichte, 1904 to date ; Spence, this Journal, Vol. III, p. 165, 1908. 6. Monographs on Bio-Chemistry. Edited by Aders-Plimmer and Hopkins. Longmans, — yreen and Co., 1908. 7. Published by John Murray for Physiological Laboratory, University of London. PROPERTIES OF OXIDIZING ENZYMES 139 The experimental work of Bach and Chodat, especially that of a = quantitative nature on the action of these oxidizing ferments, is, in our - opinion, sound, and forms an excellent basis for further work; but we - eannot agree that their experimental observations give sufficient ground for the classification they have adopted nor for belief in two classes of oxidizing ferments, the owygenases and the perowidases, which they postulate, forming together a mixture of ferments corresponding to the In our opinion there is but one class of such enzymes concerned, which, since they act only in presence of oxygen linked as a peroxide, might still be known as the peroxidases. The oxygenases do not exist as ____ ferments at all; there is no use for such a term, and it might be allowed to drop out. The oxygenases are simply preformed peroxides in juice or reagent, and not in any sense ferments. ___. + The experimental basis, and as far as we can discover the only one, for a belief in the oxygenases is that certain juices, such as that of the potato, give at once a blue colour with fresh guaiacum alone without added hydrogen peroxide, while other fresh juices, such as those of ‘Tadish and cucumber,® give absolutely no blue coloration with the fresh - guaiacum tincture until hydrogen peroxide, or some other peroxide, is added, when almost at once a strong blue is obtained. Further, if the potato juice be heated for some hours to about 60° C. _ it in most cases loses the power of blueing spontaneously, and now only gives the blue colour when a peroxide is added.? The explanation of this is easy on Bach and Chodat’s classification, which represents that there are two enzymes present in the juices, viz., oxygenase which manufactures peroxides from the material at hand in _ the juice, and a peroxidase which then activates this peroxide and causes it to attack the oxidizable body yielding the colour test and oxidine it, ‘so producing the colour. _ We submit that there is no proof that there is an enzyme or enzymes forming the class of the oxygenases and producing organic peroxides, and that the whole difference between the two classes of juices is simply that 8. Bach and Chodat, loc. cit. This destruction of peroxides (or of o oops tegen caper sp): lla ge cv wh t is usually described, for potato juice may be briskly boiled in a test-tube for half a minute without cutiexinn completely all the store of peroxide. If it is again boiled, ea ag or if it be thoroughly boiled for some minutes in a beaker so that all ta age Bee iled, the peroxide is completely destroyed. This refractiveness was discovered by Cal Cr U.S. Dept. of Agriculture, No. 18, p. 17) in the case of the tobacco oxidase, and by him attributed to part of the oxidase existing as a zymogen. There is little doubt, however, t the result is due to incomplete destruction of the peroxide and peroxidase, 140 BIO-CHEMICAL JOURNAL one has a store of peroxides and the other has not, and, further, that these peroxides are thermally unstable and are destroyed by heat. After we have put forward the evidence in favour of this view, we shall draw attention to the similarity between this simpler scheme for the activity of the oxidizing ferments, and that for the action of ‘ immune’ body and ‘complement’ in the case of haemolytic and other cytolytie sera and the cells attacked; and also for the action of ordinary hydrolitie enzymes upon their substrates. In all the above cases three things are required, viz.:—({1) a body of a ferment nature, (2) a substrate on which it can act, and (3) a body ~ which enables the ferment to act upon the substrate so as to cause hydrolysis, oxidation, or some other type of chemical reaction. Also, the ferment and the substrate are usually much more specifie to each other than is the third body, which is simpler in nature, such as an alkali or acid or a peroxide, and we suggest that complement is an activating substance of this kind. Returning to the oxidizing ferments, we may now quote the classi-— fication given by Bach and Chodat. In their general review of the subject of oxidizing ferments, published in 1903, these authors give the following list :-— ‘I. Ovygenases. Protein-like bodies which take up molecular oxygen with peroxide formation. ‘II. Perowidases, which enormously raise the oxidation power of the peroxides which by themselves oxidize very sluggishly, at the dilutions in question. ‘IIL. Katalases, which destroy peroxides with evolution of oxygen.’ In our opinion, all these three names are ill-chosen, because they break the general law that the name should, as, for example, in lactase, tyrosinase and lipase, indicate by the root of the word the substrate acted upon, and by the termination ‘ase’ the fact that the body designated is an enzyme. Now, in the first two classes named above, oxygen and peroxide respectively are not the substrates, but the bodies which the particular enzymes bring into action upon the substrates, and much more resemble the alkali in trypsin action or the acid in pepsin action. Further, in the third class, the ‘ katalases,’ there is no reliable evidence of this destructive action upon hydrogen peroxide being due to an enzyme at all. Although it is clearly a distinct activity from that of the oxidizing ferments, yet it is not an oxidation, and it is not specific, occurring, as it does, with every ferment solution of whatever type, with nearly all animal PROPERTIES OF OXIDIZING ENZYMES 141 or vegetable fluids, and with numberless inorganic catalysts. In any _ ease, it is absurd to give it a name which belongs to or includes the whole yast range of catalytic actions. Every true enzyme is a ‘katalase’ in the sense that it acts catalytically, and why a catalytic agent, which happens to act upon hydrogen peroxide, and of which it has never been ae, clearly shown that it is a specific enzyme, should be dignified with the a name of ‘ katalase’ it is difficult to conceive. Whatever the body is (or ; the large number of bodies), it is quite certain no oxidizing enzyme is in = question, since nothing is oxidized and the oxygen is simply discharged as molecular oxygen. 3 ‘The Deiter which is made by Bach and Chodat!® that the so-called oxydases are mixtures of peroxide bodies and peroxydases, appears to have led some of their English reviewers to entertain the view that they regard _ the oxygenases as unstable peroxides rather than as true enzymes producing peroxides. But that this is an error is shown by the above classification, as also by the repeated statement in their works that the oxygenases are ferments which produce the organic peroxides, and that it is only in the presence of the peroxides that the peroxidases have any action whatever. i Thus the rationale of the complete reaction of oxidation by these ferments, according to Bach and Chodat, is as follows :— First an enzyme, called oxygenase, acts upon certain substances present in the plant and forms organic peroxides; and, secondly, another and distinct enzyme, peroridase, which is entirely unable to act in absence of formed peroxide now comes into activity and causes a reaction which _ transfers the oxygen, previously attached to the peroxide by the oxygenase, to the oxidizable substrate, which may be one of several substances. ul ‘We have also looked carefully through the later papers by these authors, and have not been able to find any abandonment of the position that the oxygenases are peroxide-producing enzymes. Thus, Bach! states the oxydases are nothing else than mixtures of roxy and oxygenases, that is, of peroxide-activating and of peroxide-building enzymes. In this paper, because he obtained by alcohol precipitation, and re-dissolving in water, a solution which alone only slowly attacked tyrosin, but was strongly activated towards tyrosin by addition of small amounts of hydrogen peroxide, Bach claims to have 10. Bio-chemisches Centralblatt, 1903, and elsewhere. "IL. Berithte, Vol. XXXIX, No. 10, p. 2126, 1906, 142 BIO-CHEMICAL JOURNAL shown that the usual tyrosinase which attacks tyrosin at once is also a mixture of an oxygenase and of a peroxidase which is specifie for tyrosin. No further proof is here given of the enzymic nature of the supposed oxygenase of the tyrosinase. Again, in 1908, in a polemical paper against Chodat, who had been unable to repeat his observations as to the activation of tyrosinase by hydrogen peroxide, Bach'? states that the failure of Chodat to repeat his results was due to the use of too high concentration of hydrogen peroxide, and in this paper he once more enunciates that ‘tyrosinase, like the ‘ordinary oxydases, is composed of an oxygenase, that is, a body which ‘ produces (“ bildet”) peroxide accompanied by uptake of oxygen, and is ‘replaceable by hydrogen peroxide, and of a peroxidase, which activates ‘the so-formed peroxide, or the added hydrogen peroxide.’ There is proof given by Bach and Chodat that peroxide is present in fresh plant juice which has been treated with air,!* but none that this peroxide is produced by an enzyme. The proof as to the presence of peroxide, which is not entirely free from experimental suspicion, was obtained as follows. Air was passed through a sample of fresh juice from Lathraea squamaria containing oxydase, and at the same time a 1 per cent. solution of barium hydrate was slowly dropped into the juice. A barium precipitate was obtained which did not give the hydrogen peroxide reaction with the titanium reagent, after washing and decomposing with dilute sulphuric acid. The solution did, however, intensely blue potassium iodide and starch, and as the absence of nitrate was shown otherwise, it was assumed that an acylated hydroperoxide was present. It does not appear to us that much that is new is proven by this experiment, since the oxidase known to be present in the original juice would have similarly acted on iodide and starch solution, and there is no reason why it should not be precipitated unchanged by the baryta. On other grounds it is highly probable that traces of organic peroxides are present in most fresh plant juices. (See Expt. XX.) This does not, however, prove that they are formed either in the plant or after separating the juice by an enzyme such as the postulated ‘oxygenase’ of Bach and Chodat, and all experimental proof of such enzymic origin is hitherto lacking. | 12. Berichte, Vol. XLI, p. 216, 1908. 13. We shall show in our own experiments later that excess of air or ae as by ] in shallow layers in open vessels, again destroys the peroxide first formed, so that once more no result is obtained without added peroxide. Red See Fo ee PROPERTIES OF OXIDIZING ENZYMES 148 _ Again, the presence of the elusive oxygenase is not proven by the thermal. instability of the spontaneous blueing by guaiacum. For the peroxides are also very unstable bodies thermally, and the failure to obtain a blue with juice and guaiacum alone after heating to 50°— 60° C., and then obtaining it on adding hydrogen peroxide, may easily be due to the destruction of the thermally unstable organic peroxides, and not to that of an enzyme which produces such peroxides. In fact, the failure of the blueing after heating is all in favour of the simple presence of unstable peroxides. Because, if it were an enzyme (oxygenase) only that were destroyed by the heat, then this enzyme previously to the process of heating would have had ample opportunity to preform a good supply of organic peroxide, and although the enzyme were destroyed on heating to 50°—60°C., there would be enough pre- formed peroxide to still give the blueing afterwards. The simplest hypothesis, accordingly, is that the heating merely destroys the organic peroxides which are essential to the action of the only ferment required, viz., the peroxidase, and on now replacing this loss of organic peroxide by the simple hydrogen peroxide, the blueing is obtaimed because the essential chemical linkages are present for the peroxidase to act. In regard to the experimental fact discovered by Bach and Chodat that the juices of certain plants, such as cucumber and radish, do not give a blue with guaiacum alone, but at once give a fine blue on addition of traces of hydrogen peroxide, while other juices, such as those of potato and carrot, give a blue at once, with guaiacum alone, we can completely confirm the experimental observation, but believe it is susceptible of a much simpler explanation than that the potato and carrot contain two ferments, oxygenase and peroxidase, which act as above described, while ‘im cucumber and radish the oxygenase is absent and peroxidase only present. The oxidation in presence of added hydrogen peroxide is easily demonstrated by means of any of the colour tests, in the many vegetable juices or animal extracts or secretions which contain an oxidizing ferment. But it is an exceedingly difficult matter to trace to their true causes those cases in which a positive result is obtained without the addition of hydrogen peroxide, Such a positive result is more often obtained when the guaiacum test is employed than with the other reagents we have used for testing. This very frequent positive result is not always due to development of peroxide 144 BIO-CHEMICAL JOURNAL © in the reagent on standing, as is usually said to be the case, but is present or absent in a most capricious way. In our opinion, the variation in result is due to the presence or absence of preformed peroxide of organic nature in the particular pieces of resin from which the tincture was made, although no doubt there may be a tendency for the amount of this pe ro to increase as the reagent grows older. The only similar variation with the other test substances we have observed was in the behaviour of potato juice to p. phenylene-di-amine in giving the characteristic green without added peroxide of hydrogen when the potato juice was tested immediately after grating the potatoes. Within half an hour the same juice gave not a trace of green until hydrogen peroxide had first been added. Further, even on adding hydrogen peroxide to its juice, if the mixture was left for some few minutes before the p. phenylene-di-amine was added no green was obtained. Thus, the peroxide had been destroyed in the meantime, instead of any having been formed as would have occurred if an oxygenase or peroxide-forming ferment had been present. In this same potato juice, even when quite fresh and giving the p. phenylene-di-amine reaction, the positive result of an amethyst colour with a-naphthol could not be obtained until hydrogen peroxide had also been added. . With this one exception of the potato juice immediately on preparation, all the colour reagents except guaiacum invariably gave us a negative result until hydrogen peroxide was added. The guaiacum gave us a positive result in the capricious way described above in many instances, such as potato, carrot, wheat, oats, apple, banana; and several species of nuts. The causes of the variations with the guaiacum will presently be traced out as we followed them up experimentally, but before taking this’ up we would like to point out that in order to justify the views of Bach and Chodat, that there are two enzymes present, one of which produces peroxides, while the other activates reaction between such peroxides and the oxidizable chromogenic body, the evidence obtainable from the use of different colour-reagents must be consistent and concordant. That is to say, in the same juice one reagent must not indicate the presence of peroxidase only and absence of oxygenase, and another reagent indicate both oxygenase and peroxidase. Now this is precisely what cecurs experimentally, for with the exception of .the freshly made potato’ juice reacting positively to i i! PROPERTIES OF OXIDIZING ENZYMES 145 p- phenylene-di-amine at the same time that it reacted negatively to ‘naphtha, the evidence was conclusively negative in regard to ‘oxygenase’ throughout, save for the capricious results with guaiacum only. If now it be admitted that these variable guaiacum results were due to varying amounts of peroxide in the guaiacum, and hence afforded no evidence of ‘ oxygenase’ in the juice being tested, then all evidence for the existence of oxygenase disappears, and we are left with one enzyme, or type of enzyme only, which produces ifs effect by activating towards each other peroxide and the substrate to be oxidized. In support of the view that the different behaviour of guaiacum tincture was due to varying amounts of peroxide in the tincture itself, we submit the results of Expt. XX detailed later (see p. 158), which show _ that when means are taken to exclude peroxide from both guaiacum and _ juice being tested, a negative result is always obtained until peroxide has been added from outside. . But if this be accepted we are left only with the positive result with perfectly fresh potato juice and p. phenylene-di-amine, and since this rapidly disappeared as the juice stood, and, further, as traces of added os hydrogen peroxide similarly disappeared on standing, we are disposed to attribute this effect to traces of peroxide in the fresh juice which disappeared on standing. Tf such a ferment as the postulated ‘ oxygenase ’ had really existed .. in the potato juice, then the amount of peroxide would not have decreased a on standing, but, on the contrary, there would have been an increase all é the time, and the test would have grown stronger instead of disappearing. _ The disappearance was probably due, in part, to the substances _(‘katalases’) present in all such juices which decompose peroxides after the juice is shed from the cells, and in part due to the using up of peroxide in the oxidations brought about by the tyrosinase and other oxidizing -ferments which we have been calling peroxidases. That the rate of disappearance of peroxide is in some way connected with exposure is shown by the fact that the positive reaction with p- phenylene-di-amine is lost in a very short time (inside half an hour) when exposed to the air in a thin layer in a flat-bottomed glass dish, while it is still present after several hours in a sample of the same juice preserved in a tightly corked flask which has been completely filled. A positive reaction to a sample of guaiacum tincture which gives no colour with carrot juice is still given by this potato juice, for long after it has ceased to react with p. phenylene-di-amine. This is probably due to two reasons : 146 BIO-CHEMICAL JOURNAL first, that the guaiaconic acid is. more readily oxidized (or at a lower chemical potential) than the p. phenylene-di-amine, and secondly, that there is a conjoined effect of the minimal traces of peroxide in both reagent and juice in one case, and of that in juice only in the other. It would thus appear that all those cases in which a colour test of oxidation is obtained without adding hydrogen peroxide are due to peroxide already present in traces either in the juice or in the reagent. Also, that the amount of peroxide in the juice decreases instead of increasing on standing, and that the peroxide which is specially present in varying amount in the guaiacum reagent exists in the resin and its products quite at the beginning, and so may be present in absolutely freshly made tinctures, from which it may be removed by treatment with charcoal, ig it: It was further found that a mere trace of a reducing agent, such as a drop of dilute ammonium sulphide, or sulphuretted hydrogen water added to half a test tube full of a fresh juice showing the guaiacum test without addition of hydrogen peroxide, was quite sufficient to permanently destroy all the trace of organic peroxide present, so that now the reaction became negative, RECORD OF EXPERIMENTS The chief substances used by us for demonstrating oxidation were (1) Guaiacum in fresh 10 per cent. tincture made from the resin. (2) A 1 per cent. solution of ». phenylene-di-amine in distilled water. This gives on oxidation a fine green colour, which usually strikes out quite suddenly after a pause of several seconds; it occurred positively only after addition of hydrogen peroxide, saving the exception above mentioned of potato juice, where it is obtainable without added peroxide for a period of about half an hour in the fresh juice; after that it is only obtainable when hydrogen peroxide is also added. (3) A 1 per cent. solution of a naphthol in 50 per cent. alcohol. This reagent never gave a positive result with any of the juices, however fresh, until hydrogen peroxide was also added; it strikes a fine amethyst colour in presence of the peroxidase and peroxide; the reagent turns slowly amethyst coloured on standing, and should be made fresh. (4) Indo-phenol or Spitzer's reagent, which we prepared fresh in’all cases by mixing equal parts of the two previous solutions of p. phenylene-di-amine and @ naphthol, and of a 2 per cent. solution of sodium carbonate. This on oxidation yields a fine PROPERTIES OF OXIDIZING ENZYMES 147 purple; the test never gave a positive result unless hydrogen peroxide was ad ded, the development of colour being no greater than occurs spontaneously in the diluted reagent or in boiled juice of equal eoncentration, to which the reagent has been added in equal quantity. 5) Hydrochinon in 1 per cent. solution in water. (6) Synthesized guajacol’ (Merck). This behaves quite differently from the natural um resin, giving a strong brown colour, and on standing a brown ipit te, obtainable only in presence of peroxide. ” Guaiaconic ni In ‘making comparative tests care was taken to use as nearly as possible corresponding amounts of juice equally diluted, of reagent, and | Se pct oe peroxide where that reagent was added. In the earlier ex) ents a solution of hydrogen peroxide, the usual laboratory pe ng rth (10 vols. of HO, per cent.) ten times diluted was employed, and in the later experiments the pure perhydrol of Merck was used, first uted ten-fold, as a stock solution made up in small volumes at a time, 1 this was then again ten times diluted immediately before use. Asa general rule, about 5 c.c. of juice was taken or a given dilution (ue eee with distilled water, and to this about 1 ¢.c. of the diluted peroxide was added and 1 c.c. of the reagent being used. “Where necessary three tubes were used, of which one contained the juice. after boiling, another the juice without boiling, with reagent only added, and a third, unboiled also, to which both reagent and hydrogen In other cases ere the juice was of known character and the » of peroxide was not being proven, but rather the effect of nts upon its activity, this procedure was not necessary, and the ‘imentation was modified accordingly. t I.—-Testing of wheat for oxidizing ferments. One part of y weight, extracted with three parts by volume of distilled water , filtered to an almost clear filtrate—water in contact with the dered Bain for about one hour. Reagent 1. Boiled 2. Unboiled extract 3. Unboiled extract plus hydrogen extract : alone peroxide alone 1. Guaiacum Blue* Blue Blue 2. (eager a some Nil _ ,, Green ; Nil 3. a@ naphthol Nit © - © ~~ Amethyst Nil | 4. -Hydrochinon Nil Brown ©) © >) Nil * This result was afterwards shown to be due to insufficient boiling of the extract. 148 BIO-CHEMICAL JOURNAL Experiment II.—Similar experiment with powdered and extracted oats, giving exactly similar results. Experiments IIT and IV.—Swiss Condensed Milk and a proprietory food called Glaxo gave negative results on testing with all the usual reagents. This is to be expected, since all such foods and also canned fruits and patent foods for children and invalids are sterilized by boiling. It is a point worth bearing in mind that this absence of oxidizing ferments distinguishes all preserved foods from fresh foods. Experiment V.—Fresh milk, tested as above to guaiacum, p. pheny- lene-di-amine, a naphthol, and hydrochinon, gave a reaction only in presence of added hydrogen peroxide, except in the case of guaiacum, where a slow blueing occurred without the peroxide. Experiment VI.—Fresh carrot juice was positive to guaiacum with and without addition of peroxide, but positive to other reagents only after peroxides. In latter experiments with a guaiacum free from peroxide carrot juice was often found negative, especially after standing in air for some time. Experiment VII.—Fresh potato juice was found the most strongly positive of all the juices tested towards guaiacum; no preparation of guaiacum was used throughout which did not give a blue with it, but when both potato juice and guaiacum were deoxidized as much as possible, the blueing effect practically disappeared. Potato juice when just drawn oft is slightly positive to p. phenylene-di-amine but negative to the other tests, and turns negative to the p. phenylene-di-amine also after about half an hour. : Experiment VIII.—Potato juice was dried in an air oven at about 50 to 55° C. and then extracted with water at 48° C. for forty-eight hours. This very materially reduced the oxidizing power, a faint blueing was still obtainable both with and without added peroxide, but stronger with; hydrochinon both negative; a naphthol negative without, slightly - positive with; p. phenylene-di-amine, both negative. Experiment I1X.—Wheaten flour gave strong plus to guaiacum with and without peroxide; faint reactions with other reagents and only in presence of peroxide in each case. Experiment X.—Serum of pig’s blood, three days old, gave negative to guaiacum both in presence and absence of hydrogen peroxide; to p- phenylene-di-amine slight reaction without and strong reaction with peroxide; to a naphthol, peroxide tube strongly positive, doubtful without peroxide. PROPERTIES OF OXIDIZING ENZYMES 149 : BE Baperiment XJ.—Effect of minute amounts of acid and alkali and of id and alkaline phosphates on the oxidizing reactions. This was tested follows, using guaiacum as reagent :— ~The acid stops in minute amounts; much more alkali is required to p the oxidation, but the colour changes from blue to a 265 aa green. e were carried out with potato and carrot juices :— Fresh potato juice diluted ten-fold with distilled ete: and 5 e.c. of 3 e taken to quantity named below of reagent and then 0°5 c.c. of tincture of guaiacum added. . WM Normal control Blue at once. ike 2, Added 0-1 c.c. of iy Ha (= 359 approximately). Faint blue, very slowly deepening. Added 01 cc. of Sf NaOH. As blue as contro. | A Added 0-5 c.c. of 5 per cent. NaH,PO, (= 5 Added 0-5 c.c. of 5 per cent. Na,HPO, Blue much weakened. a Y 3 eh a _ 1. This reversed behaviour of the phosphatic solutions as compared to the acid and alkali isv -y peculiar, and difficult to understand, but it was several times observed. ai x ui fh RS * | approximately) As blue as control.! rot juice treated exactly the same way gave :— 2 “Added 0-1 0. of 2 Htc No bleting, 14 Added 0-1 c.c. of 7 NaOH. Strong blueing. | a ao 4 Added 0-5 c.c. of Fan ii NaH,PO,. Strong blueing. “a “Added 0-5 c.c. of 5 per cent. Na,HPO, Weak blueing. _ EBaperiment XII.—¥resh juice from grated radishes was taken and tested to (1) guaiacum, (2) p. phenylene-di-amine, (3) a ey agen 4) pyrogallol, (5) indo-phenol (Spitzer’s reagent). The same result was obtained throughout, viz., negative in absence ia ; hee ‘of hydrogen peroxide; positive in presence of the peroxide. There is absolutely no blueing with guaiacum alone, even with guaiacum which alone gives a good blue with potato juice. On standing over night in a . stoppered vessel the same radish juice now gives a fair blue with the same _ guaiacum. The remaining portion of the radish juice was centrifuged, first alone and then after fractional precipation, with alcohol added up to 25 per cent. in the mixture. It was found that both sediments were very _ strongly active, much more so than the supernatant liquor. . Experiment AUT. —This experiment was made with carrot juice and with apple juice. In each case the fresh juice got, as usual, by grating ~ "lass 150 BIO-CHEMICAL JOURNAL and filtering was tested alongside an extract made by drying the grated mass at 45° C. for some days. ‘The carrot gratings had been in the oven for three days and the apple gratings for eight days. The contrast in the filtrates from the fresh and the dried preparations is most striking. The dried is throughout completely negative, and the fresh positive. With guaiacum alone there is little blueing, even in the case of the fresh juices, but immediate effect with addition of peroxide; in neither case is there an effect with the filtrates from the dried materials. With p. phenylene-di-amine, a naphthol, and indo-phenol there is a very strong reaction, but only in presence of hydrogen peroxide with the fresh preparations, and nothing with preparations from the dried material. Experiment XIV.—Effeets of partial or fractional precipitation of potato juice with alcohol. The properties of the different alcohol pre- cipitates, and results of combustion of the dried alcoholic precipitates. A quantity of 320 c.c. of fresh potato juice was taken and thoroughly centrifuged. The deposit consisted of starch underneath with a thinner greenish brown layer on top like very fine mud, or ooze. The upper layer could be easily washed off from the strongly impacted starch granules underneath, It was so removed, shaken up with distilled water, re-centrifuged, and separated again from the small amount of starch mechanically removed with it at the first separation. It was once again shaken up with distilled water, and the brownish colloidal solution or suspension which frothed strongly was found to be strongly active, giving a good blue with guaiacum alone. Examined under the microscope it shows a field crowded with exceedingly minute particles much less than 1» in active Brownian movement, This deposit is like an excessively fine mud, which readily passes into suspension; it closely resembles the different fractional precipitates with alcohol, about to be described. The supernatant fluid after centrifuging was still opalescent and gave a strong blue with guaiacum, to this one-quarter of its volume of absolute alcohol was added, making a 20 per cent. alcoholic solution, in which a copious greyish brown precipitate appeared. Throughout it was found difficult and tedious to nncenl this exceedingly fine precipitate by filter and pump, and that they settle excellently and quickly into a compact mass with the centrifuge so that they can readily be separated by decantation. The precipitate so separated from 20 per cent. aleohol on shalsual up a} destroy added hydrogen peroxide, for if to a juice which is not 1 i y an oxidizing reaction with guaiacum alone one adds a minute ount of hydrogen peroxide, sufficient to give a good immediate blue on ‘urther addition of the guaiacum reagent, and if now the juice so treated 3¢ divided into two equal parts, to one of which guaiacum is added, at e giving a good blue, and the other allowed to stand for five minutes, per ‘agi is found that no 0 colour i is given by the latter owing to destruction Phau “That this is the true ee a is shown by the immediate blueing - obtained on adding more peroxide after the guaiacum has failed to give the colour in the second portion. _ By taking a large quantity of juice, adding small quantities of drogen peroxide at a time, and testing small portions at intervals after each addition of peroxide, this peroxide destruction may be followed out f aften as desired. __ This experiment further demonstrates that there is no firm linkage etween ferment and peroxide such as can prevent the latier from truction, for then enough peroxide would be permanently preserved | on to the ferment to give a blue colour at once when the substrate - (guaiacum) was added even after a time interval. Fac Experiment XVI.—Effect of reducing agents upon ferment and - owidizing reactions. : i _ The oxidizing ferment is extremely susceptible to the merest traces _ of reducing agents, such as sulphuretted hydrogen or ammonium sulphide, for the addition of a drop of diluted ammonium sulphide or sulphuretted hydrogen water to a half test tube full of potato juice will not only stop lueing with guaiacum alone, but will prevent it even in presence of a vess of hydrogen peroxide. The ferment appears to be completely coverably destroyed, either by molecular change or by firm anchora ze of the sulphide. At any rate, we have never been able to restore activity once the reducer has been added. _ Experiment XVII.—T hermo-labile nature of the perowides. 7 Except that we prefer to call it peroxide instead of oxygenase, we ean experimentally confirm the statements of Bach and Chodat, that the : substance giving oxidizing reactions without added hydrogen peroxide is a ~ Tess. stable than the ferment or peroxidase which gives rise to the oxidation __ in presence of peroxide. We have pointed out that our dried precipitates 156 BIO-CHEMICAL JOURNAL from alcohol had lost practically all their peroxide, although they had plenty when freshly thrown down and at once taken up in water. Heating to about 55° C. destroys the unstable peroxide bit leaves the peroxidase ; this occurs more quickly in some juices than in others. Thus, potato juice takes some hours, and even then although the amount is very greatly diminished a slight trace remains which is most difficult to get rid of. For example, a portion of freshly prepared potato juice was divided into eight similar portions, which were placed in an air oven kept at 55°C. on February 7th, 1909, at 12-20 p.m., and the tubes were tested one at a time at the following intervals afterwards:—Tube 1, tested at 12-45, still gives intense blue immediately; tube 2, at 1-20 p.m., blue not so intense and comes more slowly; tube 3, at 2-20, more slowly still ; tube 4, at 3-50 p.m., still a good deal of blue, partial precipitation by the heat; tube 5, at 4-30 p.m., still blue; tube 6, at 11-40 a.m. (February 8th), very faint blue only coming after prolonged shaking; tube 7, at 3-40 p.m., no longer gives blue until hydrogen peroxide has been added. Thus a period of about twenty-seven hours was required to destroy all the natural peroxide at 55° C. The same result occurs more slowly, as pointed out, on standing in air at ordinary temperatures, and here again potato requires longer than any other juice we have experimented with, taking several days, while carrot juice will be almost free within twenty-four hours. We have not investigated whether this is due to a larger original supply, or to slower reduction or greater resistance of the natural potato peroxide. Great variations are experienced of a perplexing nature both as to the rate at which the maximum amount of peroxide is developed, and the rate at which it disappears, for which we are quite unable to account. Experiment XVIII.—Effeets of germination on distribution of oxidizing ferment and peroxide. A small quantity of oats, from the same sample as the grain used in Experiment II, were sown on moist cotton wool, and left in the incubator to germinate from November 4th till November 16th, being kept moist during the period and at a temperature of 32°C. At the end of the period the sprouts were about 10 centimetres long. They were — pulled out from the oats, and the sprouts and residues of seeds were tested for oxidizing ferment with the following results :— Both gave a positive result with ordinary guaiacum tincture alone; both were quite negative to all the other coloured indicators for oxidation until hydrogen peroxide had also been added, and both contained PROPERTIES OF OXIDIZING ENZYMES 157 bd se,’ as shown by vigorous discharge of oxygen when added to peroxide (5 vols. per cent.). Experiment XIX.—Absence of oxidizing enzymes in the fresh juice of lemon, and orange ; but presence of such in seeds of lemon and orange. One of our incentives in commencing this research on oxidases was wry absence of oxidizing enzymes by reason of the sterilization, other form of preparation, from all preserved foods, vegetables, and , as also from sterilized milk and milk substitutes, such as children’s infant food. While such enzymes are present, and in considerable ‘ity, in fresh vegetables and juices, in fresh fruits eaten uncooked, This is a difference of a tangible nature in the chemistry of food, of _ these two classes, and it appeared possible that it might give some basis ( r a a better understanding of the etiology of such diseases as rickets or scurvy, the former of which is said to be associated with exclusive use of boil aaa proprietary foods, while the latter, which is, however, a distinct ondition, appears to arise from prolonged abstention from fresh vegetable oe | a I It was these considerations which led us to try those antiscorbutie vegetable juices which are accredited with the most powerful properties, such as lime juice, lemon juice, and orange juice. ‘We were greatly surprised to find that this group amongst the many _ which we tested was the only one which did not yield good oxidase _ reactions either in presence or absence of peroxide. Entirely negative results were given both by juice and rind, and the crushed seeds only in the case of lemon and orange (we were unable from lack of material to test _ the lime seeds) gave a somewhat feeble positive effect. a _ The absence of oxidase in the juice of the fruit of this group is very interesting, although we are at a loss at present to account for it. The is not simply concealed by the somewhat high acidity of the _ juices, for no more success is obtained in the testing on neutralizing the _ juice. It may be that the organic acids present inhibit the production of the ferment, as they certainly inhibit its action when added from without, and tend to destroy it. Thus, if a quantity of potato juice be mixed with the acid (or even almost neutralized but still faintly acid) lemon juice, on — now adding guaiacum a negative result is obtained; but if the acid be completely neutralized the usual blueing follows, especially on adding hydrogen peroxide. In testing the lime juice care must be taken to use a sample which 158 BIO-CHEMICAL JOURNAL has not any sulphurous acid as preservative, as this gives a transient oxidation and blueing with guaiacum even in minimal amounts. The crude juice gives not a trace of effect with any of the tests for oxidizing enzymes. Experiment XX.—Direct application of guaiacum test for pn and perowide, to fresh cut surface of vegetables. This was carried out by slicing, after thorough washing of the outer surface, and then applying an ‘old’ solution of guaiacum to one surface and a ‘new’ solution of guaiacum to the other surface. The test is a convenient one for peroxide and peroxidase distribution in different parts of the tissue, and also demonstrates that even in those plants which yield most easily the reaction with guaiacum alone, such as the potato and carrot, the natural organic peroxide is not present as such in the plant tissue, but is formed in the first few minutes after cutting from some precursor in the plant juices. It may be pointed out that although the terms ‘old’ and ‘ new’ are used in describing the results of this experiment, this is done because at the time we thought the difference was due to age of the two tinctures. Later we found (see Expt. XXI) that the amount of peroxide present in any given sample of guaiacum was more an accident of amount of impurities of vegetable origin in the piece of resin from which it was originally made up. Accordingly ‘old’ means containing more peroxide and sufficient to give direct test in presence of peroxidase, and ‘new’ means comparatively peroxide-free, and hence enables to give blueing with peroxidase in absence of added hydrogen peroxide. A potato was taken, washed thoroughly, dried, and sliced through with a clean knife. At once tincture of guaiacum was placed on the two cut surfaces; ‘old’ guaiacum on one, and ‘ new’ guaiacum on the other. The surface exposed to the ‘ old’ guaiacum blues instantly all over; that with the ‘new’ guaicum only at one or two spots and much more faintly. « The blueing with the ‘new’ tincture occurs more especially at a bruised spot or just close to the epidermis. On cutting away about a centimetre all around the peel to remove any injured portions, only a very slight blueing is obtained, for a few minutes, but comes on standing. In order to test whether the residual amount of blueing in the case of the potato were due to age, as the potato had been removed from the earth for some days or weeks, and since quite negative results had previously been got with fresh radish and fresh cucumber treated with ‘new’ guaiacum tincture, the following similar experiment was next tried. Pe ee ee eT ete ee © aE ht hy eee RT PROPERTIES OF OXIDIZING ENZYMES 159 | ha fresh clean carrot was taken, a sectional cut made across it, and m cut t surfaces were treated one with ‘old’ and the other with 1 for about a zone of half a centimetre thickness round the outside, 1 again most marked in the protoxylem, the central part is less ter stained, but the whole surface is still distinctly blue. = - The ‘new’ tincture, on the contrary, gives for over ten minutes’ time anid application no blueing except at ~ minute spots where there happen to be old bruises just under the skin; “ at about the expiry of ten i a a faint blue appears as a ring sending out radiating branches, s distinctly marking out the protoxylem. It is noteworthy that 5 is part of the section where most oxygen would probably be pr in the plant tissues. On adding to the section of fresh carrot treated with ‘new’ guaiacum neture, a minute trace of hydrogen peroxide, by touching with a fine ass rod moistened with the diluted peroxide, there is produced at once intense blueing, showing the failure to obtain blueing initially is due : to absence of peroxide and not to absence of ferment. Beperiment XXI——Effects of destruction of peroxide in both reagent is (geaiaen tincture) and vegetable juice by treatment with animal charcoal ‘This experiment was conducted in order to make a closer study of certain results of the previous experiments which show rather perplexing e variations i in behaviour towards the guaiacum test (a) with different types of vegetable juice, or the juice of the same origin with the age of the juice, and (5) taking the same juice and at the same time, variations according to whether one or another tincture of guaiacum made from the “ ay stock of the resin was employed for the test. _--———s« ith the single exception of potato juice, for an hour or so after it is ke made, giving a positive result with p. phenylene-di-amine without addition be ‘also of hydrogen peroxide, all the oxy- and amido-phenol coloured indicators of oxidation had concordantly given that a ferment peroxidase r alone was present, which required to have peroxide added in order that it might act. ___-‘The guaiacum test, on the other hand, as above indicated, gave most - _ n to a section of an showing a bruise did not, however, demon- Bear ke anes acted viesing to the aught reed of thet injured and browned cells. Still, ‘i the naturally occurring must soa an oxidizing effect upon tyrosin or similar chromo- genie substances giving colour on oxidizing. 160 BIO-CHEMICAL JOURNAL perplexing variations. Thus, even with fresh guaiacum and potato juice as fresh as it can be separated from the grated tuber, -a considerable blueing was obtained; fresh carrot under like conditions sometimes gaye a blue fainter than that with the potato, sometimes nothing; and fresh cucumber or radish juice, even with moderately stale guaiacum tincture, gave but a poor effect and nothing with fresh tincture. The most feasible explanation of these peculiar variations which occurred to us, and that which suggested the following experiments, was that there are two natural sources of peroxide, viz.:—-(a) the plant juice, and (6) the tincture of guaiacum, The summation of these two effects causes the guaiacum test to be positive, especially with a strongly peroxide containing tincture, in cases where the small amount of peroxide in the juice is insufficient yet to start the oxidation of the oxy- and amino- phenols. In further support of this, the potato juice, which of all the juices we examined is most strongly positive to guaiacum alone, reacts positively, when it is quite new and highest in its content of natural peroxide, to p. phenylene-di-amine, but just fails to appreciably quicken the oxidation of a naphthol, unless hydrogen peroxide be added, We hence had to examine separately the tincture and the vegetable juices, using mildly destructive agents for peroxides which would not destroy the ferment also. Direct reducing agents, such as sulphuretted hydrogen or ammonium sulphide could not be employed, since, as above stated, they appear permanently to destroy the ferment also, either by altering its constitution or by firmly linking on to it. An attempt to clear the guaiacum of colour by means of animal charcoal although it failed in its immediate objective, giving a much deeper green coloured solution, happily had the advantage of destroying the peroxide of the tincture, and yielding a clear filtrate which had no power of blueing until hydrogen peroxide was also added. The usual explanation of the variations in peroxide content of guaiacum tincture is that the peroxide is formed as the tincture stands, and hence, for purposes of the test, that freshly made tinctures must always be employed. ; While we are not prepared to deny that peroxide may be so formed, as the tincture stands and darkens in colour in the course of several weeks, we have clear evidence that this is not the main source of the variations, and that a quite freshly made tincture just filtered off may give marked blueing, while another sample of tincture a week old may give quite a negative result, although it was made from some pieces of the same stock of resin. PROPERTIES OF OXIDIZING ENZYMES 161 a nade-with the same absolute alcohol in 10 per cent. solution from Pia - same stock of guaiacum resin, but one was made about ten weeks previously and the other four days previously. The ‘old’ tincture gave _ fine blue with all our vegetables on the cut surfaces, as detailed in a Experiment XIX, and with the separated juices, while our ‘ new ’ tincture _ gave scarcely a trace of effect. In order to make sure that it was a _ question of ageing and accompanying peroxide formation, we determined upon making an absolutely fresh preparation, and for this purpose we some pieces of the guaiacum resin, and washed the outer surface ily with alcohol in order to remove a green powder which forms on ther surface of the broken guaiacum, and which we thought might be oxidized. These washed pieces were dissolved as far as they would _ dissolve to make a 10 per cent. tincture in previously boiled absolute aleohol, and a clear filtrate was obtained and used immediately for testing. _ To our great surprise, this gave at once a fine blue immediately with the sections of vegetables and juices without adding any peroxide what- We therefore had now a tincture just made which reacted positively _ without added peroxide, a four days old tincture made without any special precaution which acted negatively under like conditions, and a ten weeks old tincture which behaved exactly like the just made one. ig Further, a tincture made from the green powder on the outside of the pieces of guaiacum gave, like the four days old tincture, a negative result. | Tt was also nctiond that while the ten weeks old tincture and the ‘new’ tincture possessed the deep yellow brown colour of guaiacum tincture, the four days old tincture was much paler i in colour, and the _ tineture made from the surface powder was green in colour. This led us _ to boil up some of the ‘ old’ tincture with animal charcoal, in order to try to decolourize it, when we found that instead of decolourizing it went darker, and the filtrate had a green colour. - This filtrate tested now upon carrot juice gave no blueing until hydrogen peroxide was added, showing that its peroxide had been removed by the charcoal; it still, however, gave a blue with potato juice alone. ____ Boiled up once more with charcoal, and again added to potato juice, it still caused blueing, but only slowly and less intensely. This final amount of blueing was probably due to the peroxide in the potato juice itself (vide infra). Cee a i 5 a 162 BIO-CHEMICAL JOURNAL On examining now the residue left behind on the filter paper in filtering the alcohol extract of the guaiacum resin before the addition of animal charcoal, the chief source of the peroxide of the — tincture, and the cause of the variations was discovered, The greater part of this residue consisted of broken seeds and tests of seeds, and other vegetable material. These impurities evidently had been collected with the resin, and since, like all such fresh vegetable material they would contain peroxides, it can easily be understood that — they would yield peroxide to the tincture. Also, in making up tinctures from the same stock, the amount of such impurities varying in different pieces, would explain the variations in degree of spontaneous oxidizing power shown by the different tinctures in absence of added peroxide. This all the more so because the pieces of seed are quite large, and whole seeds even were seen, much larger than barley seed. It was on this account, in all probability, that the green outside powder was free from peroxide, and by accident, probably, some pieces more free than usual had been picked up for the preparation of the four days old (‘ new’) tincture. We would accordingly recommend that in preparing a guaiacum tincture for peroxidase experiments this débris be avoided, and that the filtrate be thoroughly boiled with animal charcoal and filtered. Under such conditions the filtrate can be safely used for several days at least, and any spontaneous blueing observed may be certainly set down to the action of peroxide in the juice or vegetable tissue being tested, and not in the guaiacum itself. The view that the animal charcoal acts by destroying peroxide in the guaiacum is supported by the fact that animal charcoal added in small amount to diluted (1 in 10) hydrogen peroxide solution causes an immediate effervescence. Having ascertained that the peroxide could be discharged from: guaiacum tincture by animal charcoal, we turned the method upon those vegetable juices giving blueing alone, such as potato and carrot, and found that here the peroxide could also be removed by treating in the cold with animal charcoal and filtering. Carrot juice, potato juice, and a sample of tincture of guaiacum which gave a good blue with each of them without hydrogen peroxide, were severally treated at laboratory temperature with animal charcoal and left to stand for one and a half to two hours, then they were filtered, the — guaiacum having turned green in colour. PROPERTIES OF OXIDIZING ENZYMES 163 The carrot juice so treated when now tested with the ordinary untreated-guaiacum gives a dirty greyish green instead of the previous deep blue, whereas with the charcoal treated guaiacum it no longer gives _ a trace of blue or green. Here peroxide has been completely arpa _ both from juice and reagent. The potato juice which before gave an intense blue when now, after charcoal treatment, tested with the ordinary untreated guaiacum gives a dirty greyish blue, and when both charcoal treated potato juice and ; arcoal treated guaiacum are used only a grey with the faintest fpeeeetion of blue is obtainable. This experiment, therefore, supports the view that a positive result wit! th the guaiacum test in the absence of added hydrogen peroxide, s traces of peroxide either in the guaiacum or in the plant or other ee being tested. By the charcoal treatment the guaiacum can be freed peroxide, and then the test may be utilised as a fairly delicate one for oxides naturally occurring in plants, or formed soon after the juice is Experiment XXI1—Dialysis in parchment paper. __ A quantity of 500 c.c. of fresh carrot juice was placed in a sausage gi tube of parchment paper, and dialysed against 1,500 c.c. of distilled water in a tall cylinder. Next morning the outer fluid gave with guaiacum (ordinary) alone a slow but distinct blueing. This subject, however, - ae further investigation. Experiment XXIII—Supplementary experiment on presence of , ses in bananas, in certain species of nuts, viz., Spanish chestnut, ‘almond, filbert, walnut and Brazil nut and in hyacinths. _ None of these gave positive results with the phenol or amido-phenol derivatives, or with guaiacum free from peroxide, except when hydrogen _ peroxide was added, when they reacted in varying degree. In the case of the banana, the inner surface of the peel rapidly blues a with peroxide containing guaiacum, but the pulp only blues very slowly, and the reagent applied to the cut surface shows marked veining a accompanying the course of vessels. -___ Cross sections of a Spanish chestnut, tested at once with both peroxide iz containing and peroxide free guaiacum, show blueing with the former at - once, but no blueing with the latter, even after ten minutes. The blue first produced by the peroxide containing guaiacum bleaches in a few minutes, but can be brought out again even more strongly by re-applying more of the reagent; this is repeated several times. On touching a spot 164 BIO-CHEMICAL JOURNAL on one of the sections treated with peroxide free guaiacum with a glass rod previously dipped in dilute hydrogen peroxide solution, there is an instant blueing at this spot. Brazil nut gives exactly similar results. Almond, filbert and walnut give no colour with peroxide free guaiacum. Walnut and almond give a slight colour, filbert rather more colour, with peroxide containing guaiacum. Haricot beans, old and very dry, gave no colour either with | peroxide containing or peroxide free guaiacum, but an extract gave a feeble positive effect with ordinary guaiacum alone after vigorous shaking, heightened by adding hydrogen peroxide. The green leaves, bulb, and rootlets of a hyacinth, taken fresh folk the garden were examined with peroxide free guaiacum, and only the rootlets gave a rather slow blueing with this alone, but all three parts gave a fine blue on also adding hydrogen peroxide. Experiment XXIV.—Effect of synthetic guaiacol (Merck). This reagent behaves quite differently from the natural resin, giving a deep brown colour in oxidation which is very distinctive. Tested with fresh potato and carrot juice it gives no effect until hydrogen peroxide is also added, when the mixture at once turns brown, which deepens in colour to reddish brown, and a reddish brown precipitate is thrown down. Experiment XXV .—Effect of gquaiaconic acid (Merck). This substance is contained in guaiacum resin and turns blue on oxidation. A supply obtained from Merck was found to dissolve completely to a brown solution in absolute alcohol. A 2 per cent. solution was used for testing the fresh juice of potato, carrot, apple, and cucumber. The colour obtained was identical with that given by the tincture of the resin, and, as the following results show, it contained a trace of peroxide, about equal to that in the best of the tinctures made from the resin, and distinctly greater than the tincture when freed from. peroxide by thorough treatment with animal charcoal. Potato juice gave with guaiaconic acid alone a greyish blue, deopantiig on standing; on the addition of a few drops of diluted hydrogen peroxide an immediate blue was obtained, rapidly deepening on standing. Carrot juice gave practically a negative result with the guaiaconie acid alone, there being only a slight dulling of the natural carrot colour and no trace of blue in fifteen minutes; addition of a few drops of dilute hydrogen peroxide to half of the test gave an ‘instantaneous deep blue. Bi) ae Fa 5 a PROPERTIES OF OXIDIZING ENZYMES 165 ; ae ___..-bhueing on standing; on adding peroxide also the usual deep ~ blue was obtained, increasing and showing just a shade of blue on standing; addition of hydrogen peroxide also caused a deep blueing at fiat once, Sth THE PARALLELISM IN MODE OF ACTION BETWEEN DROLYTIC ENZYMES, OXIDIZING ENZYMES, AND THE ACTIVE BODIES DEVELOPED IN IMMUNE SERA 4 “The experiments recorded in the previous section appear to us to demonstrate clearly that the whole difference between the various juices _ and other fluids showing an oxidizing action consists in the presence of 4 a variable small amount of peroxide which is chemically unstable and i. ee by the agencies recorded. a _ All the juices showing oxidizing properties possess one type of ferment ich, since it acts only in presence of either naturally present or + antifically added peroxide, may provisionally be styled a peroxidase, and there is no proof of the existence of any other type of enzyme engaged in oxidation processes. This not only materially simplifies our conceptions regarding the _ oxidizing ferments, but, in our opinion, brings the class into line both with the great division of hydrolytic ferments engaged in the processes nf of digestion and metabolism and with the active bodies in the natural me and i immune sera which combat and immunize against disease. So that “the oxidizing enzymes form a connecting link between the two classes. ah Th all three classes of enzymic action it is to be observed that three interacting bodies are required. These three are (1) the substrate on Emr whic h the ferment is to act, (2) the body which is to be combined directly or indirectly with the substrate and alter its chemical and physiological properties, a and (3) the enzyme or ferment which is to activate the reaction. In the case of the ordinary hydrolytic enzymes these are as follows :— Substrate, the foodstuffs, protein, carbohydrate, or fat; the Combining Body, % the elements of water finally, intermediately the acid or alkali, in presence of which alone the ferment is active; the Catalyst, one of the digestive or other hydrolytic ferments, such as pepsin, trypsin, diastase, zymase, lipase, &e. 15. No generic name, so for as we are aware, has yet been given to this substance usually ture than the substrate, which ix added to or taken away from the substrate i . Dasivtie actin. We suggest that it might conveniently be called the Combinate. 3 tea 166 BIO-CHEMICAL JOURNAL In the case of the oxidizing ferments: Substrate, the oxidizable substance, such as tyrosin, naturally occurring phenols in the plant, or the chromogenic indicators used in the preceding section; the Combining Body, oxygen yielded by peroxide bodies present in some form, either as simple hydrogen peroxide or as organic peroxides; the Catalyst, the enzymes, such as tyrosinase, and the peroxidase experimented with in the preceding section. In the case of the immune sera, cytolysins, etc.: Substrate, the cell or bacterium to be dissolved or the toxic or foreign substance in the serum to be attacked and rendered inert; the Combining Body, the complement, or thermo-labile substance, in the absence of which the reaction cannot proceed; the Catalyst, the specific immune body or anti-body which attacks and disintegrates the foreign cell or ‘neutralizes’ the toxic substance. Between two of these three reacting substances, viz., the substrate and catalyst, there exists usually a considerable amount of specific relationship. This specific relationship is in most cases not narrowed to a single chemical substance, but is closely confined to the members of a class of bodies possessing in their molecular constitution a certain definite grouping. Even very nearly allied groupings are quite inert to the particular catalyst, as, for example, the fermentation by organisms of certain sugars or other substances, which are stereo-isomers of others which are not attacked in the least. But given the zdentical molecular conformation at a certain portion of the molecule, there may be, and will be, attack and chemical action, although the molecular structure at other parts may be such as to render the two bodies in other respects very different physically and chemically. For example, pepsin and trypsin attack all classes of proteins, down to certain well-marked stages of hydrolytic cleavage so long as certain — connections in the molecular aggregate exist, although in physical properties, in reactions to precipitates and indicators, and even in ultimate chemical composition, these proteins are very distinct from one another. Similarly, the peroxidase ferment in presence of peroxides attacks a large number of oxidizable substances, such as those experimented with in the preceding section, but leaves other classes of oxidizable substances, both those which are more readily and those which are more ree oxidizable, unattacked. Turning to the third member of the group of three, represented Se PROPERTIES OF OXIDIZING ENZYMES 167 . : B the oxidising forments and by the complement in the immune sera, we find that this is much less specific in character. Thus, an immune dy or cytolysin of very specific character may be activated by practically yy serum, including the natural serum of the same species as the animal ‘ich had been immunized. Similarly, any body containing a peroxide \kage, organic or inorganic, will activate a peroxidase, and any type of id or alkali which increases hydrogen or hydroxy] ion concentration, spectively, will activate a hydrolytic enzyme. _ The ferment character of the reaction in the case of immune sera is @ o shown by the disproportionately large amount of complement from . sp normal serum which can be bound to lipéid, and so rendered inert 7 ad haemolysis in the second stage of the Wasserman reaction, by very small amounts of syphilitic * immune’ body. In this reaction it is clear that an active principle in the syphilitic serum acts as a catalyst or ferment, the lipéid from liver or elsewhere as a substrate, and the - complement from any serum as a combining body with the substrate under _ the influence of the catalyst. Finally, attention may be drawn to the similarity between the oxidizing ferments and immune sera in regard to thermo-stability. When an immune serum is heated for some time at 55°C. the complement is be destroyed, but the ‘immune’ body remains untouched; the heated serum, ae — is inactive until complement is added by mixing with some unheated serum which may be drawn from any animal. Quite similarly, when a vegetable juice is heated to 55°C. the _ peroxide is destroyed, but the ferment or peroxidase is untouched, and ough the heated juice is inactive as an oxidizing agent it is at once ted on adding hydrogen peroxide or other form of peroxide. .. 7, aki if a vegetable j juice be kept it loses gradually its siluatiaieoes : — of oxidizing, but regains it as soon as a peroxide is added. Tian bn abe | 168 ON THE OCCURRENCE OF A MON-AMINO-DIPHOSPHATIDE LECITHIN-LIKE BODY IN EGG YOLK By HUGH MacLEAN, M.D., Carnegie Research Fellow, University " Aberdeen. From the Department of Physiological Chemistry, Institute of Physiology, Berlin (Received March 17th, 1909) In making some investigations on the ethereal extract of egg yolk, I succeeded in separating a body of the general nature of a lecithin, but containing in its molecule two parts of phosphorus to one part of nitrogen. This compound is of the same type as that separated from heart muscle by Erlandsen,'! and called by him ‘Cuorin,’ though, as shown below, it differs in certain respects from this substance. A preliminary note giving the N and P content which characterise it as a mon-amino- diphosphatide was published some time ago,? but as this portion was prepared from a comparatively small number of eggs, no account at its properties or probable elementary composition was given. During this winter, however, I have isolated the substance Pic a large quantity of egg yolk, and having used certain modifications in the — preparation of different portions, will now proceed to give a short nenoumt of its isolation, composition and chief properties. PREPARATION The egg yolk of fresh eggs was separated from the white portion, and after being spread out in thin layers on a glass plate was dried by means of a current of air generated by a fan-like arrangement attached to a — motor. When thoroughly dry the mass was broken up into small pieces; — and finally passed through a coffee mill. In this way a very fine powder was obtained, which was carefully extracted five times with ether. The ethereal extracts were mixed together, evaporated to a fairly small volume, and treated with acetone. The precipitate was dried in vacuo over H,SO, and divided into two parts. Portion A was treated by a combination of the methods used by Stern and Thierfelder? for purifying lecithin, and by 1. Zeitschrift f. physiol. Chemie. Ba. LI, 8. 92. 2. Ibid., Bd. LVI, 8. 304. 3. Ibid., Ba. LITL, 8. 370. - MON-AMINO-DIPHOSPHATIDE LECITHIN-LIKE BODY 169 Erlandsen* for the isolation of cuorin; as will be seen, this combination ____ is rather-tedious in carrying out. * Portion B was treated by a much simpler method, which was found exceedingly easy to carry out, and capable of giving quite a pure substance. TREATMENT OF Portion A ___ This portion was again dissolved in ether and gave a markedly turbid solution. By means of the centrifuge a perfectly clear fluid was obtained, the residue in the centrifuge tubes being of a whitish colour, and with difficulty soluble in ether. The clear solution was again precipitated with acetone and dried. This process of purification was repeated five times, and by this means impurities such as cholesterin and fat were in great part got rid of. The final material gave a perfectly clear solution in ether, but only after standing for some time, being at first slightly turbid. The solution was now treated with about four times its volume of absolute alcohol, and left to stand under CO, in a closed vessel for twenty-four hours; an alcohol insoluble residue remained, which was separated by filtration. This substance was washed with cold : alcohol, and the united filtrates evaporated in vacuo to a small bulk, i precipitated with acetone, and dried as usual. This precipitate was now = treated with absolute alcohol, when a small portion remained behind, F ft which was added to the above alcohol insoluble part. Thus, the ethereal solution was divided into two parts—the part soluble in cold alcohol consisting of ‘ lecithin.’ The alcohol insoluble part was now placed in an incubator and heated for a fairly long time with alcohol at 65° to 70°C. By this means ee part of the substance went into solution, but separated again on the -___ aleohol cooling. This process was repeated three times, and finally boiling aleohol was used in an attempt to obtain an alcoholic solution that would remain quite clear after cooling. In every case, however, the alcoholic filtrate on cooling became slightly opalescent, though only faintly so. By this procedure the part insoluble in cold alcohol was divided into two parts, one of which (portion a) was soluble, the other (portion 4) insoluble in hot alcohol. Portion (a) was thoroughly washed with cold alcohol and treated as 3 described later. ihe _ Portion (6) was dissolved in ether and left to stand over night under CO,; next day a slight precipitate had settled out, the solution itself being 4. Loe cit, i i 2 ON eee eee ae 170 BIO-CLLEMICAL JOURNAL almost clear. On centrifuging, the perfectly clear solution obtained was precipitated by acetone; on again dissolving in ether it gave practically a clear solution. This solution being again precipitated, the substance was dissolved in hot ethyl acetate, out of which it separated on cooling. It was then filtered, dried and analysed. TREATMENT OF Portion B This portion was thoroughly extracted with cold absolute alcohol, whereby the greater part went into solution. This solution was evaporated to a small bulk and the syrupy residue dissolved in ether; the ethereal solution was then precipitated by acetone. The precipitate was dried, redissolved in ether and reprecipitated by acetone. By this means ordinary ‘ lecithin * was obtained in a pure condition, the ordinary contaminating substances present in the raw ethereal extract mass being relatively insoluble in cold alcohol. Residue insoluble in cold alcohol was now treated with hot alcohol exactly as described under Portion A. By this means impurities such as cholesterin and fat went into solution. This treatment with hot alcohol was repeated four times, boiling alcohol being ultimately used. The residue was then further purified by dissolving in ether, precipitating with acetone, and finally dissolving in hot ethyl acetate, as described above; it was then dried and analysed. The hot alcoholic solution on cooling deposited a voluminous precipitate of floccular masses. This was obviously composed in great part of fat and cholesterin, and was not examined. This method of separating the raw material is exceedingly simple and quite efficient. It is much more quickly carried out than the method adopted under Portion A, and can be recommended as an easy means of separation of the mono- and di-phosphatides present in the ethereal extract of egg yolk, heart muscle, and probably in other substances. In the following analyses Parts I and II were prepared as described — under Portion A. Part III was prepared as mentioned under Portion B. ANALYSIS I Nitrogen ( Kjeldahl) can substance used 1-5 c.c. yy H,SO, = 0-81 % 0-3122 , - » 2826.0. a = 0-82 9 Phosphorus (Neumann) 0-2411 gm. substance used 15-66 ¢.c. } NaOH = 3-60 % 0165, “» ww» 10Bex. , = 364% Elementary Analysis 0-1625 gm. substance gave 0-3527 gm. CO, = 59-19,% C and 0-1388 gm. H,0 = 9-56 % H e} Nit 03140 gm. substance used 1-78 ¢.c. J, H,SO, = 0-794 CMe don coed Phosphorus 0-4139 gm. substance used 26-55 c.c. } NaOH = 3-55 % 02729, » » ITBbec. ., = 356% Elementary Analysis 0-1541 gm. substance gave 0-3329 gm. CO, = 58-92 % C and 0-1296 ,, H,O = O-dl 92 H 01701, », » 03686 ,. 0b, = 59-00 C and 0-1413 ,, mp = 929%H , 01448 ,, % 0-3140 ,, == ae Cc “and 0-1217 H, H Iodine Number 0-1691 gm. substance pend Ca Iodine = 76-8 02254 ” ” ” ” ” = 77:1 Il Nitrogen 0-4836 gm. substance used 2-85 c.c. to H,80, = 0-83 % Phosphorus _ 0/2132 gm. substance used 13-82 ¢.c. $ NaOH = 3:59 % Elementary Analysis otis substance gave 0-2453 gm. CO, = 59-31 % C and 0-0963 ,, H,O = 955% H a Portion F Foriien: II Portion III Average of Cuorin | percent per cent. per cent. per hai per cent. per cent. tes (Erlandsen) - 219 — 58-92, 59:09 59-14 59-31 59-12 61-63 9-56 — | &él 9-29 94 9-55 O44 9-03 O81 0-82, _— 0-794 0-806. 0-83 O812 LO 3-60 3-64 = 355 3-56 3-59 3-59 4-46 _ ied et es 27-048 a ' ser , a comparison of the two substances is of some interest. A » to the analyses shows that they are not identical. If, for ut the difference is relatively high. Again, the O content of my is higher than that of cuorin. At first it was thought that this e in percentage composition might possibly be dependent on the ence of a certain amount of oxidation, prior to, or during, the ‘ion of the substance, and the extreme facility with which cuorin oxidation lent colour to this view. An examination of the 172 BIO-CHEMICAL JOURNAL figures, however, shows that possible oxidation does not account for the difference ; a priori the probability of any marked oxidation was unlikely, since all manipulations were so conducted so as to exclude as far as possible the presence of oxygen. Since cuorin contains a relatively higher percentage of N and P than this substance, it is obvious that the addition of more O would lower the N and P percentage, and so tend to lessen the difference in composition. For a specimen of oxidised cuorin, Erlandsen gives the following figures: —O 30°72, N 0°92, P 4°06 per cent.; while my substance gives O 27, N 0°812, P 3°59 per cent. If we consider cuorin oxidised only to the extent of 27 per cent. (instead of 30 per cent. as given), this portion must contain something over 0°92 per cent. N and something over 4°06 per cent. P. Since my substance, oxidized to 27 per cent., contains only 0°812 per cent. N and 3°59 per cent. P, it is clear that possible oxidation is not the cause of the difference. It is probable that this is really accounted for by a difference in the fatty acids of the two substances, Again, though the substances differ somewhat in their iodine figures, this difference is not great enough to satisfy the assumption that the one is but an oxidized form of the other. Whether the somewhat prolonged treatment with hot alcohol necessary for the isolation of the substance in a pure condition has any action in causing some decomposition, and so a slight change in the ultimate formula of the phosphatide is perhaps worth consideration ; if this is so, it is possible that the exact percentage composition may vary slightly, depending on the amount of hot alcohol treatment. Since, however, the different portions mentioned above yielded practically the same results, this is not very probable; in any case, it does not affect the fact that in egg yolk there is contained a well defined lecithin-like substance containing N : P in the proportion of 1 : 2. PROPERTIES This substance is obtained as a yellowish brown brittle substance, which, after being carefully dried, is easily ground to an exceedingly fine powder; it is much more brittle than cuorin, and though hygroscopic, is by no means markedly so in comparison with certain other phosphatides. In common with all lecithins it undergoes oxidation on exposure to air. It is insoluble in cold aleohol but somewhat soluble in boiling alcohol, — and thus differs from cuorin, which is said to be insoluble in boiling . ~ MON-AMINO-DIPHOSPHATIDE LECITHIN-LIKE BODY 173 alc leohol ; in chloroform, ether and petroleum ether it dissolves easily at the the ordinary temperature. In ethyl acetate it dissolves on heating, to be thrown out on cooling. From its ethereal solution it is precipitated by acetone. With water it forms an emulsion. With platinum chloride it gives double salts, and by hydrolysis gives fatty acids and glycero- _ phosphoric acid; no base of the nature of choline could be obtained. When heated to 90° to 100°C. it changes colour, but an exact melting ~ t could not be determined. After hydrolysis with weak hydrochloric acid no reducing carbohydrate was obtained. All its general properties _ show that here we have a typical phosphatide substance. | Warr Supstance Sotvusie 1x Hor Atconor AnD ieee Ovt on CooLInG This substance (Portion a, page 169) was dissolved in hot alcohol, filtered hot, allowed to cool, and again filtered. By this means a voluminous precipitate of whitish snowflake-like material was obtained, tich on drying shrunk to a very small volume. This process was peated four times. By this means a fine whitish granular powder was obtained, which was completely soluble in ether, chloroform and benzene. On the addition of acetone to any of these solutions it was, after a little time, but not immediately, precipitated. As some doubt exists as to the composition of this ‘ white substance,’ Erlandsen having found N but no P in apparently similar material, while _ Thierfelder and Stern found both N and P, I made some further investigations with the following results. _____In this particular preparation there was neither N nor P present. Melting point was easily observed being 62° to 63°C. When burned on platinum foil it left no inorganic residue. After boiling for some time rith HCl no substances of a reducing nature were obtained. Saponification with Ba(OH), failed to give any substances of basic nature precipitated by platinum chloride. From these observations it was apparent that this substance was probably of the nature of a fat. A combustion experiment _ showed this fat to be tripalmitin. Analysis 0-1068 gm. substance gave 02964 gm. CO, = 75-70 % C and 0-1179 ,, H,O = 12:35 % H Calculated jor Tripalmitin CuHy,04 = 75°86 % C and 12-24 % H I would here noel ike no ional of in purifying the above substance was made. This somewhat soluble in ‘lecithin,’ so it is doubtful if, ‘lecithin ’ ariel free from fat. ” Reale ‘lecithin’ with N : P as 1: 1 another lecithin amino-diphosphatide body—with N : P as 1: 2. This substance, as above obtained, differs somewhat in and properties from the substance, cuorin, prepared from h the difference being probably a on the presence of d acids. The other substance in the ethereal extract whi dissol alcohd] and ey ont on wae was bapa to be pure t wren 2 wey. 175 10 : )D DO-EOSIN: AS A TEST FOR FREE ALKALI IN DRIED-UP a = ANT TISSUES By A. C. HOF, Héchst a. Main. a Ciinptediented by Péafeswe P.Bhelich i (Received March 24th, 1909) a ay free dye-acid of iodo-eosin has been employed by Professor lant tissues.” Sy The dye used is iodo-eosin, the potassium salt of tetraiodo-fluorescein. soluble in ether or any other organic solvent. The free dye-acid, e * obtained as a yellow precipitate from the alkaline solution of eosin by adding hydrochloric acid in excess, dissolves easily in ether in ai any other organic solvents, but is insoluble in water. It is for this reason that the reaction can be made use of for testing free alkali in a perfectly dry tissue. Suppose we have a free hand transverse section of a dry twig of a eommon forest-tree, for example, a spruce fir, and put the section into a solution of the dye-acid of iodo-eosin that has been dissolved in ether, leave it there for some minutes, then wash out the section thoroughly with ether. The preparation brought into xylene should now be ‘amined under a low power of the microscope, when some of the istologically differentiated elements of the tissue will be stained d y red. In our section, for example, the cambium, the resin ug ‘canals and the secreting cells lining the cavity appear plainly red- 24 Iti is easy to see that these red-stained elements of the tissue must be those that contain free alkali, and, further, that their red colour is due to the red alkali salts formed. We are justified in supposing that the free alkali located in these _ tissues is acting as a dye-base as soon as the dye-acid of iodo-eosin is 1. Ehrilich-Lazarus, Die Anaemie, Vienna, 1898. 9. A. C. Hof, Botanisches Centralblatt, UX XXIII, 1900. 176 BIO-CHEMICAL JOURNAL added, yielding immediately the characteristic red dye-salt. If there is no water present the red colour remains in the tissue fixed at the exact place where it has been formed. Thus we get an exact idea of the distribution of free alkali in dry tissues. Mernops Preparation of the dye-acid of iodo-eosin.—Dissolve 1 gramme iodo- eosin, the commercial dye, in 1 per cent. potassium hydroxide, add hydrochloric acid in excess. The dye-acid precipitates at once. Filter and wash the precipitate with hot water thoroughly till the filtrate is absolutely free from hydrochloric acid. Dry the precipitate of dye-acid and dissolve it in 100 c.cm.-ether. Staining of sections—Bring the sections, either sectioned free hand or in a microtome, into the above solution of dye-acid, leave them in it for some minutes, wash out the sections carefully with ether, transfer to xylene, and seal preparation in Canada-balsam. For preserving the sections distinctly stained it is absolutely essential that the balsam used is of perfectly neutral reaction. The ordinary balsam bought from dealers in microscope supplies often reduces the dye-salts immediately. By this — reduction of the dye the colourless compound—the leuco-base or leuco- compound—is formed.* I venture to hope that the foregoing reaction, simple, effective and feasible as it is in the case of almost any drug, may be of some use to students of pharmacognosy. 3. P.G. Unna, Centralblatt fiir Bakteriologie und Parasitenkunde, Vol. ILL, 1888, 7 HE GROWTH OF THE BACILLUS TUBERCULOSIS AND -——~OTHER MICRO-ORGANISMS IN DIFFERENT PERCENTAGES OF OXYGEN : vas BENJAMIN MOORE, M.A., D.Sc., Johnston Professor of Bio- Chemistry, University. of Liverpool, axp R. STENHOUSE WILLIAMS, M.B., D.P.H., Lecturer on Public Health Bacteriology, University of Liverpool. From the Departments of Bio-Chemistry and of Bacteriology, University “a of Liverpool (Received March 31st, 1909) ‘The experiments here described were suggested by the seats of growth of the Bacillus tuberculosis in the body corresponding as they do to - situations where there is a high pressure of carbon dioxide and a low _ pressure of oxygen. It was thought from this that the bacillus might either require a certain definite percentage of carbon dioxide in the air, or might be very sensitive to high pressures of oxygen, and only grow, as in the lungs or elsewhere, where the partial pressure of the oxygen is normally much lower than in the atmosphere. The experiments did not quite justify the theory; but the theory served the most essential purpose of a theory in leading to experiment, and certain of the results have proved in some respects sufficiently interesting to warrant description. ly These experiments are still being continued, but as far as they have ——_ gone their chief results may be briefly put as follows:—-The Bacillus tuberculosis either does not grow at all, or grows very badly in the entire sence of oxygen, or in presence of a partial pressure of oxygen amor ting to 80 to 90 per cent. of an atmosphere. A number of other organisms have also been tested, and certain of these, like the Bacillus tuberculosis, cease to grow in the higher oxygen percentages, while others appeared to be unaffected by the variations in oxygen. The experiments with the Bacillus tuberculosis will first be described, and afterwards those with the other organisms. Experiment I.—Growth in large tubes with stopcocks, and analyses of the gases in the tubes after growth. Since carbon dioxide is such a heavy gas, it was thought that under the ordinary condition of culture in test tubes with cotion-wool stoppers ee a ee oe a ee 178 BIO-CHEMICAL JOURNAL there might be an accumulation of carbon dioxide in the culture tubes in which the bacilli might grow, as in the lung. Accordingly two extra large tubes of about 120 c.c. capacity were made, each with a draw-off tube for analysing the tube gases, closed by a small glass tap near the bottom; and one tube was plugged with cotton-wool in the usual way, while the other was closed above with a rubber cork, through which passed = a small glass tube, also closed by a glass tap. Each tube was inoculated on a slant surface of glycerine agar! with a strain of Avian tubercle, and the two tubes were incubated alongside of each other for fourteen days at 36° C. Then the gases in each tube were analysed by drawing samples off into a Hempel gas burette in the usual way. The tube plugged with cotton-wool showed 19°4 per cent. of oxygen and no carbon dioxide; while that closed air-tight by the rubber cork gave 18°7 per cent. of carbon dioxide and no oxygen. Hence the reply of the experiment is that there is no accumulation of carbon dioxide in growing in the ordinary way with wool-stoppered tubes. There was a good growth of the bacillus in both tubes. This appears to be contradictory to the result of some of the later ~ experiments, which show no growth in hermetically sealed tubes in absence of oxygen; but it is to be remembered that in the later experiments ordinary small test tubes were used, in which the supply of oxygen would soon be exhausted, while here, in order to get sufficient gas samples, large tubes of about 120 c.c. capacity were used, and there would be a sufficient amount of oxygen to allow of a good growth before the oxygen became exhausted. Experiment I1.—Effect of high partial pressure of oayyen. This experiment and some of the later ones were carried out in a small copper autoclave, kept in an incubator at 36 to 37°C., and filled with the desired oxygen mixture, while the controls were grown in the same incubator outside the autoclave but alongside it. The autoclave was ~ used because it was intended to pass on later to oxygen pressures higher than atmospheric, but when it was found that growth was stopped at 80 to 90 per cent. of oxygen at ordinary atmospheric total pressure, the autoclave was not found to be necessary, and hence in later experiments a glass desiccator on a ground base with a mercury seal all round outside the glass junction was used, as subsequently described. Six tubes containing cultures on glycerine agar of Bacillus tuberculosis (Avian) were used for the experiment, four being placed in the autoclave 1. Veal broth agar containing 5% glycerine, and 2% peptone, and acidity iw to phenol , phthalein. ae GROWTH OF THE BACILLUS TUBERCULOSIS — 179 ‘ith a vessel containing soda lime to absorb any carbon dioxide set free, _ while the other two were grown in air as controls outside the autoclave in - the same incubator. The autoclave was exhausted and then joined up to a gasometer holding oxygen, prepared carefully in the laboratory from potassium permanganate. The exhaustion and filling up was repeated four times, and at the | end the autoclave atmosphere was found by analysis to contain 87°5 per gent. of oxygen. The experiment was begun on June 24th, 1908, and the autoclave was opened and results noted and compared with control on ‘Fuly 8th, 1908. _--—~—sS'‘The following show the percentages of oxygen in the autoclave on q ‘different days: —June 24th, 87°5 per cent.; June 26th, 85°77 per cent.; July Ast, 85°1 per cent.; July 4th, 84 per cent.; July 8th, 63°2 per cent. re was always an analysis made for carbon dioxide, but none was found, owing that the soda lime was quite effective. The drop in oxygen in e last analysis indicates the starting of a very slow leak around the rim f the autoclave; but there was never less than sixty-three per cent. of _ oxygen, and in the earlier part the percentage was nearly ninety, 4 a On opening the autoclave it was found that the four tubes grown i inside showed practically no growth at all, while the two controls grown in air showed a moderate growth. . Experiment I11.—Growth of Bacillus tuberculosis (Avian) in ordinary q small test tubes (a) hermetically sealed, (b) stoppered with rubber corks, and (ei in controls grown in the ordinary fashion with cotton-wool stoppers. fe Eight test tubes containing the glycerine agar culture medium were inoculated with Avian tubercle on June 17th, 1908; four of these were hermetically sealed off at the upper end, avoiding any injury to the edium; two were stoppered with ordinary rubber corks; and two were sed with cotton-wool in the usual fashion. The whole eight tubes were | then incubated alongside of one another in the same incubator for twenty- one days (till July 8th, 1908), when the condition of growth was noted in each set, and the gases analysed in the hermetically sealed and in the rubber closed tubes respectively. The two control tubes showed a good growth, the four hermetically sealed tubes showed very slight growth, and the two rubber stoppered tubes showed slightly more growth than. the sealed tubes, but much less than the controls. The glass point of one of the hermetically sealed tubes being broken under water, a negative pressure was shown by an in-rush of water. The = 180 BIO-CHEMICAL JOURNAL free capacity of the tube was about 40 c.c. and 26°8 c.c. of residual gas was obtained. This contained 07 c.c. of carbon dioxide (2°6 per cent.) and no measurable amount of oxygen, so that practically all the residual gas was nitrogen. A similar analysis in one of the rubber stoppered tubes gave 29°6 c.c. of total gas, containing 3 per cent, of carbon dioxide, no oxygen, and the balance nitrogen. ; Experiment IV.—Growth of Bacillus tuberculosis (Avian) in high partial pressure of oxygen. Six tubes of glycerine agar, plugged in the usual fashion with cotton- wool and equally inoculated with Avian tubercle, were taken (July 15th, 1908), and of these four were grown in the autoclave in increased oxygen, while the other two were grown in air alongside in the same incubator. The autoclave after receiving the four tubes, and also an open vessel containing 25 grammes of soda lime, was screwed up, exhausted by a water pump, and allowed to suck in oxygen from a reservoir, the oxygen being made previously, as in all the experiments, from potassium permanganate. The exhaustion and filling was repeated five times, and the final percentage of oxygen in the autoclave was 82°6. The experiment was run for twenty-one days, viz., from July 15th to August 5th, 1908, and analyses of the autoclave atmosphere at intervals gave the following results :—July 18th, 75°5 per cent.; July 22nd, 70°9 per cent.; July 26th, 57-9 per cent.; August 5th, 50°3 per cent. There is here again a slow leak in the autoclave packing, and an oxygen percentage varying from 82°6 at the beginning to 50°3 at the close. There was no appreciable amount of carbon dioxide present throughout. Examined at the end the two control tubes show a fair growth, while the four tubes grown in the increased oxygen show practically no growth. Experiment V.—Growth in stoppered large tubes alongside cotton stoppered similar tubes. In order to examine more fully the gaseous exchanges in growth in» sealed tubes where the growth was finally inhibited, and also to examine the effect of the medium alone upon the air in the sealed tubes, the — following experiments were carried out in larger tubes than usual, the capacity of each tube being about 120 ¢c.c. Six culture tubes were taken for the experiment and treated as follows : — A.—Control. Glycerine agar inoculated with Avian tubercle and plugged with cotton-wool in the usual manner. © B.—Same as A, but plugged air-tight with solid rubber stopper. C.—Glycerine agar, not inoculated, but plugged exactly like B. GROWTH OF THE BACILLUS TUBERCULOSIS 181 ' D and E.—Same as B, and inoculated, but with a glass tube _.-~sealed at outer end inserted through rubber cork so as to be easily broken afterwards in rubber tubing to allow sample of gases to be taken for analysis. F.—Glycerine agar, not inoculated, arranged as in D and E. The six tubes were grown together in the same incubator from July 28rd till August 21st, 1908 = twenty-nine days. _ The final result was as follows :— Amount of Growth Analysis of Residual Gases Ry i “The whole surface of the medium Open to air; therefore no analysis. —s eovered with 7. B. growth. fifth of surface covered carbon dioxide 5-7 per cent. oxygen; no carbon dioxide. cent. carbon dioxide. more than D. cent. carbon dioxide. no carbon dioxide This ee shows that the control grown under atmospheric _ oxygen gives by far the best growth; that there is an up-take of oxygen by the medium alone but no output of carbon dioxide, about half the available oxygen being so used up in the time; and that in the inoculated _and stoppered tubes all the oxygen disappears, but only about one-third u of the corresponding amount of carbon dioxide appears. Experiment VI.-Growth of strains of Bacillus tuberculosis of Avian, vine and Human origin, in inereased percentage of oxygen. This experiment, instead of being carried out in the autoclave, was done in a large desiccator or bell-jar, resting on a well-fitting ground glass base, and having a glass tube and well-fitting tap above fixed in a -mereury seal was arranged all round the base by constructing a circular mound of putty all round about a centimetre high and of about two centimetres greater diameter than the rim of the bell-jar. In this way _ the slow leak of the autoclave experiments was avoided, and, as shown by’ the analyses in this and and the succeeding experiments, the per- centage of oxygen was kept high and very nearly constant throughout the necessarily somewhat prolonged experiments. _ Fair growth, roughly about one- Total volume 83-4 c.c.; no oxygen; Not inoculated; no growth. Total volume 846; 12-4 per cent. of Hardly any growth. _ Total volume 74°8; no oxygen ; 5-0 per Moderate growth; less than B, Total volume 82°9; no oxygen; 6°7 per No inoculation ; no growth. Total volume 80; 11-5 per cent. oxygen ; _ rubber cork in a wide tubulure. In order to keep a quite tight joint below, a Se eo eee ee a | il) i iia a 192 BIO-CHEMICAL JOURNAL Two tubes each were inoculated on glycerine agar with tubercle bacilli of avian, bovine and human origin, and plugged in the ordinary way with cotton-wool. These were placed in the bell-jar above deseribed along with 20 grammes of soda lime in an open flat vessel. One tube of each strain was inoculated and grown in air outside the bell-jar, but close alongside it in the same incubator, to serve as a control. The experiment was commenced on the 2nd December, 1908, and concluded on the 24th December, 1908 = twenty-two days. The per- centage of oxygen was got up at the commencement in the usual way by — exhausting and connecting with a reservoir containing permanganate — oxygen. The initial value for the oxygen percentage was 773 on December 2nd and 76°53 on December 24th, showing that there was no appreciable leak. Examination of the nine tubes at the later date shows in every case that the growth is stronger and thicker in the controls than in those grown in the oxygen. This is particularly well shown in the human strain, in which one of the two tubes shows hardly any growth and the other a very scanty growth, while there is a much better growth in the control. : The same holds for the bovine growths, but all three tubes are some- what further advanced. In the avian the growth is considerable in both oxygen grown tubes and controls, but the control is thicker and better grown. Experiment VII.—Growth of Bacillus tuberculosis (Human) in * increased oxygen percentage. ; Three tubes were used of human strain, grown as before on glycerine agar. Two were grown in the bell-jar with increased oxygen, one as control in air outside and close to the bell-jar in the same incubator. Bell-jar exhausted and refilled six times; final oxygen percentage on January Ist, 1909, was 90°5. Experiment continued till January 27th = twenty-six days, when percentage of oxygen was 889; no carbon dioxide present, . rs Examination showed that there had been no growth in the two tubes kept in the oxygen, and a moderate but quite obvious growth on the air grown control tube. As the control had dried somewhat during the twenty-six days of the experiment, in the succeeding experiment this was obviated by placing the control also under a similar bell-jar and in all respects in similar condition to the other set of tubes, except that the growth was made in air instead of in a high percentage of oxygen. But VT a GROWTH OF THE BACILLUS TUBERCULOSIS 183 it may be pointed out in regard to this present experiment, that any drying would militate against growth rather than favour it, yet the control had obviously grown, while the oxygen grown tubes had not grown. Experiment VIII—Growth of Bacillus tuberculosis (Human and Re: Bovine) in increased oxygen percentage. Four culture tubes each, of human and of bovine strains were made, “and eee ornare’ 5 in the usual manner. 3 Two tubes of each strain were placed in the oxygen bell-jar, which was exhausted and refilled in the usual way five times, the final per- centage of oxygen obtained being 87°6. Alongside this was placed another _ similar bell-jar, fitted up in the same way but filled with atmospheric air, and in this likewise two tubes of each strain were placed. The experiment was commenced on February 4th, 1909, and on _ February 24th and from that date onward a most marked and increasing difference was observable in the two sets of tubes; those in the oxygen had not appreciably grown, while the surfaces of the air cultures were ‘doited over with vigorous colonies of growth. The experiment was ‘ discontinued on March 3rd, when the percentage of oxygen was found 3 to be 75. Fe The photographs shown in fig. 1 were made from this experiment. In the photograph the four upper tubes were those grown in the oxygen, and the four lower tubes were those grown in air. In each series the two tubes on the left-hand side are bovine tubercle, and the two tubes on the right are human tubercle. In order to test whether the growth of the bacillus was only inhibited _ by the oxygen, the organisms still remaining alive, or whether they had bs been permanently destroyed by their stay in the higher oxygen per- “centage, sub-cultures were made of both bovine and human, from both | “the tubes in air and those in oxygen. The results showed a good growth = about fourteen days on the sub-cultures from the air grown tubes, while the sub-cultures from the previously oxygen grown tubes showed no growth, but turned brown where there were specks of the inoculated material on the agar. These sub-cultures were, of course, in both cases grown in air. The experiment, therefore, shows that in this case the oxygen had killed the bacteria; but the experiment requires repetition. Experiment 1X.—Growth of Bacillus tuberculosis (Human and Bovine) in increased oxygen percentage. In this experiment other micro-organisms were cultured alongside the B. tuberculosis under the same oxygen and air bell-jars, but the 184 BIO-CHEMICAL JOURNAL ' ; ‘ m 7 j Bacillu uberculosis (Bovine). . Bacillus tuberculosis (Human), ’ ; ; , ° . ) ; ; ’ “ Bacillus tuberculosis (Rovine). 4. Bacillus tuberculosis (Human). GROWN IN AIR 2. i | Bacillus tuberculosis (Bovine) ; Bacillus tuberculosis (Human). Bacillus tubercul (Bovine). 4. Bacillus tuberculosis (Human). Kia. | [uBERCLE BacitLus GROWN IN AIR AND IN OXYGEN. GROWTH OF THE BACILLUS TUBERCULOSIS — 185 growth of the tubercle bacillus is so slow that the tubes containing the tubercle bacilli had to be kept in the bell-jar during more than one a experiment on the other organisms, which grew abundantly in two or _ three days. For clearness of description the effects on the tubercle bacilli ___ are described separately, but the opening of the desiccator to remove and e — replace other organisms is noted, and the oxygen percentage at each period in the experiment was usually determined each time the atmosphere was changed. March 5th, 1909. Two tubes of Bacillus tuberculosis (human) and two of B. tuberculosis (bovine), which had been inoculated March 2nd, were placed in the oxygen bell-jar containing 90°0 per cent. of oxygen. a Four similar tubes, two of each strain, inoculated at the same time ___ and grown in the interval alongside in the same incubator, were placed in the similar control bell-jar in atmospheric air. ‘The two sets of tubes show a commencing growth in each case, but slightly advanced. ‘The two bell-jars placed alongside of each other in the incubator at 630 p-m., March 5th, the eight tubes being very similar as to growth. “The two bell-jars were opened on March 9th to remove the tubes with * étlae micro-organisms described in the next experiment (see Expt. X). The growths of the tubercle bacillus had not yet advanced far enough for comparison, so they were replaced and the oxygen bell-jar again charged by six exhaustions and refilling with oxygen from the reservoir? The oxygen percentage at this second charging was 91°7 per cent., and the tubes were not again disturbed till March 11th, when they were reopened to remove the other organisms grown in tubes alongside. The tubes were again replaced in their ‘Tespective bell-jars, and the oxygen percentage raised in the oxygen bell- ar to 88°8. ‘The bell-jars were again opened temporarily and at once , restarted on March 12th, March 16th, and finally for examination at the conclusion of the experiment on March 17th, after twelve days’ growth in the oxygen, and in the air alongside for the controls. The oxygen percentage on March 16th was 90°8 per cent.; it was not determined on March 12th or 17th. All four tubes grown in air showed good growths at the end of the ‘2. That the failure of the Bacilli to grow was not due to the f exha bell-ja¥, is shown by the vigorous growth under such conditi f B co Deke x She grown alongside, and also in subsequent experiments. ae the vs arama 186 BIO-CHEMICAL JOURNAL period; the human and bovine strains in this experiment grew at about the same rate. None of the four tubes grown in oxygen showed any growth whibiins, and the small amount of growth which had occurred. in air during the period from March 2nd till March 5th, when they were placed in the higher oxygen percentage, had turned dark brown in colour, the cultures being obviously dead. Experiment X.—Growth of other organisms—Staphylococcus albus, and— Bacillus coli—in increased oxygen percentage. March 5th, at 6-30 p.m. Two tubes of Staph. albus and two tubes of B. coli immediately after inoculation were placed in the oxygen bell-jar and grown in 90 per cent, oxygen. As controls, one tube of each a was grown in the air bell-jar alongside. March 9th, 2-30 p.m. The two bell-jars opened and growths compared. The two tubes of B. coli grown in the oxygen showed a growth quite equal to that of the tube grown in air. (See fig. 2.) On the other hand neither of the two tubes of Staph. albus grown in the oxygen showed any appreciable growth, while the control tube grown in air had a very good growth, the whole surface of the median being covered. (See fig. 3.) The six tubes were at once photographed, and the results are As in figs. 2 and 3. Experiment XI.—Growth in fiicreased oxygen percentage of Staphy- lococeus aureus, Bacillus coli, and Bacillus typhosus. March 9th till March 11th, 1909. Two tubes of each organism ‘were grown in the oxygen and in the air bell-jars respectively. Growth was commenced on afternoon of March 9th. When examined without opening on March 10th, afternoon, all six control tubes in air show good growths; in the orygen the B. coli and B. typhosus show good growths, © while the Staph. aureus shows no growth at all. : March 11th. Both bell-jars opened and tubes examined. All four B. coli tubes show full normal growth. All four B. typhosus tubes show full normal growth. The two Staph. aureus grown in the air bell-jar showed full sicsiad growth; but the two grown in oxygen showed the merest trace of growth. Experiment XII.—Growth in increased oaygen percentage of Bacillus pyocyaneus, Vibrio cholerae, Bacillus dysenteriae (Shiga), Batillus dysenteriae (Flexner), and Staphylococcus citreus. GROWTH OF THE SACTLLUS TUBERCULOSIS : f i % Ark AND IN OXYGEN 187 188 BIO-CHEMICAL JOURNAL Experiment commenced at 4 p.m., March 11th; growths examined March 12th at 4 p.m.; oxygen percentage = 88°8. Two tubes of each organism grown in air and two in oxygen. All ten tubes grown in air show a good full growth. Of the tubes grown in oxygen— Vibrio cholerae shows good growth, as good as control. B. dysenteriae (Flexner), growth as good as control. B. pyocyaneus, growth as good as control. B. dysenteriae (Shiga) shows no growth. (See, however, Expt. XV.) Staph. citreus. Both the oxygen tubes show an appreciable growth, but much less than controls. Experiment XIT1.—Growth in increased oxygen percentage of Bacillus diphtheriae, Staphylococcus citreus, Staphylococcus aureus, Staphylococcus albus, Bacillus dysenteriae (Shiga), and Bacillus dysenteriae (Flexner). Examined after twenty-four hours, the two Staph. citreus tubes grown in oxygen show a quite perceptible growth, rather more than either Staph. albus or Staph. aureus, but nothing like the growth of the controls grown in air. The two tubes of Staph. albus and Staph. aureus respectively from oxygen bell-jar show just the merest trace of growth, practically _ no growth, while the four air grown tubes show’a good growth. The two B. dysenteriae (Shiga) tubes from the oxygen show no greath at all; the two from the air are well grown (see Expt. XV). All four from B. dysenteriae (Flexner) tubes, on the other hand, show good growths both in oxygen and in air, there being no appreciable difference. None of the four B. diptheriae tubes show a good growth, but the oxygen tubes appear to be a little less than the air grown tubes. Experiment XIV.—Growth in increased oxygen percentage of Staphy- lococeus albus, Staphylococcus aureus, Staphylococcus citreus, Bacillus dysenteriae (Shiga), and Bacillus dysenteriae (Kruse). Four tubes of each of the above organisms were cultivated from March 19th (afternoon) till March 22nd (afternoon). Two of each in air and two in oxygen of 90 per cent. . All the tubes grown in the air showed good growths. Grown in the oxygen. B. coli, good growth, quite as good as controls. Staph. albus, fair growth, but not nearly so good as controls. Staph. aureus, poor growth, less than Staph. albus and not nearly so good as a i, ee Deas GROWTH OF THE BACILLUS TUBERCULOSIS — 189 7 “eontrols. Staph. citreus, fairly good growth, much better than the two _ previous, and approximately half its own controls. B. typhosus, a very . - sal growth, perhaps just a little poorer than the control. 2. dysenteriae _ (Shiga), a very poor growth, not to be compared to the control. 4 _ B. dysenteriae (Kruse), poor growth, less than one-third that of control. In this experiment, which was of longer duration than the previous ones with the more rapidly growing organisms, there was distinctly more h in the oxygen grown cultures than in the shorter experiments, but, at the same time, the inhibition of certain of the organisms was es undoubted. It was also particularly noticeable here, as was more or less - obvious throughout the whole series of experiments, that in those cases _ where the bacteria grew in a suppressed fashion, under the inhibition “Ss athe oxygen, that the cultures, instead of forming a more or less uniform __ mass or smear over the surface of the culture medium, consisted of a ~ number of very marked round colonies heaped up. In several cases where tubes of staphyloccus which had not appreci- ly grown while in the oxygen had been left on longer in air in the ou nator, it was noticed that these recovered and grew fairly well in a rather spotted and heaped up fashion. So that the shorter stay in oxygen does not appear to kill the more rapidly growing organisms in the same manner as the prolonged stay 1 in oxygen appears to kill the tubercle bacillus. To test the effects of a more prolonged stay in oxygen Experiment XV was carried out. oe Experiment XV.—More iota growth in inereased oxygen per- centage of Bacillus coli, Staphylococcus citreus, Staphylococeus aureus, roves albus, Bacillus diphtheriae, Bacillus typhosus, Bacillus 86 ae (Flexner), Bacillus dysenteriae (Kruse), and Bacillus dysenteriae a), _ Two tubes of each of these organisms were grown in oxygen of 90°5 per cent. and in air respectively, the experiment being continued from the afternoon of March 25th to that of March 30th. The following is the comparison of the two sets of growths :— B. coli, typhosus, diphtheriae show no difference in air or oxygen of any great magnitude, the air tubes perhaps slightly better grown, but difference very slight. Staph. aureus shows a good growth in air, none in oxygen. surface. eR: 2 sit ‘ Staph. alls shower a would growth in’ “air 4} re oxygen. Bay 5 B. dysenteriae (Shiga) shows romth in Doth, oxygen. oxygen. B. dysenteriae (Kruse) was nearly caval grown jin both pak! 2 TE iris 5s We desire to express our itd to as; Arthur Witenes valuable assistance in the sare ancen gated work meh sep just 191 _ THE ELECTRICAL FORCES OF MITOSIS AND THE ORIGIN OF CANCER By A. E. ann A. C. JESSUP, E. C. C. BALY, F.RS., Fellow of University College, London, F. W. GOODBODY, M.D., M.R.C.P., anp E. PRIDEAUX, M.R.C.S., L.R.C.P. (Received March 21st, 1909) Professor Hartog has recently brought forward the interesting suggestion that the phenomena of mitosis, that is to say the well-known mitotic figures, are due to the existence of a dual force, as for example, a magnetic or, better, electrical type. Without pre-judging its nature, he al this force mitokinetism. He introduces the conception of relative 8! permeability in elucidating the behaviour of this dual force in comparison with the phenomena of magnetism. He goes on to say that as the cell _ structures are all material the conception cf geometrical lines of force is adequate to explain them. He says that the effect of stresses within a mixture of substances which are of different permeability and free to arrange themselves will be to segregate out the more permeable material in strands along the lines of force. While the idea of an opposite polarity is reasonable, it is difficult to accept the polarity as being magnetic in any way, because there does not seem to be present any mechanism whereby magnetic stresses are to be produced. On the other hand, the substitution of an electrostatic difference of potential for the magnetic removes these difficulties, for it would appear that in the configuration of the protein material we have at hand all the necessary conditions for the establishment of such charges. The - NH - CO- linking which, from a physico-chemical point of view, is both acid and basic in character, or, as usually called, amphoteric, possesses residual affinity of two opposite types, and it is in the existence of these two types that we can obtain the mechanism for the establishment of electrostatic difference of potential. Although in the cytoplasm and centrosome we can find an analogy with the solvent and the dissolved and ionised salt in inorganic chemistry, yet it must be remembered that in the organic cell the phenomena must be those of colloids, and for this reason we are somewhat hampered by ignorance of the nature of colloidal substances. It is possible, however, to develop a theory that electrically charged colloids play a very important role in mitosis, a theory which leads to some very interesting results. When a crystalline salt, as, for example, sodium chloride, is dissolved in 192 BIO-CHEMICAL JOURNAL water the residual affinity of the water molecules causes them to condense round and form loose hydrate compounds with one or both of the sodium and chlorine portions. The lines of force due to the chemical combination of the sodium and chlorine are thereby weakened, and by virtue of their velocity of movement by diffusion the two ions get separated, becoming at the same time seats of positive and negative charges respectively. In — an analogous fashion the cytoplasm can resolve a discrete molecule or complex of molecules of amphoteric type into two oppositely charged portions. The cytoplasmic mass can, by virtue of its two types of residual affinity, form loose compounds with the two portions of the simpler compound. The lines of force between the two will be weakened, and the two portions can be separated and become seats of positive and negative charges respectively. It appears, therefore, a justifiable assumption that electrostatic differences of potential are established during mitosis and that the two centrosomes represent the location of two of these charges. Very much the same reasoning may be applied to the chromosomes or chromatin granules; these, by the same process as detailed above, can become resolved into two oppositely charged portions. Without in any way assuming that these differences of potential cause the phenomena of mitosis to occur, it is very difficult to believe that they are not produced when mitosis does occur. How far they act as the causae causantes is not determinable with any certainty in the present state of our knowledge of the vital processes, but our knowledge of the chemical and physical properties of the protein configuration leads us to the standpoint that the resolution of the centrosomes and chromatin granules must be accompanied by the establishment of definite electrostatic differences of potential. If now we consider the probable influence of the lines of force between two oppositely charged bodies upon a colloidal mass it will be seen that the colloid will tend to coagulate. The well-known coagulation of colloids in the presence of an ionised salt is now attributed to the alteration in their surface tension by the lines of force between the ions passing through the surface. If the view be accepted that a colloidal solution is due to the existence of a negative surface tension, that is to say, a tendency to form as great a surface as possible, the penetration of that surface by the lines of force between the ions becomes at once comprehensible when a colloidal and ionised solution are mixed together. Applying this view to the prophase of mitosis, the penetration of the nucleus by the lines of force between the centrosomes will cause the coagulation or partial coagulation of the chromatin in the chromosomes THE ELECTRICAL FORCES OF MITOSIS 193 a ll oagulation so will increase, the greater the number the lines of ree whicl s through the nucleus. Thig perhaps gives a reason for : Ts denention of the reticular structure in the chromosomes in the arliest prophase of mitosis. fis To deal next with the chromosomes themselves. There is little doubt it these consist of discrete particles or granules of chromatin, and, in | probability, each of these is resolved into two daughter particles _ charged with positive and negative electricity respectively. The _ chromosome, by virtue of the splitting of each of its particles into two, forms two daughter chromosomes, one charged with negative and the % — positive electricity. It may be argued here that the splitting » chromosomes occurs at a much later stage than the commencement ration of the centrosomes. ‘This, however, would be the result fact that during the earlier phases the chromosomes are rulating; for it is not likely that the resolution of the material can occur until the coagulation of the granule into a discrete particle with a finite surface has taken place. ‘ So ae vt it eel Se ; - “The first stage of somatic mitosis would, therefore, appear to be the resolution and separation of the centrosomes with their definite, equal | ae opposite charges of electricity. The lines of force between the two itrosomes as these lines penetrate the nucleus cause the coagulation or x n of the chromosomes. ‘The next stage is the resolution of the ivcmese granules, each into two daughter particles of equal and _ Opposite charge, and this is eventually followed by the splitting of the chromosomes. The first evidence of an incipient splitting of the chromo- 4 somes will be their polarisation; that is to say, each chromosome will be half positively and half negatively electrified and will tend, therefore, to move and take up its — in the equatorial plane of the mitotic figure (as in fig. 1). The next change in the mitosis will be the actual parting of the Les 194 BIO-CHEMICAL JOURNAL chromosomes into two daughter chromosomes, which by reason of the electrostatic attraction will migrate to the oppositely charged centrosome. When the daughter chromosomes arrive at the centrosomes the electrical charges will be neutralised. It is necessary to assume that the charges are entirely neutralised, for if there remain a balance of*positive or negative — electricity, this will tend to mount up in successive ‘divisions—a condition which it is quite impossible to accept. We are, therefore, bound to take the view that the sum of all the charges on one set of daughter chromo- somes is equal and opposite to that upon one centrosome, and that perfect neutrality of charge is established at the end of each somatic mitosis. When the lines of force cease to exist, each chromosome tends to become again de-coagulated. It at once begins to increase its surface, which it does by means of growing processes which extend until the whole nucleus appears to all intents and purposes structureless. The chromosomes, however, must preserve their individuality, although their processes appear inextricably intermingled. The application of the lines of force due to the commencement of a new mitosis will at once cause each chromosome to contract and condense until it exists once again as a dense individual, capable of being highly stained. . ++ ee pe ed We have not considered as yet several of the attendant phenomena ~ of mitosis; for example, no reason has been advanced for the separation of the centrosomes, the formation of the spindle figure, and also the disappearance and the reappearance of the nuclear wall. Considering first the separation of the centrosomes, which appears to us to be the most important feature of mitosis, it must be remembered that in the analogous case of ion formation in aqueous solution the separation of | the ions is due to diffusion. But this is quite inapplicable to the present case. It is equally necessary, however, to postulate some definite influence which separates the centrosomes—an influence which is stronger than — and quite apart from the electrical forces: for these would naturally tend to draw the two centrosomes together. In other words, the electrical _ > force cannot be the causa causans of mitosis, but must be a concomitant phenomenon. This fact cannot be too strongly insisted upon, for although ~ the conditions of a living cell would seem to be such that the vital units must become resolved into two oppositely charged masses, yet unless some definite and separate stress were present, the two oppositely charged masses would lie side by side without any electrical influence on their surroundings. As stated above, the resolution into oppositely charged masses is the natural result of loose combination between the vital unit THE ELECTRICAL FORCES OF MITOSIS 195 ‘ } | surrounding medium, and, again, these loose combinations are tl resthe result of the chemical structure of the cell materials. Finns compounds must play a very important part in the general "fife of the cell, as, for example, in the growth of the chromatin. There E ‘is no doubt a strong separative force at work—a force which would appear 3 4 0 to be connected with the mass division of the cytoplasm. It would seem a this mass division is an inherent vital property of living cytoplasm ; ; = we incline to the view that it is the real causa causans of mitosis, and _ that the phenomena described above are produced by the mass division _ taking place. There is a sound foundation for our view, for it has been ‘shown that the periodic activity of cytoplasm is independent of both 1 and centrosome. For example, in the case of a fertilized egg ded into two portions, one of which contains the nucleus and the other 10 pte behaviour of the enucleated portion is most remarkable. It s three times in succession a polar lobe at the same time that the half is dividing, becoming spherical after each period without _ At the fourth cleavage a fourth lobe is formed, which is not a ed but grows steadily larger, so that the fragment appears finally be divided into two. The activity of the enucleated half is thus not ly rhythmic in character but changes in character at the fourth cleavage when in normal development the polar lobe no longer forms a temporary structure but is permanently cut off by cell division. The _ eytoplasm, therefore, would seem to possess a power of mass division—a power which is also periodic in its action, its periodicity no doubt depending upon the cytoplasm reaching a certain stage of development during the vegetative period of the cell. The existence of this power of a — division possessed by the cytoplasm gives a reasonable explanation r the separation of the centrosomes. When the period has been reached, ‘the first feature of the phenomenon is the resolution of the centrosome om the previous division into two oppositely charged centrosomes. When the mass division begins the axis’ is determined by the position of _ the centrosomes, so that they are drawn apart. The electrical forces _ brought into play cause the condensation or coagulation of the chromo- _ somes, their resolution and migration, and, finally, when the mass division of the cytoplasm is finished the formation of two daughter cells. By the statement that the axis of mass division is determined by the centrosomes we mean that the two daughter centrosomes are separated, because one muist be in each daughter mass of cytoplasm. This fact cannot be a matter of chance, for if it were so, many more divisions of cytoplasm 196 BIO-CHEMICAL JOURNAL would occur than mitoses of the nucleus, which is absurd. It is a natural sequence that the line of cleavage of the cytoplasm is determined by the centrosomes. This would seem to be the normal order of events in any mitosis, but there is no reason why certain minor details should not be altered either as regards their character or their position in the scale of operations. Such variations need not, and do not, militate against the electrical theory in any way. For example, in many cases the centrosome in the daughter cell divides immediately after the mitosis is finished in readiness for the next division. All that the theory demands is that the centrosome divide into two; the period at which this occurs is not of any moment, but the fact that is of the greatest importance is that the two new centrosomes never get separated except during mitosis, and then only with the formation of the spindle figure. | In reference to the statement that the axis of the mags division is determined by the centrosomes, 0. and R. Hertwig! and also Roux? have noticed that as a rule the plane of division of a non-spherical cell is at right angles with the direction of the greatest diameter or extension, and Driesch has shown that if the newly fertilized egg of the sea urchin be gently pressed under a cover glass, so that it is slightly flattened, the plane of division is at right angles to the slide. The position of the plane of cleavage is determined by the position of the nuclear spindle, and this depends upon the position of the centrosomes. Moreover, in the process of cell division the egg of some animals becomes elliptie with the long axis falling in the direction of the common diameter of the amphiaster. This has given rise to the idea that it is the spindle itself which elongates the egg, but Loeb? has often noticed that the elongation, though in the ~ direction of the spindle, always occurs immediately befase the cell division. Again, the possession by the cytoplasm of a power of mass division will give an explanation of the phenomenon of streaming which is observed, during mitosis, for the streaming will merely be the flowing of the cytoplasm from the cleavage plane into two daughter masses. Inasmuch — : as the sum of the masses of the cytoplasm of the two daughter cells is less than that of the mother cell, it at once suggests itself that the mass division is caused in the first place by a loss of water from the periphery - of the cell by osmosis. 1. Untersuch. zur Morphol. wnd Physiol. der Zelle, V, 1887. 2. Breslawer Arzt. Zeit., 1885. 8. Loeb, Dynamics of Living Matter, p. 64. Columbia University Press, 1906. i. oe THE ELECTRICAL FORCES OF MITOSIS 197 To give an explanation of the formation of the asters and spindle fig: »-of mitosis is not easy. The natural view to take would be that ' these are due to the coagulation or condensation of the cytoplasm along _ paths parallel to the lines of force, using the same argument as in the ease of the chromosomes above. Furthermore, it is quite true that in - many cases the mitotic diagrams present great similarity with the lines of force between oppositely charged bodies. This view has been previously advanced by A. Fischer.' In certain cases, however, the rays from the two asters appear to cross one another—an effect which is impossible if they are simply due to lines of force. But it may be that this is only an apparent effect due to the point of view of the phenomenon. If this be a real crossing it is clear that the rays cannot be due to threads of coagulated cytoplasm; although it might be possible to look upon them as direct growths from the centrosomes, yet the former explanation would seem far more reasonable, provided that the difficulty as regards their crossing one another be surmounted. Of course it must not be forgotten that the separation of the centrosomes by the mass division of the cytoplasm will induce stresses which may disturb the position of the threads. On the whole the evidence would appear to support the view that the mitotic figures are due to the coagulation of the cytoplasm under the influence of the lines of force. In the present state of our knowledge of the chemistry of cell physiology it is impossible to account for the disappearance of the nuclear membrane during mitosis and its reappearance after the process is finished. We might say that the membrane is due to a definite chemical reaction between the nucleic acid and the cytoplasm, and that this reaction is reversed under the stimulus of the lines of force, so that the membrane disappears only to reappear when the force lines die away; but this can only be pure hypothesis. _ We may next turn our attention to the maturation divisions of the germ cells, and investigate the relations which exist between the electric charges in these cases. The results obtained are peculiarly interesting, inasmuch as it seems absolutely certain that complete electrical neutrality does not, and cannot, result from these divisions. The first fact which we are met with in these divisions is the fusion together of the chromosomes in pairs to give the meiotic gemini. In order to account for this and bring it into line with the other phenomena it is necessary to assume the existence of some form of polarity difference 1. Fixierung, Farbung und Bau des Protoplasmas, 1899, 198 BIO-CHEMICAL JOURNAL between each of the two individual chromosomes, which fuse together to form the gemini. All recent cytological research leads to the view that in each case it is one paternal and one maternal chromosome which — fuse together, producing a bi-polar twin; and, moreover, that it is not any chance pair which fuse together, but there exists some type of — . selective pairing between paternal and maternal chromosomes. For this — pairing there must exist in the cell some opposite polarity between the two members of each twin-—a polarity which miy well be of electrical type. This would point to the existence of some difference in electrical charge between the paternal and maternal chromosomes which comes into play during the long resting period of the germ cell. That an electrical attraction can be produced between paternal and maternal chromatin in the germ cell follows readily from the general theory, but its consideration may be postponed for the moment. It must be confessed that the phases of the phenomena of maturation divisions differ so much according to various observers that it is impossible to deal with more than what appear to be the most typical cases; and we must content ourselves with pointing out how the general relation between the charges is not altered in ms of the cases observed. The simplest case to deal with is when no tetrads are formed and when the first maturation division takes place between the split halves of the meiotic gemini, while the second division is a somatic division of the reduced number of chromosomes. It is necessary, in order to follow | out the distribution of the charges in the maturation divisions, that the relative values of the charges upon the centrosome and chromosome be considered. Beyond the bare statement that it is essential that at the end of each somatic mitosis the charges upon the centrosomes must be neutralised by the chromosomes nothing has been stated as to the relative values of the charges. The establishment of electrical neutrality at the close of each somatic mitosis is of a very great importance, for if by some means or other the charges were not neutralised entirely and a small amount were left over in the daughter ce qes ly this would go on mounting up | steadily in-suecessive divisions wiSa “98« apparent limit. This _is 18,1 CC course, impossible of acceptance, and therefore we are driven to the bohal gio which is indeed the simplest, that complete neutrality obtains at the end of every somatic mitosis. We must therefore equate the charge upon each centrosome to the sum of the charges upon the daughter chromosomes, and in doing this it will be convenient to speak of the charge upon each centrosome as a unit-charge of positive or THE ELECTRICAL FORCES OF MITOSIS 199 ative aw respectively. In the case, for example, of an individual with fou atic chromosomes, the sum of the charges upon the four “a daughter ey which migrate to one daughter cell must equal the F Ze charge upon the corresponding centrosome. We therefore in this would expect the average charge upon each of the chromosomes to be one quarter of the unit charge, but it may be pointed out that there is no _ @ priori reason why the charges on all the chromosomes should be equal a _ in amount, the essential condition only being that the sum of the charges on them be equal to the unit charge. a atering now to the germ cells we may cay; as above, that a definite erence of potential is developed between paternal and maternal p | on osomes; and let us say, merely for purposes of argument, that this difference of potential is half that carried by the chromosomes in somatic tosis. If we continue to deal with the case of an individual with four tic chromosomes, then we will assume the difference between paternal i c 1 end a one-eighth unit-charge of positive and snantivs electricity - respectively The result will be that the gemini will carry themselves + iste Fic. 2. _ When the gemini split again, and the two chromosomes migrate to _ the centrosomes, the charges will not be neutralised, but in each daughter cell there will be left a residue of electricity. When the meiotic gemini split they each give two halves carrying an eighth positive and negative charge respectively, and in fig. 2 the two positive halves migrate to the centrosome on the right and the two negative ones migrate to the es centrosome on the left. When the two positive halves arrive at the a negative centrosome they each bring a one-eighth unit, that is to say, one-quarter unit positive electricity altogether. Since the centrosome we ee eo, ee ae 200 BIO-CHEMICAL JOURNAL carries a whole unit of negative electricity, we have one unit negative and one-quarter unit of positive electricity, which leaves a balance of three- quarter positive electricity. Similarly at the other side there will be left a residue of three-quarter unit negative electricity. On these lines it is clearly impossible for the charges to be neutralised in the daughter cells, for the only condition under which this could be secured is that the difference of potential between the paternal and maternal chromosomes — was half a unit-charge, or twice as much as between the daughter chromosomes in the previous somatic mitoses. Such a large difference of potential is out of the question, for it would be impossible for such a charge to lie dormant through the various somatic divisions which occurred previously to the maturation divisions. We must conclude, therefore, that whatever may be the real value of the potential difference between paternal and maternal chromosomes, a residual charge is left in the daughter cells of the first maturation division, and at the same time point out that the smaller we assess the potential difference between paternal and maternal chromosomes in relation to the normal somatic charge, the greater will be the residual charge. The next division is of the ordinary somatic type, but with this difference—that there are now only half the somatic number of chromo- somes, and therefore only half the number of chromatin granules in each cell. It is quite evident, therefore, that in this somatic division the respective charges cannot be neutralised, for we have the whole mechanism ~ of mitosis but only half the proper number of chromatin granules. Following out the case above, the reduced number of chromosomes is two, and each will give two daughter chromosomes, carrying one-quarter positive and negative charges respectively, which are the normal somatic charges for the individual in question. At the end of this mitosis there will be again a balance of charge left over, for in one daughter cell there is one unit positive charge and two one-quarter unit negative charges, leaving a balance of a half-unit positive charge; similarly, in the other daughter cell there is a balance of a half-unit negative charge. A second case, which frequently occurs, is the formation of the tetrads, and in this case, as in the previous, the result is the same—a balance of charge must be left in the daughter cells. The formation of the tetrads is due, no doubt, to the somatic division of the chromosomes taking place before the first division has proceeded very far. Assuming, as before, the existence of one-eighth unit-charge upon paternal and maternal chromosomes respectively, the first stage will be the formation + Se: _-——s THE: «ELECTRICAL FORCES OF MITOSIS 201 the bi-polar gemini. These gemini will again arrange themselves - equatorial plate of the mitotic figure and then will begin to - undergo the somatie resolution in readiness for the second division. Owing to the bi-polarity being most pronounced at each end of the twin chromosomes this resolution, under normal circumstances, should begin i in the centre of each twin, which thus forms a ring, the procedure being of the true hetero-typical kind. This resolution, however, will be accompanied by the establishment of a positive and negative charge respectively on each side of the ring; and, in our case of four somatic _ ¢hromosomes, this charge will in each case amount to one-quarter unit- _ eharge. This ring formation is followed by the completion of the somatic _ splitting and the resolution of the gemini back again along the lines of the preliminary fusion. Each ring thereby breaks into four portions, which form the tetrads, and the two cell divisions rapidly take place in ‘succession. The distribution and balance of charges follow exactly the _ same lines as before, with the establishment of three-quarter unit positive and negative charges in the two first daughter cells and the further establishment of a residual half positive and negative charges alternately 1 the grand-daughter cells. The only proviso that we must make as regards maturation by tetrads is that the two divisions follow one another in rapid succession with no intervening resting stage. This, however, seems quite a reasonable position to take up, viz., that when the somatic division of chromosomes takes place with the formation of tetrads during the first maturation division, the second maturation division must follow the first at once. If the somatic resolution does not take place, or only takes place incompletely, then the charges will be the same whether there is a resting period betwen the two or not. _ Before discussing the result of the establishment of the residual electric charges in maturation, it may be pointed out that the hetero- typical resolution of the meiotic gemini with the formation of rings will only take place when all the chromatin granules in each twin are perfectly ‘uniform. We might readily imagine that the chromatin granules in one paternal chromosome, for example, are weaker in character than those in the maternal chromosome with which it fuses to form a twin. In this special case the ring would not necessarily be formed when the somatic split took place, but rather, a body of the form : <> or aS eee Sk ee ey ee ee ee z * : ee, es meta yt j s ‘ee | 202 BIO-CHEMICAL JOURNAL Moore and Arnold have described various forms of these gemini in the meiotic phases of many germ cells, and it would seem that if their existence be confirmed they can be explained by certain distributions of activity in the chromatin granules in the twins. They are, therefore, only of secondary importance as far as regards the phenomena - under consideration. It may be argued that the formation of residual charges might be prevented by the cytoplasm providing in each division centrosomes of just sufficient charge to meet the needs of such division. This, however, seems impossible of belief, for it would mean a variation in the power of the cytoplasm between very large limits in a very short space of time. Against this view, we would point out that the cytoplasmic activity of the germ cells at the time of maturation is exceedingly great, and, therefore, it seems in the highest degree unlikely that the resolving power should become half the normal value or even less, and, further, that it should vary. Although it may be said that we have arbitrarily assumed a difference of potential between paternal and maternal chromosomes and fixed it at one-eighth of a unit-charge, it must be remembered that whatever be the view taken of it, the establishment of residual charges is necessary, for only one somatic division of the chromatin granules occurs and twice the somatic number of centrosomes are brought into play. On these grounds alone, without making any assumption whatsoever as regards the existence of the potential difference between paternal and maternal chromatin being necessary to cause the preliminary fusion in pairs to form the meiotic gemini, it appears absolutely impossible for the residual charges not to be established in the maturation divisions. At the same time, as we have already shown, it is necessary to assume some type of difference of potential between paternal and maternal chromatin, and the existence of one of electrical type, as we shall presently show, is quite easy of acceptance, although its value cannot be directly estimated in the present state of our knowledge. If we, therefore, put this at half the somatic charge on the daughter chromosomes, that is to say, in our example of four somatic chromosomes, one-eighth of a unit-charge of positive and negative electricity on paternal and maternal chromosomes respectively, and follow out the mounting up of the residual charges 1 in the maturation divisions, we arrive at the following values : — In the first, or meiotic, division, the residual charges will be three- quarters of a unit positive and negative charge respectively, for the centrosomes each carry one unit-charge, and there are two chromosomes i” Of a re it CO 4> rs Em ( CJ U o <. J 7 *® ) + ° se ) a + * , e 4+ /+ % /+ e 54 4- ft ae Te Tiny. Tu /- /- 2 & ee & a Sw ss é ; As can be seen from the diagram, there will be, after the third post- meiotic division, sixteen cells, each carrying x chromosomes, and of these six will be neutral, four will have a unit positive charge, four a unit negative charge, one 2 units positive and one 2 units negative electricity — a | OR eee bs Je, eee A sr THE ELECTRICAL FORCES OF MITOSIS 215 ‘respectively.! If the distribution of charges be followed out for the ensuing divisions, it will be found that two of the cells formed always carry equal maxima of positive and negative electricity respectively, and there will continually be produced a constant ratio of neutral cells and cells with intermediate values of charge. Sooner or later the magnitude of the charges produced will cause the maximum charged cells to fuse together _ with the formation of new cells carrying 2n chromosomes, when, of course, the cycle begins again. The organism, therefore, will present the _ appearance of having two types of cell, one carrying 2 and the other chromosomes. Some of the latter will from time to time fuse together to form new cells with 2n chormosomes, and so the cycle is complete. _ This state of affairs will be the natural result of the sex differentiation being very incomplete, or, in other words, when the hereditary character- lies have not developed the maximum possible difference in polarity. - When this occurs we find the condition that, owing to the magnitude of the charges involved, only one post-meiotic division takes place, the cell _ being at once sufficiently charged for fusion or fertilisation by one of a opposite type. The whole difference between the various types of re- _ production may therefore be summed up in the conception of. sex _ differentiation, or of opposite polarity in the hereditary characteristics. It is also of some importance to note that as sex differentiation is increased the relative size of egg to sperm is increased. When the sex differentiation is small, or as we might say merely embryonic, there will be no difference in size or visible character of male and female cells. _ When the male and female chromatin begin to differ materially then we find a difference in the development of the two, the male becoming smaller than the female, until finally we arrive at the spermatozoon and ovum, _ where the ratio of size is very marked indeed. In the development of the above argument we have attributed the variation in the number of post-meiotic divisions, i.e., divisions between the reduction and the ensuing fusion, to the ratio between the driving force and residual charges established; the greater this is, the greater the number of post-meiotic divisions. We have introduced the conception of driving force merely to illustrate our point, and would now deal with this conception in more detail and consider the relation of the chromatin to the surrounding nucleic acid. There is no doubt that during the resting period between two successive divisions the chromatin must be growing - 1. Exactly the same relative values are obtained, whatever be the size of the residual 216 BIO-CHEMICAL JOURNAL ~ by means of chemical reactions between itself and the surrounding nucleic acid. Indeed, we may go so far as to say that unless this growth occurs any somatic division is not possible, for such division would result in a decrease in the active mass of chromatin in the daughter cells, a consequence impossible to accept. Now from a physico-chemical point of view it is evident that any reaction between chromatin and nucleic acid must be based upon some essential points of similarity in structure between the two, and it would seem a natural deduction to make from this stand- point that any influence tending to decrease the similarity between the two would retard the growth of the chromatin at the expense of the nucleic acid. Hence it is quite a natural sequence that a decrease in the similarity would tend to act as a deterrent to mitosis. The establishment of the residual charges in the maturation divisions, of course, means a considerable modification in the chromatin of the daughter cells, i.e., a considerable decrease in similarity between the chromatin and nucleic acid, so that we finally arrive at the conclusion that the establishment of the residual charges is a direct deterrent to mitosis. Hence the failure in the animal kingdom of the daughter cells of the post-meiotie division to undergo further divisions may be explained at once by the fact that the residual charges prevent the growth of the resulting chromatin, so that no reason or scope for division exists. We arrive, therefore, in this way at a very definite foundation for the assumption made previously that the number of post-meiotic divisions depends upon the ratio of driving force to amount of residual charges established, for we find that the driving force is essentially determined by the growth of the chromatin during the so-called resting period. The — influence of the amount of differentiation between male and female characteristics upon the number of post-meiotic divisions follows very clearly from this view, for, as several times pointed out, the amount of residual charge in the meiotic division depends directly upon this differentiation, or, as we would now state the law: The greater the ‘ number of different hereditary characteristics of opposite type the greater is the dissimilarity between the chromatin and nucleic acid in the grand- daughter cells of the reducing divisions, and hence the fewer the number of post-meiotie divisions. It may be stated here, parenthetically, that whatever view be taken of the method by which the chromatin grows or the period at which the vrowth occurs, the same conclusion is arrived at: that if the growth be stopped then mitosis cannot occur. THE ELECTRICAL FORCES OF MITOSIS 217 a It is impossible to enter fully into the various types of reproduction which are known to oceur, especially in the vegetable kingdom, but there seems no doubt that they all are capable of explanation on the theory here put forward. We have dealt with the ease of the unicellular organism with no sex characteristics, those cases in the vegetable kingdom where incomplete sex differentiation exists, and the zoological cases where the sex differentiation is complete with the two sexes as distinct as possible. All other cases seem only to be intermediate between the first and last. A very important corollary must be added to what has been said above upon the reducing divisions. At the completion of this division there is established a definite amount of residual charge in the daughter -eells. In other words, a certain amount of energy is stored in the cells, and this must result in the vitality of the cell being increased. This fact ____ is very important, for it would seem that the reducing division forms a -_ means by which the vitality or activity of the cells is renewed, for there is “no doubt that a considerable amount of energy is absorbed at the time. _ _ Hitherto we have made the tacit assumption that the phenomena _ described are those occurring in the mitosis of normal healthy cells. The _ question now arises as to what would happen if the electric charges in the somatic cells were disturbed by some means, and the equilibrium between them upset. It is very evident that important changes in the phenomena might take place, and it has occurred to us that the various types of malignant growth might very readily be explicable by their being due to the derangement of the electrical forces present during mitosis. On studying the experimental results obtained in this field we were very _ foreibly struck by the support given to our idea, and we feel more than BE justified in offering this as a reasonable explanation of malignant growths, _viz., that they are due to abnormal cell reproduction arising from a disturbance of the electrical equilibrium of mitosis. _ - One of the simplest methods of causing a disturbance of the electrical equilibrium in the cell is by the external application of an electrical stimulus. . If, for example, a somatic cell was given an added charge, ie., if the cell wall by some means were electrified, a natural sequence would be an artificially produced multi-polar mitosis as already described in the fertilisation experiments of Boveri and Loeb above. Furthermore, the daughter cells produced as a result of such mitosis will naturally have a balance of positive or negative electricity left in them, owing to the result of the asymmetric distribution of the chromosomes. Since there is no means of dissipating these residual charges, the effect of a 218 BLO-CHEMICAL JOURNAL single initial external stimulus will be handed on from generation to generation of daughter cells. There is no doubt that the periodic activity of the cytoplasm resulting in its mass division would not be interfered with by the small external stimuli referred to. The mechanism of the cell mitosis would be the same in the main, but owing to the excess of electrical energy multi-polar and asymmetric mitoses must result. Now, multi-polar and asymmetric mitoses are frequently observed in malignant growths, and, indeed, Galeotti has artificially produced asymmetric mitoses in Salamander cells by treating them with certain chemical substances.!| These substances undoubtedly acted as a stimulant to the cell either by electrification of the cell walls by virtue of the different velocities of the ions, as already shown in the case of Loeb’s experiments, or by more purely chemical means. That the action of an external stimulus is capable of producing these two pathological mitoses in cancer, by which term we understand all malignant neoplasms, is thus evident. Waller? and others have shown that electrostatic differences of potertial are a normal result of any external stimulus being applied to healthy tissue, and whether we accept Loeb’s* explanation or not that the effect is due to the migration of hydrogen ions, still the fact of sufficient potential difference being set up to deflect a galvanometer is completely established. It is possible, therefore, that an external stimulus, as for an_ example a bruise or blow, could induce sufficient electricity to derange all the neighbouring cells, that is to say, a sufficient electric stimulus could be established to start pathological mitoses. It would thus appear that, provided the necessary conditions were existent, a blow or bruise could give rise to a malignant growth. It stands to reason that the more healthy is a cell and the stronger its vitality, the greater will be its . resisting power against the effect of an external stimulus. Conversely, the lower the vitality of the cell the more liable it becomes to derangement. There is no doubt that, speaking generally, the vitality of cells must decrease with their age, so we would expect the tendency to malignant _ : growths to increase with age; in other words, we have herein a direct explanation of the age incidence of cancer. The actual change which takes place, and which we have spoken of as a decrease in vitality, would be due partly to a decrease in the active growth of the cytoplasm and partly to a decrease in the active growth of the vital units or chromatin 1. Beib. 3, Path. Anat., XIV, 2 (1893). 2. Waller, Signs of Life, p. 143. 3. Loeb, loc. cit., p. 68. THE ELECTRICAL FORCES OF MITOSIS 219 _ granules. Whereas in a healthy cell the mitotic phenomena are due to forces which-are periodically brought into play, so a decrease in these 73 forces will tend to weaken the vitality of the cell. We have shown, also, that the possibility of mitosis is dependent upon the growth of the - __ ¢hromatin, and as this certainly will decrease with the age and differentia- ___ tion of the cell, so the vitality of tlie cell will decrease with age. We ____ therefore have two causes for the decrease in the vitality of a cell. a It is not possible in the present state of our knowledge to advance any definite chemical theory for the change which is developed with age, = but it is fairly certain from cytological investigations that a stage is reached when the cytoplasmic power is not sufficient to resolve the centro- somes and chromosomes with the formation of the mitotic figures, and en amitotic division may and does frequently occur. It may, however, e concluded that the occurrence of amitosis is due to the fact that the active growth of the chromatin has fallen to a value below the limiting value for mitotic resolution of the chromatin granules while the cytoplasmic activity is sufficiently great to cause the mass division to take place. We have already directly connected the decrease in vitality of the cell with the occurrence of cancer, so the ocurrence of the amitotic divisions of cancer cells is easy to understand. As already pointed out, the age incidence of malignant growths is the natural sequence of the fact that the somatic cells must reach a certain critical minimum of vitality before they can be disturbed by any external stimulus. We may for the ; present purpose give the name of the nth generation to that generation at which the cells reach the critical minimum, i.e., when their vitality has fallen low enough to be susceptible of disturbance by the external stimulus. It is of some importance at this point to notice that when any tissue is subject to continually repeated abrasion or irritation, the resulting continuous renewal of tissue will cause the nth generation to be arrived at somewhat earlier than would otherwise be the case. From _ Waller's experiments it is a natural sequence that any irritation or stress should tend to produce an electrical difference of potential. This would seem at once to account for the occurrence of malignant growths in those parts of the body which are subject to such stresses. Hitherto we have been considering the derangement of a cell by external stimulus, but it is also possible that derangement may occur internally. If we consider the case of a cell which is highly differentiated, and, moreover, one the vitality of which has sunk very low, it is evident on Re oe ae - a at es a a Le ee a, 220 BIO-CHEMICAL JOURNAL first principles that the potential difference, actually set up when the vital — units are resolved, has fallen to a very small value. This decrease in— activity, sooner or later, as we have before pointed out, results in amitotie — division. This oceurrence of amitosis, however, demands. that all the = chromatin granules nust be at a low ebb of vitality. The formation of | 2 the loose compounds between the nucleic acid and the chromatin granules must, to all intents and purposes, have ceased, or, in other words, the growth of the vital units has become very slow. It is not essentially necessary, however, that every single chromatin granule of the cell decrease in vitality at the same rate, and the question arises as to what would happen if the vitality of the various granules differ considerably within a single cell. If we consider for a moment the effect of inbreeding and hybridisation, it is perfectly evident that by far the most probable — condition is the one specified, namely, a considerable variation in vitality among the granules of chromatin within each cell. Clearly this gradation of vitality among the chromatin granules can give rise to a derangement of the electrical equilibrium in mitosis, as can be seen from the following. Whereas in the normal mitosis of a healthy somatic cell the chromosomes give daughter chromosomes of equal and opposite charge, and as in true amitosis the chromosomes are not resolved at all, in the present case a condition may arise when some of the chromosomes are resolved while the remainder are not so resolved, owing to the vitality of the granules of the latter being very much lower than that of the granules of the former. These latter chromosomes would only be polarised, and therefore would not migrate to either centrosome; a fusion of the electrified daughter chromosomes with the polarised chromosomes would naturally ensue, and an asymmetrical mitosis would take place, with a reduction in the number of chromosomes. This would again cause a balance of residual electric charge in each daughter cell, exactly in the same way as occurred in the case of the external stimulus described above. Further, it must be pointed out that the age incidence would apply to this condition just as much as to an ordinary cell, for the derangement is caused by the vitality of the weakest granules falling below the minimum value and also by a general decrease in the activity, both being the normal results of the age of the cell. To assume for the moment that this condition of chromatin granules is possible, we may now re-state our case as it stands. An electrical theory of mitosis at once renders possible abnormal mitosis by reason of the electrical charges becoming disturbed. The electrical theory of mitosis in itself receives very considerable support tre 9 8 8 ee ee ; Flee we ‘ta 4 ; os é ; . SS g , ee El _ - — THE ELECTRICAL FORCES OF MITOSIS 221 ‘from experiments upon artificial fertilisation, from parthenogenesis, from sterilisation of the ova and spermatozoa by X-rays, etc. The disturbance of the balance of electrical quantities in the cell may arise from the _ application of an external electrical stimulus arising from a blow or from - considerable and repeated irritation or stress, It may also arise internally in the cell from a certain number of the chromatin granules being of _ such a character that they do not give daughter granules with sufficiently different charges. In both cases the result is the same. The normal equilibrium between ihe charges is disturbed and asymmetric and multi- polar mitoses occur, producing daughter cells with a reduced number of chromosomes and a residual positive and negative electrical charge. The tendency to both these disturbances is increased with the age of the cell. * i: » The main point we have arrived at is that when derangement of the ¢ occurs, the daughter cells are produced with a balance of positive ‘negative electricity and that this is accompanied by a reduction in the number of chromosomes. It is, therefore, extremely interesting that Farmer, Moore and Walker! claim to have discovered that a true reducing division occurs in malignant growths; that is to say, a division of the same type as the first maturation division of the germ cells. Although ‘these results are still sub judice, yet the existence of cells with: fewer chromosomes than the normal somatic number seems faily well established. The condition of chromatin granules such as we have postulated would give rise to cells containing any number of chromosomes between the somatic number and half that number, and would not necessitate a true reducing division. For this reason we would give the name of pseudo- reduction to this process. As was pointed out before, in dealing with the reducing division in the vegetable kingdom (that is to say, the cases where the sex differen- tiation is incomplete) the operation leads to an increase in the vitality of the daughter cells. When, owing to the decrease in the activity of a cell, the pseudo-reducing division occurs, the resulting daughter cells carry a certain amount of residual charges, that is to say, a definite amount of fresh energy has been absorbed by the chromatin. This clearly will enhance the activity or vitality of the cells considered as a whole, and therefore we are met with the condition of a new growth of cells of greatly increased vitality in a tissue of cells of low vitality. It stands to reason that the new cells will multiply with great rapidity and will be out.of co-ordination with the soma. Their growth will depend entirely 1. Proc. Roy. Soe., Vol. LXXIT (1903). 222 BIO-CHEMICAL JOURNAL upon the new lease of vitality which they have received from the reducing %. division, that is to say, it will be inversely proportional to the age sce ~~ cells before the derangement. The course followed by these new cells should be very femdi on ‘aie same lines as that given in detail upon page 214 for the cells obtained: “by " the true reducing division in the ease of an organism with small sex differentiation. The descendants of the pseudo-reducing division of cancer will produce a certain number of neutral cells, a few wile charged cells both with positive and negative electricity, and the — é remainder with intermediate charges. It is impossible to assess the relative number of the three types, since the original reducing division — was not a true reducing one, but only a pseudo one with an indefinite reduction. The maximum charged cells will soon reach the limit, which cannot be passed owing to the magnitude of the charges involved, while the remainder sub-divide indefinitely. When the maximum charged cells have reached the limit the question at once arises as to their future behaviour. The normal course would be for them to fuse together in pairs of opposite charges, with production of new cells with an increased =~ number of chromosomes. A new generation of highly active cells would thus be: formed, and thus the cycle would be complete, just as in the — botanical case detailed before. It must also be remembered that under — the peculiar circumstances owing to the reaction of the soma there isa supply of leucocytes continuously made. These leucocytes being neutral and active cells would be expected to conjugate with the maximum — charged ‘reduced’ cancer cells, since the electric potential would in this way be reduced. Now Farmer, Moore and Walker! have observed such — conjugation between leucocytes and cancer cells—an observation which strikingly confirms the theory. No doubt conjugation also occurs with the cells of the surrounding tissue, to which may partly be due the infiltration of the malignant growths. . This seems to us to account fully for the cyclical form of growth of | cancer in mice as demonstrated by the Imperial Cancer Research (No. 2, Pt. 2, 1905), but the space at our disposal prevents us from producing . further proofs for our contention. tel, An important point arises here in relation to the age and differen- tiation of the cell. It would be readily understood that the greater the vitality of any cell, the greater will be its activity in proliferation when the malignant diathesis has once been established. If we suppose, for 1. Proc. Roy. Soc., Vol. LXXII, 1903. THE ELECTRICAL FORCES OF MITOSIS 223 a ple, that either owing to the abnormal condition of the chromatin the presence of an external electrical stimulus, the electrical equilibrium is disturbed when the cytoplasmic activity is still very considerable, it will be evident that the rate of proliferation will be greater than would be the case had the cytoplasmic activity been less. In other words, the greater the amount of the activity the more rapid the growth of the tumour, the more highly differentiated the cell and the older it is before the taint is established, the less rapid will be the owth of the neoplasm. As the age of the host is increased, therefore, ss potent becomes the taint. If the development and differentiation the cell has proceeded sufficiently far before the cancerous diathesis is stablished, the smaller will be the potential differences established by the eduction in the number of chromosomes. The tumour then loses its mancy to a certain extent, and a cancer of slow growth, such as an ic scirrhus, is established. There can be no doubt that the growth of the tumour by reduction in the number of chromosomes produces, as before stated, daughter cells which are out of somatic co-ordination with he host, and the tumour grows parasitically, feeding upon its host. Resulting from this condition of parasitic growth of cells out of somatic _ ¢o-ordination, the wandering of some of the active cells from the seat of the tumour may occur. When one of these charged cells comes to rest, it possesses potential probability of reproducing itself, resulting in the growth of a secondary tumour of the same type as the primary one. The tendency to the formation of secondary growths must, therefore, depend _ directly upon the activity of the cells when the taint is first established, because, as before said, the greater the activity the greater the activity of the daughter cells of the pseudo-reducing division. _ With reference to this subject we are at once struck with the narkable facts which have accumulated during recent years with regard ie transplantation of cancerous tissue. As the Imperial Cancer Research have indicated, it is much easier to transmit any malignant “E “growth from a mouse of one locality to a mouse of the same locality than to a mouse of another locality; moreover, there is a general concensus of opinion that cancer can in no way be transmitted from an animal of one species to an animal of another. This is the natural outcome of our view on the cytoplasmic activity. _ For the more different the species of the animals in question, the more dissimilar would be their respective cytoplasms; and at a certain limit of difference, conjugation either with leucocytes or normal tissue Ve eee , ane > ae ie : ee om) : ‘ ate, , = 4, 224 BIO-CHEMICAL JOURNAL © cells would be impossible, and the engrafted tumour would not be able to survive the reaction of the connective tissue stroma. In order that conjugation may take place between a charged nucleus and a neutral nucleus or between two oppositely charged nuclei, it is essential that the limiting surfaces of the two masses of cytoplasm be eliminated at any rate during the process. This elimination of the surface layer can only occur if the two cytoplasmic masses be of specifically the same chemical nature. If, however, as a result of the two individuals having lived in different localities or having been fed with different food, their cytoplasmic material be of different chemical character, conjugation between the nuclei will be impossible owing to the difficulty of ‘ae the limiting layers of the cytoplasm of the adjacent cells. The malignancy of new growths varies generally indirectly with the age of the host. As a corollary to this it may be added that the condition of the chromatin granules may be such that the electrical disturbance occur at a very early age, e.g., in the embryo. The occurrence of sarcoma in utero is doubtless due to this condition, and such cases should be, as they undoubtedly are, exceptionally malignant. We have hitherto confined ourselves to the mere statement that the existence of chromatin granules of such a type that the daughter particles produced in mitosis have a very small difference of charge will tend to cause electrical disturbances, such as seem to occur in cancet. We may now turn our attention to the investigation of this possible condition, for if this possibility be established it will afford an explana- tion of the true origin of cancer. In any healthy race with no close inter-marriages, it would appear from first principles that the chromatin granules would be perfectly normal, if healthy female ova are fertilised with spermatozoa of a different type; that is to say, as long as father and mother are sufficiently differentiated and both healthy, the chromo- somes of their children and the chromatin granules of their descendants will be normal. If, on the other hand, close inbreeding takes place through several generations, then the chromatin granules will tend to become more and more uniform, so that they give in mitosis daughter particles of smaller and smaller potential difference. The tendency to abnormal mitosis is thereby progressively increased until a stage is reached when the incidence of the malignant growth is markedly intensified. Up to the present we have not taken into consideration the chemical side of the problem, and although the action of certain chemical 2 nM). THE ELECTRICAL FORCES OF MITOSIS 225 4 ‘substances has been instanced, as, for example, the production of asymmetric mitoses by antipyrine as observed by Galeotti, the influence of the metabolic products of the cells has not been discussed. We have dealt with the possibility of electrical stimulus and the disorganisation of cells by means of a bruise or blow, and it is a natural sequence of the electrical theory that disorganisation could be produced by a purely chemical stimulus apart from that arising electrically by the different velocities of two ions. Owing to the peculiar configuration of the protein molecule it must be sensitive to both bases and acids, and the influence of these two reactions seems of considerable importance. _ As far as is known at the present time the normal cells seem to be strongly influenced by both bases and acids, and the influence of the former seems to be one of stimulation, while acids seem to produce the opposite effect.! For example the well-known case of a nerve fibre may be quoted: if two needles be inserted into the fibre andone be connected with the copper of a Daniel cell and the other to the zinc, the nerve cells are stimulated around the latter needle and depressed near the former. It is evident that around the latter there is an excess of alkalinity and an excess of acidity around the former. Whiile several experiments giving similar results might be quoted, the above shows clearly that cells are stimulated by alkali. From a physico-chemical point of view this action of alkali is explicable if the tautomerism or dynamic isomerism of the protein molecule be considered. Recent work has shown that there is a distinct and definite tendency on the part of a - NH - CO - grouping not to exist in either of the two possible desmotropic forms - NH - C - and- N =C - I a ti 0 OH but rather as a mixture of the two in dynamic equilibrium with one another. Moreover, the chemical activity of this group as concerns either the nitrogen atom or CO group is determined entirely by this _ dynamic condition, for in a great number of cases it has been proved that a molecule in a static quiescence is singularly inactive to all | chemical reagents. In order that a molecule should be reactive it is + necessary that a certain amount of dynamic oscillation between the t residual affinities be present, and the reactivity is proportional to the amount of dynamic isomerism which exists. In the case of the - NH - CO - grouping in question the phenomenon seems to be connected with the "1. Moor) Roaf, and Whitley, Proc. Roy. Soc., B. 77, p. 102, ete., 1905, Moore and Wilson, Bio-Chemical Journal, Vol. 1, p. 297, 1906. Moore, Roaf, and Knowles, thid,, Vol. LI, p. 279, 1908. 226 . BIO-CHEMICAL JOURNAL wandering of the hydrogen atom from the nitrogen to the oxygen. Now in all such cases the dynamic isomerism is increased by the addition of alkali and decreased by the addition of acid. Since the chemical reactivity of a substance depends upon the amount of dynamic isomerism present, so the reactivity of protein must be enhanced by the addition of alkali and depressed by the addition of acid. The activity of a eell — must essentially be determined by the chemical reactivity of its components, so it is a natural sequence that alkali will stimulate and acid depress the normal functions of a cell. If we apply this argument to the special case already considered of a somatic cell whose chromatin granules vary very much in activity, it leads to an interesting conclusion. It was previously shown that owing to the potential gradient arising from the variation in the activity of the chromf’tin, a pseudo- reducing division can occur with production of cells possessing enhanced activity, or, in other words, a new growth is started. The application of alkali to a cell of the above type will stimulate the activity of all the granules present, and will consequently increase the potential gradient in the chromatin. The application of alkali would tend, there- fore, to increase the probability of the pseudo-reducing division, that is to say alkali would tend to act as a direct cancer irritant when the necessary conditions are present, the necessary conditions being the existence of a _ gradation in the vitality or activity of the chromatin granules. ; The term alkali has been used in the broad sense of any baile substance, and there seems no reason to doubt the direct connection between cancerous growth and irritation by basic substance. Two of the best known examples need only be quoted, namely, chimney-sweep’s cancer, where the soot is the irritant, and, further, the undoubted connection between tobacco smoke and cancer of the lip and tongue. Both soot and the distillation products of tobacco are strongly alkaline substances, since they contain nitrogenous bases. When once the new growth has started, the metabolism of the new cells comes into action, and it is not improbable that a chemical stimulus may arise from the hydrolytic and degradation products therefrom. It is conceivable that these products may themselves act upon the surrounding tissue cells and cause them to undergo the pseudo-reducing division. For this reason we do not wish to restrict ourselves to the statement that infiltration is due entirely to conjugation of the maximum charged cells with neutral tissue cells. The chemical stimulus arising from the hydrolytic and degradation products of the cancer cell métabolism can, THE ELECTRICAL FORCES OF MITOSIS 227 and doubtless does, disorganise the surrounding tissue cells, causing them to undergo-the pseudo-reducing division, thus infecting them with the cancer taint, The variations in the type of initial stimulus account readily enough for the various types of cancer which are known to arise in the same type of tissue. While it should not be possible from a given stimulus to _ develop more than one type of cancer cell, yet the possibility is by no means precluded of producing a new type of cancer in experimental transplantation. If a tumour be ingrafted on to a new host and if the new host were closely similar in every way fo the first host, the new tumour would grow and infiltrate without great difficulty. A slight - difference: between the two hosts would tend to increase the resistance of te _ the second to the ingrafted tumour, with the result it would become a 3 a ‘ eneapsuled. On the other hand, it should be possible to produce a new . infection by virtue of the chemical stimulus arising from the degradation products of the cell metabolism of the ingrafted tumour. ‘This new infection might give rise to a cancer of the same type as the _ original tumour, but it might also, if slightly differently differentiated cells were affected by the chemical stimulus, give rise to a different type of new growth altogether. Although the connection between cancer incidence, inbreeding, and hybridisation follows quite naturally from the theory of cytological processes advanced above, yet we feel the importance of entering more fully into the detail of a subject which, taken as a whole, would seem to open up a new field of research, namely Mendelism and environment as explained by electro-cytology. To turn to the major factor of the 7 equation, in Mendelism we see the synthetical links which bind together ____ the variations in chromatin distribution with racial index of cancer incidence. We have already pointed out that one of the fertile sources of cancer lies in the existence in the chromosomes of an individual of a certain number of chromatin granules of a poor or weakly type which tend to cause fusion of the chromosomes in mitosis with the production of daughter cells with a reduced number of chromosomes. The effect of this will be most marked when the segregation of these chromatin granules into one or other of the paternal or maternal sets of chromosomes ee oceurs. However the Mendelian segregation of these granules takes place in the maturation division of the germ cells of the mothers, their ova will on the average contain chromosomes possessing a definite number of these granules. These ova, when fertilised by spermatozoa from men of 228 BIO-CHEMICAL JOURNAL lower cancer incidence, will give rise to individuals having the same cancer incidence as the mothers, because the cancer incidence depends upon the presence of the weak granules in the one set (maternal) of chromosomes. A further reason for the maintenance of a mother’s cancer incidence in her children is the fact that the cytoplasm of their cells is entirely derived from the maternal side. The first generation resulting from hybridisation, therefore, must preserve the cancer incidence of the mothers, although the fathers have a smaller incidence. The second generation of hybridisation will, however, decrease the cancer incidence, as can readily be seen. The cells of the first generation possess two sets of chromosomes: the maternal with their weakly granules and the paternal without them. When the meiotic gemini are formed and the De Vries re-arrangement occurs the weakly granules distribute themselves, and on the average the chromosomes formed by the splitting of the gemini will have fewer of the weakly granules. The children produced from these gametes with gametes of the new stock will of necessity have a lower eancer incidence. We have, therefore, a direct connection between the Mendelian segregation of the weaker chromatin granules and cancer incidence, a fact which explains the different incidence in the children of one family, and also the frequently observed skipping of a generation by the disease. It would appear from what has already been said that cancer as a disease cannot be inherited—it is only the predisposition to the disease which is inherited, and we have shown that this predisposition must be influenced by inbreeding and hybridisation. We have emphasised the fact that for inbreeding to have any eyil effects, it must occur throughout more than one generation; it is the inbreeding of a stock already inbred that will lead to deterioration. Again, it is obvious that hybridisation in one generation cannot prevent the diathesis being handed down to the descendants, if the offspring of the first hybridisation be again inbred, for whether the pre- disposition in the inbred race is dominant or recessive to the hybrid, on again inbreeding there must be produced some pure dominants or recessives, as the case may be; on the other hand, every successive generation of hybridisation increases the immunity to the disease. After consideration for some years past of the prevailing views concerning the aetiology of cancer, we are forced to conelude that the explanation is to be found in a study of cytology and cytopathology. We put forward in this paper the view that all malignant growths are due THE ELECTRICAL FORCES OF MITOSIS 229 to a derangement of the electrostatic forces normally present in somatic mitosis, which is initiated in the first place by a definite stimulus, internal ___ or external, physical or chemical. We have shown that the susceptibility of the cell to this derangement will be increased with decreasing vitality, such as occurs with age and as the result of inbreeding. Though fully cognizant of the fallacies inherent in statistics, we venture to refer to the results of a study made by us of the geographical distribution of cancer—results which confirm the important role played by inbreeding, hybridisation, and racial immunity. We propose to publish these statistics in detail elsewhere, and will at this time only very briefly deal with the more important facts which have come to ee t. esse as the highest cancer incidence known is to be found in _ Switzerland, we have paid special attention to this country, and have made an exhaustive investigation. The data have been corrected for age * and sex constitution of the population, and the results obtained _ demonstrate clearly that it is in the isolated communities, which have been created by their geographical positions and the influence of religious antagonism, we meet with the highest cancer incidence, while in the "Passes that have served since the time of the Romans as the highways of invasion and commerce from Northern and Central Europe into Italy, we meet with the lowest. Thus the cancer incidence (54°77 per 10,000 persons living, aged 30 and over) is highest in the canton of Appenzell in Rhoden, a Catholic canton, wherein until 1848 no Protestant or even Catholic aliens were allowed to settle. It falls to 27°70 in the neighbouring district of Ober- _ Rheintal, which lies in the valley of the Rhine, in the pass which leads ay to Chur, and from thence by the Spliigen to Italy. q Z _ In the canton of Graubunden the lowest cancer incidences are in the “T passes, viz. :—-Bernina (23°56), Munstherthal (24°01), and Maloja (22°87). The cantons of Ticino and Valais have a remarkably low cancer incidence, 19°34 and 11°61 respectively. In Ticino those districts bordering < on the St. Gothard Pass (Bellinzona 12°28 and Blenio 12°60) have the wee lowest incidence, which gradually rises to its maximum in districts most remote from the St. Gothard. The same occurs in Valais, and it is the ___ district of Entremont (4°11) traversed by the Great St. Bernard Pass, which has the lowest cancer incidence for the whole of Switzerland. ‘The immunity produced by hybridisation is fully borne out by the low cancer incidence amongst the Eurasians, as reported by Dr. a ¢ ‘a : sie s = 4 a 230 BIO-CHEMICAL JOURNAL Sutherland, of the Mayo Hospital, in the Third Report of the Cancer Research Laboratories of Middlesex Hospital (p. 87), wherein he says :— ‘A striking fact is the small number of cases amongst Eurasians, who make up a large proportion of the in-patients in the Albert Victor wing of the Mayo Hospital. Only one case of carcinoma and one abdominal growth occurred out of 790 admissions for malignant disease.’ We have also made investigations as to the local origin of cancer, and we find, in comparing the cancer incidence in the various organs between the sexes and different races, that wherever any organ is specially liable to stress or excessive irritation, there is an increase in the number of malignant growths of that organ in the sex or race under consideration. Although exception may be taken to any conclusions which are drawn from purely statistical data, yet it would appear that the evidence so arrived at is overwhelmingly in support of the fact that one of the major predisposing causes of cancer is to be found in close inbreeding. Moreover, it would also appear from a detailed comparison of the organs attacked, that those organs which are subjected to stresses and irritation — are most liable to develop malignant growth. Both of these conclusions are in close agreement with the theory of electrocytology put forward in this paper. CONCLUSIONS The following conclusions are arrived at in this paper :— 1. The phenomena of somatic mitosis are readily susceptible of explanation by a simple theory of electrostatically charged colloids. 2. The simplest possible case is that when there are present in the cell hereditary characteristics of only one type, as exists in unicellular organisms. 3. When hereditary characteristics of two types occur, a reducing division is bound to occur at some period owing to the fusion of the chromosomes of opposite type. This reducing division is the forerunner of the maturation divisions of the more highly developed species. — 4. The reducing division establishes residual charges of electricity in the daughter cells, the amount of charge depending upon the amount of differentiation between the opposite types of the characteristics. 5. In the animal kingdom where the sex differentiation is complete, the reducing division only occurs normally in the germ cells, and this a rere eee eee, gS QS a , bad i es THE ELECTRICAL FORCES OF MITOSIS 231 : icin is followed by one somatic division. After this second division F Speecarther- divisions can take place owing to the magnitude of the residual & developed. 6. In the lower types, such as occur in the vegetable kingdom where the sex differentiation is incomplete, the reducing division is not confined to the germ cells, but all the cells undergo it at some period of their ‘development. The daughter cells of the reducing division give rise to an indefinite number of cells with the reduced number of chromosomes. Of these cells, in any one generation two have maximum residual charges of positive and negative electricity respectively, a fixed number are _ neutral and the remainder carry intermediate charges. The highest ged cells fuse together with restoration of the original number of re mosomes, thus completing the cycle.- The occurrence of the reducing vision endows the daughter cells with renewed vitality. fe 7. In the animal kingdom the four spermatozoa all carry different ges, one a charge equal to that of the ovum, one an equal and opposite . rg », while the other two have intermediate charges. $8. The phenomenon of sex production may be attributed to these residual charges; all the phenomena of parthenogenesis, artificial fertili- zation and sterilization by X-rays are explained by the same theory. _ 9. The distribution of chromatin granules, required by the De Vries theory, is established as a necessary consequence of the maturation division. 10. The occurrence of pathological mitoses, as the result of external —- stimulus or internal stress, is established provided that the inherent precancerous condition be already present. AL. ‘The possibility of a pseudo-reducing division of somatic cells is accounted for. __—*12. ‘These pathological mitoses result in the establishment of residual charges in the daughter cells similar to those of the maturation division. 18. The daughter cells of the pseudo-reducing division possess renewed activity. They possess potential probability of conjugation with leucocytes and normal tissue cells. 14. The direct stimulation by alkali and bases generally is found to be a normal action, and it would seem that in certain cases alkali can act as a direct cancer irritant. .15. The stimulation of the surrounding tissue cells by the degradation products of the cancer cell metabolism is possible, and to this and the 232 BLO-CHEMICAL JOURNAL facts mentioned in 14 is attributed the formation of a neoplasm with power of continuous growth. 1€ The susceptibility of the cell to these derangements is increamel with decreasing vitality, such as occurs with age, and as the result of in-breeding. 17. In close in-breeding through several generations the chromatin granules become more and more uniform so that they give in mitosis daughter particles of smaller and smaller potential difference, which markedly increases the tendency to abnormal mitosis; conversely hybridi- sation produces a maximum of cell stability and an individual with all its Mendelian allelomorphs as differentiated as possible. 18. The rate of proliferation depends upon the activity of the cytoplasm; the greater the activity the more rapid the growth, while the more highly differentiated the cell and the older it is the less rapid the rate of proliferation. 19. Age incidence, local origin, infiltration, metastases, transmission with all its limitations, and power of continuous growth are the natural outcome of abnormal cell proliferation induced by a disturbance of the electrostatic forces present in normal mitosis. | THE ESTIMATION OF PHOSPHORUS IN URINE _ By G. C. MATHISON, M.B., B.S. (Mexs.), Sharpey Scholar. From the Physiological Laboratory, University College, London (Received April 9th, 1909) _ As a preliminary to some investigations on the metabolism of orus, an examination of some of the methods that have been. employed for the estimation of P,O, in urine was carried out. In view of the probable presence of organic phosphorus compounds, Neumann’s method, as modified by Plimmer and Bayliss (1), was also*employed. First Neumann’s method, precipitation with magnesia mixture and ‘precipitation with magnesium citrate mixture,! were compared on a solution of K,HPO,, and were found to give results concordant within one per cent. For Neumann’s method 10 c.c. of urine are combusted with 10 c.c. of sulphuric acid, repeated small amounts of nitric acid being added towards the end of the combustion. The rest of the procedure is that described by Plimmer and Bayliss. _ For imorganie phosphates, about 4¢.c. of the magnesium citrate mixture are added to 10 c.c. of urine, and enough ammonia to make the mixture smell distinctly of ammonia. The mixture is well stirred and ee let stand over night, and is then filtered through an ash free paper. The _ precipitate is dried and ashed; P,O, is calculated from the ash, Mg,P,0,. Since the magnesia mixture method was found unreliable, it is not described in detail. When the three methods mentioned were applied to urine, the highest _-_—s values were given by Neumann’s method, while magnesia mixture gave : higher values than magnesium citrate. Neumann’s method or magnesium 1. This reagent is prepared as follows :—Dissolve 40 grams of citric acid in 500 ¢.c, of water, add to the hot solution 20 grams of light magnesium oxide. Cool ; add 400 c.c. of 0-880 ammonia and water to 1500 c.c. Let stand twelve to twenty-four hours, and then filter. 234 BIO-CHEMICAL JOURNAL citrate gave practically constant results, but those given by magnesia mixture showed considerable variations. The P,O, in the filtrates after precipitation with magnesia mixture or magnesium citrate was determined by Neumann’s method. Taste I—Comrartson or NEUMANN, MaGNestum CirraTE AND MAGNESIA Mixture MrtTuops Grams P,O, in 100 c.c. Urine Magnesium Citrate Magnesia Mixture Neumann Ppt. Filt. Ppt. o Filt. I (0-153 (0-144 —_ (0-151 {0-005 3 (0-156 (0-143 0-011 (0-156 (0-004 ae ( 0-223 0-013 7+ _ I (0-224 (0-204 (0-017 {0-228 (0-006 ¥* (0-230 {0-207 (0-019 (0-242 (0-012 IV (0-160 {0-153 0-016 (0-160 {0-004 me ( 0-166 (0-151 — ( 0-164 (0-007 The sum of the magnesium citrate precipitate and filtrate values corresponds fairly with the Neumann value; but the sum of the magnesia mixture precipitate and filtrate values is considerably higher. This discrepancy will later be shown to be due to errors inherent in the magnesia mixture method. The magnesium citrate results are very constant, even when quantities varying from 2c.c. to 10 c¢.c. of the solution are used to 10 c.c. of urine. The precipitate is insoluble in 1: 3 ammonia, for the results are not affected by prolonged and frequent washing. To obtain evidence of complete precipitation of phosphates, Scott’s reagent (2), which is capable of detecting 0°005 milligram inorganic P,O,, was applied to the filtrate after separation of the citrate precipitate. No reaction was obtained. It was found, however, that in the presence of citrates, Scott’s reagent was far from delicate, so that the desired evidence had to be obtained in a less direct way, which will be described later. The non-agreement between magnesium citrate and magnesia mixture values required some explanation. Neumann’s method, applied to the ash from citrate precipitate, showed the theoretical amount of P,O, to be present; applied to the ash from magnesia mixture precipitate, it showes less PO, than the theoretical, the amount being variable. THE ESTIMATION OF PHOSPHORUS IN URINE — 235 p TI—Estneation GRAVIMETRICALLY AND BY NeuMann’s MEeruop oF SAREE y Grams P,O; in 100 e.c. Urine Megeeses. Oyen Citrate Magnesia Mixture et ee, Gravimetric Neumann Gravimetric Neumann I 0-151 0-149 0-160 0-154 u 0-144 0-143 0-153 0-148 at (0-207 - — 50-228 {0-217 "(0-204 0-204 (0-224 — (0211 IV 0-194 oo 0-270 0-211 vi 0-226 0-222 0-235 0-227 _ If the magnesia mixture ash is dissolved in acetic acid, the addition of potassium oxalate shows the presence of calcium, which accounts for art of the error. _ By adding magnesia mixture to the filtrate after precipitation with gnesium citrate, a further precipitate is obtained. This contains a le calcium, and also some phosphorus. The P,O, was estimated in sh gravimetrically and by Neumann’s method. : I1l—Torat P,O,, Ivoreantc P,O, ann PO, PRESENT IN THE *AppitionaAL’ MaGnesta Mixtcre PRrecrprrate Grams P,O, in 100 c.c. Urine ‘ Additional ’ Ppt. with | wi aceon Oe PN Total Inorganic Gravimetric Neumann by difference . (0-224 | 0-204 (0-025 (0-016 0-003 (0-230 (0-207 (0-020 (0-012 0-011 ir 0-220 (0-194 0025 0018 0-008 0-218 (0194 is ot snilgey 0-244 —- (0-226 (0-028 (0-010 (0-008 0-241 0-222 (0-024 (0-011 (0-009 IV... 0222 «=—-0-208 0-014 0-006 loo1s This additional precipitation of P-containing substances suggested that magnesium citrate had failed to precipitate all the inorganic phosphates. Seott’s reagent was applied to about 50 milligrams of the additional precipitate, and failed to give any reaction. Thus evidence was afforded of the complete precipitation of inorganic phosphates by magnesium citrate. The inaccuracy of the magnesia mixture method is thus due to two factors, first, that some calcium is precipitated; second, that some of the organie phosphorus is precipitated. 236 BIO-CHEMICAL JOURNAL Ammoniacal solutions of barium chloride and caleium chloride have been used to precipitate phosphates, Since these reagents also precipitate sulphates, they are not convenient for quantitative estimations in urine. But in the filtrate from these the organic phosphorus of urine can readily be determined by Neumann’s method. Indeed, this direct determination is much more readily performed in the barium chloride filtrate than in that from magnesium citrate, since the combustion of the latter is a matter of considerable difficulty. Taste [V—Comparison or Orcanic PO, Vatves py Dirrerent METHODS Grams P,O; in 100 c.c. Urine Tote!’ Tnorgaai —— (etidaind (Citrate) Total, minus _Citrate BaCl, Inorganic Filtrate Filtrate ee 0-222 0-020 0-018 0-016 fn (a) oe 0-148 0-013 0-011 0-011 Il... = 142 0-127 0-015 0-012 0-013 IV... 0180 0-170 0-009 0-009 0-011 V 0-196 0-183 0-010 0-015 0-011 The ies Bet, agreement of these methods affords further oxide of the accuracy of the citrate method for inorganic phosphates. The difference between Neumann and magnesium citrate values represents the amount of phosphorus present in organic combination. The uranium acetate method was found useless for accurate work. Répiton (3) has shown that uranium acetate solutions must be standardized for the particular phosphate which is being estimated. Thus the method is inaccurate in a solution of mixed phosphates. Taste V—Comparison oF NEuMANN, MaGnesitum CITRATE AND URANIUM AceTATE! VALUES Tincture of Cochineal as a warning and Potassium Ferrocyanide as final indicator. Grams P,O, in 100 c.c. Urine Neumann Mag. Cit. | Uran. Acet, I {0-229 -- {0-213 | 0-233 0-205 (0-217 I 0-201 0-187 {Oro ie 0-166 0-156 0-166 IV 0-204 0-194 {Cite ze — 0- y (0-196 0-183 (0-192 ett (0-198 _ (0-186 Wis bax 0-154 0-142 0-160 1. This uranium acetate was standardized against the K,HPO, solution originally employed to test the different methods. THE ESTIMATION OF PHOSPHORUS IN URINE — 287 The uranium acetate values are usually somewhere between the total and the inorganic values, but may be above the total. Any attempts to _ determine organic P,O, by methods involving the use of uranium acetate must give incorrect results. Tue DISAPPEARANCE OF ORGANIC PHOSPHORUS In the course of the investigation it was several times noticed that a | duplicate samples left some days before being analysed, gave lower values ____ for organic phosphorus than samples analysed immediately; in some a __ @ases no organic phosphorus was found. It was thought that this might _--——ibe due to the destruction of organic phosphorus compounds, perhaps by ____ the enzymes present in the urine. # Samples of urine were kept in aseptic flasks, in an incubator, at 87° C., a little toluol being added. Small quantities were removed at Haletvals of a few days, care being taken to cool the flask so as to obtain , the correct volume, and estimations of inorganic P,O, made. In other eases ammonia was added to the urine, which was ‘giailaly incubated. _ Owing to the precipitation of phosphates it was impossible to obtain uniform samples, so the organic P,O, was estimated in the filtrate from 50 c.c. of urine after treatment with magnesium citrate. The following are a few of the results : — — —«T:ss Jan. 12. 250.0. Urine + toluol at 37°C. Total P,O, = 0-196 grams in 100 c.c. Urine a Inorganic P,O, = 0-183 a i Total P,O, = 0-282 grams Inorganic ,, = 0-255 ” 20. Organic P,O, = 0-007 ,, Feb. 27. No trace of Organic P,O, present » 15. Inorganic PO; = 0-187 grams in 100 c.c. Urine » 23. i » 20-194 o ” on SO » = 0-194, * ta No phosphorus present in filtrate Il Feb. 13. 250 o.¢. Urine + toluol, at 37°C. Total P,O, = 0-247 grams in 100 c.c. Urine Inorganic P,O; = 0-230 __,, Feb. 27. Organic P,O; = 0-009 grams in 100 c.c. Urine Mar. 25. can + ae OO0T Fi, If Feb. 13. 2500.0. Urine + 10¢.c. Ammonia at ‘3T°C. Feb. These results show that the organic phosphorus compounds are partially or completely broken down when urine is kept at body temperature. This decomposition is accelerated by ammonia. It is, therefore, important not to leave urine standing too long after adding the magnesium citrate mixture, and also to make estimations of organic P.O, in fresh urine. 238 BIO-CHEMICAL JOURNAL As it has been suggested, though without adequate proof, that glycerophosphoric acid is the form in which organic phosphorus is present in urine (4), 10 c.c., containing 1°2 grams organic PO, were neutralized and added to urine containing a known amount of organic P,O,. “The urine was left in the incubator for a month. The increase of inorganic phosphates was no greater than could be accounted for by the breaking down of the pre-existing organic phosphorus compounds; the glycero- phosphoric acid escaped destruction. eb Tue Resvuvts or DiArysis It was thought that by dialysis it might be possible to remove > the inorganic phosphates and leave organic phosphorus behind. Urine in quantities of 200 c.c, was dialysed for some days, the dialysing fluid being changed three times a day. In some cases continuous dialysation was employed during the last twenty-four hours. Nearly all the phosphorus dialysed out in the first twenty-four hours; at the end of four days no phosphorus could be detected in the residual fluid. By the addition of Folin’s ammonium sulphate and uranium acetate reagent to this fluid a nitrogen-containing substance was precipitated, but it contained ‘no phosphorus. Filtrates obtained after precipitating urine with magnesium citrate were similarly dialysed, but no phosphorus-containing substances remained in the tube. The addition of Hedin’s tannic acid reagent to urine occasionally produced a slight precipitate, but no phosphorus could be found in this. The organic phosphorus compound of urine is thus not identical with the protein-like material ‘sometimes present. SuMMARY 1. The estimation of total phosphorus in urine is most conveniently carried out by Neumann’s method as modified by Plimmer and Bayliss. 2. Inorganic P.O, is best estimated by precipitation with magnesium citrate mixture, incineration and calculation from the weight of the ash, Mg,P,0,. This method is shown to precipitate inorganic phosphates completely. 3. Magnesia mixture gives incorrect and variable results, partly owing to the precipitation of calcium, partly to precipitation of a portion of the organic phosphorus. THE ESTIMATION OF PHOSPHORUS IN URINE 239 4, Organic P,O, can be determined either by subtracting the _inorg vnic from the total P,O,, or directly by applying Neumann’s method ) the iltrate after precipitation of inorganic phosphates by magnesium trate or barium chloride. 5. The uranium acetate method is unsuitable for accurate work. atements as to the existence or non-existence of organic phosphorus in e urine based on uranium acetate estimations are valueless. 7. It is important to make the determinations of organic P,O, in h samples of urine, as the organic compound is partially or pletely decomposed in the course of a few weeks, or, if ammonia be in a few days. i. _ The organic phosphorus compound is readily dialysable, and is a“ ot prec’ aihgas by sian that precipitate traces of protein. ‘is ~s it is with ses pleasure that I acknowledge my indebtedness to Dr. : rs Plimmer for his freely given help and suggestions as to methods I i is investigation. 240 ON THE NITROGEN-CONTAINING RADICLE OF LECITHIN AND OTHER PHOSPHATIDES By HUGH MacLEAN, M.D., Carnegie Research Fellow, University of Aberdeen. From the Department of Physiological Chemistry, Institute of Physiology, Berlin Part II (Received April 13th, 1909) In a former article! it was shown that only about 42 per cent. of the nitrogen of heart muscle lecithin could be accounted for when estimated in the form of the choline-platinum-chloride salt. When a sample of trade lecithin was treated in a similar manner it was found that the yield was considerably higher, being equivalent on an average to about 76 per - cent. of the total nitrogen of the substance. The comparatively low choline content of heart lecithin suggested the probability of part of the nitrogen being represented by some other N-complex differing from choline in its precipitation properties. As the method of preparation of the trade lecithin could not with certainty be ascertained, lecithin was prepared from egg yolk; an analysis of this lecithin agreed in general with that obtained from the heart, but, as will be shown below, the amount of nitrogen that could be accounted for as above described was considerably greater than in heart lecithin, and a good deal lower than in trade lecithin. This curious result seemed to point to the probability of these different lecithins being really different bodies, at least with regard to the manner of combination of their nitrogen, and in order to test this a long series of experiments was made. In this paper it is proposed to deal with the results of hydrolysis of egg lecithin, and at the same time describe certain experiments made with a view to ascertaining whether possibly any circumstances were present that might reduce the final output of choline, even assuming that all the nitrogen was present as this base, in accordance with the ordinary formula for lecithin. PREPARATION OF EaG LEciTHIN Lecithin was prepared from three different portions of eggs. The method adopted was essentially the same as that described in my former papers,” and need not be repeated; in every case (with one exteption) 1. This Journal, Vol. IV, p. 38. 2. This Journal, Vol. [V, pp. 47 and 168. er: oa Be, “ 2 Beat 4 a, RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 241 I used the lecithin obtained from the ethereal extract of egg yolk, the usual] precautions being adopted to exclude, as far as possible, air and light during the process of preparation. In one set of eggs a curious state of affairs was noticed; these eggs (100) seemed to be perfectly fresh and were all rather large, it being naturally thought that this would ensure a greater yield of lecithin. On extraction, however, it was found that the ether showed quite an abnormal increase of fatty matter, but only a trifling amount of substance precipitated by acetone; after treatment with five consecutive portions of fresh ether, the extract still contained much fat, and the combined yield of lecithin was so small that after purification the total amounted only to a few grammes. On subsequent extraction with alcohol the lecithin yield ieee was also very much below the ordinary average. In this particular case it would seem as if there was a great increase of fatty matter at the expense of the lecithin; unfortunately, I had not an opportunity of further investigating the nature of this substance, but a comparison of the relationship of fat to lecithin in the egg, and perhaps in other organs, together with an examination into the nature of this fat, would, in the light of the above observation, probably be of some interest. A curious point with regard to the lecithin here obtained was that it differed greatly in general appearance from lecithin obtained from other eggs; it was from the beginning quite dark brown in appearance and not so plastic as is generally the case, despite the fact that the greatest precautions were taken to prevent oxidation; in general it looked more like a specimen that had been exposed for some time to air than the freshly extracted material, which is usually precipitated as a plastic, more or less whitish mass with a slight brown tinge. The amount of lecithin here obtained was so small that it was not made use of for this investigation. The first sample purified gave the following figures on analysis; comparison with heart muscle lecithin shows a marked similarity in elementary composition : Egg Lecithin Heart Lecithin N (3 experiments) Average 1-876 %, 1-87 % P32 ” és 3-95 % 3-95 % © 1 experiment » 818% 66:29% Hl ” » 106% 10-17 %, : N:P = 105:1 242 BIO-CHEMICAL JOURNAL Hyprotysts or LEecrrnHin ae | (a) In alcoholic solution of barium hydrate-—Uere about 1 gramme — lecithin was boiled for varying periods of from two to six hours with 100 c.c, methyl aleohol containing 5 grammes Ba(OH),. After separating certain products of decomposition, as formerly described, the purified aleoholic extract was treated with alcoholie-platinum-chloride solution, and the precipitate washed, dried and weighed as usual. Two experiments taken at random from a long series gave the following results; percentages are expressed in terms of actual amount obtained, as against theoretical amount calculated on N present :— 1-0268 gm. Lecithin boiled 3 hours = 0°2766 gm. Choline-platinum-chloride = 65-4 % 0-8989 ” a) > 6 ” = 0°2448 . ” ” = 66%, Here, as in experiments on heart lecithin, it was invariably found that the residue obtained after hydrolysis contained nitrogen, despite the most prolonged and careful washing; experiments described later on suggest that this nitrogen is probably not of choline nature; in the two experiments given above, this insoluble N amounted to 6°08 per cent. and 913 per cent. respectively of the total nitrogen contained in the amount of lecithin used. In six experiments in which this residual nitrogen was estimated, the average percentage of the total lecithin. nitrogen found in the residue was 6°74 per cent. (b) In watery solution of Ba(QH),.—In this case the bydrolaale was carried out in general as above described, certain modifications being adopted in order to obtain the end alcoholic solution of choline as free as possible from impurities. The time during which boiling was continued varied from three to five hours, but in one experi- ment this was extended to eighteen hours. A reference to the result shows that this prolonged heating had a comparatively trifling effect in destroying choline, and thereby lowering the percentage figures. In general, the results stand in accord with those obtained by the use of alcoholic solution; the following three experiments suffice to show this : — 0-8403 gm. Lecithin boiled 3 hours = 0-2246 gm. Choline-platinum-chloride = 64-8 % 07821 ,, —s, + 2. eee .. e: # = 65-2 %, O7411 ,, ” » 18 ,, = 0-1920 ,, a» 9 = 63% Thus, it is seen that this specimen of lecithin when hydrolysed in a watery or alcoholic solution of Ba(OH), gave practically similar results, varying on an average from 65 to 66 per cent. of the theoretical amount. Here, as with alcohol, the residue obtained after filtration of the hydrolysed solution invariably contained some nitrogen. a RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 243 In a few experiments a special attempt was made to render this residue free- from nitrogen, but without avail. After filtration the 2 3 ~ substance was thoroughly broken down, returned to the flask, and boiled _ for fifteen minutes with 50 to 80 ¢.c. H,O. Again it was broken down and _ treated as before, this process being repeated three times; afterwards it __was carefully washed on the filter with water almost at boiling temperature. . Despite this prolonged washing, examination in every case revealed the ; * of a distinct amount of nitrogen in the residue. fe) In mineral acid solution.—In almost all the text-books, both . Sela: and modern, there appears the statement that lecithin while easily - uit up by the action of an alkali, is but very slowly attacked by acids. In _ testing this, however, it was found that such is not the case, at any rate * th regard to the splitting off of choline. After boiling with a weak acid ¢ a comparatively short time, choline seemed to be completely separated off, and as the use of acid in this connection possessed certain advantages some experiments were carried out with it. At first sulphuric acid was used, but the difficulties experienced in getting rid of it after hydrolysis was completed rendered the method somewhat cumbersome, and instead of sulphuric acid use was made of hydrochloric acid. Here it was thought _ that removal of the acid previous to precipitation by platinum chloride _ would not be necessary, though after evaporation of the watery part of the solution it was obviously present in fair concentration, and some experi- ments (described later) showed that this assumption was correct. In all these hydrolytic processes it is an advantage to avoid if possible the use of barium, owing to the great difficulty of getting rid of it _ completely afterwards. With an alcoholic solution this is a matter of ___— very great difficulty, and even with water, from which separation is much —— more easily obtained, it is sometimes found to be present in traces towards __ the end of the operation of purifying. The danger in the presence of barium results from the fact that this substance forms a double salt with platinum chloride, which may be thrown down with choline-platinum- chloride. It is true that this barium salt is fairly easily soluble in alcohol, but in spite of this fact, great care must be taken to wash the platinum chloride precipitate thoroughly with cold aleohol when there is is any chance of barium being present. If this is not attended to somewhat pre variable results, difficult to account for, may be obtained. In all my ey experiments in which Ba(OH), was used, particular care was taken to ensure the thorough washing of the platinum chloride precipitate in order bes: to dissolve out any traces of the double barium salt that might be present. 244 BIO-CHEMICAL JOURNAL When this precaution is neglected, it is quite possible that traces of barium may sometimes account for the apparent percentage of platinum being somewhat above the theoretical amount when the salt of choline-platinum- chloride is ignited. With HCl, however, the separation of barium is got rid of, and, so far, a great simplification introduced. Experiment showed that all that is necessary is to boil the lecithin for some time with a weak HCl solution, filter, evaporate to dryness, dissolve in absolute alcohol and precipitate directly with platinum chloride. This method takes up very much less time than when done with Ba(OH),, and is to be recommended when estimating the choline of lecithin; here the choline is present from the beginning in the form of tlie chloride compound, and as such is less likely to undergo decomposition than when present in the free state in alkaline solution. As pointed out above, however, the action of Ba(OH), on choline in saturated watery or alcoholic solution is not of much importance, the loss in a sample boiled for eighteen hours with 5 per cent. Ba(OH), in H,O amounting only to about 2 per cent. of the total choline present. Im my experiments the HCl hydrolysis was performed as follows :— About 1 gramme substance was taken and boiled for varying periods with 100 c.c. of a 10 per cent. watery solution of hydrochloric acid (10 c.c. HCl sp. gr. 1°81 + 90 c.c. HO), boiling being continued with the aid of a reflux condenser for from two to five hours. The fatty material separated out as an oily mixture, and could not be conveniently filtered off while the solution was hot, but on cooling formed a solid mass, which was easily separated from the liquid. This fatty residue was returned to the boiling flask, and again boiled with 100 c.c. H,O for ten to fifteen minutes; the mixture was then allowed to cool and filtered as before, the process being in many cases repeated three times. The total filtrates mixed together were now evaporated to dryness on the water bath, and the residue extracted with absolute alcohol, filtered, evaporated to small bulk and precipitated directly with platinum chloride. It was then left to stand till next day, and the precipitate then filtered, washed, dried and weighed as usual. . The obvious object of the above prolonged washing of the fatty residue was, if possible, to render it nitrogen free. This, however, could not be accomplished, and it was found quite unnecessary to return the residue to the flask more than once, as further washing did not lessen the nitrogen content. RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 245 | lt is noteworthy that in whatever manner lecithin is split up the residue always contains a certain amount of nitrogen, and since this residue is composed practically of fatty matter, it suggests the probability of this nitrogen being in close relationship with the fatty acid radicle of the lecithin body. The following is an example of the figures obtained with HCl :— 0-7601 gm. Lecithin boiled 1} hours = 0-2012 gm. Choline-platinum-chloride = 64-2 % IE ge ip . 3 » = O2222 ,, a S = 65-1 %, O91 , * oa = 02425 ,, ” ” = 645% The its are practically identical with those obtained when barium eeeinte was used, and other samples of egg lecithin gave almost similar _ results. One sample, however, differed somewhat from the above in its ij nitrogen percentage,' and in this case a slightly larger yield of choline was obtained when calculated on the total nitrogen present, though when : “calculated on the weight of substance it was precieatty 3 in agreement with the above sample. A trade lecithin, having a nitrogen percentage of 177 and a N : P _ ratio of almost exactly 1 : 1, was now taken, and samples treated along- side of the egg lecithin as follows:—About 1 gramme of this lecithin and a similar amount of egg lecithin were hydrolysed under exactly similar conditions as parallel experiments; they were then treated as nearly as possible in the same manner, the same amount of fluid being used to wash the residue, &e., and the choline precipitated in the usual way. _ One experiment was done using alcoholic Ba(OH),, another with watery Ba(OH),, and a third with HCl. The following results were obtained, and clearly prove that the two lecithins, though having a fairly similar elementary composition, are certainly not identical substances ie rite regard to their nitrogen complex :— Fluid used for Percentage of choline Percentage of choline Difference hydrolysis ofegglecithinfound oftradelecithinfound in percentage Alcoholic Ba(OH), 65-8 80 14-2 Watery Ba(OH), 64-6 79-6 15 | ow) 65-3 80 14-7 na Again, heart lecithin differs from these results in a greater degree than they differ from each other; a comparison of the three substances Me gives the following figures with regard to choline recovered as the double ae platinum salt :— ene Heart lecithin = 42 %, Egg lecithin = 65 % Trade lecithin = 80 % 1. This sample gave an average of 1-77 %(, N. 246 BIO-CHEMICAL JOURNAL From these results it is obvious that so-called lecithins are really bodies of different composition, despite their general agreement when viewed from the results of elementary analyses. Retation or N or Env Fiurrate to CHo.ine-PLATINUM-CHLORIDE ACTUALLY OBTAINED As in the case of heart muscle lecithin, some experiments were done in order to test directly what proportion of the nitrogen in the end filtrate —i.e., the end alcoholic solution obtained after hydrolysis, and purified as much as possible from other decomposition products and ready for precipitation with platinum chloride—could be obtained as the double platinum salt. In some respects this direct estimation gives more definite results than the ordinary lecithin estimations described above, for it is known exactly what amount of the nitrogen appearing in the end solution is actually precipitated as choline. To determine this, hydrolysis was performed as before, and the alcoholic solution ultimately obtained divided into two equal parts. One part was used for N estimation, and the other precipitated directly with platinum chloride. In three experiments it was found that 23 per cent., 24 per cent. and 26 per cent., respectively, of the N actually present was not recoverable as choline-platinum-chloride. — All these results prove beyond any doubt that the nitrogen of the end filtrate is not present as choline, and suggests strongly that in lecithin the generally accepted formula is insufficient for the facts obtained. In the light of the above results, two considerations present themselves— (1) That the nitrogen of the lecithin is really not all present in the form of choline or similar basic compound; or (2) That all the nitrogen is really present as choline, but that some interaction with the chemicals employed during hydrolysis, or with some of the substances of hydrolytic decomposition, gives rise to some unknown nitrogenous complex which is not precipitated by platinum chloride. In order, therefore, to strengthen the probability that lecithin N is not all present in the form of choline, it was necessary to thoroughly investigate this point; for this purpose pure choline chloride was used, and the following experiments performed : — RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 247 Exrertments wirn CHoLine CHrormpe Samples of the choline-platinum-chloride salt obtained in the above ey ents were mixed together and dissolved in water, filtered and re-crystallised. The typical crystals obtained were again dissolved in ‘a f, and the process of re-crystallisation twice repeated. On ignition ly the theoretical amount of platinum was obtained. A strong : Watery solution of the crystals was then treated with HS to remove the _ platinum, the fluid being heated during the passage of the gas in order Ba to ensure complete separation. The mixture was then freed from platinum | sulphide by filtration, and the filtrate, which was water clear, evaporated ; ‘dryness on the water bath. Residue was then dissolved in absolute cohol, and this solution used in the experiments. _ The object of these experiments was to find out what percentage of ie choline chloride actually present in the alcoholic solution could be vovered as the double platinum salt after the solution had been treated th Ba(OH), in alcoholic, and in watery solution, and with HCl, exactly in the hydrolysis of lecithin. : : For each experiment an equal amount of the above solution was and the following tests performed : — A. For Nitrogen | 5 c.c. choline solution was directly run in to a Kjeldahl flask and the N-content estimated with the following results :— (1) Gave 9-24 mgr. N equivalent to 0-2031 grm. Choline-platinum-chloride @ . o%.,, Re » ©2081 ,, ” * _ Here in both experiments the results were absolutely identical. ; B. For Choline-platinum-chloride by Direct Precipitation 5 c.c. directly precipitated by 10% alcoholic platinum chloride solution, allowed to stand Result = 0-1961 grm. Choliae-platinum-chloride Se but 5 ¢.e. evaporated to about 2 ¢.c. before precipitated with platinum chloride :— Result = 0-1968 gm. Choline-platinum-chloride From the above it is seen that the amount of choline actually present in the solution was, on the nitrogen calculation, equivalent to 0°2081 gramme of the double platinum salt, and calculated on direct precipitation - about 01964 gramme, taking the average of the two experiments. This slight difference may be accounted for by the difficulty of absolute exactness in allowing for the necessary reduction of nitrogen formed from oe r. 248 BIO-CHEMICAL JOURNAL the chemicals used in the Kjeldahl estimation, coupled with the ordinary experimental errors and perhaps a slight loss due to traces of the choline remaining in solution, It would seem that evaporation of the choline containing fluid to very small bulk is not necessary, as the above results with volumes of 5 c.c. and 2 c.c., respectively, are practically the same. As a result of all these figures, it is clear that 5 c.c, of this solution ought to yield at least 0°1960 gramme of the double salt when precipitated by platinum chloride. C. Choline Solution Boiled with Different Reagents to Imitate Hydrolysis of Lecithin (1) Watery solution of Ba(OH),.—To 100 c.c. of a 5 per cent. solution of Ba(OH), in water, 5 c.c. of choline solution was added and the mixture boiled for three hours, a reflux condenser being used. After cooling the solution was filtered, the filter paper thoroughly washed, and the combined filtrates treated with CO, to precipitate the barium. Mixture was now filtered and the barium carbonate residue thoroughly washed with hot water. Filtrates were united, a few drops HCl added, and evaporated to dryness. Residue was then dissolved in alcohol, evaporated to small bulk and precipitated as before. Gave 0-1963 gm. Choline-platinum-chloride 9° 0- 1 959 bad ” °° ° (2) Methylic alcohol solution of Ba(OH),.—This was carried out as above, with a few modifications mentioned in a former article, to ensure the removal of the barium. Owing to an accident, only one experiment was completed; it gave— 0-1957 gm. Choline-platinum-chloride (3) Hydrochloric acid.—(a) To 100 ¢.c. of a 10 per cent. watery solution of HCl, 5 c.c. choline chloride was added and the mixture boiled for two hours; it was then filtered and the filter paper thoroughly washed. Filtrate was then evaporated to dryness over the water bath, residue dissolved in absolute alcohol, filtered, evaporated to a few c.c.’s and precipitated as usual. (6) Here a 5 per cent. HCl solution was used, and boiling was continued for one and a half hours; otherwise it was identical with above. (a) Gave 0°1970 g.m. Choline-platinum-chloride (d) ” 0-1951 ” ” ” ~ ee eee oe ae Bb) ve = =f} RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 249 ; It is thus obvious that HC] when used as above does not interfere to _ any extent with choline chloride. The only point in the manipulation at which some action might be expected is during evaporation to dryness of ‘the dilute acid solution, for it is obvious that towards the end the concentration of HCl is very great; however, this apparently exerts no destructive action. _ The following gives an indication in tabular form of the results obtained :— ai CHoLIne-PLatTiIncM-CHLORIDE IN GRAMMES _ Galeulated from Found by direct Found, using Found, using Found, using __N found precipitation HCl alcoholic Ba(OH), watery Ba(OH), 09031 0-1961 0-1970 0-1957 0-1963 0-203! 0-1968 0-1951 sae 0-1959 The above experiments were carried out with every possible ae precaution in order to ensure parallel results, and a comparison of the figures shows that the greatest difference between any two amounts only a to 0-008 gramme, the average difference being very slight indeed. When _-__ it is considered that a rather long process of manipulation, including . several filtrations and the necessity of changing from vessel to vessel (evaporation, &c.), was involved the results agree remarkably well, and show conclusively that the loss of choline-platinum-chloride in the experi- ments on lecithin is not due to any destructive action on the part of the hydrolysing agenis; further, they indicate that the necessary manipu- Pea. lations can be conducted with little or no loss, though in doing this the ie a greatest care is necessary. -_ a # ~ a In view of the above results there remained only the possibility, already mentioned, that some inter-action between choline and some other product of lecithin decomposition might ensue, and so yield a nitrogenous complex of obscure nature which was not precipitated by platinum chloride. In order to test this a similar solution to the above was used, but to the Ba(OH), or acid mixture some of the known products of lecithin decomposition were added before boiling. From the results obtained it is obvious that no such inter-action takes place. The experiments were carried out as follows: — 250 BIO-CHEMICAL JOURNAL ExrPEerRIMENtTs with Lecirnin Decomposition Propucrs For these experiments a solution of choline chloride similar to above was used, but of somewhat different strength; 5 c.c. gave on direct precipitation 0°2200 gramme of the double platinum salt. (a) 5 c.c. of this solution was added to 100 c.c. water containing 2 c.e. glycerophosphoric acid. This mixture was boiled for half an hour and 5 grammes Ba(OH), added; boiling was continued for two hours, and after cooling a whitish residue was obtained on filtration. This residue was thoroughly washed three times with 100 c.c. boiling water, being each time returned to the flask and boiled for ten minutes; finally it was washed with hot water on the filter. Filtrates were then treated with CO,, and the usual manipulations performed. (b) This experiment was conducted exactly as above, only that Ba(OH), was present at the beginning of boiling. Results were as follows : (a) Gave 0-2173 gm. Choline-platinum-chloride (b) ” 0-2161 ” ” ” A number of experiments were now made with different amounts of other known decomposition products, such as glycerine, phosphoric acid, oleic acid, &e. The average results obtained showed that only a trifling loss was accounted for by the presence of these materials, and as their introduction gave rise to marked difficulties in the way of filtration and washing of residues, this small loss cannot be held to account for the great loss in lecithin experiments; in any case this loss did not amount to more than 6 per cent. of the total, and since much greater quantities of above products than would ever be obtained as the result of the hydrolytic decomposition of lecithin containing a similar amount of choline (equivalent to 0°2200 gramme platinum salt) were used, much more difficulty was experienced in filtering than is the case with lecithin. The following experiment is sufficient to show that, even when excessive quantities of substances representing lecithin decomposition products are used, the final yield of lecithin is not materially decreased. 5 c.c. choline solution was mixed with the following substances :— Glycerine iy Tee Ys she L2C,0, Glycerophosphoric Acid ses Ses in) TSS, Phosphoric Acid ay ii ‘us .. O05 grm, Stearic Acid ... os ety sas «» O05 grm. Oleic Acid... es oad i .. O05 e.c, ‘ RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 251 and the mixture boiled in a 5 per cent. watery solution of Ba(OH), for _ four hours; it was then treated in the ordinary way to remove impurities. ‘a . In this case the difficulties in filtration were very great, the experiment taking a very long time owing to the fatty nature of the residues obtained. _ The final yield, however, gave 0°2110 gramme choline-platinum-chloride, or about 95°5 per cent. of the total obtained on direct precipitation, showing _ definitely that when choline is really present as such at the beginning, such treatment does not materially lessen the final amount obtained. Here again it is seen that the residue obtained on boiling choline with ma: ‘Ba(OH), and other substances such as the above products gives a residue whieh can be rendered N-free. From this it must be assumed that the “nitrogen remaining in the first and other residues in the hydrolysis of ecithin does really not represent nitrogen of choline, but is present in the rm of another nitrogen complex. ; The result of all the above experiments seems to show definitely that 4 if choline were really present in lecithin to the amount represented by the nitrogen content, a much greater yield would necessarily be obtained on eo and precipitation with platinum chloride. Since little or no evidence of the breaking down of choline can be Gitined experimentally under the conditions present in the hydrolysis of lecithin, it is but fair to assume that a similar state of matters holds good for lecithin itself, and that the results actually obtained gave a very fair indication (allowance being made for slight losses due to possible defects of the method) of the amount of choline actually present as such in the original substance. A consideration of these facts shows the absolute : _ futility of endeavours made at one time to estimate the amount of lecithin __ present in an organ in terms of the yield of choline obtained. It is obvious ____ that this method would give widely divergent results when applied to, say, ee heart lecithin and egg lecithin respectively. a: Thus the accepted formula for lecithin does not account for the facts, and in view of the differences existing between apparently similar lecithins but derived from different sources, can hardly be taken as a representation of any lecithin as obtained by the best methods at our disposal at present. It was next thought that an investigation of some of the salts of lecithin such as the cadmium chloride compound might yield some information; the results obtained strengthen the above view. 252 BIO-CHEMICAL JOURNAL CapmiuM-CHLORIDE-LECITHIN 82°53 grammes lecithin were dissolved in absolute alcohol, and to this an alcoholic solution of cadmium chloride was added till no more precipitation occurred. After standing for eighteen hours precipitate was filtered off and thoroughly washed with cold alcohol, filtrate and wash alcohol being preserved. It was then dried and weighed, and yielded 36°41 grammes lecithin-cadmium-chloride. Filtrate and wash alcohol together was now carefully evaporated to dryness and gave a total residue of 2°64 grammes, part of this obviously consisting of cadmium chloride. This residue (which may be termed Residue A) was now treated with water in order to dissolve out the cadmium chloride, and was extracted till the solution gave no evidence of the presence of cadmium on the passage of H,S. . On evaporating down this wash water it was tested and found to contain a good deal of nitrogen. A mere trace of phosphorus could be detected, the relation of N : P standing as 141 : 1. Here it was obvious that some nitrogen must have been split off from the lecithin; on boiling this watery solution with HCl and treating in the usual way with platinum chloride, no precipitate could be obtained, indicating that this nitrogen was not present in the form of choline. After this extraction Residue A weighed only 1:16 grammes, so that 1-48 grammes must have gone into solution, the greater part of this being eadmium chloride. Thus, the total amount of substance obtained from — 32°53 grammes lecithin after removal of excess of cadmium chloride was 36°41 grammes + 116 grammes = 37°57 grammes. The remaining portion (1:16 grammes) was now thoroughly dried and analysed, with the following results : — A (1°73 9 Nitrogen i ay $I average 1-75 % Phosphorus 2-18 %, On treating in the usual way for choline, only about 20 per cent. of the theoretical yield was obtained, but the small amount of substance rendered it difficult to get quite an accurate result. Since it is well known that lecithin is not entirely precipitated out of alcoholic solution by cadmium chloride, this residue might be expected to be composed of lecithin-cadmium-chloride; the analysis shows plainly, 4 RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 253 however, that something more containing a higher percentage of nitrogen and a lower percentage of phosphorous than this compound must have been BZ present as well. The choline-platinum-chloride found probably represents the choline of the double lecithin salt, while the remaining nitrogen was nt in some other form. All these points go to strengthen the conclusion hitherto suggested, that the nitrogen of lecithin is not all represented by choline or such basic body. Awanysis or Lecrrury-CapMiuM-CHLORIDE In the light of the above results it was of interest to ascertain what percentage of the nitrogen in the double cadmium salt could be recovered ‘in the form of choline-platinum- -chloride. Since, in the manipulation described, nitrogen was found which did not seem to represent choline oh uitrogen, it was naturally concluded that the percentage of nitrogen actually representing choline in the double cadmium salt ought to be wah somewhat higher than in lecithin. This was found to be the case. - The following figures for N and P were obtained. Nitrogen {iaie &f average 1-415 % ; Phosphorus : 3104 | average 3-095 % q N:P 21:1 From these figures it is, of course, easy to deduce the theoretical yield of cadmium-chloride-lecithin that should be obtained from a given quantity of lecithin. In the above sample the average nitrogen content ____ of the lecithin used was 1-876 per cent., and of the cadmium salt 1°45 per went. If all the nitrogen of the lecithin were contained in the cadmium ____ salt a simple calculation shows that 1 gramme lecithin contains sufficient nitrogen to yield 1°294 grammes of the cadmium salt, and, therefore, the quantity used (52°53 grammes lecithin) ought to furnish 42-093 grammes lecithin-cadmium-chloride. The amount actually obtained after removal of excess cadmium chloride was, as mentioned, only 37°57 grammes— another préof that all the nitrogen present in the lecithin did not go to ‘i form the double cadmium salt. e Although, as shown, a certain amount of the lecithin-cadmium- f chloride remained in solution, this was much too small to account for the loss of about 45 grammes calculated on the theoretical amount, using nitrogen as a basis. 254 BIO-CHEMICAL JOURNAL Hyprotysis or Lecrrarn-CapMiuM-CHLORIDE The salt was treated with watery solution of Ba(OH), in the usual way, and the choline calculated as choline-platinum-chloride with the following results : — a 0-6348 gm. Salt = 0-1470 gm. Choline-platinum-chloride = 74:5 % 05587 ” Sa cd O17T77 ” ” ” = 75% These figures show that the cadmium salt of lecithin yields about 10 per cent more choline-platinum-chloride than lecithin itself does, results being in both cases calculated on the original N-percentage of the substance. This again shows that some nitrogen, which was not present as choline, must have been thrown off from the lecithin, otherwise the results cannot be explained. On the other hand, it is obvious that this lecithin salt must, like lecithin itself, contain a good deal of nitrogen which is not present as choline. It is intended to again recover this lecithin from the salt, and to estimate its choline content; then to repeat the process of precipitation and analysis in order to ascertain whether this splitting off of nitrogen would be in evidence on a second precipitation. It is quite possible that this particular part of the nitrogen may be present as an impurity in the form of some other complex not really belonging to the lecithin molecule; if so, it must have the same general properties as the lecithin itself, both in regard to solubility in ether and precipitation by acetone, and is likely present in many so-called lecithins. Of course it represents only a comparatively small part of the total lecithin nitrogen. In the hydrolysis experiments carried out with lecithin-cadmium- chloride mentioned above, it was found that the residue obtained gave, as usual, a distinct indication of the presence of nitrogen; as before it was found quite impossible to get rid of this however prolonged washing was attempted; as in lecithin, it seems certain that this residual insoluble nitrogen has nothing to do with choline. Since it has been shown that on the hydrolysis of lecithin a considerable percentage of nitrogen actually present in the end alcoholic solution is not precipitated as choline-platinum chloride, thtis nitrogen ought to be present in the filtrate after the separation of the platinum chloride precipitate. That the substance obtained was really the double platinum salt of choline is indicated by the results of ignition experi- ments, which generally gave a residue of platinum but slightly wide of the theoretical amount; it is, of course, quite likely that in some cases, fa = ~ x Se z RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 255 at any rate, the salt was not of absolute purity, but any admixture of other material must have been so slight as to be of little or no importance in judging of the general results. ‘To substantiate this some experiments were made in order to ascertain if the amount of nitrogen used up actually corresponded to the amount necessary for the quantity of choline-platinum-chloride isolated. This was found to be so, the remainder of the nitrogen being present in the filtrate. A number of these final filtrates were now united, some water added, and the excess of platinum separated by means of H,S, the solution being _ heated during the passage of the gas. It was then filtered from platinum sulphide and the clear filtrate evaporated to dryness; residue was then dissolved in a smal] quantity of absolute alcohol and again treated with platinum chloride; in each case a slight precipitate was obtained, but it invariably proved to consist of a mixture of a small amount of choline-platinum-chloride with some of the barium salt of platinum chloride. This was in turn filtered off and filtrate again treated with HS, as above. The final solution obtained was then treated with various general precipitating reagents, but no definite substance could be isolated _ in this way. Experiments in this direction are at present being carried out, but it seems certain that the excess nitrogen of lecithin is not present as an ordinary basic compound ; on the other hand there is some evidence that part of it, at any rate, is present in the form of an amino-acid, but this will be entered into in a later communication. The purpose of the present investigation was to ascertain the reason why in the hydrolysis of lecithin the actual amount of choline isolated should invariably fall so far short of the theoretical amount, and also to obtain some definite evidence with regard to the correctness or otherwise of the generally _ accepted formula with regard to its nitrogen distribution, In the light of the above experiments it seems to me that the reason for the discrepancies mentioned depend on the fact that different lecithins contain varying amounts of choline; also that the ordinary formula cannot be accepted. Again, it is likely that many of the specimens formerly examined contained mixtures of other phosphatides besides _ lecithin, and so gave even lower results than recorded above. With regard to other phosphatides examined mention may be made of two—the mon-amino-diphosphatide isolated from the heart muscle by Erlandsen, and from eggs by the writer. As mentioned in a former paper these two substances differ somewhat in their general analyses, but im each case no definite evidence of choline or any other substance of basic nature could be obtained. On hydrolysis in the usual way, platinum 256 BIO-CHEMICAL JOURNAL chloride may give a slight often ill-defined precipitate, but the amount is so exceedingly small even when a large quantity of the substance is used, as to make it impossible to say what it really consists of. It would seem to be more of the nature of an impurity, and is in no way equivalent to the amount of nitrogen present. With the comparatively small amounts of substance at my disposal it is difficult to make any definite statement, but so far, in the case of the egg phosphatide, I have failed to obtain any substance of a basic nature in the ordinary sense though many experiments in this direction have been made. At present a large supply of heart ‘cuorin’ has been prepared, and it is hoped that some future experiments with large amounts of substance may settle the point. The general results of the above investigation and the chief con- clusions inferred in the light of these results may be shortly summed up as follows :— SUMMARY From different lecithins (heart, egg, etc.) prepared with the greatest care, and having a ratio of N : P as almost 1 : 1, different amounts of choline are obtained under similar conditions. On the other hand the amount obtained from any given specimen is practically constant, the result being the same whether watery or alcoholic solution of Ba(OH),, or watery solution of hydrochloric acid be employed. From this it is obvious that these different lecithins, though showing somewhat similar figures as the result of elementary analyses, are not identical in composition, particularly with regard to their nitrogen distribution. A long series of experiments with choline chloride has shown that if choline is really present at the beginning of hydrolysis, a very large percentage of it can be ultimately recovered as the double platinum salt, and the presence of substances corresponding to the known decomposition products of lecithin do not, by means of any more or less obscure reaction with the choline present, prevent this base being ultimately recovered as the double platinum salt; in one experiment containing excessive amounts of all the known products of hydrolytic*decomposition of lecithin, there was a loss of only 5 per cent. of the choline, obviously due in great part to the difficulty experienced in thoroughly washing the bulky residues present. . Since these insignificant losses entirely fail to account for the small yield of choline obtained from lecithin, it is necessary to assume that the whole of the nitrogen of lecithin is not represented by choline. In some lecithins, however, more of the nitrogen present is actually |) Tee Ne ee RADICLE OF LECITHIN AND OTHER PHOSPHATIDES 257 represented by choline than is the case in other lecithins, a comparison _ of the amount of choline-platinum-chloride salt obtained from heart and egg lecithin, respectively being roughly 42 per cent. and 65 per cent, while from a trade lecithin 80 per cent. was obtained. Results substantiating the above were obtained on precipitating egg lecithin with cadmium chloride and examining the filtrate and salt; in the sample examined some nitrogen was split off by CdCl, in a form soluble in H,O; this nitrogen did not represent choline. The cadmium- leeithin-chloride salt contained 10 per cent. more choline (based on its N-content) than the lecithin from which it was obtained. This practically proves that some of the nitrogen originally present in the lecithin was not present as choline. Other evidence with regard to direct estimation of nitrogen and m4 in different filtrates all points in the same direction, and lends : additional weight to the inferences derived from a consideration of the above experiments. Whether lecithin is really a chemical unit, or a mixture of two or more substances having the same properties does not affect these facts. CoNcLUSIONS 1. In the light of the results obtained, it is the opinion of the writer that the generally accepted lecithin formula, i.e.— i O« (acid radicle) CH - O° (acid radicle) CH, OH— > PO (N. in form of choline or possibly other base) - O in which the whole of the nitrogen present is represented by a base— . choline—is incorrect, and can no longer be accepted; probably part of this ‘ nitrogen at any rate is present in the form of amino-acid. 2. Lecithins obtained from different sources differ, often to a great extent, with regard to the percentage of nitrogen actually represented by choline, and though often giving somewhat similar results on elementary analysis, cannot be regarded as identical chemical substances. 2 3. These results fully explain the general failure of former j investigators to obtain anything corresponding to the theoretical yield : ; of choline from lecithin. 258 A POLARIMETRIC STUDY OF THE SUCROCLASTIC ENZYMES IN BETA VULGARIS By R, A. ROBERTSON, M.A., JAMES COLQUHOUN IRVINE, D.Sc., Pu.D., axnp MILDRED E. DOBSON, M.A., BSe., Carnegie Scholar. From the Chemical and Botanical Research Laboratories, United College, University of St. Andrews ; (Received April 16th, 1909) Notwithstanding the large number of exhaustive investigations which have been recently carried out on the sugar beet the actual mechanism of the sugar synthesis seems to be practically unknown. Two alternatives seem possible, the simpler of which is that both glucose and fructose are formed in the leaf region and are there subsequently transformed into sucrose which passes by means of the leaf meristem directly to the root: The alternative explanation, that the disaccharide is a degradation product of starch, seems unlikely. Little or no starch is found under normal conditions in the growing leaf and, moreover, such a change would involve the partial transformation of maltose or glucose into fructose. This would necessitate a fundamental change in the sugar molecule, which seems improbable. Again, according to Grafe, no definite sugars have ever been isolated in the degradation products of cellulose, and thus the latter does not seem to be a likely source of the sucrose, The first alternative is supported by many arguments. Friedrick Strohmer in liis valuable contribution to the Wiesner Fest-Schrift! shows conclusively from the result of his own experiments and those of A. Girard that the formation of sugars is restricted to the leaf region and, moreover, that the roots of adult plants contain no reducing sugars save in the early stages of growth. It must be maintained that the evidence seems to be increasing that formaldehyde is one of the initial substances formed during the natural process of photo-synthesis of sugars, and it would thus appear that a close parallel can, in this case, be drawn between natural and artificial 1. Uber Ausspeicherung und Wanderung des Rohrzuckers in der Zucker-Riibe. SUCROCLASTIC ENZYMES IN BETA VULGARIS — 259 i synthesis. Fenton’s recent successful reduction of carbon-dioxide to _ ____ formaldehyde' and the earlier work of Usher and Priestley,? who showed that the same reaction proceeded in plant cells, has strengthened the argument considerably. There seems litile doubt that the artificial sugars obtained from formaldehyde contain large quantities of ketoses, ‘s and thus is can be readily understood that in the leaves of the beet the _—s«swR@essary constituents for the formation of cane sugar may be formed. The final stage of the process, the condensation of the glucose and fructose, although theoretically a simple reaction, is one which the organic chemist has been unable to duplicate. Perhaps the most notable example of the comparative failure of ordinary chemical methods to produce naturally-occuring compounds is to be found in the meagre results which have attended attempts to ‘_ synthesis disaccharides in the laboratory. Moreover the methods followed ____ im the few successful cases must of necessity be widely different in their ae nature from those of the natural process. In view of these facts Grabe’s statement that the fundamental réle in the carrying out of these | reactions must be ascribed to enzymes seems justifiable.* | Assuming that cane-sugar is formed by enzyme action, two alternative theories may be offered. The reaction may be occasioned either by a special enzyme, termed the associating enzyme, capable of } condensing glucose and fructose, or the hydrolytic action of invertase * may be a reversible change which is capable under suitable conditions of s producing sucrose from the constituent sugars. This latter view is ee upheld by Gonnermann and Stoklasa, and brings the reaction into line with the cases of reversible zymolysis studied by Croft Hill. This idea has also recently received considerable support in the results obtained by Kohl,* who subjected invert sugar solutions to the action of yeast ____ extract rich in invertase. A series of titrimetric estimations showed that an -equilibrium point was reached, presumably owing to the partial 3 formation of sucrose. The result is interesting, but is not altogether in agreement with the nature of reversible change, as apparently conditions were realised in which both the fructose and glucose contained in the invert sugar completely disappeared on continued action. Pantanelli,°® in a paper on a similar topic, contributes the remarkable statement that 1. Trans. Chem. Soc., Vol. XCVIII, p. 687, 1907. 2. Proce. Royal Soc., Vol. LX XVIII, B, p. 318, 1906, F “3. Macchiati, Comp. Rend., Vol. CXXXYV, p. 1128. 3 4. Beth, Bot, Centralblatt, Vol. XXTLU, 1, p. 64b-640, 1908. 5. Rendiconti Accademia Lincei, 5, Vol. XVI, pp. 419-428, 1907. 260 BIO-CHEMICAL JOURNAL concentrated solutions of invert sugar undergo partial reversion when preserved at room temperature, ‘ particularly when the solution is feebly alkaline.” The reaction was accelerated by the addition of fungus revertase. The first conclusion seems to have been arrived at by the observation that the reducing power of the solution diminished. In view of Lobry de Bruyn and van Ekenstein’s work’ on the interconversion of hexoses in the presence of alkalies the result seems capable of a sae interpretation. Although opinion, in the meantime, seems undivided in attributing sucrose formation in plants to enzyme action, it would appear that it is still doubtful if the action is due to a definite specialised enzyme or is merely the result of reversible zymolytie change. The following research was undertaken in the hope that the biochemical formation of sucrose by enzyme action could be detected polarimetrically, and for evident reasons the sugar beet was selected for experiment. The adoption of the polarimetric method seemed in this case specially advisable as affording a more accurate index of alteration in the composition of sugar mixtures than any method based on the quantitative use of Fehling’s solution. In view of the fact that the chemical activity of many enzymes is increased by the presence of other enzymes, no attempt was made to separate, even approximately, the individual enzymes from the various mixtures obtained. The total product was tested in its action towards suitable optically active substrata, and in view of the well-defined selective action of enzymes, the method seems justifiable. As far as possible, substrata were used which would readily react and would give large polarimetric differences. The optical activities were determined with an instrument displaying a tripartite field and sensitive to y}, of a degree. Throughout the work standard two-decimetre tubes were used; these were provided with a water-jacket and all the determinations were made at 20°C., accurate to y°. As a rule the sodium-flame was used, except in special cases where, on account of the unavoidable turbidity of the solutions, the incandescent light was substituted. Some difficulty was occasionally experienced in filtering the solutions successfully, but this was overcome by shaking with finely- divided ignited silica, a method which we found did not introduce any experimental error. For the purposes of the present investigation only the examination of the enzymes capable of reacting with carbohydrates and glucosides 1. Ree. trav. chim. Pays-Bas, Vol. XTX, p. 1, 1900. SUCROCLASTIC ENZYMES IN BETA VULGARIS — 261 was necessary, and accordingly in the first place the general nature of the soluble enzymes present in the leaves of the adult plant was studied. The plants used weighed on an average about 650 grams. The leaves were detached at the junction of the stem, and after being finely divided, were macerated with water in a sterilised mortar. The pulp thus obtained was mixed with a large excess of water (two litres), and kept in a thermostat at 30° ©. for five hours, during which time the mass was kept thoroughly _ mixed by means of a powerful mechanical stirrer. The liquid was then filtered under pressure and the bright-red filtrate rapidly diluted with _ four times the volume of alcohol. The mixed enzymes were thus obtained ‘in the form of a grey precipitate, which was filtered off, washed with queous alcohol and dried in a vacuum. The dry product gave very little ganic residue on ignition, and was devoid of any action on Fehling’s on even on prolonged boiling. The solubility in water was slight, “gram of the substance requiring about 700 c.c, of water to effect mmplete solution at 25°C. The aqueous solution, which became turbid on the addition of traces of alkali, showed practically no activity when examined in a two-decimetre tube. This result was unexpected, but ‘se ms to be due to compensation, as enzymes of opposite activity were af er ards shown to be present. In testing for the presence of probable enzymes, standard solutions of the active substrata were mixed with excess of the solid enzymes, and the mixture sterilised by the addition of a few drops of toluene. The initial specific rotation was determined without delay, after which the liquid was kept in a thermostat at 30°C., polarimetric readings being taken every twelve hours. In each case a control experiment, in which no enzyme was used, was conducted under parallel conditions, and results were only accepted as positive when these control solutions showed no alteration in rotatory power. ‘The following table shows the “i st principal results obtained : Enzyme Substrate (a) initial (a) final Diff. Be IL. Invertese ...Sucrose * ... + 666° ... + 644°... 2-2° a °2. Emulsin ...Amygdalin .. — 344° .. — 344°... nil Mca? 3. Diastase =... Stareh ods - eee a EP se 4. Maltese _,..a-methyl-ghicoside +157°5° ... + 1284° .... 291° 5. Lactase — ... Lactose see + 63-1° ere 0-3° *In view of the possibility that the enzyme action might be inhibited by the hydrocyanic ade (ase from amygdalin, other substrata were afterwards used but a negative Welt was In the case of Experiment 3, the dilute starch paste used was rapidly liquefied and gave a dextro-rotatory filtrate. The concentration of 262 BIO-CHEMICAL JOURNAL dissolved matter was estimated by evaporation of an aliquot part of the solution and weighing the residue dried at 100°C. In this way it was shown that the specific rotation of the solution gradually decreased from +- 108°, when the first reading was made, to the constant value + 52°. This ultimate complete conversion into glucose through the intermediate formation of a more highly rotatory compound is in agreement with the idea put forward by Payn! that the hydrolysis of starch in the joint presence of diastase and maltase passes through the following stages :— Starch —> Dextrine —> Maltose —> Glucose The chemical activity of the leaf enzymes used in the above experiments was somewhat disappointing, few positive resulis being obtained. This seems to be due to the method of preparation and purification which would yield only the more soluble enzymes. A similar result has already been obtained by Brown and Morris? in a study of the enzymes present in foliage leaves. It seems in fact to be a general experience that careful filtration of the original aqueous extract is not desirable in the preparation of enzymes as the product obtained displays very little reactive power. Better results are, as a rule, given by straining the liquid extract of the macerated tissues through fine muslin, and a similar method was therefore adopted in the case of the leaves of the adult beet. PREPARATION OF MIxEpD ENZYMES FROM THE LEAF The leaves were macerated with water and extracted in the ~ thermostat as already described, the resulting liquid being strained through several folds of fine muslin, On adding a large excess of alcohol to the turbid filtrate, a brown sludge separated from which the supernatant liquid was decanted away. After washing well with absolute alcohol, the insoluble matter was filtered under pressure and washed with 50 per cent. alcohol until the washings were inactive and ceased to reduce Fehling’s solution. The purified precipitate was then diluted with water so as to give a mixture containing 1 per cent. of the dry enzymes, and the liquid was rendered antiseptic by the addition of a little toluene. A filtered sample was optically inactive and did not reduce Fehling’s solution. This method of obtaining the enzymes suspended in water neces- 1. Comp. Rend., Vol. LIII, p. 127. 2. Trans. Chem. Soc., Vol. LXII, p. 604, 1893. SUCROCLASTIC ENZYMES IN BETA VULGARIS — 268 __ sitates a modification in the preparation of the test-solution. Where possible, 20-per cent. aqueous solutions of the substrata were prepared, and 25 c.c. introduced by means of a pipette into a standard 50 c.c. flask, which was then diluted to the mark with the homogeneous enzyme sludge. The control solutions were similarly prepared, the dilution to half the original concentration being of course made with water. The experimental solutions were filtered through several baryta filters and the optical rotations determined without delay. All the solutions were kept in the thermostat at 35°C., the rotations being determined at regular intervals. The following positive results were obtained :— Substrate used c (a) initial (a)”"” final difference @methylgiucoside ... 9995 ... + 1570° ... + 1306° 2. 27-4° ss: Buerose sa 10-006 dea + 66-2° ... + 314° ... °° 348° Inulin 7 POS tn hs SB 88 4-2° Starch ati — oa — = + 60° ... — ‘The results confirmed those already obtained with the purified enzymes, but the actions were much more rapid. Although marked optical = differences were observed in a few hours the experiments were in each ease continued for twenty days. The joint presence of maltase and diastase was confirmed, while inulase and lactase were again shown to be absent. In this case, however, evidence was obtained that invertase was present, as shown by the following figures :— Experiment—Concentration of sucrose = 10-0060, initial (a)?” = + 66-2° final (a)? = + 31-4° Control— 7 ; = 10-0060, initial (a)? = + 66-6° final (a) = + 63-2° ie 3 The result was verified in a duplicate experiment, but in no case was complete inversion obtained. EXAMINATION oF THE ENzymMEs FROM THE Stem AND Roor ReGIons After removal of the leaves, the roots were separated from the short stem region and the enzymes isolated as described above in the form of an aqueous sludge. Prolonged washing with dilute alcohol was, of course, necessary to completely remove sugars and also the red colouring matters. In addition, the bulky solid residue left in each case on the muslin filter after extracting the macerated organs with water was My utilised in the following manner:—The material, after being washed a with water, was spread on sheets of filter paper and dried in a current of a " -.. ' wer. : J ra 264 BIO-CHEMICAL JOURNAL air at 30°C. After several days’ treatment, the brittle residue was powdered finely in an agate mortar, again digested with a large excess of water, filtered and dried. In this way we were able to avoid the extensive decomposition which results when attempts are made to desiccate the tissues without in the first place removing all soluble organic matter. A similar method has been used by Brown. and Morris in demonstrating the presence of diastase in foliage leaves’. We thus obtained four preparations, viz.:—stem-sludge, solid stem, root- sludge, and solid root, all of which were found to be capable of promoting hydrolytic changes. The average yields obtained were :— Weight of stem used .. 133 grams. Weight of solid stem obtained ... 6-5 grams. Weight of root used ... 520 grams. Weight of solid root obtained ... 19 grams. As usual each preparation was found to be inactive and to be deyoid of any action upon Fehling’s solution. In preparing the test solutions the method already described was adopted, that is, an aliquot part of the solution was diluted to half the concentration with the stem or root sludge. In the case of experiments with the solid stem or solid root, half a gram of the powdered tissues was added to 25 c.c. of the test solution, which was then diluted to 50 c.c. with water. The control solutions were subjected to a similar dilution without the addition of any enzymes. Sterilisation was effected by the addition of a little toluene or chloroform, and heating was conducted in the thermostat at 30° to 35°C. The optical changes were complete in a few hours, but the values given below were those obtained after seven days’ treatment. Test ror INVERTASE Substrate = sucrose (‘ Kahlbaum’). ¢ (initial) = 5-000 c (final) = 2-500 Stem sludge ... initial (a) = + 66-10° ad final (a) = — 128° Solid stem. » (a = + 66-80° wo (a a seer Root sludge... » (ay = + 66-10° sii » (ay = + 618° Solid root ss, » (a) = + 66-10° pes » (ay = + 620° A well marked difference therefore exists between the results obtained with the root and stem preparations. In the former the optical changes observed were small and did not differ in any marked degree from those shown by the contro] solutions. It would thus appear that while invertase is present in the stem region it is absent in the root. The result agrees with the general 1. Loe. cit. SUCROCLASTIC ENZYMES IN BETA VULGARIS — 265 organs” during the period in which reserve material is being laid down. Our observation is, however, at variance with those obtained by ~Gonnermann and Stoklasa.! Test ror Emvtsin ie ae Substrate = B-methylglucoside ¢ (initial) = 4-060 ¢ (final) = 2-030 a Stem sludge ... _ initial (a) = — 335° or final (ap = — 22-5° = elites lw OM ae. » (@)y = — 221° Root sludge. » (ae = — 335° a » (aj = — 22-7° - Solid root oes » (ap? = — 335° “ul » (a) = — 25-1° ‘The above results were obtained twenty-four hours after commencing i experiments ; meanwhile the control solutions suffered no alteration E ‘rotatory power. As in each case the liquids reduced Fehling’s 28 actively after treatment with the enzymes, the results are a | accepted as indicating the presence of emulsin in all the preparations. As = Ee : a confirmatory test, salicin was substituted for S-methylglucoside as a seach substrate. In each case the result was the same, the specific rotations of the solutions diminishing about 14° in twenty-four hours, and the presence of both glucose and saligenin was detected in the resulting liquids. Test ror MALTASE Maltase was found to be present in both the root preparations, but was absent in the stem as shown by the following typical results :—- ele ‘Substrate = a-methylglucoside ¢ (initial) = 4-9992 ¢ (final) = 2-5005 —— Bolid root... initial [aR + 156-0" final [af + 1400° diff. = 160° = Solid stem... Ps + 156-0° hes + 154°, 0-6° = Substrate = Maltose (Kahlbaum) ¢ (initial) = 5-0010 —_¢ (final) = 2.5005 A Solid root ..._— initial fa” + 1321° final [a + 968° diff. = 353° . Solid stem... - + 132-1° ” + ie", = «(3-7° * In using maltose as a substrate we took the precaution of adding a minute trace of caustic soda to the solution in order to promote mutarotation. The optical changes observed are therefore not due to stereochemical alterations in the sugar. deat) ’ 1, Czapek's Biochemie der Pflanzen, Bd. I, p. 375. 4 266 BIO-CHEMICAL JOURNAL Test ror DrasTase Substrate Starch solution c (initial) approx. 0-50 Stem sludge ... no initial a taken oes final [ap + 440° Solid stem... ‘ " aes ” + 32-0° Root sludge ... = ” asi a + 240° Solid root... “ 9 at i + 28-0° The final concentrations were determined by evaporation of a measured volume of the solutions and weighing the residue dried at 100°C. All the products reduced Fehling’s solution actively even in the cold. We are in the meantime unable to explain the fact that the end values obtained are lower than those calculated for glucose; no 8-glucose was present as the rotations remained permanent on adding a trace of alkali. The concentration would of course only be approximate, but any experimental error thus introduced would be insufficient to account for the discrepancy between the calculated value for glucose and those experimentally found. Possibly inactive products may have been formed, or a partial conversion into other hexoses may have taken place. It will be noticed that in the case of enzymes extracted from the leaf the hydrolysis of starch resulted in the formation of a reducing sugar which displayed the correct rotatory power for glucose. SuMMARY OF THE DistRIBUTION oF SucrocLAstic ENZYMES IN THE ADULT BEET . (1). Znvertase——Invertase was not detected in the carefully purified mixture of enzymes obtained from the leaf. When, however, the enzymes are obtained by filtering through muslin and are afterwards washed free from reducing sugars, invertase is then found to be present. Both the aqueous sludge containing the enzymes of the stem and also the powdered stem contain invertase. Similar preparations from the root contain no invertase. (2). Diastase was found in all the preparations examined. (3). Maltase is present in the leaf and root regions, but was not detected in the stem preparations. (4). Znulase seems entirely absent in the leaf region. Positive results were obtained with all the preparations from the stem and root, but the optical changes observed were irregular. (5). Emulsin—The enzymes obtained from the leaf gave negative results using amygdalin as a substrate; the stem and root preparations on the other hand gave positive results with salicin and #-methyl- glucoside. (6). Lactase was absent in every case. 7 SUCROCLASTIC ENZYMES IN BETA VULGARIS 267 Arremptep Reverstste Zymonysis By THE Action or Beer EnzyMEs The methods adopted in the attempted condensation of glucose and fructose by enzyme action were similar to those successfully applied by Croft-Hill' in the enzymatic synthesis of maltose from glucose. As sucrose is incapable of forming an osazone, and as no suitable insoluble derivative is known, the separation of which would serve to detect the formation of the sugar in the presence of monosaccharides, recourse was again had to a polarimetric method of following the reaction. The plan adopted was to subject concentrated sterilised solutions of invert sugar to the action of the various enzyme preparations, polarimetric readings being taken at intervals. Change in rotation cannot, of course, in itself at be considered evidence of sucrose formation. Other factors may play a a « i including the possible self-condensation of both glucose and fructose. experiments were therefore extended by hydrolysing the products and ascertaining if the optical value for invert sugar was reproduced. This joint evidence of a fall in laevo-rotatory power due to enzyme action, and a subsequent increase to nearly the initial value of hydrolysis, would justify the conclusion that condensation of the hexoses had taken place. In the absence of positive results indicating self-condensation of glucose or of fructose this double change in rotation would be significant of the presence of sucrose, or at all events, of a glucosidic compound containing both glucose and fructose. Before commencing our observations several preliminary experiments were necessary. The preparation of a suitable solution of invert sugar, displaying the maximum rotatory power, presented some difficulty. In the first place pure crystallised glucose and fructose (‘ Kahlbaum’) were dried until constant in weight, the former at 110° C., and the latter in a vacuum. Equal weights of each sugar, accurate to 1 milligram were then dissolved in water in a standard flask and made up to the mark at 20°C. After allowing ample time for mutarotation to take place, the rotations were determined in a two- decimetre tube (t = 20°C.). The result was, however, unsatisfactory, as the values obtained were not very uniform and were invariably too low. This is shown in the following table : — (ce. for glucose = 20-0010) . 0° 9° sa \¢. for fructose = 20-0013) (@), = — 21-2 (c. for glucose = 20-0015) . ; 2. efor fructose = 20-0006) (2), = — 11 (¢. for glucose = 20-0006) ai ig s ic. for fructose = 20-0003) (2), = — 168 1. Loe. eit, 268 BIO-CHEMICAL JOURNAL As the correct value for invert sugar is (a), = — 247° it would appear that this method of directly weighing out the sugars is inapplicable, probably owing to the difficulty in obtaining fructose in an anhydrous condition. Although the solutions referred to above were used in preliminary experiments, we prepared our invert sugar solutions by the method recommended by Maumené.' Pure sucrose (Kahlbaum) in quantities of 150 grams was dissolved in water (250 c.c.), and heated in sealed flasks at 106° C. for fifty hours. The resulting liquid gave (a) = — 241° calculating on the complete conversion into glucose and fructose. The effect of hydrolytic agents on this solution was then studied in order to ascertain if any sucrose remained unaltered. A test portion was diluted to half the concentration with water containing varying amounts of hydrochloric acid, and the optical changes on heating observed. It is of course well known that hydrochloric acid, even in dilute solution, readily decomposes fructose, and consequently suitable conditions of acid, concentration, and temperature had to be determined which would suffice to hydrolyse sucrose and yet be without action on fructose. Using solutions containing 5°3, 2°6, and 13 per cent. of acid the laevo-rotation of the invert sugar diminished steadily when the liquid was maintained at 50°C. At 20°C., however, although a similar fall in laevo-rotation was recorded in the case of the strongest acid solution, a constant value was ultimately obtained. On the other hand, the solution containing 2°6 per cent. of acid remained unaltered in rotatory power when kept for six days at 20° C. This result proved the complete absence of sucrose, and gave the desired conditions for carrying out a polari- metric test for sucrose formation in the subsequent experiments. In the first place the precipitated enzymes obtained from adult leaves were used, but, as was expected from the feeble activity of the preparation, no positive results were obtained. Even after an interval of two months the optical activity of an invert sugar solution was found to be quite unaltered by the action of the enzymes. Recourse was there- fore had to the more active enzyme preparations previously referred to as the ‘stem sludge’ and the ‘ root sludge.’ Only small optical changes were, however, observed, but these were in the right direction, and were supported by the evidence of hydrolysis. The results of one typical experiment may be quoted :— Initial concentration of invert sugar solution = 40-0100 Concentration when diluted with stem sludge = 20-0050 Initial specific rotation of the mixture = — 24-7° After 350 hours’ action at 35° = — 21-2° Decrease in laevo-rotation due to enzyme action = 3-5° Increase in laevo-rotation due to subsequent hydrolysis = 2-1 1. Journal des fabricants de sucre, Vol. XX X1,p. 46. ba EIS ee in. et de SUCROCLASTIC ENZYMES IN BETA VULGARIS 269 Control experiments showed that the enzymes were without action on either glucose or fructose solution alone. It would thus appear that the glycolytic enzymes described by Stoklasa are without action on concentrated glucose solutions. The above data represents about the average result obtained in five different experiments, and _ eorresponds with the formation of approximately 4 per cent. of sucrose. _ The addition of maltose did not effect any appreciable improvement in _ the process and, moreover, prevented the hydrolysis being studied. - It must be admitted, even if the above results can be correctly _-_-—_—aseribed to sucrose formation, that the magnitude of the changes are E i small. It appeared possible that this might be due to alteration in the nature of the associating enzyme once the storage of sucrose had ceased, we, i: and, consequently, in another series of experiments, young plants, which had not begun storing, were used as the source of the enzyme preparations. _ The isolation of the mixed enzymes was carried out as already described, but no differentiation into leaf and root region was made, the entire plants ____ being macerated and extracted. The sludge finally obtained consisted of a mixture of 445 grams of the mixed enzymes diluted to a litre with _ The following experiment showed that the sludge possessed associating powers : — 70 cc. of invert sugar solution giving (a) = — 23°7° were introduced into a standard 100 c.c. flask and made up the mark with the homogeneous sludge. A portion filtered at once gave in a one-decimetre tube the rotation a, = — 686°. The solution was then kept at 35° C. in a thermostat for a week, when it was found that the rotation had diminished to — 595°. The subsequent optical changes were only small, and the constant value observed was a = — 570°. The total change in a therefore amounted to + 116°. Expressed in specific rotations the change becomes (a)®” = — 23°99 —> (a)®” = — 19°89, and is, moreover, in the right direction. The subsequent hydrolysis was carried out by diluting a test portion to half the original concentration with water containing sufficient hydrochloric acid to give a 2°6 per cent. acid solution. The initial rotation was (a)®* = — 19°3°, and on stand- = ing at 20° this gradually altered to the constant value a (a) = — 227°, the actual change in a being — 0°99°. This result ae can only be explained on the assumption that a glucosidie product had been formed possessing a less laevo- or more strongly dextro-rotation than invert sugar, and which is, moreover, capable of undergoing hydrolysis to give an equimolecular mixture of glucose and fructose. Assuming = ae = a te is" A ee a... 270 BIO-CHEMICAL JOURNAL that sucrose is the only glucosidic product thus formed, the result indicates the production of about 6 per cent. of the disaccharide. The above observation was controlled by a duplicate experiment in which a sample of the invert sugar solution was diluted with water in place of the enzyme sludge and kept in the thermostat for an equal time. No appreciable — alteration in rotatory power was, however, observed even after three months. It was also shown that the sludge was without action on glucose or fructose solutions alone; only in the joint presence of both sugars were any optical alterations observable. These results are summarised below :— Nature of Experiment Length of tube Initial Final Behaviour on in decimetres ae Ane A hydrolysis Invert sugar and enzyme 1 — 6-86 — 575 16-6 — 555° —> — 654° sludge Control sol. of invert 1 —14-43 —14-37 O-4 No change sugar Glucose (20%) and 2 20-21 20-13 O-4 No change enzyme sludge Fructose (20%) and 2 —34-65 —34-80 O-4 No change enzyme sludge The column marked A contains the optical changes expressed in percentages, and it will thus be seen that a marked difference exists between the first experiment and the subsequent controls. The result was verified in a duplicate series of experiments in which the treatment with the enzymes was continued for three months: once wore the control solutions gave perfectly negative results, but the invert sugar solution underwent similar alterations to those quoted above :— Initial (a) = —24-05° Final (a) = — 18-10°, Diff. = + 5-95°. Change of rotation on hydrolysis (a) =—i81° -> —,27°2°, A slight irregularity will be noticed in the permanent end value obtained on hydrolysis. This we are in the meantime unable to explain, but nevertheless it is evident that the rotation changes are of the same nature as those discussed above, and are in agreement with the idea that about 6°6 per cent. of sucrose had been produced in the reaction. It is of course premature to claim the above results as a successful bio-chemical synthesis of sucrose, but it is difficult to find any other explanation of the optical changes other than that already suggested, viz., that the aldose and ketose combine to form a glucosidic derivative, and sucrose is the only compound of this type now known. Se ae ee ee ee ee ee py a NP on Sees ~_-\i as . = oe. (pM oue, ad ay eee ad P SUCROCLASTIC ENZYMES IN BETA VULGARIS 271 Discussion OF THE ReEsvu.ts vi earns soneiderations of interest to the plant physiologist accruing from - this work group themselves around the following heads : — (a) The topography and work of the enzymes. as (6) The synthesis of a glucosidic compound, presumably sucrose, by the action of beet sludge on invert sugar. i (ec) The difference in magnitude of the results obtained in vivo as fe coe geeg with that in vitro. ex (a) 1. The topography of the enzymes here worked out may be Leaf Stem Root + + 0 + on + - + wi 0 - 0 7 + 0 + + A’ significant tie in oe enzyme distribution is the absence fi of invertase from the beet root. Kastle and Clark! found that a x a inulin- and starch-producing plants such as the artichoke .. and the potato, even in the tubers, when the inulin and starch were undergoing storage, invertase was present in larger quantity than either inulase or diastase. In the beet root, when sucrose is being stored the appropriate enzyme for its formation and hydrolysis is absent. Since in the artichoke and potato respectively the inulin and starch are formed in loco, the conclusion to be drawn, on analogy, in regard to the _ beet seems to favour the hypothesis that sucrose is not formed in loco in _ the root, but is only stored there after undergoing translocation as sucrose _ from the other organs of the plant. % iN (a) 2. The varied associations of the enzymes in the different plant = are is a striking point. To take the case of diastase, which occurs in all paris of the beet, it is found that in the leaf it is associated with invertase and maltase, in the stem with emulsin in addition, while in the root a third variation in the environment of activation of diastase is produced by its association with maltase, inulase and emulsin. The questions which naturally suggest themselves are as to whether the diastatic activity is affected by the different environment in each case, --—s and as to whether a given combination of enzymes may not inhibit the e appearance of some other enzyme which @ priori might be expected to be present. This latter bears on the absence of invertase in the root in an 1. Amer. Chem. Journ., Vol. XXX, p. 422, 1003. 272 BLO-CHEMICAL JOURNAL environment containing an association of diastase, maltase, inulase and emulsin. That some interaction of the enzymes of an acceleratory, retardatory or inhibitory nature occurs is quite in agreement with the doctrine propounded on the result of experimental research by several investigators?, (a) 3. Besides the differences in the environmental spheres of activation resulting from the varied association of the enzymes themselves in the different plant organs, other factors enter to complicate the result. For example, the associated enzymes of the leaf are subjected to different light conditions from those in the root; in the leaf to alternation of light and darkness, in the root to continuous darkness. That light affects the results in the case of invertase action is shown from the recent researches of Kohl,? who has found that in darkness inversion gives place to the opposite process sooner than in light. Girard’s discovery® that the amount of sucrose in the beet leaf is much increased towards evening may bear directly on this point. Other factors affecting the result are, the presence of products of hydrolysis, differences in the osmotic pressure in the cells of various organs, and differences in the reactions of the cell contents. In the last two cases the ‘intensity factor’! would be affected, on the one hand by alteration in the concentration of the enzyme and on the other from the acceleration or retardation of the enzyme action due to increased or diminished acidity. (b) 1. Of the experiments on reversible zymolysis the first series made with pure enzyme extracts of beet leaves on invert-sugar substrates gave no positive results. It is, however, a common experience that pure enzyme extracts are less active than sludges. This was strikingly shown in the negative results obtained in testing for invertase by the action of the leaf extracts on sucrose, whereas, when the sludge was substituted, invertase was proved to be present. The presumption is that by the action of the leaf sludge on invert sugar, reversible zymolysis would have taken. place. The second series of experiments with stem sludge on invert sugar gave figures corresponding with the presence of 4 per cent. of sucrose—a_ result to be expected from the ascertained presence of invertase in the stem. The third series with the sludge of seedling plants gave the higher figure (6 per cent. sucrose), indicating the greater activity of the plant at the beginning of the storage as compared with the diminished activity of 1. Bayliss, The Nature of Enzyme Action, p. 657, and references. 2. Abstr. Bot. Centr., Vol. CVITI, p. 137, 1908. 3. Compt. Rend., T. XCVIT, p. 1305, 1883. 4. Visser, Zeit. f. physik. Chem., Vol. LIT, p. 283, 1905. BA “a a : i “ pie. 3 Fa 4. 2 SUCROCLASTIC ENZYMES IN BETA VULGARIS 278 the older plant at the end of the storage period. The reasons for regarding the products as sucrose have already been stated. (b) 2. That the cane-sugar stored in the beet root is formed from antecedent monosaccharides by reversible zymolysis in the organs containing invertase (viz., in the leaf and stem), and thence translocated as such seems highly probable. The alternative view that the sugars travel downwards as monosaccharides and are subsequently condensed into the disaccharide! meets the diffusion difficulty. The absence of invertase from the root as here shown, and the results of Strohmer’s researches proving that practically no reducing sugars occur in the root, while, according to Girard?, sucrose is present in all parts of the plant in the earliest stages ___ f development, militate against this view. (6) 3. In relation to the translocatory difficulty in the first view, reference may be made to the researches of Hanstein® and Puriewitsch* on the diffusion of disaccharides in the maize endosperm, to Peklo’s recent discovery® that the sieve tubes of the beet serve as_ the sugar-conducting channels, and to Pfeffer’s dictum on the great regulatory faculty of the cell cytoplasm in relation to the materials to be diffused. (6) 4. That the metabolism of the sucrose during the second spring to supply the young shoots takes place in the stem® is supported by the fact that invertase is found in that organ. (c) 1. The apparently relatively unlimited quantity of disaccharides capable of being formed in the living plant is in marked contrast to the small results obtained experimentally in vitro. This is not surprising when it is remembered that the reaction is presumably a reversible one. _In vitro the equilibrium stage attained is permanent owing to the fact that the reaction products are not removed, while in the plant the equilibrium phase is only momentary owing to the continual removal of the products as they are formed. Maquenne, Compt, Rend., 1. CXXI, p. 834, 1805, Compt. Rend., 'T. CLL, 1887, Flora, p. 419, 1894. Ber. d. Bot. Gea., p. 206, 1896. Bot. Centr., Vol. CVIIL, p. 239, 1908. Strohmer, loc. cit. ere ep 274 THE OUTPUT OF ORGANIC PHOSPHORUS IN URINE b By G. C. MATHISON, M.B., B.S. (Mexn.), Sharpey Scholar. From the Physiological Laboratory, University College, London (Received April 22nd, 1909) The existence of organic phosphorus compounds in normal urine has often been asserted and as often denied. In a previous paper (1) I have shown their undoubted existence. The present investigation deals with the quantity of organic PO, excreted in the urine of healthy persons on an ordinary diet. The results obtained by many previous workers are un- reliable owing to the employment of unsuitable methods. Ehrstrom (2), Gumlich (3), Keller (4), Le Clere and Cook (5), and older workers such as Lépine (6) and Zuelzer(7) attempted to determine organic P,O, by methods entailing titration of urine with uranium acetate. This method gives a value greater than inorganic, indeed sometimes greater than total P,O,, so that it is valueless for the determination of organic P,O,. — Le Clere and Cook (5), whose dictum is quoted with approval by F. G. Benedict (8), state that there is insufficient evidence of the existence of organic phosphorus, despite the fact that in twenty-four hours’ urine from a dog they obtained a difference of 0°093 gram P,O, between total and inorganic, in rabbits a difference of 0°055 gram. Even though one agree with these workers that the method they employ is not sufficiently exact to afford evidence of the existence of organic phosphorus, one cannot agree that it is sufficiently exact to afford evidence against such existence. . Oertel (9) was the first to use a sound method of estimating organic phosphorus. He precipitated phosphates by means of calcium chloride, and determined organic PO, in the filtrate. This was evaporated to dryness, fused with KOH and KNO,, precipitated with ammonium molybdate, dissolved in ammonia, re-precipitated with magnesia mixture, incinerated and estimated as pyrophosphate. The process is thus very lengthy and involves several manipulations. Oertel obtained values for the output of organic P,O, in twenty-four hours ranging from 0°12 gram (5 per cent. of total P,O,) to 0°03 gram, (15 per cent, of total P,O,). He considers 0°05 gram to be the usual ~ ORGANIC PHOSPHORUS IN URINE +275 _ quantity. Mandel and Oertel (10), employing the same method, found in the twenty-four hours’ urine of three individuals an average of 0°024 gram organic P,O,, equal to about 2 per cent. of the total. Bornstein (11) made some observations on the output of inorganic and organic PO, on an ordinary diet and on plasmon. He estimated organic PO, in the filtrate after barium chloride, by a method somewhat similar to that of Oertel. His results are here summarised :— Percentage of Nitrogen Total P,O, Organic P,O, P,O; as Organic Average of l4days ... one 14-0 2-09 0-058 2-8 Highest values sins — 14-2 1-82 0-16 8-8 Bornstein himself considers that there is some mistake in the highest gee, and rejects them in his average. Bock (12) used methods entailing uranium acetate titration, but also fin some cases estimated total P,O, by Neumann’s method, and inorganic P 2U, with calcium chloride or barium chloride. In rabbits he found as much as 0°29 gram of organic P,O,, equal to 11 per cent. of total, in _ twenty-four hours’ urine, in cats from 0°04 gram, 26 5 per cent., up to 0-20 gram, 11 per cent. The present observations were carried out on the urines of healthy 3 individuals on an every day diet during January, February and March. . All these persons followed sedentary occupations. The estimations were -——s garried out by the methods described in a previous paper (1). 4 Samples of urine from different individuals gave the following values : — Tanne I—InorGanic ann OrGanic Puosruorvs Grams P,O, in 100 c.c. urine ” Tnorganic P.O Percentage of Total P,O, PO, pee cee PO, as By difference In filtrate Organic ° 8. 0-092 0-086 0-006 0-004 55 K (0-124 O117 0-005 0-005 40 re : (0-121 . p (0-148 ( 0-145 (0-003 5-0 | ? (0-145 , 0138 (0-007 0-007 ps: M | 0-008 0-090 0-008 O-O10 9-0 a : > . “ee “ee 0-009 Since these results were considerably higher than those usually cited, further observations were made on the daily output of different individuals, usually over a period of several consecutive days. 276 BIO-CHEMICAL JOURNAL Taste [I—Ovrrevr or Organic PO, ry 24 Hours Grams P,O, : : wf ’ Subject Day Quantity a Po, OF. FOr One Po," M. act.25 = I 1320 — 3-00 0-303 10-1 _ Ir 1030 — 2-30 0-155 6-7 _ i Il 1000 — 2-43 0-180 75 — a IV 1240 — 2-75 0-148 5-4 ~ ie V 1070 _ 2-45 0-225 9-0 — Average — — 2-58 0-202 77 _— M. aet.25 I 1200 16-44 2-41 0-168 7-0 6-8 II 1140 12-77 1-90 0-114 6-0 68 Il 1200 15-28 2-45 0-120 4-9 6-2 ve IV 1050 14-59 2-05 0-121 6-2 Tl : V 1500 15-90 2-05 0-180 78 6-9 i VI 1050 13-46 2-31 0-140 6-0 65 Average — 14-74 2-19 0-140 6-3 6-7 L.aet.27. I 1100 10-18 2-10 0-104 5-0 4-9 : I 1230 9-76 1-90 0-041 21 5 ; I 1400 11-02 2-41 0-063 2-5 4-6 Average — 10-32 2-13 0-068 3-2 49 D. aet.40 = 1850. 16-63 2-84 0-27 9-5 5-9 a II 1150 14-78 2-61 0-01 0-4 5-6 uj Iu 2050 16-43 2-77 0-15 55 5-9 Average oe 15-94 2-59 0-14 51 58 P.aet.30 =I 1700 13-71 2-32 0-223 10-0 59 Organic P,O, averaged 0°15 gram per diem, equal to 62 per cent. of total P,O,. The highest output was 0°35 gram, the lowest 0:04 gram. It will be noticed that the N : P,O, ratio was fairly constant in any one individual, but that it varied greatly in different individuals. The values obtained for organic phosphorus are considerably higher than those cited by most other workers, even by those who employed accurate methods. This difference must be ascribed in the latter cases to individual variations in output. : Tue Errecr or INGEesrion or GiycerorpHosPHoRIC ACID Although the subject was not on a rigid diet, it was thought worth while to try the effect of adding a large amount of organic phosphorus, in the form of glycerophosphoric acid, to the diet. For several days an approximately similar diet was adhered to, except that on one day glycerophosphoric acid was added. ‘Two series of observations were made on the same subject. ORGANIC PHOSPHORUS IN URINE 277 Tasie Ll]—Krrecr or Incestion or GrycerorHospHoric Acip (i) Glycerophosphoric acid (Merck) containing 1-44 grams Organic P,O,, 0-075 grams Inorganic P,O, added to diet early on sixth day. . Subject Day Nitrogen Total P,O, Organic P,O, 20s = ma -~‘Maet.25 LIV 14-99 2-24 0-146 6-4 6-5 fat (average) » Vv 13-46 2-07 0-140 6-0 65 ” Vis 13-6 2-76 0-165 6-0 438 ae vil 13-77 2-29 0-162 7-0 6-0 Si (ii) Sodium Glycerophosphate, containing 2-4 grams Organic P,O, and 0°125 grams Inorganic ____ PO, added to diet on second day. oo I 14-2 2-47 0-168 6-8 5-7 bs Ii* 10-44 3-73 0-113 3-0 2-8 ae a Il 10-55 0-16 O-174 8-0 4-9 i IV 14-63 2-74 0-150 5-3 5-3 ‘The increase of organic P,O, is well within normal variations—no significance attaches to it. The same might be said of the increase of total P,O,, but for the marked alteration in the N : P,O, ratio. It is obvious that a great part of the ingested SAvcarcullinaphite has been _exereted as inorganic phosphate; it is probable that a considerable portion was not absorbed and would be found in the faeces. The experiments of Bergmann (13) are of some interest in this connection. He injected into a dog subcutaneously several grams of organic P.O, in the form of glycerophosphoric acid. He found a marked increase in the inorganic P,O,, none in the organic. He used titration methods whieh would only show large changes. The increase in inorganic P,O, was so great, however, as to leave no doubt that the glycerophosphoric acid had been decomposed here without intervention of alimentary ae processes. It has been asserted that many organic phosphorus compounds are absorbed as such. To test the probability of this assertion, I have subjected sodium glycerophosphate solutions to the action of active preparations of pepsin, of trypsin, and of fresh pancreatic juice, both with and without enterokinase.' The solutions were incubated for weeks at 39°C. Inorganic phosphates were estimated at the beginning and at intervals during the experiment. In no case was any increase in the inorganic phosphates found; the glycerophosphate remained unchanged. . 1, The juice was obtained from dogs after injection of secretin. It was used without enterokinase because some authors have asserted that enterokinase destroys the lipase present. 278 BIO-CHEMICAL JOURNAL It is probable, therefore, that the ingested glycerophosphate in the experiments detailed above was absorbed unchanged. As the glycero- phosphate used was synthetic it does not follow that natural glycero- phosphoric acid is unaffected by digestive processes. Tue Errecr or Exercise This was investigated on two occasions. The urine was collected over four or five days, an approximately regular diet being taken during this period. On the second day a sharp twenty mile walk was taken, on the other days no exercise beyond leisurely walking a couple of miles. The walk was followed on both occasions by slight stiffness, but beyond this no fatigue was felt. Taste LV —Errecr or EXercisé Subject Day Quantity Nitrogen Total P,O, Organic P,O, Percentage P,O; _N as Organic PO; M.aet.25 I 1440 15-10 2-769 0-181 6-5 5-5 e: * 1550 15-23 2-81 0-168 5-9 54 Fy Itt 1200 13-96 2-58 0-114 4-4 61 a IV 1320 16-01 2-93 0-146 5-0 5-4 V 1510 14-30 2-71 0-110 41 53 5 I 1360 13-09 2-18 0-163 1-4 6-0 = II* 1710 14-01 2-44 0-171 7-0 57 Me Il 1450 15-48 2-70 0-120 47 57 . IV 1250 13-94 2-67 0-134 5-0 5-2 *Twenty mile walk during first half of this day. These figures do not show any increase of organic P,O, after exercise. The diet was not sufficiently rigid to enable any deductions to be drawn from the nitrogen and total P.O, figures. No statement can as yet be made as to the origin of the organic phosphorus of urine. As far as can be gathered from the present results and from a long series of observations, on which Dr. Aders Plimmer is at present engaged, the quantity of organic P.O, is not affected by food. It is thought that some indication of its origin may be given by investigation of pathological conditions in which gross changes in lymphoid or nervous tissues are present. ORGANIC PHOSPHORUS IN URINE 279 SuMMARY " aT, Organic phosphorus compounds are normally present in the urine. Contrary statements are due to the employment of incorrect methods. 2. In young adults on an sanlikiog diet the organic P,O, was usually more than 01 gram per day. Occasionally it fell titer this, and in one case it reached 0°3 gram. f ,, 3. The percentage of the total PO, present in organic combination anes considerably from day to day. In the cases examined it averaged 6 per cent. of the total. ae 4. The addition of a large quantity of organic phosphorus in the form of glycerophosphoric acid to the diet had no distinct effect on the m output of organic P,O,, while it increased the total P,O; output. _ Glycerophosphoric acid was not broken down by gastric or pancreatic digestion in vitro, so it was probably absorbed unchanged. | 5. In the observations made, vigorous exercise was not followed by increased output of organic P,O,. 6. The N_ : P,O, ratio was fairly constant in any one individual on a fairly regular diet. It differed greatly in different individuals, and also in the same individual when the diet was irregular. REFERENCES Mathison, This Volume p. 233. Ehrstrom, Skand. Archiv., p. 83, 1903. Gumlich, Zeitech. /. physiol. Chem., Vol. XVITI, p. 508, 1894. Keller, Zeitsch. /. physiol. Chem., Vol. XXIX, p. 146, 1900. Le Clere and Cook, Journ. Biol. Chem., Vol. II, p. 203, 1906-7. Lépine, Comp. Rend. Acad. des Sciences, Vol. XCVIII, p. 238, 1884. Zuelzer, Semiologie des Harns quoted by Keller. F. G. Benedict, Metabolism in Inanition, 1907, p. 410. Ocrtel, Zeitech. {. physiol. Chem., Vol. XXVI, p. 123, 1898. Mandel and Oertel, New York Univ. Bull. of Med. Sciences, Vol. I, p. 165, 1901. Bornstein, Pfliger’s Archiv., Vol. CVI, p. 66, 1904-5. Bock., Arch, /. Exp., Path. u. Pharm., Vol. LVIII, p. 236, 1907. Bergmann, Arch. /. Exp. Path. u, Pharm., Vol. XLVII, p. 76, 1901. Plimmer and Bayliss, Jour. of Physiol., Vol. XX XIII, p. 439, 1906. PERE Seesueaeere 280 ON THE RELATIVE HAEMOGLOBIN-VALUE OF THE RESISTANT ERYTHROCYTES DURING THE HAE- MOLYSIS OF BLOOD WITH HYPOSMOTIC SODIUM CHLORIDE SOLUTION, AND ON THE PERMEABILITY OF THE ERYTHROCYTES TO WATER AS A FACTOR IN THE PRODUCTION OF HAEMOLYSIS By U. N. BRAHMACHARI, M.A., M.D., PH.D., Lecturer in Medicine at the Campbell Medical School, Calcutta. (Received May 10th, 1909) In a previous paper! I have pointed out that the dark coloration described by Wright, and obtained by mixing one part of blood with two parts of a progressive dilution of saline does not represent the point of complete haemolysis. This point is obtained in the observations of McCay? and my own observations by mixing one part of blood with two parts of on to aS saline solution. It may, for the sake of convenience, 40 50 be called Wright’s haemolytic point. This point probably represents the stage at which a large number of the erythrocytes undergo haemolysis as the result of osmosis and rupture. The corpuscles that do not haemolyse at Wright’s haemolytic point will be termed in this paper the resistant corpuscles. By quantitatively estimating the amount of dissolved haemoglobin in 20 cb.mm. of the supernatant fluid obtained after centrifugalisation of a mixture of blood and two volumes of 2 ‘saline solution, where 2 is any . x number from 20 upwards, I have made out the curve of haemolysis with hyposmotie saline solutions (see fig. 1). | From the curve below it will be seen that the very beginning of haemolysis starts with an saline solution. Then the degree of haemolysis i N N . N NV sees suddenly increases from 50 to 30 saline. From 30 to a5 = it is somewhat gradual, while from Po upwards it increases very slightly with the higher dilutions. 1. Bio-Chemical Journal, Vol. TV, p. 59, 1909. 2. Ibid., Vol. III, p. 97, 1907. P a 81 x ‘OINGXTU posnjraguoeo oy} Woay pny guvyeusedns oy} Jo “wut “qo OZ UI UIqo[ZoueRY Jo Junowe oq} Suyvuryse Aq UMvrp Suyoq oarno oy) ‘uomNjos [OWN ooursod = < SL oe; 282 BIO-CHEMICAL JOURNAL The fact that some of the erythrocytes haemolyse with higher dilutions of saline than others leads to the conclusion that either they are less permeable to water or they can bear the tension of distension from osmosis better than others, and therefore do not rupture so readily. I have, however, already pointed out that osmosis and rupture alone cannot explain the whole phenomenon of haemolysis with hyposmotie saline solutions, and that one has to take the question of mass action into consideration in explaining it’. The presence of erythrocytes containing partially discharged haemoglobin among the sediment corpuscles goes against the theory of rupture. The relation of the amount of haemoglobin in the resistant corpuscles to the total amount in the sample of blood under examination appears to me from observations in health and disease to have an important bearing, and I would suggest that this be called the relative haemoglobin-value of the resistant erythrocytes. It may be expressed as the quotient obtained by dividing the amount of haemoglobin in the resistant corpuscles by that of the total blood. | The method by which I estimated the amount of haemoglobin in the resistant corpuscles is described as follows:—In all cases the blood was haemolysed with two parts of a saline solution, with which in the case of healthy individuals Wright’s haemolytic point is with certainty obtained. After thoroughly mixing 5 cb.mm. of the blood with 10 cb.mm. of x saline, the mixture is centrifugalised as thoroughly as possible, and then the sediment is washed several times with N saline till the 10 supernatant fluid at the top is perfectly colourless. The sediment is now dissolved in a small quantity of distilled water with the addition of a drop or two of chloroform, and then the amount of haemoglobin is estimated by a Haldane’s haemoglobinometer. In those cases in which _ the amount of haemoglobin in the resistant corpuscles is less than 10 per cent., 10 or 20 cb.mm. of blood is taken and then treated with 20 or 40 cb.mm. of x saline respectively, and the amount of haemoglobin in the resistant corpuscles is then estimated. This number divided by two or four, as the case may be, gives the amount of haemoglobin in the resistant corpuscles of 5 cb.mm. of blood. The accompanying table gives the relative haemoglobin-value of the resistant corpuscles in the blood of some of my students as well as in some cases of anaemia in my wards :— 1. Loe, cit. —_— eeseeees HAEMOLYSIS OF BLOOD TasLe I1.—Heatra Haemoglobin in ie tedanent Corpuscles in 5 cb.mm. of blood ERESESER ANAEMIA _ Tasre I. 3 Haemoglobin in the Resistant Co cles in 5 cb:mm. of blood Taste III Haemoglobin in | the Resistant Corpuscles in 5 cb.mm. of blood Relative Haemo- globin-value of the Resistant Corpuscles 0-336 0-416 0-416 0-391 Relative Haemo- globin-value of the Resistant Corpuscles 0-200 0-133 0-285 0-277 Relative Haemo- globin-value of the Resistant erythrocytes 0-350 0-413 0-467 0-417 Relative Haemo- globin-value of the Resistant erythrocytes 0-571 0-547 283 It will be seen from the above tables that while in health the relative haemoglobin-value of the erythrocytes varies within small limits; in anaemia it varies within much wider limits. Thus, in some cases, it is 284 BIO-CHEMICAL JOURNAL much below the normal, in others it is almost the same as normal, while in others again it is above the normal. In kala-azar it is generally below the normal, while in ankylostomiasis it is above the normal. The forms of anaemia in which this value is increased or diminished and its clinical significance can only be determined by further investigation. PERMEABILITY OF THE ERYTHROCYTES TO WATER AS A FACTOR IN THE Propvuction oF HAEMOLYSIS An explanation may here be offered as to the cause of the differences of the haemoglobin-value of the resistant corpuscles in health and disease. It is possible that the resistant corpuscles are less permeable to water or can bear the tension of distension better than those that haemolyse. This permeability, or the power of resisting rupture, is probably altered in anaemia, being increased in some and diminished in others, while in others again it remains normal. The entrance of water into the erythrocytes may, therefore, to some extent, be dependent upon their specific permeability, and this may be independent of the force of osmosis. So, also, their power of resisting rupture from distension after the entrance of water into their substance may vary in the different erythrocytes. That these are important factors in the phenomenon of haemolysis is borne out by the following facts :— If one part of human blood is mixed with one part of ee saline solution and then treated with two parts of = saline, we find that the amount of haemolysis is much greater than when the pil saline contains 1 per — 10 cent. formol. The presence of formol cannot in any way change the concentration of the salts in the corpuscles, and its action must result either in increasing the resistance of the erythrocytes to rupture from osmotic distension or diminishing the permeability of water. Similarly, again, when blood is allowed to crenate between the slides for twenty-four hours or more, and then treated with = saline soiution, we find that some of them still remain crenated. Now, if crenation were simply due to osmosis, then the corpuscles would swell up and lose their crenation by re-absorption of water when ‘treated with * saline solution. The fact that some of them do not lose their crenation shows that they have become less permeable to water. In other words, along with crenation the outer portion of the erythrocytes undergo some changes, as a result ir. HAEMOLYSIS OF BLOOD wel do not allow the free passage of water into their structure | osmosis. The same is also borne out by the fact that ) gipaiolaer aaa -eceur in the blood in some forms of This cannot be due to any stronger concentration of saline in 1, as in anaemia the organ _ Benes much rt egies increased : m (see Tables IV and V). ee Ryyokeoniina YRAmeaiow 2 Panes se 7 8 48 RTE. SBE #t?; oT : i Hioegiiaeds s lat iogosln od fy 6 - . eae - ia o 4 rs é : radpune (itv ihe ey DY Mea dee tit Se rear 4 Average — 05850% —0-6727% 06727 % © 0-6435% 08772. % = 0-7019% +] o pare sai ‘ cPahae : I 1 ; j : é i os Letriy) & Sei ’ { i ) teu i x | - J ‘ , ‘ Ae WABasni ton aa: ° eVisit ebhiitsd Ae oF ain fry ( Missa! ,uitele Fyrvt aa ita ett eae se Scotk Hs evi Vey ATR (fp hie i v? ; : vvi ) oA GT r H Wis ; ; tate a. a) #) | j ocd ‘ rer (3 ie ait 4f acpi ti th - PAM brati < 286 THE ISOLATION OF THE CONIUM ALKALOIDS FROM ANIMAL TISSUES, AND THE ACTION OF LIVING CELLS AND DECOMPOSING ORGANS ON THESE ALKALOIDS By WALTER J. DILLING, M.B., Cu.B. (Abdn.), Carnegie Scholar in Pharmacology. From the Laboratory of the Institute of Pharmacology, University of Rostock, Germany (Received May 10th, 1909) The literature on the isolation of the conium alkaloids is very limited, and the only treatises requiring mention are those of Harley! and Zalewsky.? Zalewsky’s method consisted in extracting the organs with acidified water, followed by alcohol, and in shaking out the final residue with petroleum ether after making it alkaline with ammonia. This author — has given no quantitative results, and from the fact that he frequently describes scaffold-like crystals as occurring in the final residue, one is inclined to believe that the residue in many cases consisted of ammonium chloride, which gives dense precipitates with phospho-molybdie acid, as he describes. PRELIMINARY OBSERVATIONS On account of the volatility of pure coniine, it is impossible to obtain a satisfactory result unless the base is converted into one of its salts which are not volatile at 100°C. The hydrochlorate of coniine is very suitable for the purpose, as it crystallises in long, double-refracting, silky needles, which are easily recognisable. Coming to the estimation of the amount of coniine present, there is a choice of three methods, namely :—(1) The weight of the residue; (2) estimation of pure coniine by titration with normal acid using suitable indicators, such as iodeosin, haematoxylin, cochineal, lacmoid, or congo red; (3) estimation of the alkaloid or its salts by titration with some precipitating reagent, such as Mayer’s solution. In the following experiments I have used a combination of the first and third methods, and, in applying the latter, I have estimated the amount of the alkaloid by the number of drops from a capillary tube required to 1. Old Vegetable Neurotics, pp. 19 and 80, 1869. 2. Untersuchungen tiber das Coniin. Dissert., Dorpat, p. 17, 1869. CONIUM ALKALOIDS FROM ANIMAL TISSUES 287 titrate the residue which was acidulated with a definite quantity of dilute hydrochloric acid. This method gave in test cases results which were correct to the fourth decimal place in grams. It will be noted that in many of the experiments the weight of the residue and the amount of alkaloid present, as estimated by Mayer’s reagent, do not agree; this is due to the fact that one cannot get a pure alkaloidal residue without going through processes which would entail serious loss of the alkaloid. In such cases the amounts estimated by Mayer’s reagent may be taken as correct. I. Isonarron or ContineE By DISTILLATION WITH AN ALKALI _. The distillation method I have used was that ordinarily employed for such purposes; I have not adopted the method of distilling in hydrogen gas, as I found this unnecessary, also it is not readily applicable to the distillation of animal organs. When coniine hydrochlorate is distilled with sodium hydrate, I have found that a certain amount of ammonia is present in the distillate, and also that the crystals of coniine hydrochlorate re-obtained from the distillate are not nearly so strongly double-refracting as they usually are. If 10 mg. of coniine hydrochlorate is used, the whole of the coniine distils over in the first 12 to 20 c.c. of fluid, and the alkaloid, when estimated by Mayer's reagent, was present to the amount of 9°5 mg. On the other hand, by distilling the coniine hydrochlorate with sodium carbonate, almost no ammonia is produced, and the crystals obtained from the distillate are strongly double-refracting, and, again, when using 10 mg. of the salt the whole of the coniine is contained in the first 20 c.c. of fluid distilled, and this amounted in test cases to exactly 10 mg. Experiments done with sodium bicarbonate showed a more marked production of ammonia but no effect on the refraction of the crystals. When, however, coniine has to be isolated from organic matters there is always found in the distillate a large amount of ammonia, and, on evaporating the distillate with hydrochloric acid, ammonium chloride forms the greatest proportion of the residue. This salt I have removed by treating the residue with a mixture of two parts of absolute alcohol and one part of ether, or with pure chloroform, which is much more satisfactory. The organic matters were distilled immediately. after the addition of the alkaloid. 288 BIO-CHEMICAL JOURNAL (1) Jsolation from Urine The residue, left after evaporation of the neutralised distillate, is usually so large that it requires to be treated two or three times with the above solvents. 200 c.c. human urine f 200 c.c. human urine with 10 mg. coniine . with 10 mg. coniine hydrochlorate hydrochlorate Residue from chloroform ... Double-refracting, needle- ~_ ly double-refracting, like crystals P -like espace and iregular crystal, not double-refracting* Weight of residue iss 0-007 gms. a 0-013 gms. ies Estimated alkaloid by Mayer ... 0-0054 gms. ose 0-0083 gms. On boiling Mayer’s ppt. with sodium-hydrate one ... Distinct smell of coniine ... Strong smell of coniine From the above experiments it will be seen that 83 per cent. of 10 mg. of coniine hydrochlorate can be regained from urine. (2) Isolation from Blood Forty c.c. of calf’s blood with 5 mg. of coniine hydrochlorate was distilled with 100 c.c. of water and excess of sodium carbonate. The crystals of the residue being not quite typical, they were made alkaline by sodium hydrate and the freed base shaken out with ether and re- converted into the hydrochlorate. The crystals obtained were long, transparent, double-refracting needles. A solution of these in acidified water gave dense precipitates with Dragendorff’s and Rohrbach’s reagents, and also with phospho-wolframic and phospho-molybdic acids, and, on boiling with lime water, a strong smell of coniine was. given off. This shows that a sufficient amount of 5 mg. can be regained from 40 c.c. of blood for identification purposes. (3) Isolation from Liver 100 horse’s liver 100 gms. horse’s liver 100 horse’s liver th 10 mg. coniine with 10 mg. coniine with 10 mg. coniine hydrochlorate hydrochlorate hydroe ’ Residue from chloro- Long, needle-shaped = Long, needle-shaped § Pure, long, needle- form crystals, strongly crystals, strongly shaped Be ba ve double-refracting double-refracting strongly ble- refracting Weight of residue... 0-0045 gms. 0-005 gms. 0-0085 gms. Estimated alkaloid by Mayer ee oes 0-00416 gms. 0-005 gms. 0-0083 gms. On_ boili pt. with sodium hydrate ... Strong smell of coniine Strong smell of coniine Strong smell of coniine These results demonstrate that one can obtain back from liver not less than about half and not more than 83 per cent. of the coniine salt. 1. As to what the other crystals present were, one cannot give any opinion. Certainly they did not seem to affect the titration by Mayer’s reagent. at a BEY as ne re a a ae . > CONIUM ALKALOIDS FROM ANIMAL TISSUES 289 (4) Isolation from Spleen} Seen ’s spleen wi = Ae horse’s sp with . coniine hydro- chlorate Residue from chloroform 0 oat ave , transparent, double- eae needles Weight of residue Wee ood eee si 0-0085 gms. On boiling Mayer’s ppt. with sodium hydrate ... Strong smell of coniine Thus 83 per cent. of alkaloid can also be regained from spleen. Il. Exrracrion or Contrne By TREATMENT WITH ALCOHOL AND SvuBsEQUENTLY SHAKING OvT witH ETHER The finely minced organs containing the alkaloidal hydrochlorate _ were extracted three times with fresh portions of absolute alcohol. After filtering, the alcohol was evaporated off and the residue again extracted with fresh alcohol; this was filtered, evaporated, and the residue treated with water, any insoluble matter being removed. The watery solution was then made alkaline with sodium hydrate, and the freed base removed by shaking out with ether. In earlier experiments, the ether was evaporated at a low temperature, but in later cases it was acidified with hydrochloric acid before evaporation, in order to obtain the crystalline hydrochlorate. Urine was treated by the same process except that it was evaporated to syrupy consistence with excess of hydrochloric acid, before adding alcohol. When dealing with fatty organs, the ether frequently separated as a muddy layer. This trouble has been avoided either by previous shaking of the still acid water with ether or by evaporating the muddy ether which has separated from the alkaline water with hydrochloric acid, and extracting the residue with water, filtering, and evaporating the watery solution. (1) Eztraction from Urine 100 c.c. human urine 100 c.c, human urine with 10 mg. coniine with 10 mg. coniine hydrochlorate h orate Residue from ether sav .. Double-refracting, needle- Double-refracting, needle- like crystals and some like crystals and some Weight of pe rte oe matter Estimated alkaloids by Ma jun 0-0046 gms. ége 0-0054 gms. On boili Mayer's ppt. with sodium hydrate Ome .« Nosmell of coniine* .-» Distinct smell of coniine The minced spleen containing the alkaloid was first treated with tannic acid in presence Cl to precipitate with albumin as much of the fatty matter as possible. The filtrate was then rather easier to distil. Tannic acid tot Guabealie of EICL does not precipitate coniine salts. 2. The probable explanation of this is that since I had been on with pure coniine just before and as the nose becomes rapidly insensible to this smell, I had failed to detect the slight odour which must have been present. 2 =- 290 BIO-CHEMICAL JOURNAL (2) Extraction from Blood Fifty c.c. of calf’s blood with 5 mg. coniine hydrochlorate. The blood was first acidified and coagulated by heat, the coagulum was filtered off, and the filtrate evaporated to a syrup and treated as with urine. Residue from ether.—Long, double-refracting, needle-like crystals, which, when dissolved in acidulated water, gave a dense precipitate with Dragendorft’s reagent, but only slight precipitates with phospho-molybdic and phospho-wolframic acids, and Rohrbach’s reagent; on boiling the solution with excess of lime water a distinct smell of coniine was evolved. (3) Extraction from Liver The following table will give some idea of the results which are obtained when the coniine is isolated as a free base. It also shows that by the method of extraction one may isolate substances from liver which show alkaloidal reactions. =e ie eeaged ners prt fhe Pig’s liver 80 gms. without alkaloid Slight yellow ppt. Faint white ppt. Faint yellow ppt. - Wb je + 10 mg.coniine HCl Good yellow ppt. Slight white ppt. Slight yellow ppt. - + 10 mg.coniine HCl Good yellow ppt. Faint white ppt. Faint yellow ppt. In the following results the ether was acidified before evaporation. = horse’s liver with go horse’s po 10 mg. coniine Tichyadcoblacete Residue from ether A ... Transparent, needle-like ... gpoemeine 5 double- | crystals, slightly double- gp Arr ner arranged refracti along with in sheaves some resinous matter Weight of residue eos ee 0-003 gms. PP — Estimated alkaloids by Mayer... 0-0021 gms. ob 0-00125 gms. On boiling Mayer’s Ppt. with sodium hydrate .»» Faint smell of coniine .«-. Slight smell of coniine (4) Eatraction from Spleen ~ One hundred grams horse’s spleen with 10 mg. coniine hydrochlorate. Residue.—Long, double-refracting, needle-like crystals with some resinous matter. Weight of residue.—0°009 grams. Estimated alkaloid by Mayer.—0°0029 grams. On boiling Mayer’s precipitate with sodium hydrate.—Distinct smell of coniine. —————— —— es le CONIUM ALKALOIDS FROM ANIMAL TISSUES 291 (5) Eatraction from Kidney _ Fifteen grams rabbit’s kidney with 10 mg. coniine hydrochlorate. Residue.—Long, double-refracting needles, which, when dissolved in acid water, gave dense precipitates with alkaloidal reagents and gave off a strong smell of coniine when boiled with lime water. III. Precrerratioy or THE ALKALOID BY MEANs oF PuosrHo-WotrraMic Acip As phospho-wolframic acid gives precipitates with coniine hydro- chlorate in presence of hydrochloric acid up to dilutions of 1 : 10000, the following method was adopted for the isolation of the alkaloids by this means:—The organs containing the alkaloid were coagulated by heat, the coagulum removed, and the filtrate treated with phospho-wolframic acid and some dilute hydrochloric acid till complete precipitation had occurred. The precipitate was filtered off, washed with water containing some phospho-wolframic acid, and then drained free of excess of fluid. The partially dried precipitate was rubbed up in a mortar with excess of barium hydrate and the freed alkaloid extracted with absolute alcohol. After being acidified with hydrochloric acid, the alcohol was evaporated off and the residue extracted with chloroform, filtered, and evaporated, when one ought to obtain the hydrochlorate of coniine pure. Any barium hydrate which dissolves in the aleohol may be removed by means of carbon dioxide or by extracting the residue of chlorides as above with chloroform. Urine was treated directly with the acids, but it is safer to precipitate it twice, since the first precipitate is very copious. (1) Precipitation from Urine 100 c.c. human urine 100 c.c. human urine with 10 mg. coniine with 10 mg. coniine hydrochlorate hydrochlorate Residue from chloroform --- Double-refracting needles ... Double-refracting needles Weight of residue te ove 0-008 gms. 7 0-006 gms. alkaloid by Mayer ... 0-0042 gms. ai 0-0046 gms. On boiling Mayer's ppt. wi sodium hydrate... «» Faint smell of coniine ... Distinct smell of coniine (2) Precipitation from Blood Fifty ¢.c. calf’s blood with 5 mg. coniine hydrochlorate. Residue.—Long, needle-like crystals, strongly double-refracting. Weight of residue.—0°004 grams. Estimated alkaloid by Mayer.—0'0017 grams. On boiling Mayer's precipitate with sodium hydrate.—Faint smell of coniine. 292° BIO-CHEMICAL JOURNAL (3) Precipitation from Liver 100 horse’s liver 100 gms. horse’sliver 100 horse’s liver with 10 mg. coniine — with 10 mg. coniine wi nydrcodlonenie hydrochlorate hydroc lorate Residue from chloro- Long, needle-shaped Double-refracting, orga double- form crystals in sheaves ; needle-like fs crystals strongly double- crystals er on fatty refracting matter Weight of residue... — 0-008 gms. 0-005 gms. Estimated alkaloid by am Mayer 0-0037 gms. 00025 gms. 0-0005 gms, ' On boiling Mayer's ppt. with sodium hydrate Distinct smell of coniine Smell of coniine Faint, primer: recognisable smell (4) Precipitation from Spleen One hundred grams horse’s spleen with 10 mg. coniine hydrochlorate. Residue from chloroform.—Long, double-refracting, needle-like crystals, | Weight of residue.—0'008 grams. Estimated alkaloid by Mayer.—0'0021 grams. On boiling Mayer’s precipitate with sodium hydrate.—Distinct oni of coniine. (5) Precipitation from Kidney Fifteen grams rabbit’s kidney with 10 mg. coniine hydrochlorate. Residue from chloroform.—Double-refracting, needle-like crystals which, dissolved in acid water, gave dense precipitates with alkaloidal reagents and gave off a strong smell of coniine when boiled with lime water. ITV. Precrprration spy Kravut’s REAGENT This process proved in my hands quite useless for coniine, as the precipitate was of such a nature that it passed readily through a filter, and, again, it was found impossible to get rid of the iodine completely. CONCLUSIONS ON THE ISOLATION OF CONIINE It will be observed that, in the case of the distillation, the best results are in three cases 83 per cent., while with the alcohol and ether process and precipitation method the results vary considerably, and in most instances the results are considerably below 50 per cent. If one makes an average of all the figures one finds that the average return by distillation is 65°7 per cent., while the other two methods only show half — 1. This liver proved very difficult to filter after boiling. CONIUM ALKALOIDS FROM ANIMAL TISSUES 293 this amount. Again, the distillation method can be carried through in at most three hours, while the other two occupy eight hours at least, and the alkaloidal salt obtained in the end is much purer in the case of “distillation, as may be surmised by the small differences between the weights of the residues and the amounts of alkaloid found by Mayer's e ‘ reagent. Taking everything into account, only one conclusion is possible, og that, for practical purposes and satisfactory results, distillation is the 7 . most valuable method for isolating coniine from tissues. Action or Livine CEetts on ContTINE Im order to ascertain whether living cells had any power of decomposing coniine or in any way interfering with its recognition in the animal organism after death, finely minced liver containing coniine d lorate was mixed with 100 c.c. normal saline solution, to which had been added 1 c.c. of chloroform and 1 c.c. of toluol to prevent fe = a8" C. for a definite period. The alkaloid was re-isolated by distillation. gms. rabbit's 60gms. rabbit's 30 gms. rabbit's 190 gms. horse’s 100 gms. horse’s bade with 20 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg. coniine hydro- coniine hydro- coniine hydro- coniine hydro- coniine hydro- chlorate chlorate chlorate chlorate _ Time on water bath ... 16 hrs. 18 hrs. 24 hrs. 12 hrs. 18 hrs. Residue from ane pee double- Long needles, Some long, A few needle- A few long, *releacting slightly double- like crystals, double- double- refracting not double- refracting a crystals refracting needles refracting crystals = Weight of residue .- 00065 gms. 0-003 gms. 0-010 gms. 0-002 gms. 0-002 gms. Estimated alkaloid ‘i _ Mayer... «... 00065 gms. 00021 gms. 0-0037gms. No ppt. 0-0004 gms. “7 with oh cop Sg lem ... Distinct smell Faint smell of Distinct smell No smell Faint smell of es of coniine coniine of coniine coniine ; _ All these livers were re-distilled with dilute sulphuric acid, the s distillates neutralised with sodium carbonate and evaporated to dryness. | To the residue absolute alcohol and excess of concentrated sulphuric acid by. were added, and in all the above cases a very strong smell of butyric ether Ye was evolved, mixed in some cases with the smell of acetic ether. A test experiment, done with liver alone, gave a distinct smell of butyric acid, zs but not so powerful as that of the above cases. Conelusions.—If one takes the average amount of alkaloid which may be regained from liver by distillation as 58 per cent.—a low estimate— it will be seen from the table that there is a remarkable loss of alkaloid 294 BIO-CHEMICAL JOURNAL after the cells of the liver have been allowed to act on it even for a short time. ‘The powerful smell of butyric acid would seem to suggest that the coniine may probably be broken up into this substance. Whether this suggestion is correct or not, I am unable as yet to say, but I hope to follow this up shortly. ¥ Action oF Decompostne Trssurs on CoNIINE To ascertain whether decomposing tissues had any influence on coniine, minced liver with the alkaloidal hydrochlorate was mixed with 100 c.c. of normal saline solution and left in a water bath at 38° C. for a definite period. The alkaloid was isolated by distillation. 60 gms. rabbit’s 160 gms. horse’s 100 gms.horse’s 100 gms. horse’s 100 gms. horse’s liver with 10 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg. liver with 10 mg. coniine hydro- coniine hydro- coniine hydro- coniine hydro- coniine chlorate chlorate chlorate chlorate Time on water bath ... 18 hrs. 24 hrs. 24 hrs. 24 hrs. 24 hrs. Residue from chloro- : form +» «se Some trans- Dae. Detiasemnent Needle a lle nt refracting needles, crystals, not ouble- needles, not some double- needles, not refracting typical of double- refracting typical of crystals, coniine refracting, and not coniine which were hydro- others not deliquescent jac very deli- chlorate, c quescent and very and very deliquescent deliquescent Weight of residue - 0-009 gms. 0-004 gms. 0-01 gms. 0-003 gms. 0-009 gms. Estimated alkaloid by c Mayer aoe - 0-005 gms. 00021 gms. 00-0025 gms. 90-0021 gms. 0-0008 gms. Boiling Ppt. with sodium hydrate ... —? Peculiar smell, —) Peculiar smell, Strong and resembling resembling peculiar ammonia ammonia sm ; ammonia — Conclusions.—It will be apparent that the maximum amount of alkaloid found after twenty-four hours was 2°5 mg., but the crystals of the residue were only partially double-refracting and, at the same time, very deliquescent, while the smells evolved in all cases were ammonia-like. In Experiment III it should have been easily possible to obtain the ; uranium nitrate reaction for coniine, but this failed; in Experiment I, however, a positive reaction was got, probably since the quantity of liver was small and the time for decomposition short. I am, therefore, of the opinion that no coniine was present in the distillate from the latter four experiments, but that the substance obtained was a body possessing — 1. On treating the Mayer’s precipitate in this case with sodium carbonate, carbon eee = uranium nitrate, and shaking up with toluol—no red colour was obtained in the liqui 2. The same reaction in this case, however, gave a red colour in the toluol which would indicate that coniine was present. re CONIUM ALKALOIDS FROM ANIMAL TISSUES 295 some of the characters of coniine hydrochlorate, while lacking others. _ The suggestion is possible that many of these crystals were cholin bydro- chlorate, which is double-refracting and very deliquescent. This salt also erystallises in needles, and when boiled with sodium hydrate a smell of trimethylamine is given off which would correspond closely to the smell perceived above. The cholin hydrochlorate must have passed through the __ filter in a deliquesced state along with the chloroform extract. IsoLATION OF CONHYDRINE , The isolation of conhydrine proved more difficult, on account of the fact that it cannot be distilled from watery solutions, even when these are saturated with calcium chloride or under pressures as low as 10 mm. of _ mercury. Conhydrine is also less soluble in ether than in water, and _ chloroform, in which it is readily soluble, gave unsatisfactory results, so _ that it was discarded in favour of repeated shakings with fresh ether. 3 = conhydrine forms a very deliquescent hydrochlorate, and was _ therefore isolated as the free base, care being taken to evaporate solutions ____ of this at low temperatures. With reference to the process of precipitation, ___ phospho-wolframic acid is again the only suitable method, but its delicacy only reaches to dilutions of 1 : 1000. The extracts of the organs were ‘therefore evaporated to small bulk before adding the reagent, and urine was first precipitated with lead acetate in presence of hydrochloric acid, in order to avoid bulky precipitates with the reagent. For the estimation of the amount of alkaloid recovered, phospho-wolframic acid was _ substituted for Mayer’s reagent, as the latter is not sufficiently delicate. (1) Extraction from Urine by Alcohol and Ether 5O0c.c. human 100c.c. human 100c.c. human 100c.c. human 200 c.c. human : v wi urine with urine with urine with urine with — 10 mg.conhy- 1l0mg.conhy- 1l0mg.conhy- 10 is ate conhy- 10 mg. conhy- = drine rine rine rine + drine ether - Double- Needle-like, Double- Double- Double- 4 i double- refracting, refracting refracting, needles and refracting needle-like needles and needle and a the erystals crystals flat, oblong plate -like ae: having crystals, crystals, the one angle cut having an latter having vps out angle cut an angle cut ea? es out out ss Weight of residue - 0-008 gms. 0-005 gms. 0-012 gms. 0-015 gms. 0-014 gms. Maid by phosp.- 7 wolf. acid ... 0-0012 gms. 0-0017 gms. 0-0045 gms. 0-0075 gms. 0-0037 gms. hydrate ... Distinct smell of Distinct smell Strong smell of Very strong Strong smell of eonhydrine of conhy- conhydrine smell of ydrine rine 296 BIO-CHEMICAL JOURNAL (2) Extraction from Liver ih: Alcohol and Ether . s. horse’s liver "4 horse’s liver 100 horse’s liver sas 10 mg. conhy- Ome. conhy- with 10 mg. Mus 1 drine Residue from ether ... No shies! Neill fuadiale Double- pe or double- crystals of ob bad form with one . . the angles cut out Weight of residue --- 0-002 gms. 0-003 gms. 0-009 gms, 3 Estimated alkaloid by ; i phosp.-wolf. acid . 0-0005 gms. 0°00075 gms. 0-0037 gms. On si sang, Be ppt. with ydrate - Faint smell of Distinct smell of Strong smell of conhydrine conhydrine conhydrine (1) Precipitation from Urine by Phospho-wolframic Acid 100 c.c, dog’s urine 100¢.c. humanurine 100 ¢.c. human urine — with 10 mg. conhy- with10mg.conhy- with 10 a conhy- drine drine Residue from chloroform Colourless, imperfect, Imperfect, double- double- double-refracting refracting crystals Ima, dub crystals Weight of residue --- 0-004 gms. 0-006 gms. ~ 0-009 gms. Estimated alkaloid by are -wolf, acid =... 0-0015 gms. 0-0025 gms. 0-006 gms. ppt. with : pariirey i al -.. Faint smell of conhy- Distinct smell of Strong smell of conhy- drine . eonhydrine = — drine (2) penises from Liver by Phospho-wolframie Acid oo horse’s liver 100 gms. horse’s liver 100 gms. cow’s liver — 10 sheng conhy- with gin conhy- with 10 mg. conhy- drine Residue from chloroform lar, mee ive No crystals Tra’ t needles, racting crystals double-refeaaia and very deli- quescent Weight of residue --» 0-004 gms. 0-003 gms. 0-005 gms. Estimated alkaloid by phosp.-wolf. acid... 0-0012 gms. 0-0025 gms. _ 0-0025 gms. On boili pt. with sodium hydrate ... Distinct smell of Distinct smell of oe smell of - conhydrine conhydrine ydrine CONCLUSIONS ON THE ISOLATION OF CONHYDRINE The figures given above show that, by neither of these two methods are results got which are in any way constant. The higher figures are those obtained from later experiments and show probably about the limit — attainable by these processes. One can, however, say that conhydrine can | be isolated from both urine and liver in quite appreciable amounts, even when the quantity added consists of only 10 mg. in 100 c.c. or grams of substance. 0 ees eS eee et CONIUM ALKALOIDS FROM ANIMAL TISSUES 297 Action or Livinc Ceits on ConHYDRINE ee “The method used was the same as that described for coniine. The alkaloid was extracted by alcohol and ether. 100 gms. cow’s liver srs horse’s liver a . horse’s liver with 10 conhy- 10 conhy- 10 mg. conhy- dino dtine drine Time on water bath... 18 hrs. 38 12 hrs. Residue from ether ... Thin gummy layer, No crystals A few double- no refracting a like crystals a Weight of residue ... 0-011 gms. 0-003 gms. 0-012 gms. alkaloid by _ phosp.-wolf. acid ... 0-00076 gms. 0-001 gms. : 0-0037 gms. On bol : i . Doubtful smell of Faint smell of conhy- Distinct smell of eonhydrine drine conhydrine Action or Decomposinc Tissues on CoNHYDRINE _ The alkaloid was treated in the way described for coniine and isolated by means of the alcohol and ether process. 100 gms. horse’s liver with 100 gms, horse’s liver with 10 mg. conhydrine 10 mg. conhydrine Time on water bath oe 18 hrs. 18 hrs. Residue from ether --- Gummy skin, no tals ... Resinous residue and some = double-refracting, oblong crystals Weight of residue _ 0-008 gms. hs 0-008 gms. Estimated alkaloid by ’ phosp.-wolf. acid i. 60-0015 gms. eee 0-002 gms. sodium 5 sega ..» Faint smell of conhydrine ... Distinct smell of conhydrine Conclusions.—The above results show that, on account of the fact that the method of isolation does not give constant returns, it is impossible to draw any definite conclusion as to whether or not conhydrine is affected by the action of living cells or decomposed tissues. IsoLaTION oF PssuD0-CONHYDRINE On account of the small amount of this alkaloid which I possess, I have limited this research to a very few experiments, which, however, show fairly satisfactory results. The difficulties encountered in the isolation of pseudo-conhydrine are the same as those detailed under conhydrine, and they were avoided by the same methods. Pseudo- conhydrine cannot be distilled from watery solutions. ar 298 BLO-CHEMICAL JOURNAL (1) Extraction from Urine by Aleohol and Ether 60 ‘p human oe 100c.c. human urine 200 ¢.c. human urine wit mg. o- with 10mg. pseudo- with 10 * sanlericles yes conhydrine conh Residue from ether _... Double-refracting, Double-refracting, alae needle-like small, needle-like small, needle- crystals crystals Weight of residue _... 0-014 gms. 0-006 gms. 0-007 gms. Estimated alkaloid by phosp.-wolf. acid ave 0-0062 gms. 0-0035 gms. 0-0017 gms. On boiling ppt. with sodium hydrate --. Strong smell of Distinct smell of Faint smell of pseudo-conhydrine —_ pseudo-conhydrine —_ pseudo-conhydrine (2) Extraction from Liver by Alcohol and Ether 100 horse’s liver 100 gms. horse’s liver — with 10 mg. pseudo- with 10 pseudo- conh ae conhytenas Residue from ether ... eA No distinct crystals --- Small hair or needle-like crystals, double-refracting Weight of residue... ave 0-002 gms. -. 0-007 gms. Estimated alkaloid by phos.- wolf. acid ... bed ue 0-0005 gms.* «» 0-004 gms. On boiling ppt. with sodium hydrate... soe ‘ No smell of alkaloid .-- Distinct smell of pseudo- , conhydrine (1) Precipitation from Liver by Phospho-wolframie Acid 100 grams horse’s liver with 10 mg. pseudo-conhydrine. Residue from chloroform.—No crystals. Weight of residue.—0°002 grams. Estimated alkaloid by phospho-wolframic acid.—0°0015 grams.. On boiling precipitate with sodium hydrate.—A faint smell of pseudo- conhydrine. Conclusions.—The results indicate that, with urine, it is possible to isolate 35 per cent. of the alkaloid added, while, with liver, about 40 per cent. can be regained. The single experiment done with phospho-wolframic acid shows a fairly good return for that method, namely, 15 per cent. Action or Decomposinc TissvuEs oN PsEUDO-CONHYDRINE One experiment was done with this alkaloid by leaving 10 mg. in 100 grams of horse’s liver in a water bath for twelve hours, as described for coniine. The alkaloid was extracted by alcohol and ether. Residue from ether.—A few, irregular, double-refracting crystals in a gummy matrix. 1. Liver difficult to filter. CONIUM ALKALOIDS FROM ANIMAL TISSUES 299 2 Weight of residue.—0°003 grams. Estimated alkaloid by phospho-wolframie acid.—00007 grams. On boiling precipitate with sodium hydrate.—No smell which resembled pseudo-conhydrine. No definite conclusion can be drawn from this, since, in one case, as small a quantity was regained from fresh liver, although that was due to difficulties in carrying out the process. ScuMMARY 1. The most satisfactory method for the isolation of coniine from wall animal tissues is that of distillation. é. ifiy 2. Coniine appears to be decomposed both by the action of living i a cells and by decomposing tissues. » a: Conhydrine and pseudo-conhydrine can be isolated from animal tissues by extraction with alcohol and by precipitation with phospho- wolframic acid, but these methods do not give sufficiently constant results to allow of any definite conclusions being drawn as to the action of living cells or decomposing tissues on these poisons. I have, finally, to thank Professor Kobert of Rostock for procuring for me the materials used in this research and for his kind assistance and advice. I wish also to acknowledge my indebtedness to Professor MacWilliam and Professor Cash for their courtesy in advising me with regard to the arrangement of the results. 800 SOME OBSERVATIONS UPON THE ERROR IN THE OPSONIC TECHNIQUE’ By ERNEST E. GLYNN, M.A., M.D. (Canras.), M.R.C.P., Lecturer in Morbid Anatomy and Clinical Pathology, University of Liverpool, Pathologist, Royal Infirmary, Liverpool, anv G. LISSANT COX, M.A., M.B., B.C. (Canran.), Holt Fellow in Pathology, University of Liverpool. From the Department of Pathology, University of Liverpool (Received May 14th, 1909) INTRODUCTION Anyone who has compared the figures obtained for the opsonic index of the same serum as estimated quite independently by two observers, will be aware that the difference between their results is often considerable even after extensive experience of opsonic technique, and when the precaution has been taken of enumerating many leucocytes. We have recently calculated a large number of indices three times, employing tubercle bacilli and staphylococci, and consider that a detailed account of the experimental errors in our own work, together with a résumé of the errors obtained by other workers, may be of some interest to those engaged in this line of research. There also arises the larger question: is the Wright technique, even including all its most recent modifications, so hopelessly inaccurate that no deductions whatever can be drawn from it? | In a paper at present in the press? we have detailed the results of calculating eighty consecutive indices, three times, ie., 240, with staphylococcus, and forty consecutive indices, three times, i.e., 120 with tubercle. The staphylococcus indices were calculated on seventeen different days, and the tubercle indices on ten different days, almost invariably twelve indices, or four sets of three, on each day. The technique of Wright and Douglas was adopted. One half of the indices calculated were the result of comparing the degree of phagocytosis obtained with different sera, but the same leucocytes, i.e., they were opsonic indices; the other half the result of comparing the degree of phagocytosis obtained with different strains of leucocytes, but the same sera; these we have called ‘ Cytophagic Indices.’ 1. The greater part of this paper formed a portion of a Thesis for the degree of M.D. Cantab. 2. Journal of Pathology and Bacteriology, Vol. XIV, No. 1. ERRORS IN THE OPSONIC TECHNIQUE 301 _ According to the Wright School, the latter indices should always be unity, because the “phagocytic power of corpuscles from different sources ’ _ is‘thesame."' We have demonstrated that the inherent phagocytic power of corpuscles is not always the same, and the cytophagic indices in our series of observations vary from about 1°2 to about 17. This fact, however, does not affect the present question, viz., the _——s aeeuracy of the Wright technique for measuring phagocytosis, and we have included all our figures, both of opsonic and of cytophagic indices, __—s im order to increase the number of observations available for statistical ; ‘The Method by which the indices were calculated is briefly as follows :— A sample of serum and of corpuscles were drawn from three normal i “men, *G,’ ‘L,’ and ‘ A,’ i.e., three samples of each were prepared. Twelve _ separate phagocytic mixtures were put up from these, and the counts of D ‘ined enabled us to calculate twelve indices, three opsonic indices for __ *A’ and for ‘L’ respectively, and three cytophagic indices for ‘A’ and : for ‘L’ respectively; the washed leucocytes and serum of ‘G’ furnishing the control. ae The method by which these indices were calculated from the various _ combinations of sera and leucocytes is tabulated below. Tasce I, Grvine THE various ComBINATIONS OF WasHED Levucocytes oF ‘G,’ ‘ L,’ ee ap ‘A,’ UsED IN THE Puacocytic Mrxrures, AND THE MEeTHODs oF CALCULATING - i tHE Inpices, ‘G's’ Serum or LevucocyTes BEING USED As ‘ ConTROL’ Source of Serum in Source of Leucocytes Number of phagocytic mixture in phagocytic mixture phagocytic mixture A. A.) : A. A. ) “** . A. G. 2. A. L. 3. ia G. A. 4. . Ga. G.) 5 : G. G.) : 4 G. L. 6. i . 7 i + : 7 L. G. 8. - L. L.) L. Li 9. Fractions used for the caleulation of ‘ A’s’ three opsonic and three cytophagic indices :— A's opsonic indices ane te ; ; A's cytophagic indices eid ; : ; | 1. Practitioner, May, 1908, 302 BIO-CHEMICAL JOURNAL The same principle was adopted in the calculations of ‘ L’s’ indices. It is clear that ‘ A’s’ three opsonic indices were obtained from six distinct phagocytic mixtures; therefore, any difference between the three indices is due not only to errors from counting, but also to errors in ‘putting up” the phagocytic mixture, preparing and staining the films. This is a more thorough and practical way of ascertaining the degree of error inherent in Wright’s technique than that of recounting the same slide—a method adopted by some observers. It will be noticed that the counts obtained from phagocytic mixtures 1,5, and 9 are really the mean of two separate estimations. This tends to increase the accuracy of our indices somewhat, and gives us a slight advantage in comparing our errors with those of other writers. The same holds good for the other sets of triple indices. |The reasons for this somewhat complicated method of calculating the indices, together with an example fully worked out with figures, are given elsewhere. The bacterial emulsion was prepared as recently recommended by Fleming.! It is important to note that the washed erythrocytes of these three individuals were not agglutinated by any of the combinations of sera employed. Fleming’ who recently contributed a valuable paper on the accuracy of the opsonie technique from Wright’s* laboratory, states that ‘a diminution in the number of washed corpuscles in the opsonic mixture causes an increased amount of phagocytosis.’ In order, therefore, to eliminate errors from this source and ensure that the relative amounts of normal salt solution and washed corpuscles taken up in the opsonic pipettes were the same in all the triple estimations, the tubes containing them were placed vertically between the palms of the hand and vigorously rolled to and fro immediately before a quantum was removed; thus the corpuscles and salt solution were always well mixed. Ineubation—The phagocytic mixtures, consisting of equal parts of corpuscles, serum and bacterial emulsion in salt solution, were placed for fifteen or twenty minutes in a patent incubator at 37°C. This apparatus consists essentially of a metal box filled with water, into one side of which two horizontal and parallel rows of narrow metal tubes are inserted. Each tube is open to the air in front, but is surrounded by water which is maintained at a constant temperature by a single flame of gas. 1. Practitioner, May, 1908. 2. Fleming, Practitioner, May, 1908, p. 618. 3. Wright, Lancet, 1907. ERRORS IN THE OPSONIC TECHNIQUE 303 It was found, even when the apparatus was most carefully regulated, that the temperature of the tubes in the centre of the upper row was liable to be half a degree C. or more higher than in the periphery of the lower a row, on account of deficient circulation of the water. This defect was completely remedied by constantly agitating the water in the incubator with a rotating paddle. We do not know whether a difference of }°C. between the individual tubes will exert any appreciable effect upon the amount of phagocytosis, but it is advisable that all scientific apparatus should be as perfect as possible, especially in the technique so full of pitfalls as the opsonic technique. The readings of temperature were taken with a microscope from a thermometer graduated in tenths of a degree C. After incubation the contents of the a pipettes were remixed and three films prepared from each one. | The smears were made in the usual way by placing the drop at one , end of the slide and drawing it out with another narrower slide held at an i? angle, great care being taken to make the termination of the smear aa ; rectangular as possible. (Vide diagram.) DIAGRAM OF SLIDE wiTH BLOOD SMEAR. Staining.—The staphylococcus films were stained by Leishman’s method, using accurately measured quantities of stain and distilled water, and rocking the slides at intervals to ensure uniform staining—a very important point. The tubercle films were fixed in saturated corrosive sublimate solution, and stained by pouring upon them boiling carbol fuschin, decolourised in 2 per cent. sulphuric acid, washed with 5 per * cent acetic acid, and counter-stained in methylene blue. Counting.—The number of bacteria phagocytosed by sixty polymor- phonuclear leucocytes were enumerated in two out of the three films made from each phagocytic mixture. Thirty cells were taken from the two borders beginning at the corners B 2 and passing backwards to B1. Thus the same part of every film was examined. All the eosinophiles and all ” polymorphs with indistinct edges owing to damaged cytoplasm were 304 BIO-CHEMICAL JOURNAL eliminated from the counts. Clumps of more than three leucocytes were also omitted. When the batch of films was finished, the figures were added up, and if there was a deviation from the arithmetic mean of the counts of any two duplicate films of more than 5 per cent., another sixty leucocytes were taken from the third uncounted film and an average struck from the three sets of figures. Half the total number of leucocytes counted (120) were selected from one film and half from another, in order to diminish the errors due to improper smearing, unequal staining, or inaccurate counting, and, lastly, because it is obviously advantageous, when there is a marked difference between the figures obtained from the two films, to count an additional sixty from the third film. If the figures when added up were contrary to expectation, recounts were not made, nor were an extra sixty cells counted in the hope that ‘all might be well.’ In fact, no recounts were made except in the case of some half dozen slides, because the counter was interrupted or found his attention wandering. No counts were discarded on the grounds that the films were improperly stained. The usual number of polymorpho- nuclear leucocytes counted was sixty, but occasionally eighty or fifty, according to the strength of the emulsion. The number once fixed by an examination of the first film was invariably the same for all the batch. As duplicate films were used, it is clear that the total number of cells counted from each phagocytic mixture was usually 120, sometimes 180 or 100. Elimination of Auto-suggestion—In order to eliminate any auto- suggestion in counting, all the stained films, usually twenty-four in all, were placed in numbered compartments, the figures upon them being rubbed off. An attendant handed these unnumbered films indiscriminately to the observer, who thus remained ignorant of which films he was counting. Every film was counted by the same individual, E. E. G. STATEMENTS OF THE Wricut Scuoot Recarpinc Errors or TecuniQue Three definite statements have been made by the Wright School regarding the accuracy of the method. 1. In 1907 Wright! said the error ‘in the case of normal bloods in the hands of a good worker ’ is ‘ rarely greater than plus or minus 5 per cent.’ 1. Lancet, p. 427, August 17, 1907. ERRORS IN THE OPSONIC TECHNIQUE 805 2. Next year Fleming,+ who had carefully investigated the matter, concluded that ‘duplicate estimations of the tuberculo-opsonic index of tuberculous patients can be performed, the results differing from each other by less than 20 per cent., except in rare instances (two in fifty-two)’; that is to say, he admits that the limits of error are at least twice as wide as those laid down by Wright. We have examined the figures given on Table VIII, in which thirty- eight duplicate indices are calculated by two observers putting up separate opsonic mixtures; fourteen pairs were from normal and twenty-four pairs (not twenty-six as stated by Fleming) from tuberculous persons. We find that the average difference between the fourteen pairs of normal indices is 0'076, the maximum being 0°29, and between the twenty-four pairs of tuberculous indices 0°068, the maxima being 0°21 and 0°20. The ee average difference between the whole thirty-eight pairs is 0'071; nine ee = ee = a hal out of these, or 25 per cent., differed by more than 10 per cent., and two, or 5 per cent., by more than 20 per cent. _ 3. Fleming stated in 1908 that ‘two practised observers can count same slides and obtain results in almost all cases within 10 per cent.’ a _ Analysing the figures in Fleming’s table, VII, we find that the average difference in forty-one indices obtained by two observers A and B counting the same slides works out at 0°064 per cent. Seven of these differed by - more than 10 per cent., the maximum difference being 20 per cent. The comparison of counts from the same slides is, however, a very unsatisfactory way of testing the accuracy of the method, for all errors associated with putting up the phagocytic mixtures are excluded. Meruops or CatcuLatinc Errors _ The errors in this paper have been estimated in three ways. Method A.—We have calculated the percentage deviation from the arithmetic mean of the two counts of duplicate slides made from the same phagocytic mixture. It has already been mentioned that usually sixty cells were counted on each slide, and that if there was a deviation from the arithmetic mean of more than 5 per cent. another set of sixty cells was counted from the third slide. Method B.—The simplest and most practical way of estimating the degree of error in opsonic work is to compare the difference between indices calculated from the duplicate phagocytic mixtures. We have already alluded to Fleming’s paper, where the average difference between 1. Practitioner, p. 634, May, 1908. 806 BIO-CHEMICAL JOURNAL thirty-eight pairs of indices calculated by two observers from duplicate phagocytic mixtures is 0°07. In our paper three sets of indices have been caleulated from triplicate phagocytic mixtures; in order, therefore, to compare our results with those of Fleming we have taken the mean of the difference between each of the three arranged in pairs. For example, on July 20th the three indices of A were :—(1) 0°778, (2) 0889, (3) 0°825. The difference between 1 and 2 is 0'11, between 1 and 3 is 0°05, and between 2 and 3 is 0°06. Now, 011 + 0:05 + 0°06 = 0°22. The mean of the differences of the three pairs is, therefore, 0°07. Our error so calculated is 0°17 for all duplicate staphylococcus indices and 0°15 for all duplicate tubercle indices, which compares rather unfavourably with Fleming’s figures of 0°07 for tubercle, though it is within his 20 per cent. limit. As previously mentioned, half our indices were opsonic, the other half we have called cytophagic; that is to say, they were a comparison of the phagocytic power of different strains of leucocytes put up with the same serum. For some reason, probably accidental, the error in the cytophagic indices, calculated by Method B, is slightly less than in the opsonic. The figures being—staphylococcus, 0°14 and 0°20, and for tubercle, 0°14 and 0°15 respectively. Method C.—Krrors have been estimated in another way also, by taking the maximum deviation from the mean of the triple estimation. For example, the mean of the three indices 0°778, 0°889, and 0°825 is 0°831. The maximum deviation from the mean is, therefore, in this case 0'058. The application of this method to Fleming’s figures would give 0°035 for the average maximum deviation for the mean. These methods of estimating the error of technique may be rather elementary for a statistician, but they have the merit of being easily understood by the non-mathematical mind. Method B is the most valuable; it deals with indices, not counts, gives the most consistent results, and can most easily be compared with duplicate estimations of other workers. | LS eee ee a ee eee a “4s TT Wn. oe ‘ a al ERRORS IN THE OPSONIC TECHNIQUE 307 TABLE ‘IT, SHowrne Averace DirreRENCE BETWEEN DupticaTe AND TRIPLICATE Inpices or THe Same Serum BuT From Serarate Puacocytic MIXTURES A8 CALCULATED FROM THE Resutts oF DirFERENT OBSERVERS No. of | Approximate Approximate Observer Average indices number number of Organism figures counted per cell .. Two 007... 16 100 2 Tubercle ‘Ais op O10. 6 100 2,3 rf art O12... 6 100 2-0 ; c es O10. 6 100° 2-3 fs Sie ah O15 .... 120 120 825 peri Aa a... 2G, O17 ... 20 120 35 ... Staphylococeus - Strangewayst F.G. & W 036,045 36 ... 50 1-2... Tubercle ey We Cee we he ss al F. G. 051,055 18 ace Eee * Nore.— exact strength of emulsion employed by s workers (in Table VIII) be ascertained, but we assume it is similar to that in Table VII, viz, approximately 2. . pp- 630, 633, May, 1908 + See page 313. } See page 309, Table IIT. Various UnravourasLe Opinions UPON THE ACCURACY OF THE Orsontc TECHNIQUE " Many writers, especially in America, have recently impugned the - aecuracy of the opsonic technique. After working at the tuberculo-opsonic index for several months and eliminating clumping and fragmentation of the bacilli in the emulsion by exposing their culture to direct sunlight, Jeans and Sellards! conclude that ‘the limits of error in our technique, at least, are so great as to _ render the method inapplicable for clinical work.’ It may be added that _ they followed the method of Wright as closely as possible. -____ Moss,? who superintended very extensive comparative tests carried out by three observers simultaneously, comparing the percentage and _ greatest percentage variations from different counts, concludes that ‘ none __ of the present methods of estimating the opsonic content of the blood seem sufficiently accurate to be of practical value.’ _ ~—s«éDr. Bolduan,’ of the Department of Health of the City of New York, ‘a found that, ‘for reasons not yet understood, duplicate and triplicate tests made on the same serum at the same time and under apparently identical conditions often yield widely divergent results.’ Simon‘ speaks of the ‘ phantastic curves’ and ‘absolutely absurd’ results which Wright’s index sometimes gives. Thomas® remarks ‘ that, aside from technical difficulties, the question Bulletin of the Johns Hopkins Hospital, p. 234, June and July, 1907. Bulletin of the Johns Hopkins Hospital, p. 234, June and July, 1907. Long Island Medical Journal, Vol. 1, No. 10, p. 6. Journal of Experimental Medicine, Vol. 1X, No. 5, pp. 488, 489. Journal of American Medical Association, p. 1249, October 12, 1907. Peer 308 BIO-CHEMICAL JOURNAL of personal equation evolved in opsonic determinations is so serious as to practically nullify the value of the method in most instances.’ Potter’ concludes that Wright’s method of estimating the opsonie indices in bacterial infection is hardly accurate enough to compensate for the amount of time involved in its application. Jiigens,* discussing the well-known difficulty of phagocytosis of clumped bacilli, quotes an example where an index was raised from 0°95 to 113 by the inclusion of two out of one hundred leucocytes which had ingested twenty-seven and twenty-nine cocci respectively. W ork of Fitzgerald, Whiteman and Strangeways.—A valuable enquiry into the accuracy of the opsonic index was undertaken by Fitzgerald, Whiteman and Strangeways,® in 1907, on account of ‘the very unsatisfactory and discordant results’ obtained in the pathological laboratories of Oxford and Cambridge Universities. One of the investigators (Whiteman) had received instruction in the technique at St. Mary’s Hospital, London, and had been engaged for a year in the estimation of indices, and was under the impression that: the error in his work was seldom greater than 10 per cent.’ In the first part of their research they made counts and estimated indices from phagocytic mixtures and smears prepared by themselves, and obtained very unfavourable results. The most important of all the statistics are those in Table IV, page 124, which give various opsonic indices obtained by two observers, who put up the opsonic mixture and calculated the indices of the same sera absolutely independently. Two capsules of serum were taken daily from a tuberculous patient, viz., R.1 and R.2, and from two normal persons used as controls, viz., W.1 and W2 and F.G.1 and F.G.2.. They were numbered by a disinterested observer S. to eliminate the unconscious influence on the results of knowing which blood was being dealt with. As a rule these observers F.G. and W. put up the blood within a short time of each other; the washed corpuscles were usually taken from the same individual F.G. For the sake of simplicity we have modified the headings in Table IV and have not quoted all the indices calculated from the various combinations of sera, including those by using S.’s control, and have added columns showing (1) the variations between the duphicats indices of different observers, and (2) the variations between the duplicate indices of the same 1. Journal of American Medical Association, p. 1815, November 30, 1907. 2. Berliner Klinischer Wochenschrijt, p. 641, May 30, 1908. 3. Bulletin jor the Study of Special Diseases, Cambridge, Vol. I, No. 8. ERRORS IN THE OPSONIC TECHNIQUE 809 ers. The sera from R.W. and F.G. were divided into two capsules purposes of convenience, so all four indices on February 4th, for example, ‘in which W.’s serum was taken as control, should be theoretically the same. a TABLE ILI, SHowrne VaRIATION IN THE Opsonic INDEX OBTAINED IN CoMPARISON ia or THE Same Sera BY DirFERENT OBSERVERS e Wik cam LGpecais ie mixtures put u Opsonic mixture put u ao coe & akiedioes anlosinded - |:Vacia- Variations in | Vtia- and indices calculated. be : by F.G tions in indices of tions in by W Capsules of at rors 2 bp “or Capsules of ier moe same same 4 | ~~ serum from observer . observer serum from which indices | Index | F. G Fr G&W Index | which indices | __ ealculated. -G. & W. parr He ay t Control Control Patient CRi CW. | 156 | 959 | 070 | 045 | oo, | O86 | CW. CRI jc C.W.2 1-06 0-20 | 0-95 061 | CW2 CR2 P/ CRI CFG1| 147 | 455 || 006 | 002 | ogg | 141 | CFRGI CRI | GR2 CF.G.2 | 0-95 0-46 | 0-54 0-93 | CFG2 CR2 2 {CRI CW.l | 075 | ogy || O10 | 050 | goog | 085 | CW. CRI Sp oms CW2 | 1-35 0-38 | 0-22 113 | CW2 CR2 2 (CRI CFG1| 109 | 4,, || 062 | 009 | og | 171 | CFGI CRI Re CR2 C.F.G.2 | 1-60 O-1l | 0-42 18 | CF.G2 CR2 . CR. C.W.1 146 | os 0-47 | O72 | 905 0-99 | CW.l CRI a CR2 C.W.2 | 0-89 0-10 | O15 074 | CW2 CR2 | |GR1 CFG1| 090 | gig || 060 | 047 | os | 153 | CFGI CRI | GR2 CF.G2) 1-06 0-17 | 0-33 073 | CFG2 CR2 HOR CW. | O49 | oye | O33 | O13 | oo, | O82 | CWI CRI CR2 CW.2 | 0-95 0-38 | 0-08 087 | CW2 CR2 22 {CRI CW. | 072 | p99 0-25 | 016 | 9.99 047 | CW1l GRI toms CW.s | 1-61 1-14 | 105 056 | CW2 CR2 >/GRI CFG1/ O83 | 44, | O21 | O31 | ogs 104 | GF.G1 CR! GF.G.2 | 1-21 0-17 | 0-69 552 | OFG2 CR2 Total 4:59 12-83 3-25 SUMMARY Average difference between nine duplicate estimations by the same observer F.G.=0'51, by the same observer W.=0'36, and between 36 _ duplicate estimations by the two different observers =0°36. __ These writers also give an exactly similar series of indices, calculated from capsules C.W.1 and C.R.2 and C.W.2 and C.R.1, instead of from capsules C.W.1 and ©.R.1 and C.W.2 and C.R.2. 310 BIO-CHEMICAL JOURNAL We find in this series the figures are, nine duplicate estimations by observer F.G.=0°55, by observer W.=0°40, and 36 duplicate estimations by the two observers =0'45. This table has been analysed at length for three reasons :— 1. It gives the figures which can be most satisfactorily compared Bs, with ours. 2. The figures compared are the indices, i.e., the results of the method, which is a more practical test than statistics of the percentage differences between the highest and lowest counts obtained from various slides, ete. 3. As the indices compared are calculated from duplicate phagocytic — mixtures the errors due to ‘ putting up,’ as well as counting, are included, which is again a more practical test of the technique than a comparison of indices obtained from two observers counting the same slides. The table shows that the average difference between duplicate indices calculated by F.W. and S. is more than twice as great as our own and more than four times as great as that of the workers quoted by Fleming. This inaccuracy may be partly ascribed to inexperience in technique, the - enumeration of only fifty cells and employing too weak an emulsion, points which will be referred to later. Other tables, II, III, XII, give the phagocytic counts obtained for two different capsules of the same blood, and their percentage differences, and also the percentage difference between the highest and the lowest phagocytic counts obtained each day with successive sets of fifty or one hundred cells. Thus on February 4th, 1907, F.G. found two counts from duplicate phagocytic mixtures were 65 and 36 respectively; the difference thus being 29. Now there are three possible ways of calculating the percentage difference of these figures :— I. By placing the lower figure 36 in the seaman: 29 x 100 II. By placing the higher figure 65 in the denominator. 29 x 100 III. Placing the mean of the two figures (50°) in the denominator. 29 x 100 505 = B7-4 It is clear that in I the error is largest and in IT smallest. er ERRORS IN THE OPSONIC TECHNIQUE $11 a Fitzgerald, Whiteman and Strangeways frequently calculate the percentage differences between the figures by placing the lower figure in ____ the denominator, thereby making the error as great as possible. But they have no right whatever to assume that the lower figure is more correct than the higher. As it is impossible to determine which figure is more correct, the only reasonable method would have been to place the mean of the two figures in the denominator (Vide III). Fitzgerald, Whiteman and Strangeways by adopting Method I have made their errors appear a as high as possible. In Tables IT and III, Fitzgerald, Whiteman and Strangeways show “the phagocytic counts obtained for two different capsules of the same blood and their percentage difference.’ For example, on 4th February, 1907, the counts obtained from two different capsules of the same serum eS (fifty cells counted) were :- - a We Capsule I.—36. tak Capsule II.—65. ‘oad Now these writers estimate the percentage difference between these _ two counts as 80°6, in the manner we have described on page 310, but the percentage difference from the arithmetic mean would be, however, 28°7. As all our percentages have been calculated by the latter method, we have re-calculated the figures given by Fitzgerald, Whiteman and Strangeways in their Tables II and III, and can therefore compare them directly with our own. Taste IV, SHowrmnc Percentage DevIATION FROM THE ARITHMETIC MEAN OF Two Puacocytic Counts Opratnep From Two Different CapsULES OF THE _ Same Serum Observer No. of duplicate Organism No. of cells A percent- ; ettatlons counted on each age deviation slide from the arithme- tic mean F. G. ea 22 -» Tubercle ae 50 “ 14:3 w. hee 51 ase fe sai 50 <3. 14-9 E. G. née 30 td e dda 120 et 5-3 E. G. wie 79 ie Staphylococcus ... 120 nv 7:3 On account of the ‘inconsistent results’ obtained by Fitzgerald, Whiteman and Strangeways in their own experiments, a new series of observations was made by them upon eight slides, one from a normal and ; seven from tuberculous persons, prepared in another laboratory, the i: reputation of which should guarantee the opsanic technique being 312 BIO-CHEMICAL JOURNAL ‘unquestionable.’ From these slides several thousand leucocytes were counted in sets of twenty-five by one observer, F.G, The normal slide was_ unknown. In Table XII they give the percentage difference between the highest and lowest phagocytic count obtained on each slide in consecutive — sets of 25, 50, 100 and 500 cells, and. found if averaged for single cells to be 128°77, 70°82, 34°6 424, respectively, the maximum difference in the figures for 500 cells being 9°9 and the minimum 0'1. ‘They state that this table proves clearly that ‘the percentage difference on the results decreases enormously the greater the number of the cells that are counted.’ If the observers had adopted the usual method of calculating per- centage differences these figures would have been much more favourable. However, taking them as they stand, it is clear that if 500 cells are counted their technique will give fairly accurate results, though, of course, no account is taken here of errors due to putting up the phagocytic mixtures. Analysing further the counts of these eight slides, the observers show that the more cells counted the more the figures for the normal and tubercular sera tend to approximate. When the index is based upon 1,000 cells for the normal and control sera, the seven indices are 0°78, 1°04, 0°88, 0°86, 0°109, 0°98, 0°95 (Table XI, p. 136). They point out, further, that ‘if only fifty cells are counted this might in most cases be sufficient to account for the differences recorded between normal and tubercular blood.’ The suggestion obviously is that if sufficient cells are counted Wright’s positive and negative phase would cease to exist. There is considerable force in this objection. But two facts must be remembered: firstly, that the normal limit for tubercular indices is probably, as Fleming has pointed out, 09 and 11, not 0°8 and 12. ~ Second, it is quite possible that either the control serum was not normal or that the tubercular sera were drawn ata time when the indices happened to be nearly within the normal limits. The occurrence, however, of a positive or negative phase has been noted by numerous independent observers again and again, and cannot be disproved by the examination of eight slides, even if they came from a laboratory above suspicion, and a thousand cells were counted from each. Greenwood! has recently published a paper entitled ‘A Statistical View of the Opsonic Index.’ His conclusions, which are based upon Strangeway’s counts of these eight slides and six slides counted in Wright’s Laboratory, are the following :— | 1. Proceedings Royal Soc. Medicine, Vol. II, No. 5, p. 154. ERRORS IN THE OPSONIC TECHNIQUE 313 (1) Phagocytic distributions are markedly asymmetrical. (2) This asymmetry, although reduced, is not removed by emulsions of (from the experimental standpoint) maximal thickness. (3) The mode of a phagocytic distribution is a more reliable constant than the mean. (4) A corollary of (1)—Positive and negative deviations will not occur in random sampling equally often. Another set of figures which may compare with the results of Fleming and ours are those of Lloyd Smith, Radcliffe and Crossley. (1) Here thirty-two duplicate indices for tubercle were estimated by two observers counting the same slides. Analysing the figures obtained from their charts I and II, we find the average difference between the duplicate indices is 0°26, the maxima being 1-1, 06 and 0°4. To the table we have also added some figures by French. Taste V, SHowine AVERAGE DIFFERENCE IN INDICES OBTAINED BY DIFFERENT OBSERVERS RECOUNTING THE SAME SLIDES Paper by Observer Average Numberof Approximate Approximate Organism difference pairs of number of number of indices cells counted _ bacteria compared per slide per cell Fleming ne 21s 9066.0 0 Sa 100'** ‘Ja 2 =... Tubercle Smith* eka 6 xa 0-26 ami inert ? eas bB1...3 5 French+ re nce 0-09 ane ee OO. isa | Spe = le 3 . 103 pe + ohlsyin® | a Pe ae a *. Lancet, July 18, 1908. +. Practitioner, 1906, p. 70. t. This is from an example, purposely given, of a very bad result. Dr. Hort? sent capsules of the same blood drawn from tubercular or non-tubercular persons to ‘experts’ in ‘well-known laboratories’; 5 2a frequently duplicate capsules of a particular blood were sent to the same expert ‘unknown to him.’ Hort gives the result of three duplicate observations on three sera by the same observer T., the average difference being 0°10; and a similar number by the same observer O. and the same observer B., with an average difference of (12 and 0°10 respectively. The figures obtained from examining the same serum in different laboratories were much more divergent, especially in the case of expert A., whose indices were usually far too high. For example, Test 3, observer = aan en 1. Lancet, July 18, 1908, 2. Practitioner, p. 70, 1906, 3. B. M. J., February 13, 1909. 314 BIO-CHEMICAL JOURNAL ¥e tuberculo-opsonic index, 1°34; observer T., 0°67; observer T., 0-56. 2 Test 6, tuberculo-opsonic index, observer A., 2°20; observer O., 0°96; observer O., 0°82; observer B., 0°82. A. made no duplicate observations. The average error of T., B., and O. is slightly less than our own, though their figures are based on six — indices instead of 120. It is noteworthy, however, that their emulsion yielded about 2°3 bacilli per cell, but that of expert A. only 11. Some Sources or Error vi Assuming that the technique is carefully performed and none of the fallacies recently described by Fleming, such as agglutination of the erythrocytes, are introduced, two sources of error require special attention—the emulsion and the counting. The Emulsion Quality of the Emulsion.—We were’ surprised to find that the counts of tubercle bacilli were rather more accurate than those of staphylococci, in spite of the fragmentation, beading, and comparatively large and frequent clumps obtaining in the former organism and the small size of the clumps, rarely more than three cocci, in the latter. , Quantity of Bacilli in the Emulsion—The quantity of bacilli in the emulsion is perhaps even more important. The figures published by Fleming show that the Wright School employed in 1907 a tubercle emulsion yielding about two, and a staphylococcus emulsion about three bacteria per cell in normal serum. In Tables VI and VII we summarise the average errors in the four sets of triple indices estimated on the different days with different bacterial emulsions. The errors are grouped according to the strength of the emulsion employed each day. Thus, twelve indices with staphylococeus were estimated on July 23rd, May 28th, and August Ist, respectively, and on each of these three days the average number of staphylococci per leucocyte was less than 19. The average, however, for the three sets combined is 1°4 staphylococci per cell. The average error for this strength of emulsion, as calculated by Methods A, B, and C is 6°5, 0°25, ete. (See first line of table.) 1. Reyn and Kjer-Petersen of Copenhagen (Lancet, 1908, March 28, p. 919) in a most valuable paper criticise Wright’s theory and technique rather severely, but we are unable to compare any of their results with ours. ERRORS IN THE OPSONIC TECHNIQUE 315 ‘Tasie VI, Ixiusrrarine Trae Retationsare Berween Error anp STRENGTH OF Se EmuLsIon I—SrapnHyLococeus ALBus INDICES SLIDES F : = Error—Method A Error—Method B Error—Method C A per- Average No. of indices EmvuLsion par Sager Average difference maximum upon which _ No. of bacteria per cell tion the between triple indices | deviation from ~ ari taken in pairs arithmetic are ‘Limits of Average mean of mean of triple . based counts cell counts counts 19 6-5 0-25 0-32 0-13 36 n 2 and 2-9 5-1 0-17 0-19 0-13 60 m3and3-9 3-4 3-7 0-17 0-20 0-14 60 4and49 3-7 4-5 0-14 0-22 0-13 24 Sand5-9 5-1 3-5 0-13 0-16 0-11 12 B 6-3 40 0-11 0-15 0-10 48 rag: 3-5 48 0-17 0-21 0-12 240 “f ; Il—Tusercie 19 1-7 2-5 0-22 0-22 0-16 12 n2and2-9 2-5 6-7 0-16 0-24 0-14 48 a3and 3-9 3-3 6-2 0-16 0-15 0-13 24 é 4-6 5-7 0-09 0-13 0-08 verage 3-2 5-9 0-15 0-19 0-12 120 Vil—Summary or Precepine TABLE I—Srarny.Lococcus ALBus : INDICES SLIDES ‘ ~ Exror— ¥rror— . Error— Method A Method B eit ont c Average tage Average tage Average difference diminution maximum No. of Seviation between triple indices in error deviation indices __—_—s Evuision from the taken in pairs on 120 from upon which No. of bacteria per cell —_ arithmetic cell —_ arithmetic _precedi Ne 5 = — of counts mean of figures ofgroup Average du te p= ee triple are based Pu et count (i) on 120 (ii) on 60 all percell (60 cell cell counts cell counts (on 120 cell ; counts) counts) Between land2-9 1-9 5-6 0-20 0-24 16-6 0-13 96 Between 3and49 3-9 41 0-16 0-21 23-8 0-13 S4 Between Sand 6-9 60 3-9 0-12 O15 20-0 0-10 60 Average 3-5 48 0-17 0-21 0-19 0-12 240 . Il—Tusercie Between land 2-9 23 5-9 0-18 6-23 2-7 O15 60 Between 3and49 41 59 0-12 0-14 14:3 0-10 60 Average 3-0 59 O-15 0-19 210 0-12 120 ~ 316 BIO-CHEMICAL JOURNAL These tables demonstrate that there is a most definite connection between the error and the strength of the bacterial emulsion. This is especially obvious when large numbers of indices are grouped together, and when the error is calculated by Method B, which is the most practical and useful of the three methods. Naturally there are discrepancies, particularly in Table VI, but when it is remembered that we are dealing with two separate organisms, that the homogeneity of the emulsion, the quality of the film, and the accuracy with which they were counted by the observer (E. E. G.) necessarily varied somewhat from day to day, and, lastly, that the methods of calculating errors are somewhat rough, the fact that so few discrepancies occur is all the more striking. The following deductions may be drawn from Table VII, in which the indices are arranged in the largest groups. 1. The error steadily diminishes as the strength of the bacterial emulsion increases as shown especially by Method B. 2. An emulsion of tubercle bacilli yielding about two or four bacilli respectively per cell, gives approximately as accurate results, as an emulsion of staphylococci yielding four or six cocei per cell. That is to say, the emulsions of staphylococcus should be somewhat stronger than those of tubercle. This also has been recommended by the Wright School. We give below the percentage diminution in the error by increasing the strength of the emulsion. Tubercle emulsion increased from 2-3 to 4-1 per cell, diminishes error from 0-18 to 0°12 = 33% Staphylococcus _,, » 1939 ,, rm 0-20 to 0-16 = 20% . * » 89to6 ss 0-16 to 0-12 = 25 % These figures indicate, as far as our work is concerned, that increasing the concentration of a tubercle or staphylococcus emulsion from two to four bacteria per cell diminishes the error by about 25 per cent. The inaccuracy of Fitzgerald, Whiteman and Strangeways’ work must partly be ascribed to the employment of an emulsion averaging only, as a rule, one to two bacilli per cell. Lloyd Smith and his co-workers also employed a very weak emulsion, averaging, if one may judge from page 148, about one tubercle bacillus per cell in the control. Allusion has already been made to the fact that in Hort’s figures observer A., who obtained the most divergent results, employed the weakest emulsion. The importance of a strong emulsion is confirmed by an analysis of ERRORS IN THE OPSONIC TECHNIQUE 317 _ Fleming’s figures, in which two observers, A and B, calculated forty-one duplicate indices by counting the same slides. Separating the indices into two groups, viz., those calculated from counts giving more than two bacilli per cell and less than two bacilli per cell, we find that in twenty- seven pairs of indices the average error was 0°07, and the strength of emulsion 1°6 bacilli per cell, and in fourteen pairs the average error was 0°05, but the strength of emulsion 2°4 bacilli per cell. In our thirty-six observations (Table VI) with emulsions averaging 4°6 tubercle bacilli per cell, i.e., about twice the strength used in Wright's laboratory, gave an average error of 0°09, i.e., almost equal to Fleming’s error of 007 for indices from duplicate phagocytic mixtures. This fact indicates that one of the main factors in the superiority of Fleming’s workers lies in tlie greater uniformity of their emulsion, prepared, no doubt, from a strain of tubercle bacilli specially selected after continuous Probably the less homogeneous the emulsion the stronger it must be. There must be, however, an optimum strength for every emulsion above or below which the accuracy of the counts diminish. This optimum strength will depend not only on the nature of the organism and the tendency to clump, but possibly on the personal equation of the observer. All the facts, then, favour strong emulsion and support the conclusions of Greenwood,’ who, arguing from statistical considerations alone, stated ‘ that it is better to work with tolerably thick emulsion giving an average for normal serum of not less than three bacilli per cell.’ Owing to the laborious nature of the research from which the figures are obtained, most of the counting was done after the completion of the technique, so that, unfortunately, we did not realise the great importance of employing strong emulsion till too late. It must be remembered, however, that a high phagocytic count per cell demands considerably more time than a low one. Counting Method of Counting Fleming holds that it is a ‘ great mistake’ to count an ‘arbitrary ’ number of leueocytes, for the observer should display some ‘ intelligence,’ and the number counted should depend on the regularity of the count. This system is not a fair test of the accuracy of the opsonic technique unless the observer, having decided upon the number of leucocytes which give a true estimation of the phagocytosis of each slide, resolutely adheres 1. Practitioner, May, 1908, p. 645. 818 BIO-CHEMICAL JOURNAL to his decision, even though he find the indices eventually calculated are contrary to expectation, or differ considerably from those of another observer. If such should happen, an enthusiastic and optimistic worker is exposed to the insidious temptation of either recounting the suspected slide or counting an additional number of cells in the hope that all may come right. Perhaps our error might have been less had we counted in the manner suggested by Fleming, but then we might have unconsciously imprerrs our results.! Fitzgerald, Whiteman and Strangeways appear to have counted any polymorpho-nuclear cells indiscriminately on any part of the slide. Moss noted that the highest counts occur towards the end of the slide, and particularly is this evident if larger groups are considered. He attributed this result to a sorting out process when making the smear, whereby the largest cells tend to be left to the end. As previously stated, we made it a rule to count thirty cells on pa vf the two borders of a film from the end of the smear, where the leucocytes are most abundant, passing backwards towards the beginning. Analysing the results of this method, it appears in a consecutive series of 311 slides that the number of bacteria in the first ten of the thirty cells averages 2'7 per cent. less than in the last ten cells counted. In another series of 100 slides the figures were 2°6 per cent. less for tubercle and 1°9 per cent. less for staphylococcus. These results contradict Moss’s statement, but he divided his slides into zones 1 em. wide and counted 150 cells in each zone. Our figures, however, suggest the advisability of counting as far as possible corresponding portions of every slide. We believe that leucocytes should never be selected from the middle zone of the smear (AA in the diagram), for here, the film being thicker, the leucocytes tend to be contracted, so that their cytoplasm stains more intensely, and the contained bacteria are more difficult to count. Of course all damaged leucocytes in which the outline is indistinct, owing to damaged cytoplasm, must be rigorously excluded from the counts. Number of Cells Counted We have already drawn attention to the fact that the technique of Fitzgerald, Whiteman and Strangeways, as tested by their own figures in Table II, is much less accurate than our own, but they enumerated the bacteria in only fifty cells, selected apparently indiscriminately, and 1. See footnote, p. 321. : ~~ 4 ERRORS IN THE OPSONIC TECHNIQUE 319 employed an emulsion yielding about two bacteria per cell, while we counted 120 cells, selected with method, and the emulsion averaged 3°2 bacteria per cell. In order to equalise the conditions as far as possible, we have re- calculated our indices from figures obtained by counting the first sixty cells only, instead of the whole 120. (See Tables VI, VII.) These tables demonstrate that even with a weak emulsion yielding on the average 2°3 bacteria per cell, our average error is 0°23 for tubercle as compared with their figures of 0°51 and 0°55, 0°36 and 0°40, 0°36 and 0°45. Even after making due allowances for the slight advantage to us of enumerating sixty instead of fifty cells, of using some phagocytic mixtures in duplicate (see p. 302), and from the somewhat stronger emulsion, it is clear that our work is decidedly more accurate than theirs, as it is less accurate than that of Fleming. Ii will be noticed that on one occasion the error appears less on the Bae a first sixty than on the total 120 for tubercle. This is an accident, and would be more than neutralised by the counts of the second sixty cells; thus, on July 20th, the average error for staphylococcus was 0-09 with 0 cells, 0-05 with the first sixty, but 0°135 with the second sixty. The following conclusions may be drawn from perusal of Table VII :— 1. The error diminishes by doubling the number of cells counted in the case of both tubercle and staphylococcus by some 20 per cent., but not by a half. This agrees with Greenwood’s comment— the belief that a sample of fifty cells is twice as good as one of twenty-five indicates a somewhat primitive state of knowledge.’ 2. Sixty cells with an emulsion of four bacteria per cell gives approximately as accurate results as 120 cells with an emulsion yielding _ about two bacteria per cell, in the case of both tubercle and staphylococcus. (Sse Chart, p. 320.) Fitzgerald, Whiteman and Strangeways speak of the absolute necessity of ‘enumerating at least 1,000 cells.’ But ‘even’ by so doing a percentage difference of 25 might occur. If this statement were invariably true, then Wright’s technique is practically useless, not only for the estimation of opsonins, but also for any experimental work in which degrees of phagocytosis are compared. The figures given below, however, should convince the most hostile critics that Wright’s technique measures something! They represent four sets of indices, each estimated nine consecutive times on the same day, August 10th, 1908. These indices are calculated from twenty-seven 320 BIO-CHEMICAL JOURNAL separate phagocytic mixtures, nine being used as controls in series I and II, and a second nine in Series III and IV. 120 cells were counted, and there were no duplicate estimations. ‘A’ ‘L’ I Itt I IV Opsonic indices Cytophagic indices Opsonie indices Cytophagic indices 1-34 ies 0-90 ee 1-36 sch 0-98 1-08 Ps 0-79 be 1-20 a 0-86 1-05 er 0-78 — 1-13 7 0-85 1-04 pie 0-76 sia 1-08 Pp 0-85 1-02 os 0-73 “— 1-05 ae 0-84 1-00 oye 0-73 a 1-03 ah 0-83 1-00 abe 0-73 iby 1-03 “Sh 0-77 0-96 cas 0-62 ws 0-93 it 0-73 0-86 sat 0-58 sae 0-88 va 0-63 Average 1-04 0-73 1-08 oe 0-81 If we have exhibited some of the work of Fitzgerald, Whiteman and Strangeways in an unfavourable light, it is because we believe that we have discovered the main reasons for their imperfect technique. They deserve the best thanks of everyone interested in opsonic work, for the courageous publication of their results. V2 g Bact frer Kel. 1572 25 3 $5 4 4G 5 5S 6 Soa Toons 20 Ds Siu , eed hlinenvrdius' “i SS eodatts oe se a ERRORS IN THE OPSONIC TECHNIQUE 821 SUMMARY AND CONCLUSIONS 1. The accuracy of the opsonic technique of Wright and Douglas has been vigorously impugned by many writers, notably Simon, Bolduan, Strangeways and Greenwood, 2. In 1907 Wright stated that the error in estimating the tubercular opsonic indices for normal bloods is rarely more than plus or minus 5 per cent. 3. Next year Fleming pointed out that duplicate estimations of tuberculo-opsonic indices usually differ from each other by less than 20 per cent., provided the observer counts ‘ intelligently.’ 4. We have estimated a large number of consecutive indices with tubercle and staphylococcus three times, and find that the average difference between each set of triple indices taken in pairs is 0°15 with tubercle and 0°17 with staphylococcus. These figures may be compared with 0-07 obtained by Fleming’ and 0°51 and 0°55, 0°36 and 0°40, 0°36 and 0°45 obtained by Fitzgerald, Whiteman and Strangeways in a series of duplicate estimations with tubercle. 5. We adopted all the precautions recently advised by Fleming in 7 our work. The possibility of auto-suggestion influencing our counts was eliminated. ; §. The accuracy of our technique was closely dependent upon two variable factors:—{a) The number of cells counted; (6) the strength of the bacterial emulsion. 7. The error steadily diminishes as the strength of the bacterial emulsion increases. By increasing the concentration of a tubercle or staphylococcus emulsion from two to four bacteria per cell the error diminishes about 25 per cent. 8. An emulsion of tubercle bacilli yielding about two or four bacilli respectively per cell gives approximately as accurate results as an emulsion of staphylococcus yielding four or six cocci per cell. 9. The error diminishes by doubling the number of cells counted from 60 to 120 in the case of tubercle and staphylococcus by some 20 per cent. 1. Greenwood concludes that ‘Dr. Fleming's counts show signs of not being random sam but selections.’ We are not clear whether this criticism applies to these figures from the itioner. (Greenwood, Proc. Roy. Soc. Medicine, Vol. I, No. 5, p. 151.) 322 BIO-CHEMICAL JOURNAL 10. Sixty cells with an emulsion of four bacteria per cell give approximately as accurate results as 120 cells with an emulsion of about two bacteria per cell, in the case of both tubercle and staphy- lococeus. | 11. Assuming the truth of Wright’s dictum that the opsonic index is an index of the ‘ power of phagocytic response’ (although we claim to __ have demonstrated elsewhere that this dictum is not true), the inaccuracy of the opsonic technique is such that at present we attach no importance to an index between 0°8 or 1°2 (even estimated by an expert) in the diagnosis and treatment of disease, unless the observation had been repeatedly confirmed. 12. We entirely disagree with those crites who appear to maintain that the technique of Wright and Douglas is practically useless as a means of comparing degrees of phagocytosis. : 323 THE RELATIONSHIP OF DOSAGE OF A DRUG TO THE , SIZE OF THE ANIMAL TREATED, ESPECIALLY IN REGARD TO THE CAUSE OF THE FAILURES TO CURE TRYPANOSOMIASIS, AND OTHER PROTOZOAN DISEASES IN MAN AND IN LARGE ANIMALS By BENJAMIN MOORE, M.A., D.Sc., Johnston Professor of Bio- Chemistry, University of Liverpool. From the Department of Bio-Chemistry, University of Liverpool (Recewved June 11th, 1909) It is an almost universal custom at the present time in describing scientific work of a pharmacological or therapeutic nature intended either to establish a lethal or a curative dose, to state the dose as so much per kilogram of body-weight of animal or man employed as the subject of _ experiment or treatment. ‘Many of the observers who use this method of expressing results are aware and state that it is only roughly accurate, and that experiments must be made from species of animal to species of animal on account of idiosyncrasies. The object of this note is to point out, that, quite apart from idiosyncrasies, and alteration in the species of animal, this method of stating dosage in reference to body-weight is not only inaccurate, but rests entirely on a wrong principle for many kinds of drugs, which, even in the same animal species, act upon two individuals of different size, not proportionately to their weights, but proportionately instead to their body-surfaces or, in other words, proportionately to the two-thirds powers of their weights, which leads to quite different doses. For example, the dose of many drugs which can be given to children or infants to preduce a given therapeutic result, is often many times larger than the proportionate dose for an adult, on the basis of body-weights. There are a few cases such as the preparations of morphia where the child has a marked idiosynerasy or sensitiveness to the drug, but in the majority _ of cases the balance is entirely in the other direction, and an adult of say 150 pounds weight cannot be given 15 times-the dose of an infant of 10 pounds, but much more nearly a dose of 6 times as much, which is the two-thirds power of the ratio in the two weights. Not only is this relationship of importance in regard to the method of expressing dosage and determining the approximate dose in man or large animals, from experiments made upon smaller animals. It is also §24 BIO-CHEMICAL JOURNAL of the utmost importance in that it naturally sets a limit to our power of | applying therapeutic agents against disease in larger animals, and allows a cure with ease in smaller animals, which is difficult or impossible in man or large animals, simply because they are large and not because of any particular sensitiveness to the drug. For example, it is perfectly easy to cure trypanosomiases by atoxyl or other organic arsenical preparations, or better still by a proper combination of arsenic and mercury, in small animals such as the mouse or rat. The difficulty increases with rabbits, but in a large number may still be surmounted, as also in small monkeys. But with donkeys, cattle, horses and men, the difficulty is enormously increased, the trypanosomes can scarcely be driven out from the circulation by such sub-lethal doses as it is possible to give, recurrence svoner or later takes place and the animal or man succumbs. This is the common experience of all workers in the fight against the trypanosome group of diseases, and is particularly well seen in the work of Moore, Nierenstein and Todd upon treatment by atoxyl followed by mercury. Like other workers we were in all cases able at once to drive the parasites out of the peripheral circulation in rats with atoxyl and by then giving mercury were able to keep them permanently from recurring in a high percentage of cases (about 60 per cent.). But when we came to test this greatly improved result as seen in rats to the larger mammalia, our results took on quite another complexion, and of 15 donkeys we were unable ultimately to save one. In the first case, on account of the limitations in the relative dose we could not satisfactorily and at once with a single dose drive the parasites out from the peripheral circulation. In some of our experiments for considerable intervals no parasites could be found even by most careful examination, but since in other donkeys similarly treated, a small number of parasites kept persistently present and finally became inured or ‘Fest’ to our drugs, we could not be free from suspicion, that the parasites in very small number so as to escape actual observation, were always present, and that our practically complete failure with the large animals, using a method of treatment almost perfect for the rat, was due chiefly to this cause. That is to say, in the large animal the dose cannot be given proportionately to the body-weight and so the trypanosomes cannot all be killed. Exactly the same results follow for the admirable treatment by antimony, introduced first by Plimmer and Thomson, good results follow and often a high percentage of absence of recurrence in rats and small animals,’ but in large animals and man the percentage is much lower, although some cases of cure have been noted. 1. I can completely confirm this result from a large number of experiments made in collaboration with Dr. T. T. Bark in this laboratory. We found that even smaller doses than recommended by these authors drove the parasites out. Also a careful search of all the heavy metals, elements of the phosphorus group, and rare elements, gave negative results, except in the cases of antimony and arsenic only. RELATIONSHIP OF DOSAGE TO BODY-SURFACE 325 A like result has been obtained with other organo-arsenical 4 _ compounds. In all these cases it is to be observed (with the possible exception of the dog') that the difficulty is not one of idiosynerasy either to parasite or drug in a particular animal species, but that definitely in each case, and from each group of observers, we have the uniform report that small animals are easily treated and large animals including man are difficult of treatment. It is hence of peculiar interest to review the situation from the general point of view, and try to find whether there is any physico-chemical basis which may supply an explanation. This is particularly so, since a side-light is cast upon the general bio-chemical question of what regulates the maximum therapeutic dose possible with such drugs in these diseases due to protozoa or other micro-organisms. The establishment of the minimum lethal dose of atoxyl proves at once that this dose is not proportional to the body-weight, but relatively falls off very rapidly as the body-weight is increased. Thus, a large sized rat of say 140 grams weight can safely be given abont 0-4 c.c. of a 5 per cent. solution of atoxyl, that is about 0°02 gram. Now, if the dose could be given proportionately to body-weight, a man of 70 kilograms weight, since he weighs 70,000 grams or 500 times as much as the rat, ought to be able to safely stand 500 times as much atoxyl, that is 0°02 x 500=10 grams or in 5 per cent. solution =200 c.c. As a matter of fact, he not only cannot stand approximately this quantity, he can only stand a small fraction of it, the highest dose which can be given being one gram or one-tenth of the above amount calculated from relat've body-weight, and this is not an idiosyncrasy of man, but is true for all large animals. If now instead of hody-weights, the two-third power of the body- weights be taken for the purpose of estimating the relative doses. We have that the ratio of weights is 1:500 and the two-thirds powers of this ratio gives the ratio approximately of 1:63, and if 0°02 grams is the dose for the rat, on this basis the dose for the man will be 0°02 x 63=1°26 grams. Now this very closely represents the amount found by actual experimentation, as 1 gram of atoxyl has frequently been given as a therapeutic dose in man. When the cause of this relationship is further investigated, it is found 1. It may also be noted that the dog has a very small area of intestine relatively to its body-weight. 326 BIO-CHEMICAL JOURNAL to lie in the selective action of arsenical and antimonial compounds upon cells lying upon certain surfaces of the body. . These substances are selectively taken up by epithelial surfaces. It is well known that arsenic and antimony and indeed most of the heavy metals such as zinc, silver and copper, when taken by the mouth act as surface irritants upon stomach and intestine. It is less well known and only for certain heavy metals, but is none the less a general law, as experiments have shown me, that all the heavy metals are selectively taken up by the cells lining the intestine, and kill by extensive inflammation of the intestinal mucosa. Thus I have found that after subcutaneous injection, of soluble salts of Jead, tin, bismuth, iron, copper, nickel, cobalt, or antimony, there is extensive inflammation of the gastro-intestinal tract, often accompanied by multiple hemorrhage and ulceration. The same inflammatory processes in the intestine are found after all forms of arsenic including atoxyl. This shows that the intestinal mucous membrane acts as the excretory organ for these foreign substances, and hence takes them up selectively from tne circulation. Accordingly the minimal lethal dose or the full therapeutic dose will be limited by the concentration to which that dose gives rise in the epithelium cells of the intestine. Now, placing on one side variations in length of intestine, and variations in length of intestinal villi, this means that the maximum dose is proportional to the area of intestine which the animal possesses. Hence, if for simplicity we suppose that the animal from small size grows symmetrically bigger in all proportions so as to form a bigger animal which is a fae simile of the smaller, then we must inevitably have from purely geometrical considerations of the dimensions of solid figures, that when the length, or linear dimensions are say doubled, the surfaces external and internal of the body increase four-fold, and the cubic dimensions, whch are of course proportional to the body-weight, increase eight-fold. Put otherwise this means that the dimensions of surfaces such as area of skin, lung area and intestinal area increase as the two-thirds power of the body-weights. Now a great number of drugs including, as above mentioned, atoxyl and all salts of heavy metals attack cells spread out on a surface and hence the bigger animals cannot stand proportionately large doses, and, for example, an animal eight times as heavy as another cannot be given RELATIONSHIP OF DOSAGE TO BODY-SURFACE — 327 eight times the dose, but only 84, that is approximately four times as mich. . a But the result of this is that the concentration of the drug in the tissue fluids and blood of the heavier animal will only be approximately _ one-half of that in the smaller animal. Suppose now that both animals have been affected by trypanosomes and we have been attempting to kill them in both, then it is obvious that there is far more margin to come and go on in the smaller animal than in the larger one The trypanosome is swimming about in the blood plasma of the ee. animal, it may be a rat in one case and a horse in the other, the factor sit ‘has to deal with is the concentration to which the drug can be raised in that plasma, it is indifferent what the host may be. But as regards the host, the drug is taken up by the intestinal cells selectively, and the < ‘amount of drug which can be given is not determined directly by the - concentration in the plasma, but by the number of columnar intestinal cells, that is by the area of intestine which is proportionately greater in the smaller animal. ) . ; This fact that the surfaces are relatively larger in smaller animals than in larger animals, has been thoroughly appreciated in regard to body- temperature and its variations. A small animal is more susceptible to changes of temperature than a large one, it requires more food proportionately to its weight than a larger animal, and for the same reason its temperature goes up and down more readily because the fly-wheel of weight is smaller relatively _ to the amount of surface through which heat exchanges are going on, that is the working part of the engine. It is also obvious since the surfaces are the working parts of the body, and these are relatively greater in _ proportion to the amount of material re-acting (¢.e. body-weight) that all the processes are quickened up in the small animal, so that there is an exceedingly rapid heart-beat and respiration in all small mammalia. In cold-blooded animals and invertebrates, a different chemical constitution in the protoplasm with a different reactivity and speed of oxidation sets a new limit. Returning to the pharmacological aspect, we find that this same peer Gs factor of relative surfaces and volumes is in operation, but has not hitherto § been sufficiently recognised, and that it lies fundamentally at the bottom : of the difficulty of treating man or large animals. A careful consideration of the experimental facts known regarding the action of atoxyl and similar therapeutic agents upon trypanosomes has 328 BIO-CHEMICAL JOURNAL led me to the belief, that the intestine not only acts as above pointed out in limiting the possible therapeutic dose, but also that the cells of the intestinal mucosa, are essential intermediaries in preparing or elaborating in a bio-chemical fashion from the atoxy] or other substance administered, a substance which is peculiarly deadly for the trypanosome. Wri, This is shown by the fact so well known to all workers upon & trypanosomiasis, that substances can be found out by the score which even in minimal concentration are deadly to trypanosomes in saline in a watch-glass, yet these substances can be subcutaneously injected into animals infected with trypanosomiasis without producing the slightest obvious effect wpon the parasites. . The explanation here probably is that the said substances are thrown out of solution by the plasma proteins, which are not present to protect the parasites in the experiment with the trypanosomes in saline in vitro. A still more remarkable and instructive experiment, well known also to workers on the subject, is that trypanosomes may be treated in vitro in saline, with concentrations of atoxyl, and other trypanocidal drugs, ‘many times higher than the lethal concentration required to destroy them after subcutaneous injection in the animal, without producing the slightest apparent effect upon the parasites, which go on moving about vigorously for hours. The inference is obvious, viz.: that there must be formed intra vita by some bio-chemical process, an organic body especially lethal to trypanosomes. Now it has been shown that the seat of bio-chemical activity after administration of arsenic or antimony is the intestinal mucosa, and it would seem probable that this is also the situation of manufacture of the trypanocidal virus. If this be the case, it is clear that the larger animal is doubly handicapped, for in the first place the therapeutic dose is lowered because of the relatively smaller number of intestinal cells to take up the drug, and their consequent rapid poisoning and death of the animal if the attempt be made to give the relative dose; and secondly, that this relatively smaller number of cells or laboratories must first act upon the drug before it can be turned out as a trypanocide upon the parasites. These reflections suggest certain lines of experimentation and treatment, which, since I am not myself in a position to experiment upon larger animals, I here outline for others who may desire to test them. In the first place, an attempt might be made to prepare the trypanocidal material by giving gradually increasing doses of atoxy! in RELATIONSHIP OF DOSAGE TO BODY-SURFACE 329 healthy animals, and then using an extract of the intestinal mucosa in full doses as a therapeutic agent in man if it were found to be active in small animals. Such an extract of the intestinal mucosa ought to be active against trypanosomes in vitro. Secondly, it appears to me that an attempt might be made to increase the production of anti-trypanocidal virus by giving atoxyl by mouth simultaneously with subcutaneous injection. Lastly, as a kind of opposed treatment, the attempt might be made to increase the possible subcutaneous dose by giving an antidote by the mouth to the arsenic or antimony just before injection, so as to protect _ the intestinal cell at the first pressure of the drug in the circulation due to the subcutaneous injection. SUMMARY 1. In the case of substances which act by stimulation or Seifsmmation of surfaces, such as the intestinal tract, the maximum dose is proportional not to the body-weight, but to the two-thirds power of the body-weight. 2. This leads to important differences in dosage in man and large animals. © 3. It also shows that the possibilities of treatment are diminished by : a natural means in man and large animals. These animals have naturally ___ less intestinal, and other, surfaces per unit of weight; accordingly they ean only take up proportionately less drug, and if any remedial substance is manufactured by the surface cells, they can only manufacture relatively less of this than the smaller animal. 4. Also in general terms, uptake and output of poison or infection _ are relatively more rapid in the small animal. The small animal and child are hence at the same time more susceptible to onset of infection, and have more power of recuperation when infected. Nore.—The practical suggestion may be made that for the great majority of drugs the method of stating dosage as so much per kilogram should be abandoned. The following method is suggested :— The dose for an animal of an observed weight should be determined, then by taking the two-thirds powers of the two weights, the dose for an animal of, say, 1 kilogram, can readily be calculated. This should be "-BIO-CHEMICAL stated se tiecliaie Mend doe kilogram From this dose, the dose for an a kilogram animal, ‘not multiplied by 4, oe t -th which i is 16. Here it is 3 obvious ae the usual: baal therapeutics, the dose for low weight and nips vel fall and rise in direct proportionality to the w lessened ratio. The heavier person requires a little proportionately to increased weight, and vice versa. "PROPOSALS FOR THE NOMENCLATURE OF THE LIPOIDS* ‘By OTTO ROSENHEIM. From the Physiological Laboratory, King’s College, London (Received June 16th, 1909) _ The term ‘lipoid’ was used fifty years ago by Kletzinski, but has only obtained importance since Overton (1901) re-introduced it in connection with H. Meyer's and his own theory of narcosis.1 It is now generally accepted as a generic name for all those ‘ fat-like ’ constituents of animal‘or vegetable cells which can be extracted by means of ether or _ similar solvents. If we accept this definition which from its origin is mainly a biological one, we are forced to include amongst the lipoids substances which have very little in common from a chemical point of ‘The fundamental importance of these substances in biological processes is being more and more demonstrated by exact experimental investiga- ___ tions, especially in relation to problems of immunity. These investigations have drawn attention to the incompleteness of our chemical knowledge __ of these substances, and stimulated research on their chemical constitution, a thorough knowledge of which seems to be the first essential condition for the elucidation of the biological questions. Li Unfortunately there still exists a great confusion in the nomenclature . 3 of these substances. This confusion is mainly due to three reasons: | (1) Substances which are evidently identical have received different names from different observers; (2) the same name has been applied to different substances; (3) several names which have no chemical meaning are used ‘in a general physiological and histological sense. ah r In order to arrive at a uniform nomenclature, it seems desirable (1) to omit, for the present, all those names which have been given to 7 ‘substances which are either insufficiently characterised, or the existence : of which has not been verified by later researches; (2) to dismiss altogether 7 the names given to substances which do not represent definite chemical compounds, and (3) to adopt, in the case of different names for the same + ‘4 substance, those names which were proposed by the original discoverer. Ms, The following noménclature is based on the classification introduced a by Thudichum, the value of whose pioneer work in this field is now iam: recognised, after it has been neglected for nearly thirty years. A paper read before the Physiological-Chemical Section of the Seventh International ES of Applied Chemistry, May, 1909. 1, Overton, Studien tiber die Narkose, Tena; 1901. 332 BIO-CHEMICAL JOURNAL This classification offers the advantage of easily allowing for extension and of having been already adopted in its main outlines by the majority of modern workers (Schulze, Winterstein, Hammarsten, Erlandsen, Bang, Thierfelder, Frankel, etc.). We may distinguish three large groups of lipoids: (I) The Cholesterin group (free from both chjecaed and ss nitrogen). (II) The Cerebro-Galactosides (free from phosphorus but con- taining nitrogen). (III) The Phosphatides (containing both phosphorus and nitrogen). (1) The Cholesterin group. The main representative of this group is cholesterin. The name cholesterin has the advantage of long and inter- national usage to recommend it in preference to ‘ cholesterol,’ which latter name only represents one characteristic of the latter substance, namely, its alcohol nature. This group also includes the vegetable cholesterins, usually called phytosterins. Pigments such as /ipochromes, as well as odoriferous substances which have not yet been chemically characterised may also be provisionally included in this group, for at any rate they resemble cholesterin in being free from both phosphorus and nitrogen. (II) The Cerebro-Galactosides represent a group of nitrogenous phosphorus-free substances which are characterised by the fact that its members furnish galactose on hydrolysis. The general name of this group which was first used by Thudichum, is preferable to the shorter name ‘Cerebrosides’ since ‘ cerebrose’ is identical with galactose. Two sub- stances belong to this group, namely, Phrenosin and Kerasin, the latter of which has not yet been so well studied as the former. In adopting the name Phrenosin a number of names must be discarded, which have given rise to a great deal of confusion. Phrenosin was recognised by Thudichum as the main phosphorus-free constituent of the mixture ‘ Protagon ’ (=Cérébrote of Couerbe, 1854; Cerebric acid, Fremy, 1841), and he proposed this name in preference to ‘ Cerebrin,’ as the latter term had been ee to widely different substances. The name ‘cerebrin’ was originally given by Kiihn (1828) to a mixture of phosphatides and cholesterin; it was used by Gobley (1850) — for a substance which, according to its preparation (boiling with sulphuric or hydrochloric acid), must have been a partially hydrolysed product, still containing phosphorus. Miiller (1858) for the first time applied this 1. The general name, ‘ Sterin.” has been proposed for this group by Abderhalden. Apart — from other objeetions, the possible confusion with ‘Stearin,’ from which the name is derived, will probably stand in the way of its general acceptation. NOMENCLATURE OF THE LIPOIDS 333 name to a phosphorus-free substance, obtained by a process in which brain had been coagulated by means of baryta or lead acetate. Miiller’s _ ‘cerebrin’ probably represented also a partially hydrolysed product and has not since been obtained. Some time after Thudichum described phrenosin, Gamgee obtained the same substance and called it provisionally *Pseudo-cerebrin.” Parkus (1881) and Kossel and Freytag (1893) again used the name ‘cerebrin’ for a phosphorus-free substance similar to phrenosin, in the preparation of which, however, boiling baryta had been used. Finally Wérner and Thierfelder (1900) gave the name ‘ Cerebron ’ to a phosphorus-free substance, which they prepared mainly by fractional __ erystallisation from so-called ‘ protagon,’ and which was found to be _ identical with Gamgee’s ‘ pseudo-cerebrin.’ It seems certain that ‘cerebron ’ and ‘ pseudo-cerebrin’ are identical with phrenosin, and it is therefore desirable to retain the original name 4 scorn, and to dismiss the others. __ Kerasin was first obtained by Thudichum. Parkus described a similar substance, obtained by his baryta process, which, however, he called ‘ Homocerebrin.’ The name kerasin was again used by Kossel and Freytag for their preparation, which resembled that of Thudichum’s. The substances called by Bethe ‘ amino-cerebric-acid glucoside ’ is also probably identical with kerasin. ie (III) The Phosphatides. This name was proposed by Thudichum e for a group of substances which contain both phosphorus and nitrogen. | Tt was re-introduced by Schulze and by Ilammarsten, and is now generally accepted, the name ‘lecithans’ proposed by Koch not being - general enough. Thudichum demonstrated the existence of a whole series of these substances, where formerly the presence of only one, namely ‘Lecithin,’ has been assumed. He introduced the principle which guides me ~ modern workers in the classification of these substances. The _ phosphatides, according to this, may be divided into several sub-groups, according to the ratio of nitrogen to phosphorus contained in them. For the present the constancy of this ratio (after repeated re-crystallisations and fractionations) offers the only index for the chemical entity of these substances. We may distinguish the following sub-groups : — (1) Monoamino-monophosphatides ..........N: P = 1: 1 (2) Diamino-monophosphatides —............ B:P = 23.1 (3) Triamino-monophosphatides ............. N: P=8:1 (4) Triamino-diphosphatides .................. Ni: P= 3:2 (5) Monoamino-diphosphatides .......,....... BLP «= ): 3 834 BIO-CHEMICAL JOURNAL It is obvious that this list may be easily extended as —— possessing a different N : P ratio are isolated. (1) Monoamino-monophosphatides. This sub-group comprises Lecithin and Kephalin. he term ‘lecithin,’ which was formerly given toa i mixture of phosphatides, should be restricted to those monoamino-mono- phosphatides which are soluble in ether, and not precipitated from their solution by alcohol. The nature of the fatty acids contained in them is still uncertain, but oleic acid seems to be the characteristic one. Kephalin (or kephalins) first described by Thudichum, is soluble in ether, and precipitated from this solution by absolute alcohol. It probably contains as its characteristic fatty acid a still more unsaturated acid than oleic acid. (Zuelzer has given the name Myeline to a substance obtained from egg yolk, which is evidently similar to or identical with Thudichum’s Kephalin). Another substance belonging to this group has recently been isolated from ox pancreas by Frankel and Pari. This monoamino-monophosphatide which has been called Vesalthin, furnishes on hydrolysis myristie acid, besides an unknown unsaturated fatty acid and a base different from choline. Thudichum includes in this group another substance called by him Myeline. In view of the fact, however, that this substance has been very little studied, and in order to avoid confusion with the general term ‘myelination ’ as used in the physiological sense, it seems desirable for the present to dismiss the name altogether. (2) Diamino-monophosphatides. | The main representative of this group is Sphingomyelin. This phosphatide, which, in distinction from the waxy lecithin and kephalin, is a crystalline white substance, was also first obtained by Thudichum. It represents the main phosphatide of the so-called ‘ protagon’ mixture. Its oceurrence in brain (and probably in the cortex of the adrenals) has been confirmed by Rosenheim and Tebb, who prepared it by a new method. A similar diamino-monophosphatide was obtained as a cadmium salt by Erlandsen from the heart muscle, and by Thierfelder and Stern from egg yolk. (3) Triamino-monophosphatides. A phosphatide belonging to this group has recently been isolated by Frinkel and Bolaffia from egg yolk. They gave the name Neottin to this triamino-monophosphatide. (4) Triamino-diphosphatides. A substance belonging to this class is described by Frankel and Nogueira, who obtained it from ox kidney. (5) Monoamino-diphosphatides. The first phosphatide of this group was discovered by Erlandsen in heart muscle, and called by him Cuorin. NOMENCLATURE OF THE LIPOIDS 835 Recently MacLean demonstrated the presence of another member of this group inegg- yolk. Both substances are highly unsaturated and yield on hydrolysis a base different from choline. In this classification the lipoids which contain sulphur, although possibly of considerable importance, have not yet been considered, as they have so far not been isolated in a sufficiently pure state to warrant their group as Cerebro-sulpho-galactosides, or if found to contain phosphorus as well, in the third group as amino-sulpho-phosphatides. Itis proposed to discard the following names : — Cérébrote Cerebric acid Mixtures of various lipoids. Protagon Cerebrin Pseudo-cerebrin Identical with phrenosin. Cerebron Hlomocerebrin— identical with kerasin. Myelin—for reasons explained above. | ‘The following table contains a list of the substances which are included by the proposed classification. I. The Cholesterin group. 1. Cholesterin. 2. Phytosterins, 3. Lipochromes, etc. Il. The Cerebro-Galactosides. 1. Phrenosin. : 2. Kerasin. : IL. The Phosphatides. am 1. Monoamino-monophosphatides N: P = 1: 1. a. Lecithin (or Lecithins). 6. Kephalin (or Kephalins). ec. Vesalthin. 2. Diamino-monophosphatides N: P = 2: 1 Sphingomyelin. 8. Triamino-monophosphatides N: P = 3: 1. Neottin. 4. Triamino-diphosphatides N: P=8: 32 =1: 2. 5. Monoamino-diphosphatides N : P Cuorin. es In the sisal’ state of our Land desi to defer the introduction of new names until a com) hydrolytic cleavage products makes it evident that the sv is fundamentally different from those previously describe of the lipoids (except cholesterin) in a chemically pure, 3 impossible at present as that of the proteins and we just as in the latter case, mainly on the results of c further information about these substances. = - vi hutersee avr t oe inees e. Par a a ys i WAY Bet eee oe Fe 837 A COMPARISON OF THE METHODS FOR THE ESTIMA- TION OF TOTAL SULPHUR IN URINE By STANLEY RITSON, A.K.C. From the Physiological Laboratory, King’s College, London Communicated by Prof. W. D. Halliburton, F RS. (Received July 16th, 1909) It is well known that urine contains sulphur, not only in the form _ of sulphates, both inorganic and ethereal, but also in the form of less highly oxidised organic compounds, generally spoken of (following __E. Salkowski’s! suggestion) as ‘neutral sulphur. The organic { compounds in question are very diverse and include thiocyanic acid and he ; its salts,? eystine and closely related bodies,* taurine and tauro-carbamic acid, methyl merecaptan,5 ethyl sulphide,® thiosulphuric acid,’ sulphurous acid,’ urochrome,® oxyproteic acid,!° uroproteic acid,! and uroferric acid.!? The quantitative determination of the total amount of sulphur present in the urine depends upon the fact that this neutral or unoxidised sulphur can be converted by the aid of suitable oxidising agents into sulphuric acid. The various methods in use differ mainly in the different oxidising agents chosen. They may-be arranged in three groups :— . 1. Virchow’s Archiv., Bd. LVIII, 8. 472, 1873. _—__— 3. Leared, Proc. Roy. Soc., Lond., Vol. XVI, p. 18, 1870; R. Gschleiden, Tageblatt d. 47, : prune gy deut. Natur, 7 u. Aertzte in Breslau, | 4 (quoted from E. Abderhalden’s Lehrbuch "= d. physiol. Chem., 11 Aufl., 8. 343, 1909); I. Munk, Virchow’s Archiv., Bd. LXIX, 8. 354. Sp 3. Stadth Zeitsch. }. physiol. Chem., Bd. TX, 8. 125, 1885; E. Goldmann u. E. ibid., Bd. XII, 8. 254, 1888. 4. E. Salkowski, loc. cit. 5. M. Nencki, Arch. /. exper. Path. u. Pharmak., Bd. XXVIII, 8. 206, 1891. | se cco gem neliee etiaresdtett J. J. . J. Abel, loc. cit., for d Presch found in case of typhus that a compound which isolated from the urine in small. uantities =v rg acid on distillation with acids. the case IT can find in w * Faerie / uric acid has been mopens 3 in urine of man. Quoted from A. Heffter, Ergebnisse d. Asher and Spiro), Jg. I, Abt. I, 8. 458, 1902. Striim in case of fever, bane rs Heilk., 1876. (Quoted from Dixon Mann, Physiol. 8. iy and Path, of Urine, 2nd Edit., p. 18, 1908.) Ps 9. St. Dombrowski, Zeitsch. /. physiol. Chem., Bd. LIV, 8. 204, 1907-8. or Aller peng pe tt ghee ge Soups ote ie ie Wap sage No. 33, 8. 577, 1897; K. Panck, Ber. ¢. Dest. chem. Gesslioh. Jg. XXXV, 8. 2059, 1902; St. go i, Bt. Dombrowski u. K. Panek, Zeitach. }. physiol. Chem., Bd. XLV, 8. 83, 1905 ; F. Pregl, Pfliiger’s Archiv., Bd. LXXV, 8. 87, 1899. bf 11. Max Cloetta, Archiv. /. exper. Path. u. Pharmak., Bd. XL, 8. 29, 1897. 4% 12. 0. Thiele, Zeitsch. /. physiol. Chem., Bd. XXXVII, 8. 251, 1903. 338 BIO-CHEMICAL JOURNAL 1. Methods in which the residue left upon evaporation of a certain definite volume of urine is effected by fusing with a mixture of sodium carbonate and saltpetre. These are merely applications to the urine of Liebig’s original method of sulphur estimation. Full directions as to — the procedure are given in Savelieft’s' and Moreigne’s? papers. 9 oxidising agent. Schulz* and Mohr* have described methods utilising this reagent. Schulz originally carried out the process in a special closed apparatus, believing that volatile sulphur compounds might be formed, but in his last communication on the subject he states that this is not the case, and now carries out the oxidation in an open Kjeldahl flask. Konschegg,° who also uses fuming nitric acid, adds a small quantity of potassium nitrate, probably in order to effect more complete oxidation. 3. Methods in which the oxidation is brought about by sodium peroxide. The introduction of this reagent, for the purpose of sulphur estimations, we owe to Hempel.® It was subsequently used by Hoehnel,? Glaser® and by Asboth,® for the estimation of sulphur in organic materials. Ina later paper Asbéth,!° who subsequently employed sodium peroxide for the estimation of sulphur in organic compounds, suggested that the method might also be useful for the determination of the total sulphur in urine.!! Modrakowski!* simplified the method by omitting the addition of sodium carbonate to the peroxide, which formed a part of the original process. More receptly Folin! has modified the Asbéth- 1. Savelieff, Virchow’s Archiv., Bd. CX XXVI, 8. 197, 1894. 2. Moreigne, Bull. de la Soc. Chim. (3), XI, 975, 1894. 3. H. Schulz, Pfiger’s Archiv., Bd. LVII, 8. 57, 1894; also ibid., Bd. CXXI, 8. 114, 4. P. Mohr, Zeitsch. /. physiol. Chem., Bd. XX, 8. 556, 1895. 5. A. Konschegg, Pfliger’s Archiv., Bd. CX XIII, 8. 274, 1908. 6. W. Hempel, Zeitsch. /. anorg. Chem., Bd. TI1, 8. 193, 1893. 7. M. Hoehnel, Archiv. d. Pharm., Bd. CCXXXII, 8. 225, 1894. 8. ©. Glaser, Chemiker Zeitung, Jg. XVIII, 8. 1448, 1894. : A. von Asbéth, Chemiker Zeitung, Jg. XTX, 8. 599, 1895. A. von Asboth, ibid., Jg. XTX, 8. 2040, 1895; A. Edinger recommended sodium vxide for the estimation of o anically combined sul hur before ory xiv Ronen Fis uesd an aqueous solution of the peroxide. Zeitsch. f. analyt. Chem., 1895; Ber. d. Deut. chem. Gesellech., Jg. XXVIII, 8. 427, 1896. 11. §. Lang used the Asboth method to estimate total sulphur in urine, but gives no control analyses. Zeitsch. /. physiol. Chem., Bd. XXIX, 8. 305, soto. 12. G. Modrakowski, Zeitsch. /. physiol. Chem., Bd. XX XVIII, 8. 561, 1903. F. Clark, Journ. Chem. Soc., 1893, I, p. 1093, had previously suggested that sodium carbonate was unnecessary. Almost immediately after Modrakowski’s paper, Neumann and Meinertz (Zeitsch. }. physiol. Chem., Bd. XLITI, 8. 38, 1904) advised the admixture of potassium carbonate. 13. O. Folin, Journ. of Biolog. Chem., Vol. I, p. 155, 1906. See also T. B. Osborne, Journ. of Am. Chem. Soc., Vol. XXIV, p. 142, 1902; J. A. Le Clerq and Dubois, ibid., Vol. XXVI, p. 1108, 1904; W. L. Dubois, sid. Vol. XXVIL, p. 729, 1908. 2. Methods employing concentrated fuming nitric acid as the ' TOTAL SULPHUR IN URINE 339 - _Modrakowski method in several particulars (especially with regard to _ the addition of a little water before the final fusion in order to obtain - complete fusion with the aid of comparatively little heat, and to protect the crucible against corrosion). In all these methods the fusion is accomplished by heat applied to the exterior of the crucible, preferably by means of a methylated spirit burner. Pringsheim, however, utilising an observation of Parr’s,! showed that the oxidation may be most easily brought about by the introduction of a red-hot iron nail into the mixture. He first employed this method for the estimation of the halogens, phosphorus and arsenic,? and later, _ following Konek? and other observers,‘ to the estimation of sulphur® in ‘organic combination. Recently, Abderhalden and Funk® have applied Pringsheim’s method to the estimation of total sulphur in the urine. __It is obvious that in any trustworthy method, the results obtained ‘with the same urine should agree infer se; and in the comparison of different methods, the method which gives the highest results will in the absence of other indications be the most correct. On both these points there are differences of opinion with regard to the different methods: thus Osterberg and Wolf? state that while the method of Asboth- _ Modrakowski, carried out according to Folin’s directions, invariably gives higher figures than the Schulz method, the results obtained by the latter method do not agree well inter se; Konschegg states that his modification of Schulz’s procedure gives considerably higher figures than the latter; Abderhalden and Funk claim that the Pringsheim modification in their hands also gives higher figures than Schulz’s method; and Gill and Grindley® say that Konschegg’s figures are invariably higher than those obtained by both Osborne’s and Folin’s modifications. ‘Taking all these statements into consideration, and bearing in mind 4. that no corroborative observations have yet appeared on the Pringsheim bi method, it seemed advisable to investigate the degree of accuracy of the = various methods in a series of urines. 1. 8. W. Parr, Journ. Am. Chem. Soe., Vol. XXII, p, 646, 1900. - ee 2 Ae oo ok Ber. d. Deut. chem. Gesellach,, Ig. XXXVI, 8. 4244, 1903: Amer. Chem. Journ., Vol. XX XI, p. 386, 1904; Zeitech. |. angewand, Chem., Bd. XVII, 8. 1454, L904, 3. F. von Konek, Zeitsch. /. angewand. Chem., Bd. XVI, 8. 516, 1903. 4. C. Sundstrom, Journ. o are ees Vol. XXV, p. 184, 1903; J. D. Pennock sage ae eegieap der peer XV, p. 1265, 1 5. H. H. Pringsheim, pagel! ad et OO Jg. XLI, 8. 4267, 1908. 6. E. Alderhalden and C. Funk, Zeitsch. /. physiol. Chem., Bd. LVIII, 8. 331, 1908. 7. E. Osterberg and C. G. L. Wolf, Biochem. Zeitech., Bd. IX, 8. 307, 1908. 8. F. W. Gill and H. 8. Grindley, Proceed. of Soc. of Biolog. Chem., Vol. VI, p. 11, May, 1909. ois ene 340 BIO-CHEMICAL JOURNAL In comparing the different methods, I have taken as my standard of agreement the one adopted by Abderhalden and Funk, namely, variations of 0:1 milligr. on either side of the mean value, when the sulphur is estimated in 10 c.c. of urine. re As there seemed to be no necessity of confirming the correctness of the old Liebig method,! I limited myself to the following, viz.:—those a of Schulz, Konschegg, Asbéth-Modrakowski, and Pringsheim (Abderhalden-Funk modification). Schulz’s Method. The following are the analytical results I have obtained with four different urines: Urine A 5 c.c. urine gave 0-0358jgr. BaSO, = 0-0098 gr. sulphur in 10 c.c. urine me i: » 0-0362 gr. » = 0-0100 gr. ‘i Ac ie Urine B 5 ¢.c, urine gave 0-0348 gr. BaSO, = 0-0096-gr. sulphur in 10 c.c. urine ” ” 0-0353 gr. ” => 0-0097 gr. ” oo ” Urine © 5c.c. urine gave 0-0510 gr. BaSO, = 0-0140 gr. sulphur in 10 ¢.c. urine ” ” ” 0-0501 gr. ” = 0-0138 gr. ”? ” ” Urine D 5je.c. urine gave 0-0154 gr. BaSO, = 0-0042 gr. sulphur in 10 ¢.c. urine ios as » 00150gr. ,, =00041 gr. ,, oe res it will be seen that (contrary to Osterberg and Wolf’s statement) the figures obtained agree very well inter se, the maximum variation being only 0-2 milligr. Mohr, who used a method almost identical with that = of Schulz, also obtained concordant results. My results by the Schula method are, however, lower than those obtained by other methods, as " will be immediately seen.? Rr? Konschegg’s Method. 'The following are my analytical figures :— Urine A 5c.c. urine gave 0-0378 gr. BaSO, = 0-0104 gr. sulphur in 10 c.c. urine ” ” ” 00375 gr.» = 0-0103 gr. ” ” ” Urine B * 5c.c. urine gave 0-0369 gr. BaSO, = 0-0101 gr. sulphur in 10 c.c. urine ” ” ” 0-0384 gr. ” = 00106, ° ” ” bh Urine C 5.0, urine gave 0-0528 gr. BaSO, = 0-0145 gr. sulphur in 10 c.c. urine ” ” ” 0-0524 gr. ss = 0-0144 gr. ” ” ” Urine D 5c.c. urine gave 0-0165 gr. BaSO, = 0-0045 gr. sulphur in 10 c.c. urine ” ” ” 0-0163 gr. ” = 0-0045 gr. ” ” ” 1. See however O. Folin, Journ. of Biolog. Chem., Vol. I, p. 156, 1906. 2. This confirms the statement of Konschegg, Osterberg and Wolf, Abderhalden and Funk (loc. cit.) and of Sherman who used the nitric acid method for the estimation of sulphur in organic compounds. (Journ. of the Amer. Chem. Soc. Vol. XXIV, p. 1100, 1902). TOTAL SULPHUR IN URINE 341 | Pringsheim’s Method (Abderhalden and Funk's modification). The _ following are my analytical figures : — Urine A 10 c.c. urine gave 0-0740 gr. BaSO, = 0-0102 gr. sulphur in 10 c.c urine ” ” ni 0-0744 gr. ow ; = 0-0102 gr. ow ” ” Urine B 10 c.c. urine gave 0-0798 gr BaSO, = 0-0110 gr. sulphur in 10 c.c. urine ” ” ” 0-0800 gr. ” = 0-01 10 gr. ” ” ” Urine C 10. c.c. urine gave 0-1032 gr. BaSO, = 0-0142 gr. sulphur in 10 c.c. urine ” ” ” 0-1043 gr. ” = 0-0143 gr. ” ” ” Urine D 10 c.c. urine gave 0-0322 gr. BaSO, = 0-0044 gr. sulphur in 10 .c. urine ” ” » 00318 gr os = 0-0044 gr. ows ” ” Urine E 10 c.c. urine gave 0-0756 gr. BaSO, = 0-0104 gr. sulphur in 10 c.c. urine 5 c.c, ” ” 0-0383 gr. ” = 0-0106 gr. ” ” ” It will be seen, in confirmation of what Abderhalden and Funk state, that the figures are very concordant. If the figures obtained by the Konschegg method are compared with _ the above obtained by the Pringsheim method, it will be observed that the Konschegg figures are a little higher (1 to 2-1 per cent.) than those obtained by this method, and 5°2 per cent.! on the average higher than those obtained by Schulz’s method. Asbéth-M odrakowski Method. Were different results were obtained, according to differences in the details of the modus operandi. In most of my estimations I carried out the process in the following manner :— I added the urine gradually to the sodium peroxide to avoid loss by spitting,” evaporated down on the water bath to complete dryness, and subjected the residue to complete fusion over a spirit burner, maintaining it in the fused condition for ten to fifteen minutes; cooled; added 1-2c.c. of distilled water, and more peroxide and again subjected the mixture to complete fusion, prolonging the fusion for twenty to thirty minutes. I used between three and four grams of sodium peroxide for the preliminary fusion and eight grams for the final; the quantity of urine used was 25 c.c. Using this method I obtained, in all the estimations I performed, agreement between the individual determinations made on the same urine, and also higher results than by any of the other methods.* On the average the figures were 8 per cent. higher than those - actin ard ale eee wach ing Aone by Konschegg himself. 2. Recommended by Modrakowski, loc. cit. 3. M. A. Deamouliére, Journ. de Pharm. et de Chim, (6th series}, Tome XXIV, p. 294, 1906, states that Moreigne’s and Modrakowski’s methods give accurate results in estimation of total sulphur in urine; the vremerinme serthdapmelland Mee! By Ret deen joomla ~~ sd ieee aoe h cen figu (really sight! ee ae onstration, the same Tes wer) as Na,CO,—KNO, method, accords with my results. . ‘ 342 BIO-CHEMICAL JOURNAL given by the Konschegg method. I was unable to detect any evolution of sulphuretted hydrogen, either during the evaporation or the subsequent acidification of the fused mass, such as Gill and Grindley cc The following are my analytical figures :— Urine B 25 c.c. urine gave 0-2036 gr. BaSO, = 0-0112 gr. sulphur in 10 ¢,c. urine ‘ . ” we 0-2053 gr. ” = 0-0113 gr. ” ” ” - Rated . Urine © a 25 c.c. urine gave 0-2724 gr. BaSO, = 0-0150 gr. sulphur in 10 ¢.c. urine ” ” 0: 2798 gr. ” = 0-01 54 gr. ” ” ” Urine D 25 c.c, urine gave 0-0966 gr. BaSO, = 0-0053 gr. sulphur in 10 ¢.c. urine es » 0-0970gr. , =00053¢gr._,, pe 2 This aaaa’ as I have already described it, is in the main identical with that described by Folin, except that he does not mention the preliminary fusion, and maintains that ten minutes’ final fusion is sufficient. The number of analyses I have carried out according to his directions is not great; they agree well inter se, but they are never higher than those obtained by Pringsheim’s method, and in the one case where © I can make the comparison they are lower than those obtained by Konschegg’s method.' The following are my analytical figures : — Urine A 25 c.c. urine gave 0- Ay BaSO, = 0-0102 gr. sulphur in 10 c.c. urine ” ” ” 01858 gr. os = 0-0102 gr. ” ” ” Urine E “a 25 ¢.c. urine gave 01858 gr. BaSO, = 0-0102 gr. sulphur in 10 ¢.c. urine eee ” » 01880 gr. 5, = 0-0103 gr. ” ” ” Putting all the results together, the following table shows the mean figures expressed in grams of sulphur per 10 c.c. urine. Method Urine A Urine B Urine C Urine D Urine E GIEER © cobcn sdoavecccnkudvac coensiaee 0-0099 0-0097 0-0139 0-0042 ~ Konschegg sheesh eigdmagiiia na odteed 0-0104 0-0104 0-0145 0-0045 — i RIE Yana vectcabasvudeobadenta 0-0102 0-O11L0 0-0143 0-0044 0-0105 ’ Asboth-Modrakowski ............ — 0-0113 0-0152 0-0053 — ’ Asboth-Modrakowski (Folin) .... 0-0102 _ —- = 0-0103 From the above table it will be seen that Schulz’s method gives the lowest, whilst the Asbéth-Modrakowski method gives the highest figures; the Pringsheim, Konschegg and the Folin’s modification of the Asbéth- Modrakowski method all give intermediate figures. _ Conelusion. The sodium peroxide method carried out according to Asbéth-Modrakowski (as described above) gives the highest figures in the estimation of the total sulphur in the urine, and must therefore be considered to be the most trustworthy of the methods at present in use. — 1. Gill and Grindley, loc. cit., state that the difference is even greater than I have obtained. 343 ber THE USE OF BARIUM PEROXIDE IN THE ESTIMATION _ OF TOTAL SULPHUR IN URINE By STANLEY RITSON, A.K.C. From the Physiological Laboratory, King’s College, London Communicated by Prof. W. D. Halliburton, F.R.S. (Received July 16th, 1909) ——— Although the Asbéth-Modrakowski method gives the best results for the estimation of total sulphur in urine (see preceding paper), it has the disadvantage of being somewhat lengthy. In metabolic experiments, where it is necessary to make a large number of estimations, it is essential that a process should be adopted which can be carried out rapidly. From this point of view the Pringsheim method seemed to be the best, _ provided it could be modified so as to give figures equal to those obtained by the Asbéth-Modrakowski method. With this end in view it occurred to me that the use of barium peroxide, in the fusion of which a higher _ temperature is obtained than with sodium peroxide, might lead to a more complete oxidation. The introduction of a barium salt would have the additional advantage of shortening the method, as the barium sulphate is formed during the actual process of the oxidation.' I therefore tried fusing with barium peroxide instead of sodium peroxide, but found that fusion did not take place easily. This difficulty was overcome by the addition of sodium peroxide to ____ the barium peroxide in the proportion of seven to one. The process was __ then carried out exactly as in the Pringsheim method, the details of ____ this new method being as follows :— Applying this idea to the Konschegg method, I found that the addition of barium slices’ does not give any better results than when it is absent. The following are my mean ee co ee oop pee 10 oe. urine :— * Method Urine A Urine B Urine C Urine D Konschegg ... Fs .. O-0104 0-0104 0-0145 0-0045 Barium-nitrate-nitric acid . 0-0102 0-0109 0-0144 0-0042 The addition of barium Tati in the Asbéth-Modrakowski method did not yield con- cordant results, and as the method is afin ig 2 I did not pursue it further. In the literature the only references I find with regard to the utilisation of a barium salt in the estimation of sulphur were :— (2) H. Weidenbusch, who, at Liebig’s suggestion, estimated sulphur in albuminous materials by fusing » paste formed of the substance, barium nitrate and nitric acid. He gives ae a ber « Ann., Bd. LXT, 8. 370, 1847. pate um peroxide to estimate sulphur in cit S308, sub . — finally fusing with sodium peroxide, Zeitech. /. analyt. Chem. Ba. Ba, XXXI EM Ot a a ae a CMT ety AY, ee ee ey a a B44 BIO-CHEMICAL JOURNAL Ten c.c. of urine were measured into a nickelled steel crucible, as recommended by Pringsheim,! and made alkaline with sodium carbonate. After the addition of 0:4 grm. lactose, the mixture was evaporated down, on the water-bath, to a syrupy residue. Without further drying, the residue was carefully mixed with 8 grms. of the oxidising agent, consisting — of 7 grms. of sodium peroxide and 1 grm. of barium peroxide.? The crucible is next immersed up to three-quarters of its height in distilled water contained in a larger porcelain crucible or basin. A red-hot iron nail is introduced through the hole in the lid, and in a few seconds the reaction is completed. When the crucible has cooled down sufficiently it is overturned into the water, the basin being covered by a clock glass. The contents of the basin are then transferred quantitatively into a 500 c.c. Erlenmeyer flask and raised to boiling point. Concentrated hydrochloric acid is added gradually to the boiling fluid until the ferric oxide (derived from the iron nail) has gone into solution. A small excess of hydrochloric acid and a few c.c. of alcohol are then added, and the boiling continued for a short time. This serves to drive off the chlorine, which is always formed by the action of the excess of sodium peroxide on acidifying the solution with hydrochloric acid. It was noticed that under the above conditions the oxidation (as judged by the absence of carbonaceous particles) is complete, whilst without the use of barium peroxide filtration is very frequently essential in order to remove particles of carbon. The barium sulphate which is now present in the form of a granular precipitate is then collected on a weighed Gooch crucible, dried, ignited, and weighed as usual. It was found as a further advantage of the barium peroxide addition that the barium sulphate precipitate settles and filters very easily, probably owing to the physical conditions under which it is formed. Using this method my analytical figures were the following :— Urine B 10 ¢.c. urine gave 0-0857 gr. BaSO, = 0-0118 gr. sulphur in 10 c.c. urine ” ” ” 0-0837 ” 0-0115 ” ” ” 2) Urine C 10 c.c. urine gave 0-1112 gr. BaSO, = 0-0153 gr. sulphur in 10 ¢.c. urine ” ” ” 0-1122 ” = 0-0154 ” ” ” 1. The crucible, together with the perforated lid, is obtainable from Messrs. Kohler, Leipzig. 2. A certain amount of care must be exercised in the addition of the begining. Aten I found it advisable to add about 0-1 to 0-2 gramme at a time at the addition of about half the required amount, the remainder may be added in a Paste Fs The barium peroxide used was tested for sulphur with negative results. TOTAL SULPHUR “IN URINE 345, = Urine D “10 c.c. urine gave 0-0472 gr. BaSO, = 0-0065 gr. sulphur in 10 ¢.c. urine ” ” ” 0-0488 ” = 00067 ” ” ” _ From this it will be seen that the results agree well inter se. If we pare the mean figures obtained by this method with those obtained out the use of barium peroxide (Pringsheim method) it will be seen ve considerably higher results. Compared with the Asbéth- \ditions the oxidation is even more complete than in the Asboth- cowski method. ere Urine Bs Urine ~——dUrrine D Pringsheim method =... —«.. O00 0-0143 0-0044 Asbéth-Modrakowski method... 0-0113 0-0152 0-0053 New method 3 we OOLIT 0-0154 0-0066 346 A CONTRIBUTION TO THE BIO-CHEMISTRY OF HAEMOLYSIS :— (a) CHANGES IN SOLUBILITY OF THE LIPOIDS IN PRESENCE OF ONE ANOTHER, AND OF CERTAIN > UNSATURATED ORGANIC SUBSTANCES. (6) THE BALANCING ACTION OF CERTAIN PAIRS OF HAEMOLYSERS IN PREVENTING HAEMOLYSIS. (c) THE PROTECTIVE ACTION OF SERUM PROTEINS AGAINST HAEMOLYSERS. (d) THE EFFECTS OF OXYDISING AND REDUCING AGENTS UPON HAEMOLYSIS. By BENJAMIN MOORE, M.A., D.Se. (R.U.1.), Johnston Professor of Bio-chemistry, University of Liverpool; FREDERICK P. WILSON, M.D. (Liverpool), saxn LANCELOT HUTCHINSON, M.D. (Liverpool). From the Department of Bio-chemistry, University of Liverpool (Received July 22nd, 1909) The subject of haemolysis, and the relationship of lipoid substances to this process of laking of the blood corpuscles, is one which is at the present time exciting very general attention from physical chemists, biological chemists, and clinicians alike because of its important relation- ships to the chemistry of colloidal solutions on the one hand, and of its valuable applications to the diagnosis of disease on the other. In earlier papers more directly concerned with the subject of the digestion and absorption of fats, it was shown in 1897 by Moore and Rockwood! and in 1901 by Moore and Parker? that the salts of the bile, and the products of fatty cleavage of a lipoidal nature, possessed when in common solution some kind of an affinity, apparently of a physico- chemical nature, which had the effect of increasing the solubilities of the fatty acids and soaps. This remarkable change in solubilities was shown by Moore and Parker to extend to other lipoids, such as lecithin. It was further demonstrated that the unsaturated oleic acid and its sodium soap had a 1. Proc. Rey. Soe., Vol. LX, p. 438, 1897; Journ. of Physiology, Vol. X XI, p. 58, 1897. 2. Proc. Roy. Soc., Vol. LXVIII, p. 64, 1901. BIO-CHEMISTRY OF HAEMOLYSIS B47 most peculiar effect in increasing many times the solubilities of the fully saturated palmitic and stearic acids and their sodium soaps. These when in a state of purity were found to have practically a zero solubility in either water or bile salt solution. | Moore and Parker showed in the case of lecithin, which they prepared _ from egg yolk, that the lecithin, when it was added to a solution of bile salts or to bile at body temperature, did not form an emulsion or fine suspension as in the case of treatment with water, but gave instead a Fae water clear solution. This solution was then more effective than the se! “veda in dissolving other lipoids. oe tes - They also found, probably on account of this mutual effect upon : _ effective solvent than a considerably stronger solution of the separated i cand re-dissolved bile salts. PD _ These earlier results on mutual solubility appear to us to possess a ‘bearing, which will be pointed out later, upon the process of haemolysis of the lecithin-containing corpuscles by other lipoids, such as sodium - oleate, the bile salts, and saponin-like bodies, and for this reason we quote here certain of the figures given by Moore and Parker which definitely show the mutual effects. +The solubility of the fatty acids and soaps was found to be as follows :— ‘Oleic Acid : solubility in distilled water less than 0-1 per cent. ; solubility in 5 per cent. bile salt solution, about 0-5 per cent. ; solubility in 5 per cent. bile salts plus one per cent. lecithin, 4-0 per cent.’ pe _ * Palmitie Acid : in distilled water less than 0-1 per cent. ; in 5 per cent. bile salts, about ___ O1 per cent. ; in 5 per cent. bile salts plus 1 per cent. lecithin, 0-6 per cent.’ ____ * Stearic Acid : in distilled water less than 0-1 per cent. ; in 5 per cent. bile salts less than ’ O-1 per cent. ; in 5 per cent. bile salts plus 1 per cent. lecithin, 0-2 per cent.’ Sodium Oleate : in distilled water, 5-0 per cent. ; in 5 per cent. bile salts, 7-6 per cent. ; in SPUN UeaA hile. salts glue 1 for cent. lecithin, 11-6 per cent * Sodium Palmitate : in distilled water, 0-2 per cent. ; in 5 per cent. bile salts, 1-0 per cent. ; in 5 per cent. bile salts plus 1 per cent. lecithin, 2-4 per cent.’ * Sodium Stearate : in distilled_water, 0-1 per cent. ; in 5 per cent. bile salts, 0-2 per cent. ; in 5 per cent. bile salts plus 1 per cent. lecithin, 0-7 per cent.’ * Lecithin. “Pure” lecithin is practically insoluble in water, the addition of as little as 0-1 per cent. causes an opalescence and further additions give rise, as is well known, to a kind of emulsion. But when lecithin is added to a 5 per cent. solution of bile salts, or to bile, the appear- ances observed are quite different.’ *The lecithin dissolves to a clear brown-coloured solution and the amount taken up is surprising ; thus a 5 pot cent. solution of bile salts takes up no less than 7 per cent. of lecithin 848 BIO-CHEMICAL JOURNAL — | is at a temperature of 37°C. On cooling, part of the lecithin is thrown out of solution as a finely suspended precipitate or emulsion which glistens with a silky lustre when the test-tube containing it is shaken so as to set the fluid in motion. At ordinary room temperatures of 15° to 20° a considerable amount of lecithin, 4 to 5 per cent., is, however, still retained in solution.” “AS SP be, a ‘The power of lecithin in increasing the solubilities of the fatty acids and soaps, in great part why lower solubilities are obtained in experimenting with pure bile salt solutions, in than with bile. The lecithin naturally occurring in bile thus increases the solvent power of that fluid in the intestine for fatty acids and soaps.’ We have quoted at length these earlier experiments upon the mutual effects of different lipoids in common solution upon one another, because they appear to us to have some bearing upon haemolytic phenomena. For example, sodium oleate or sodium linoleate have a strong laking effect upon the red blood corpuscles. Now the red blood corpuscles contain lecithin, but the above experiments ‘show that the presence of lecithin in solution increases the solubility of oleates. In haemolysis of this type it is hence obvious that the converse result is being obtained and that the oleates or linoleates are laking the corpuscles, because lecithin is more soluble in presence of the oleates or linoleates. We shall also see that the bile salts and the members of the saponin- digitalin group of glucosides are all unsaturated compounds like the oleates and linoleates, and that they increase by their presence lipoid solubilities, and hence are powerful laking agents. These results upon solubility were confirmed and extended in several papers by Pfliiger! and others, and Pfliiger laid particular stress upon the effect of the presence of sodium carbonate and of oleic acid and oleates in : raising the solubilities of the other constituents. The above experiments upon solubility of lipoid materials and their derivatives may now be considered in relationship to haemolysis. ° | A very considerable portion of the stroma of the red corpusele is lipoidal in character, that is to say, is soluble in ether or similar solvents. The amount is placed at one-third of the dry weight by Pascucci,? and of this a large amount consists of mixed lecithides, containing unsaturated fatty acids in the molecule. Accordingly, any constituent in a serum or suspending saline which possesses the property of increasing the solubility of these lecithides must tend to lake the corpuscles by dissolving up the stroma. Such an action, as shown by Moore and Parker, is possessed by the bile salts, and they accordingly act as powerful haemolysers. 1. Arch. f. d. ges. Physiol. Bd. LXXXII, 1900, 8. 303, 381; LXXXYV, 1901, 8. 1; LXXXVIITI, 1902, g 299, 431; XC, 1902, 8. 1. 2. Hofmeister Beitriige, 1905, Vol. VI, p. 543; Iscovesco (* Les Lipoides,’ p. 13, 1908) places — the amount of lipoids in the dried corpuscle at a lower value than one-third. BIO-CHEMISTRY OF HAEMOLYSIS 349 This haemolytic power of the bile has long been known qualitatively ; it has just now been followed out quantitatively in this laboratory by MacLean and Hutchinson with the most interesting results, recorded in the paper immediately succeeding this one.! In the same fashion, we have seen that oleic acid and oleates were found experimentally to raise the solubilities of the practically insoluble i palmitates and stearates in the presence of bile salts. Also, even in the __ absence of all bile derivatives, the solubilities in water obtained by Moore and Parker for the separate sodium salts of the acids, oleic, palmitic, and ___ stearic on the one hand, and for the mixed sodium soaps of naturally --—s geeurring fats of pig, ox and sheep on the other, clearly show that the _ presence of sodium oleate increases the solubility of the other soaps. These experimental results must be the basis of the results obtained _by many observers that sodium oleate is a powerful haemolyser, while, as ae demonstrated by Noguchi,? the sodium palmitate and sodium stearate aS aes inert. A chemical point of great importance is that both the oleic acid and the bile acids are unsaturated bodies containing in each case doubly-linked __ earbon atoms in an open chain, and this suggests the general law, first enunciated by St. Faust and Tallqvist,? that the haemolytic property is _ associated with this absence of saturation. At the outset of our work, we were unfortunately unaware of the a existence of St. Faust and Tallqvist’s paper, and we must express our Px regret that for this reason we were unable in a preliminary communication to do justice to their most interesting work upon the subject. These authors, in following out in a highly interesting fashion the causes of a pernicious anaemia due to the intestinal parasite, ___—C Botriocephalus latus, were able to separate from the dried bodies of the ___— parasites a material consisting to a large extent of an unstable compound _ £ cholesterin and oleic acid. This substance was shown to be a cholesterin ester of oleic acid of the type first separated from blood serum by Hirthle.® This cholesterin-oleic ester had a most powerful haemolytic effect even in small quantities, and on further testing the matter, St. Faust and Tallqvist discovered that the haemolytic action was due to the oleic acid, and that sodium oleate gave a like result, while saturated soaps or their esters gave no effect upon the blood corpuscles. 1. See page 369. 2. Noguchi, Journ. of exper. Medicine, Vol. VIII, p. 92, 1906. 3. Arch. f. exper. Path. u. Pharm,, Vol. LVIS, p. 370, 1907. 4. 5. Journ. of Physiol,, Proo. Physiological Society, March, 1909. . Zeitach, |. Physiol. Chem., Bd, XX1, 1895-6, 8. 331. 850 BIO-CHEMICAL JOURNAL This result led the authors to the generalisation that such haemolytic action was associated with the want of saturation of the oleic acid. This was tested by employing other unsaturated acids such as acrylic, tiglie, cinnamic and erucic acids, and it was found in each case with the free — acids there was marked haemolysis, although in the case of the sodium __ salts of tiglie and cinnamic acids there was no haemolytic activity. St. Faust and Tallqvist further demonstrated in support of their view, that when acrylic acid is hydroxylated into hydracrylic acid the latter is almost without action upon blood corpuscles. Now one of these acids has a double bond which has been split up in the other, as shown by the formulae given below, and this is probably the cause of the difference in activity. Acrylic acid. Hydracrylic acid II if 2 Coon COOH Strongly haemolytic Not haemolytic We have ourselves been able in the present series of experiments to demonstrate that an unsaturated glucoside with strongly haemolytic properties, isolated by Moore from the seeds of Bassia longifolia (Mowrah - seeds), called ‘Mowrin,’ loses its haemolytic properties when it becomes saturated by bromination. It is difficult to explain why the sodium salts of tiglic and cinnamie acids do not haemolyse, for they are unsaturated compounds. It may be that a certain conformation of molecule in addition to the double linkage is necessary in order to confer upon the haemolytic molecule certain physico-chemical properties which we shall subsequently see all these : haemolysers possess, and that the want of saturation really confers laking power because of the physical properties of solution, ete., which attach themselves to it, and not because the double bond is broken to allow a firm combination between the two compounds. St. Faust and Tallqvist do not appear to have tested other acids and salts than those mentioned above, nor to have proceeded to the further generalisation that a similar lack of saturation characterises other laking agents such as the bile salts and saponins. Believing that the view is one of somewhat far-reaching importance, we have in the present experiments tested it with a number of haemolytic substances, and have always found that substances of this nature which were haemolytic were also unsaturated or possessed of a good deal of residual chemical affinity. BIO-CHEMISTRY OF tlAEMOLYSIS 351 Connected with the above two points of haemolytic power and want of chemical saturation, these bodies—often of widely different origin and chemical constitution—possess always a well-marked group of common properties, physical and physiological, which are so striking when placed in juxtaposition as to indicate that they and the laking process are all closely connected together and have a common cause. Further, these common properties, which will presently be stated, are such that in spite of the fact of chemical unsaturation running through the whole group, it is difficult, or indeed impossible, to draw any definite conclusion as to whether these bodies act by forming a feeble labile chemical union, or by physically altering the properties of the solvent so a, that it now dissolves the lipoids. The energy phenomenon at play is evidently one due to interaction between dissimilar chemical molecules, but whether it consists wholly, or as an initial stage, in a lowering of the surface tension at the interface bes between lipoid and solvent, or whether there is first a labile chemical union between lipoid and unsaturated acid causing an accumulation on the inter- ae face, so leading to a negative surface tension and hence to solution of the ___ lipoid and to haemolysis, it is impossible to say in the present state of our knowledge. It may be remarked, however, that the combating physical and chemical hypotheses are not so very widely apart as the two camps of adherents suppose, for in either case, the interaction is between dissimilar molecules or aggregates at an interface, and this does not differ widely from chemical action. We do not know what are the initial ‘ physical ’ stages of ‘ chemical ’ combination. It might be asked, what is the nature of the energy change which eatises accumulation of a dissolved substance on an interface and lowering _ of surface tension, if it be not chemical attraction and the preliminary stage of a chemical reaction? Leaving these more abstract considerations of chemical combination versus physical adsorption, we may now return to the characteristic chemical and physiological properties of the group of haemolytic agents which we are discussing. Prorertizs or HAEMOLYSERS Physical Properties—All these substances are colloidal in aqueous solution, although some of them diffuse very slowly through parchment paper; they do not crystallise out of aqueous solution, and they give rise to thick syrups as they are evaporated down to more and more concentrated 352 BIO-CHEMICAL JOURNAL solutions. Even in dilute solution they all froth easily, showing petenly that the surface-tension is lowered,! Chemical Properties —All show a great tendency to form — compounds, which are very easily hydrolysed by dilute acids. For example, the bile salts with amino-acids, such as glycocoll or taurin; ; oleic acid with cholesterin to form cholesterin esters, and with glycerol to form fats; saponin, digitalin, mowrin, and other haemolysers of that type are glucosides; the lecithides are not only conjugated compounds themselves, but unite in feeble union with a vast number of substances of biological origin, such as snake venoms and tox-albumens. This property of conjugating chemically is, as we shall see, of the utmost importance in connection with haemolysis, where it also occurs, and may cause active haemolysis or an anti-phase, according to how it is directed. . Physiological Properties—Vhe physiological properties of the whole group are closely related, and are, no doubt, dependent upon the above physical and chemical properties. Thus the soaps, the bile salts, and the whole saponin-digitalin group, are characterised by a very intensely bitter taste. Introduced directly into the circulation, they are all poisonous, and all affect the heart, causing slowing and stopping. ‘This is in all proba- bility due to a common cause, viz., combination with the heart lecithides. That same physico-chemical property which attacks the red blood corpuscles by means of the attraction for its lecithin and causes haemolysis, causes attack, always of a common type with minor variations, upon the heart, due here also to chemical attraction between soap (sodium oleate), bile salt, saponin, mowrin, digitalin, or what not, of this large group of unsaturated bodies on the one hand, and the heart lecithide on the other. So variations in reactivity are caused within the heart cell, and accom- panying modifications in heart beat. Here it is to be remarked that it is an integral change inside the cell of which the lecithide is a vital part that occurs, and is not a mere question of altered permeability of a lipoidal membrane. . These peculiar properties are shown in varying degree by different members of the group, but taken together they form a good set of characteristics for a very widely distributed’ group of substances all possessing haemolytic properties. 1, This is known to be so with the soaps, experiments with other haemolysers are in progress, as BIO-CHEMISTRY OF HAEMOLYSIS 353 Tue Batanctnc Action or HAEMOLYSERS ~-Oné of the most interesting experiments in haemolysis is that of Sachs and Altmann,! demonstrating that two bodies, each of which is strongly haemolytic in itself, can be so admixed in common solution that no haemolysis whatever results, the two haemolysers balancing each other. Thus, it was found that when sodium oleate was added in just the proper quantity to a strongly active haemolytic serum no haemolysis resulted, and that as the amount of oleate was gradually increased above this balancing amount, the mixture gradually became haemolytic again. This result has been stated to be due to the neutralising of comple- “a ment, the sodium oleate acting as an anti-complement. We think, however, that there is clear evidence against this view. In the first place, as we shall see later, an ordinary serum which is not haemolytic to the corpuscles being used, it may, in fact, be their own serum, is strongly ee. _ protective against the haemolytic action of sodium oleate. =_ We have followed this question up in detail, as shown by the protocols ae 3 of our experiments, and have successively removed or destroyed (a) immune body, (4) complement, and (c¢) the lipoids from the active - serum. In all cases we have found that no one alone of these substances is responsible for the neutralizing of the haemolytic activity of the sodium oleate. The further fact that not only is the laking power of the sodium oleate destroyed, but also the natural activity of the pig’s serum, or the invoked activity of a specially sensitized serum, appears to us to clearly demonstrate that the soaps of the unsaturated fatty acids, oleic and linoleic, possess a selective affinity for the immune body, or actively laking, substance, of these haemolytic sera. That is to say, in the active _ Serum the immune body and the sodium oleate or linoleate combine and _ mutually destroy each the other's laking power, so that the mixture in due proportion is quite inert upon the blood corpuscles. But in case the immune body has been inactivated by heating, then the sodium oleate or linoleate is still captured and held by the serum proteins, so that no laking oceurs until much more of the oleate or linoleate has been added than would have sufticed to cause complete and rapid laking in a saline suspension, where there is no protein to present a counter-attraction and binding agent, so that the first trace of oleate or linoleate at once attacks the lecithides and other lipoids of the corpuscles, When the serum proteins are present, although inactive themselves, they 1. Berl. klin. Wochensch., pp. 494, 609, 1908. 354 BIO-CHEMICAL JOURNAL form binding material for unsaturated lipoids, such as the oleates and linoleates. Yao The action of lecithin and cholesterin of a similar type can be. explained on similar lines rather than on the view that these substances wy aig behave as active anti-complements, lee On the other hand, as our experiments also prove, two lipoids of nearly allied nature which do not therefore combine with each other, or mutually adsorb each other, such as oleate and linoleate of sodium, show no balancing action whatever, but produce a distinctly additive effect. So that whether shown by the smaller amounts which will produce complete laking in a given time in presence of each other, or better by observing the laking times of two minimal amounts of oleate and linoleate separately, as compared with the time spent for laking with the halves of these amounts acting in consort, the result always comes out that the haemolytic effect consists of the two added factors of oleate action and linoleate action; there being no reduction whatever due to action between the -two haemolysers, such as is seen between either of them and the active haemolytic body of a sensitized serum or a serum naturally haemolytic. We may hence enunciate the law that if two given haemolysers are capable of combining or adsorbing with each other, they will tend to balance each other, and the effect on corpuscles will be less than either acting alone; but if no adsorption is possible between the two, the effect in common solution upon the corpuscles will be the sum of the effects of the two. Errects or Ox1pizinc and Repucinec AGENTS uron HaArMoLysis These experiments were suggested by analogies between the mode of action of the peroxidases and haemolytic serum, in that heating to 56° C. destroys the tissue peroxides and so stops the action of the _ peroxidases in a somewhat similar way to that in which heating to 56° C. inactivates a haemolytic serum by destroying complement. The results of experiment showed that it was not possible to replace the destroyed complement of an inactivated serum by means of hydrogen peroxide or other form of peroxide, so that the haemolytic agent or immune body can hardly be regarded as a peroxidase ferment. Yet the experiments yielded the very interesting information that addition of an alkaline reducing agent, sueh as ammonium sulphide, even in very small amount, entirely inactivated an active serum, and contrariwise an oxidizing agent, such as hydrogen peroxide in alkaline . — ae BIO-CHEMISTRY OF HAEMOLYSIS 355 solution, very much increased the haemolytic power. ‘The peroxide alone, _ or lrydrogen sulphide alone, in absence of alkali, had very little action; but the addition of a trace of ammonia at once produced the inhibiting action in the ease of the sulphide, or favouring action in the case of the peroxide. . In view of the fact that ammonia and other alkalies by themselves possess a laking effect, it may be emphasized that the amounts being used lay below the laking amounts when used alone, as shown by control experiments. EXPERIMENTAL Mretruops AND REsULTS The sodium salts of five fatty acids were taken, namely, the sodium salts of stearic, palmitic, erucic, oleic and linoleic acids. In this list the first two are sodium soaps of saturated fatty acids, a 3 : belonging to the acetic acid series; sodium erucate and oleate belonging ; _ to the acrylic series, are unsaturated sodium soaps, each having one doubly- _ linked carbon atom in their formula; sodium linoleate belonging to the linolie acid series is still more unsaturated, having three doubly-linked carbon atoms in its constitution. To test the haemolytic power of these soaps, solutions varying in strength from 0°01 M to 0°001 M were made up by the simple procedure of weighing out the requisite amount of pure free acid and neutralising with the calculated amount of decinormal alkali. Thus, taking C,,H,,0, as the formula for oleic acid, this gives a molecular weight of 282, which is equivalent to 0°282 grams in 100 c.c. for a centimolecular solution. This weight of oleie acid was therefore weighed out in a beaker and __— neutralised with 10 c.c. of 011M NaOH, and the volume made up to —___—«* 100 e.c. by adding normal isotonic saline solution. The weaker molecular strengths were made up by adding proportionally more normal saline. The other sodium soaps were made up in a similar manner. For the experiment a series of test-tubes were taken, and in each was placed a certain known quantity of the sodium soap, whose haemolytic properties it was desired to test, and 1 c.c. of a 5 per cent. emulsion of sheep's red blood corpuscles; the volume of each tube was then made up to 5°5 c.c. by adding normal isotonic saline solution. When the contents of the tube were completed, they were placed in a thermostat at a temperature of 37° C, and observations made. It should be mentioned that in this and the subsequent experiments the emulsion of sheep’s red blood corpuscles was made by defibrinating 356 BIO-CHEMICAL JOURNAL fresh blood and then washing and centrifuging the corpuscles three me in ree saline, and finally the washed red blood corpuscles were made up into a 5 The results of several experiments with the soaps above mentioned — gave the following results : — In the tubes containing: 0°2 c.c. = sodium stearate. M : ; 02 o.c. Too. sodium palmitate. M . 0°2 c.c. Too sodium erucate. M_ 0°8 c.c. 1000 sodium sruaite, M 3 0°2 c.c. 00 sodium oleate. M . 0°4 c.c. 1000 sodium oleate. M . 0°2 c.c. 1000 sodium oleate. tee 0-4 c.c. 4000 sodium oleate. M . . 02 c.c. 00. sodium linoleate. M Tore 08 c.c. 7000 sodium linoleate. M a 0:4 c.c. 1000 sodium linoleate. M / : 0-2 c.c. 1000 sodium linoleate. M , : 0°4 c.c. 4000 sodium linoleate. It will be seen that in the case of the saturated sodium soaps of stearic and palmitic acid no haemolysis was observed with the above strengths, ‘fia but with very much weaker strengths of the unsaturated soaps complete r haemolysis was obtained, and that sodium linoleate, which has the greater number of doubly-linked carbon atoms, possesses also the strongest laking per cent. emulsion in normal isotonic saline. (eae « met No haemolysis in 24 hours. No haemolysis in 24 hours. Complete haemolysis in 24 hours. No haemolysis in 94 hours. Complete haemolysis in 1 hour. Complete haemolysis in 8 hours. Slight amount of laking after 20 hours. Merest trace of laking after 20 hours. Complete haemolysis in 16 minutes. Complete haemolysis in 50 minutes. Complete haemolysis in 153 minutes, Complete haemolysis in 20 hours, Slight amonnt of laking in 24 hours ae oe ee action, completely haemolysing the sheep’s red blood corpuscles within twenty hours, even in a concentration equivalent to 0:000004 M. oleate, which has one doubly-linked carbon atom, is also a powerful haemolytic agent, but not so active as sodium linoleate, though more Sodium powerful than sodium erucate, which is an equally unsaturated soap, but mad with a different molecular constitution, and with corresponding physical ce eee IE OS ney eerste 2 Le Oa eo BIO-CHEMISTRY OF HAEMOLYSIS 357 _ properties showing less typically the common character of the class of i On the same grounds, some observations were also made with a _ glucoside mowrin and the sodium salt of mowrie acid, one of the products prepared from the glucoside by hydrolysis. These preparations, which are a unsaturated bodies, were prepared by Moore from the seeds of Bassia ____ dongifolia, commonly known as Mowrah seeds. ms The following are the haemolytic results obtained with these substances, using 1 c.c. of 5 per cent. emulsion of sheep's red blood corpuscles and a total volume of 5°5 ¢.c., and following the same M 1 c.e. "00 Mowrin. Complete haemolysis within 1 minute. ¥ ; M i oe 22'S 0.0. 10000 ” Complete haemolysis within 4 hours. = M Ue $x 05 cc. Too00 ” No haemolysis in 24 hours. at M 1 cc. Too Sodium Mowrate. . Complete haemolysis in 15 minutes. M . 1 cc, 1000 PP ts Trace of haemolysis in 20 hours. ae : ue No haemolysis - “hod O75 c.c, 1000 ” ” ysis. It will thus be seen again that these unsaturated bodies, especially the glucoside mowrin, are also powerful haemolytic bodies. . If, however, the sodium salt of mowric acid is brominated, the haemo- lytie action is markedly weakened, for instance, 1 c.c. of 0°01 M sodium -__- mowrate haemolyses in fifteen minutes, but of an exactly similar quantity if the brominated sodium mowrate be used, the time required in this case ____ for complete haemolysis is five hours. _ ————s*Tn view of the fact that there are present in the normal organism _-—s many ~=unsaturated haemolytic lipoids, it is of interest to note the . protective action that the animal's serum is able to exert on behalf of its own red blood corpuscles. In order to study this action as regards the three unsaturated haemolytic soaps used in the previous experiments, a series of test-tubes were taken in which various haemolytic quantities of these soaps were placed, and 3 c.c. of fresh sheep’s serum added to each tube; the serum and soap were then incubated together for half an hour at 57°C., after , which the sheep's corpuscles were added and the tubes replaced in the . thermostat; no haemolysis occurred in any of the tubes, even though " 358 BIO-CHEMICAL JOURNAL 1 c.c. O'01 M of each soap was used, which amount alone would in the case of sodium oleate and linoleate have laked an equal quantity of sheep’s red blood corpuscles almost instantaneously, and eight minutes would haye _ sufficed for sodium erucate. whe Further observations showed that 0°5 c.c. sheep’s serum will exactly a protect 1 c.c. 5 per cent. emulsion of sheep’s red blood corpuscles against 0°35 c.c. of 0°01 M sodium linoleate. Sheep’s serum will also protect its own corpuscles against the natural haemolytic action of pig’s serum; for example, we found that le.c. of 5 per cent. emulsion of sheep's red blood corpuscles is completely haemolysed by 0°5 c.c. fresh pig’s serum within an hour, the addition, however, of 2 e.c. of sheep’s serum will completely inhibit this action. Cholesterin also has an anti-haemolytic action, though not very marked, 1 c.c. 0°002 M cholesterin emulsion being able to inhibit the action of 0°7 c.c. 0001 M sodium oleate. Difficulty was experienced in obtaining the cholesterin in a suitable medium to work with, as the solvents of this compound, such as acetone, etc., are mostly haemolytic. In these experiments, therefore, an emulsion of finely suspended cholesterin in normal saline was used, its strength being approximately 0°01 M. This protective action of serum and cholesterin will be again referred to later on, when we shall have pointed out an action which Sachs and Altmann first described in the case of sodium oleate, and which they termed the behaviour of sodium oleate as anti-complement. __ Experiments are described below showing not only that this action can be extended to other unsaturated-soaps, but also that it is, to a certain extent, independent of the presence of either complement or amboceptor, and therefore the term ‘ anti-complement’ has been omitted and the word ‘balancing’ used in its place, as more accurately. describing the action. For not only is the haemolytic property of the pig's serum on sheep's red blood corpuscles gradualiy inhibited as the amount of soap increases, but also after this action has been completely balanced, the serum on its own part further inhibits the haemolytic action of the soap. Pig's serum is naturally haemolytic for sheep’s red blood corpuscles, but if to pig’s serum is added a certain quantity of sodium oleate there is an inhibition of haemolysis, and a point can be found where, owing to interaction between these two substances, no haemolysis occurs, although” the quantities used of each haemolytic agent are such that if either was usel separately complete laking of the sheep’s red blood corpuscles would ensue, 1. 4 q ‘ BIO-CHEMISTRY OF HAEMOLYSIS 359 For instance, tubes containing :— ___ @. -1-¢.e. sheep's red blood corpuscles—0°5 c.c. pig's serum—4 c.c, normal saline gives complete haemolysis within half an hour. - M b. 1 c.c. sheep’s red blood corpuscles—0°7 c.c. 100 sodium oleate—3°8 c.c. normal saline results in complete haemolysis in 3 minutes. But :— M e. 1c.c. sheep's red blood corpuscles—0°7 c.c. 100 sodium oleate—0°5 c.c. pig’s serum —8-3 c.c. normal saline results in almost complete inhibition of haemolysis. The same holds good for sodium linoleate, and sodium erucate, _— the quantities _ vary in each case. | The results of an experiment are shown graphically in fig. 1. Many similar experiments were carried out giving parallel results : — Tm all cases the sodium salt of the acid, the serum and saline were incubated together for three-quarters of an hour at 37° C. prior to adding the sheep’s red blood corpuscles. When the contents of the tubes were completed they were again placed in the thermostat at 37°C., and observations made from time to time. f The slight variations in these results are probably due to variations in the ‘titer’ of the different supplies of pig’s serum that were used, as the solutions of oleate and linoleate of soda were the same in each experiment, and, moreover, it may be mentioned that the difference between the balancing points for the two unsaturated soaps is constant in all experi- ments, in each the amount of 0°01 M sodium linoleate required to balance 05 c.c. pig’s serum being 13 ¢.c. in excess of the quantity of sodium oleate required for the same result. It is interesting to note that although according to the previous experi- ____- ments sodium linoleate alone isa stronger haemolytic agent than sodium oleate, yet it does not seem to be as powerful as the oleate in balancing the action of pig’s serum. These experiments, while showing the balancing action of sodium oleate and linoleate, seemed also to point to the existence of another. inhibitory action which might be independent of the existence of comple- ment in the pig’s serum. Some pig’s serum was therefore inactivated by heating it at 56°C. for half an hour, and after making sure that the serum was completely inactivated, exactly similar experiments were earried out as before. It was then found that the haemolytic action of the sodium by ; linoleate was inhibited up to the same point when using the inactivated as “e0uBleq sour Aq} oe SMOYS peop oy, 9*“suoTOR or Ajoureeq nssalions 2104} Buouvyeq ur oyvefoul] UNTpos pus UINJes 8,31d 043 Jo UOKoN a peti wre rep 2 @AOgE OWL ‘o'0 G JOOUMNIOA FULySHOD B UTVUTVUT OF OUITCS [BULIOU yuOLOWgNS + ozvo[our, WNIpos _ yo ‘@a0q¥ Pozyworpul SB “*9"D OF uy syunoure + urnaos $84 *9°9 G.0 + 0 °q ‘1 8,deoys "9°9 T poupeyuoo osvo yous uy poforduro eINgXIY KOAWAG S,OIg NI ALVA IONITT WAIGOg AO NOILOY ONIONVIVG “T “OLT —s : <3 bo = 2 2 2 2 © 2 2 ° 2° or or oOo © @ 7 oa or ~ wo bo = we r a < seneeeee ; + + | ae a ¢ T ae Ht aH a=} AH i G — ee a oO Het i = ° + | i AH Hm 8S (6B — j 2 3 | S He sh a S ° je! Seeseest = t+ — +4 > pot 33 5 co seent Gc 5 ’ bt e 4 © tESSH TEESE: S ol = Seen : a = 3335133 B3is3tt: i 9. 2 ees| : = Ptritt : seen t 2 * 4 * i= eaner ’ 2 bY +4 °o 3333 . sss * sae : = o> + >4 - be “— HF : A ee ses H sesstsasesssesssssss | ‘ +4 +44 4 + oes eee H sasseesaes : oe aes 8 aa ’ : .es ® ® . se tr rhe t oan rt +4 secneee es 23 2 6 : <= sas + ® e 4 = ~ sae . hth : TITT +444 £223 HE ihe : Or BIO-CHEMISTRY OF HAEMOLYSIS 861 when using the fresh serum; that is to say, whereas 1 c.c. 0°01 M sodium _ linoleate will alone haemolyse 1 c.c. of 5 per cent. emulsion of sheep’s red blood corpuscles almost instantaneously, yet when used with either (5 c.c. of the fresh or the inactivated pig’s serum its action was almost completely inhibited. As a further step, some pig’s serum was taken in which both the complement and the amboceptor had been removed. This was accomplished by heating some pig’s serum to 55° ©. for half an hour. to destroy the complement, and then adding excess of sheep’s red blood corpuscles (fresh pig’s serum being powerfully haemolytic for sheep’s red blood corpuscles), incubating the mixture together for an hour at 37°C. and then centrifuging; the clear supernatant serum being again treated with sheep’s red blood corpuscles until by tests it was evident that all the ambocepior had combined with the corpuscles, and the clear serum contained neither of its two haemolytic factors. The effect of this serum on the haemolytic properties of sodium “4 - lineleate was then tested in an exactly similar way to that used in the ___ immediately preceding experiments, and it was found that even thus depleted it maintained its protective power intact, and, moreover, that this factor was not in any way diminished. With these results before us, it will be at once apparent that although in the fresh state pig’s serum is a powerful haemolyser of sheep’s corpuscles, yet if we remove from this serum one or both of its haemolytic factors it then exerts a powerful protective action on the red blood corpuscles, an action which in the case of the sheep’s red blood corpuscles is more potent than that exercised by the animal’s own serum, for it will be observed that 0°5 c.c. of sheep's serum was able to protect 1 c.c. of a 5 per cent. emulsion of sheep’s red blood corpuscles against the haemolytic action of 0°35 c.c. 0°01 M sodium lir.cleate, while under similar conditions 0°5 c.c. of inactivated pig’s serum was able to completely inhibit the haemolytic action of 0°6 c.c, 0°01 M sodium linoleate on a similar quantity of sheep’s red blood corpuscles. We have already mentioned that cholesterin has the power of inhibiting, up to a’certain point, the haemolytic action of various soaps. The question arises, is the above protective action due, as Iscovesco thinks, to cholesterin 7 | An attempt to investigate this by trying to extract this inhibitory body from pig’s serum by means of ether gave a negative reply to this question. To carry out this experiment 100 c.c. of fresh pig’s serum was poured ‘into a separating funnel and 150 c.c. of ether added, the contents being then shaken up together for fifteen minutes, allowed to stand, and then the 362 BIO-CHEMICAL JOURNAL serum which collected in the lower part of the funnel was withdrawn. This process was repeated three times, using fresh ether each time. The _ three portions of ether were then collected into one flask and the ether slowly distilled off at a temperature of 37° C., the last traces being removed _ by means of asuction pump. The residue containing ether extractives wis 4 then shaken up with 30 c.c. of warm normal saline, forming ins a white opalescent soapy emulsion. The action of this emulsion was tried on some sheep’s red blood corpuscles, but it was found neither to have any haemolytic action alone nor any inhibitory action against other laking agents. The serum, which had been carefully separated from the ether, was then placed in a flask and all trace of ether removed by bubbling air throug), it was then tested with sheep’s red blood corpuscles, and it was found that although now it had no haemolytie power, yet its protective action was intact. | The result would appear to be evidence indicating that although z cholesterin undoubtedly has considerable inhibitory power, yet it does not >. account for the protective action of the serum, as the extraction with ether : would probably have removed the greater portion. : _ F Appitive Errect on Harmorysts or Two Cioseiy ALLIED HAEMOLYSERS WHICH CANNOT THEREFORE CoMBINE witH Eacu OTHER B re The preceding experiments show that two dissimilar haemolysers, 4 such as the haemolytic substance of pig’s serum for sheep’s corpuscles and oe sodium oleate or linoleate, so far from supplementing, balance each other. — i The present experiment demonstrates that sodium oleate and sodium lincleate uscd in common solution aid each other, the effect being approximately additive. Thus, using the same technique as previously described :— I. Sodium oleate, 1:25 ¢.c. of 0-001 M + sheep’s r. b. c. 1 ¢.c, of 5 per cent. emulsion dye + normal saline to 5¢.c. Result—complete laking in 26 minutes. ae II. Sodium linoleate, 1 c.c. of 0°001 M + sheep’s r. b. c. 1 c.c. of 5 per cent. emulsion - + normal saline to 5c.c. Result—complete laking in 19 minutes. III. The same quantities of the two together, viz., sodium oleate 1-25 c.c. of 0001 M + sodium linoleate 1 c.c. of 0-001 M + sheep’s r. b. c. 1 c.c. of 5 per cent. emulsion + saline to5c.c. Result—complete laking in 11 minutes. IV. Half the initial amounts of the two sodium salts gave the following results, viz, :— Sodium oleate, 0°62 c.c. of 0-001 M + sodium linoleate 0:5 c.c. of 0-001 M + sheep’sr. b. c. 1 c.c. of 5 per cent. emulsion + saline to5c.c. Result—laking in 28 minutes. | There is accordingly no balancing here, and the effects of the two used in common solution are practically a purely additive function. PIO-CHEMISTRY OF HAEMOLYSIS 363 When sodium linoleate and fresh lecithin are used as a pair of haemolysers,-a very distinct balancing action is obtained. Thus, 0°2 c.c. _ of 0°01 M sodium linoleate laked 1 ¢.c. of 5 per cent. emulsion of sheep’s red blood corpuscles in four minutes; but when exactly the same amount _ of the sodium linoleate is first treated with 0°5 c.c. of an emulsion - -—s ¢orresponding to 001M lecithin, the haemolysis is delayed for about -__ two and three-quarter hours. This illustrates very clearly the constituent in the red blood corpuscle which is attacked in haemolysis, and also shows that the haemolyser and =" the lecithin of the corpuscles enter into at least a quasi-combination, so limiting the amount of corpuscle haemolysable by a given amount of _ haemolyser. wl he _ ee omaars or Oxipizinc AND Repvucine AGENTs To HAEMOLYSIS _ Hydrogen Peroxide and Haemolysis.—At the outset of this investi- an attempt was made to test whether complement could be replaced by hydrogen peroxide. _ Pig's serum, which, as is well known, has a haemolytic action on sheep's red blood corpuscles, was inactivated, and to different dilutions of is ‘Serum vary ing strengths of hydrogen peroxide were added. Repeated experiments yielded discordant results; a strength of hydrogen peroxide - which on one occasion caused haemolysis, failed to do so a second time. Investigation showed that the hydrogen peroxide had an acid reaction, and this was probably the cause of the uncertain results. On using dilutions of Merck’s perhydrol, much more uniform results es were obtained. Except in very strong solutions—-up to 1 in 50 in normal _ saline—perhydrol had no haemolytic action on fresh sheep’s red corpuscles. Added to inactivated pig’s serum it had no complementary _ action, and, indeed, the haemolytic actioa of the stronger solutions seemed ; aa be inhibited by the serum. Another oxidizing agent, quinone, was tried in varying strengths in a similar way. No haemolytic or complementary action was obtained. Added to fresh pig's serum, neither perhydrol nor quinone interfered with its haemolytic power. : : Similarly, oxidase containing solutions from fresh vegetable juices ee: could not be used as substitutes for complement. ~ Thus, we have been unable to trace the nature and action of comple- ment in haemolysis, but our experiments led us incidentally to certain observations upon the effects of reducing and oxidizing substances on haemolysis which are here recorded. a bas. we ss ee ial ms; at “ee pe ae | : 364 BIO-CHEMICAL JOURNAL Errecr or Repuctnc AGENTS oN COMPLEMENT We next tried the effect of various dilutions of ammonium sulphide on the haemolytic action of pig’s and other sera. The stock ammonium sulphide used for dilutions was of a strength 0'4 M. Table I gives the details of the experiments. The results show that in dilution up to 1 in 1,000 the ammonium sulphide inhibits the haemolytic action of the sera used. Equivalent solutions of hydrogen sulphide and ammonia in normal saline have not such inhibitory action. In all experiments the tubes were all made up to 5 c.c. with normal saline. The sheep’s red corpuscles were used in the form of a 5 per cent. suspension in normal saline. Fresh guinea-pig’s serum was used as complement. The anti-sheep haemolytic rabbit’s serum was of such a titre that le.c. of a 1 in 1,500 dilution dissolved 1 c.c. of a 5 per cent. suspension of sheep’s red blood corpuscles in half an hour. Taste I 1 c.c. of fresh pig’s serum + 1 ¢.c. ammonium sulphide (dilution lin 50) + 1 e.c. r. b. e.—No haemolysis % asi (dilution 1 in 100) Ms af Pa ¥ (dilution 1 in 200) e- e, 9 ” (dilution 1 in 300) ~ Partial haemolysis 1c.c. of inactivated serum) +1 ¢.c. ammonium sulphide (dilution lin 50) + 1 ¢.c. r. b. ¢. —No heelys + O'Le.c. complement | pepe 2 il eo = a (dilution 1 in 100) Cs 3 ks (dilution 1 in 200) = Haemolysis F ” ” (dilution 1 in 300) its ae f 1 c.c. inactivated rabbit's serum (dilution 1 in 20)) +1 ¢.c. ammonium sulphide + 1 ¢.c. r. b. ¢.—No pm + 0°1¢.c. complement j (dilution lin 50) ” ” (dilution 1 in 100) ~ a aa - a (dilution 1 in 200) ~ Slight haemolysis — * a (dilution 1 in 300) He Complete haemolysis 1 c.c. fresh pig’s serum + 1 c.c. r. b. ¢. Haemolysis 1 c.c. inactivated pig’s serum + 0-1 ¢.c. plclinteehdain 45 ice tate a 1 c.c. inactivated rabbit’s serum + 0-1 c.c. complement + 1 ¢.c. r. b. c. ht be 1 c.c. inactivated pig’s serum + 1 ¢.c. r. b. ¢. No haemolysis 1 c.c. inactivated rabbit's serum + 1 c.c. r. b. c. : 1 c.c. ammonium sulphide (dilution 1 in 50) + 1 ¢.c. r. b. ¢. All tubes were made up to 5 c.c. with normal saline. The sera and ammonium sulphide were incubated together at 37° C. for half an hour, r.b.c. emulsion then added, and whole incubated for one hour. BIO-CHEMISTRY OF HAEMOLYSIS 365 A comparison of the effects of equivalent strengths of sodium hydro- oxide, ammonium hydrate, and ammonium sulphide on sheep’s r.b.c. are _ given by Table IT.. Tasre IT w NaOH + 1c.c. r. b. c, — Haemolysis in half an hour 2. O65 c.c. =e NH,OH + lc.c. r. b. c. — Slight haemolysis next day l.. 0506.0. =~ 3. OD e.c. X. (XH,)8 + Lee. r. b. c. —- No haemolysis All tubes made up to 5 ¢.c. with normal saline. | In Tables III, IV and V are shown the ‘ balancing ’ action of sodium hydrate with fresh and inactivated serum; also the influence of perhydrol on this action. All tubes were made up to 5 c.c. with normal saline, incubated at 37° C. for two hours, and results noted. Tasre III le.c. fresh pig’s serum + 1 c.c. r. b. ¢ Haemolysis N » * + 0-5¢.c. 0 NaOH ... ane nae . | No haemolysis N a + 0-5 c.c. Te NaOH __.... =e a aa Bs " ” ” + 0-5 ¢.c, Sy NaOH eee oes see see Haemolysis ” ” + 0-5 c.0. naon ese eee eee oss ” N . ” ” + 0-5 c.c. 30 NaOH ... dee ‘a ue fe i. N fo 1% 05 c.c. 75” NaOH + loo.r. boo. ... a mt aa te ike ii ‘ N a4 0-5 c.c. 5 NeOH + leer. boc. ... te & te oan ti we ” : = ; 05 c.0, “gp NaOH + Loc. rb. c. . cee ce tee nee nee nes « Pata). : 05 cc, “g5 NaOH OS eo oe ll. O5e.c. x Naot + locr. bo, ... as nae eae “on See ae 1. Tasix IV 1. 1... fresh pig’s serum + 1o¢.c.r, boc... eae exe ee yes Haemolysis a 2. O5c.c. x NaOH + 1o.c. r. b. c. Be ST eke.” | es ie . 3. Le.c, fresh pig’s serum + 0-5 0.0. ~*~ noon + loo. r. bo. sie No haemolysis 4. ” ” ” + Loe. web = aes Haemolysis ql te Sd aileeion) 5. ” ” ” + Leo, perh A a Rag Deri tm 6. 1.0. perhydrol (1 in 30 dilution) + 1.0. r. b. ©. ioe, am oe No haemolysis ¥; ” (1 in 60 dilution) + 1 ¢.c. r. b. o. iad: |. a oe a ‘ oa a ii cd Tee « en pe 366 BIO-CHEMICAL JOURNAL a TABLE V N 1 c.0, inactivated pig’s serum + 0-5 ¢.c. 7 NaOH + le.c.r.b.c. Slight ies ns 4 hours ” se ” " . i ae ¢.c, = oe Haemolysis in 9 lig (i in 60 dilution) . & +a + leo. rb. ce. + Lec. perhydrol No haemolysis ye (1 in 30 dilution) a. is ss » + 1¢.c, perhydrol No haemolysis (1 in 60 dilution) The haemolytic action of ammonia in various strengths on sheep's r.b.c. is shown in Table VI. Taste VI N 4 l. B3e.c. 70 ammonia + le¢.c.r. b,c. ... ar Haemolysis in 2 or 3 minutes 2. 2c.c. es ro bie sie ” 3. 1-5c.c. -: » tee bes Partial haemolysis in 2 hours 4. leo.e. pa " a aoe Slight haemolysis in 2 hours 5. OF5c.c. ,, » sos oa No haemolysis in 2 hours 6. 0-5 ¢.ce. ” ” eee "eee ” ” All tubes were made up to 5c.c. with normal saline. Table VII gives the effects of adding ammonia to fresh and. inactivated pig’s serum, and of variations in the technique. é Taste VII ae l. lee. aT Ye ammonia + 1 c.c. fresh pig’s serum ... —... ... Slight haemolysis next day 2. 0°75 c.c. o = pes is ... Very slight haemolysis next day 3. 0-5 c.c. s cc} ‘e a aaa 99 * i 4. lose. * ammonia + 1 c.c. inactivated pig’s serum ... ... Pale brown colour next day 5. 0-75 c.c. ” ” oo ” ” 6. 0-5c.c. ” ” ” ” Tubes made up to 4 e.c. with normal satiaes incubated for one hour at 37° C., then 1 c.c. r.b.c. added to each tube and incubated for two hours, — N Act 1 c.c. 70 ammonia + 1 ¢.c, fresh pig’s serum + 1¢.c.r.b.c. Slight haemolysis next day =~ 8. 0-75 ¢.c. Pa ‘9 ... Very slight haemolysis next day 9. 0-5 c.c, ” ” eco ” ” 10. Ic.e. 77 ammonia + 1 c.c. inactivated pig’s serum + 1 c.c. r. b. ¢. Pale brown colour . ll. 0°75 ¢.c. : ” ” ” ” ” 12. 0-5c.c. ” ” ” ” ” Tubes made up to 5 c.c. with normal saline and r.b.c. at onée, and incubated for two hours. - BLO-CHEMISTRY OF HAEMOLYSIS 367 Having found that quinone alone did not much influence haemolysis, “we next tried the effect of quinone with ammonia added. The alkali promptly turns the quinone black, or brown in weak dilutions, probably turning it into hydroquinone, which then undergoes some further change. Quinone added to a dilution of ammonia, which by itself had no haemo- lytic action in two hours, promptly turned black and produced instant haemolysis. Inactivated pig’s serum inhibits this action to some extent. - Hydroquinone alone has no haemolytic effect, but with ammonia produces the same results as quinone. The results obtained are set forth in Table VIII. Peery! |, Taste VIII Jo *mmonia +loor. bo. ... ae See ee or sal een «» No haemolysis wy om ” + 1 c.c. 0-1 % quinone ee eee — nee ties Dark brown color Haemolysis in 2 ” ” va ° eas se ans Si ... Brown colour No haemolysis in ” ” ” » + 1¢.c. inactivated pig’s serum... Black colour ; No haemolysis | ¢.¢. 0-1 %, quinone + 1 c.c. r. b. c. ve os Be apd ae ae a an ph = 0-1 % hydroquinone + 1 c.c. r. b, ¢. be Fe fo ade ae he * oa 4 N ” ” ” re + 1 c.c. inactivated pig’s serum Fe Ea OS cc. 0 ammonia + | ¢.c. r. b. c. + 1 ¢.c, 0-1 % hydroquinone ot pie ... Black colour Haemolysis i, ” ” * Pa + 1c.c. inactivated pig’s serum Brown colour . No haemolysis Ole. Jo *mmonia + loc. r. b,c, + 1e.c, 01% hydroquinone ... 4. «ss» Dark brown colo om No haemolysis ah ” ” ” ” + lc.c. inactivated pig’s serum Brown colour ag No haemolysis All tubes made up to 5 c.c. with normal saline. SuMMARY OF REsULYTS 1. The substances concerned in haemolysis, including thereby both the haemolytic agent outside and the substance attacked within, have a powerful mutual effect upon one another's solubilities. 2. Instances are given of such increased solubilities, and the favouring effects upon haemolysis noted. As a result of such increased solubility lecithides are dissolved out from the mass of the corpusele, so setting free the haemoglobin also, and laking is the result. 3. It is noted that all the haemolytic class of unsaturated soaps of 368 BIO-CHEMICAL JOURNAL fatty acids, saponin, mowrin, digitalin, the various bile salts, possess common physical, chemical and physiological properties, and are all unsaturated biddise capable of bromination, ete. 4. The similar action upon the heart of the haemolytic bodies i is si probably due to combination between these and the heart lipoids. — ilies 5. Although want of saturation exists, it is probable that the first fundamental step is a ‘ physical’ one of lowering of surface tension with accompanying tendency to solution. But no hard and fast line can be drawn between so-called physical and chemical action. 6. The balancing action of haemolysers is discussed, and it is shown that this is not obtained with closely similar haemolysers, where instead an additive action is seen. This suggests that balancing is due to a combination or interlocking of the two haemolysers, whereby nothing is left free to touch the corpuscles. 7. Where sera and such haemolysers as sodium oleate balance, the first call is between the active body of the serum and the oleate; next, in absence of the active body or of complement, the serum proteins alone, although not active in themselves as haemolysers, possess a superior binding power over the corpuscles for the oleate, and hence act as protectors, so that much more oleate in excess must be added before the corpuscles are attacked. Accordingly, as is well known, a mere trace of oleate suffices to break down corpuscles in saline suspension, but in serum suspension many times more oleate must be added before any result is obtained. 8. It follows from this that oleates, etc., do not act as anti-comple- peep ments, and ought not to be described as such; it is most.probable that they possess no specific relationship whatever to complement. 9. Sodium oleate can also be balanced by lecithin for similar reasons. 10. Under conditions specified, and in alkaline solution, oxidizing agents favour haemolysis, and reducing agents restrain it ; but an oxidizing agent alone cannot replace complement in an active haemolytic serum, — and it is not probable that complement has the nature of a peroxide body. 369 ‘OBSERVATIONS ON THE HAEMOLYTIC ACTION OF + CERTAIN BILE DERIVATIVES By HUGH MacLEAN, M.D., Carnegie Research Fellow, University of Aberdeen, anv LANCELOT HUTCHINSON, M.D. (Liverpool). From the Bio-chemical Laboratory, University of Liverpool (Received July 22nd, 1909) Our knowledge of the processes involved in the laking of red blood _ corpuscles is at present in an obscure and unsatisfactory condition, and _ despite the amount of research carried out on haemolysis and the number __ of theories advanced to account for different results, many cases still remain unexplained. ___ In connection with this subject, one very interesting point has lately __ been advanced by St. Faust and Tallqvist, and afterwards by Moore; these ____ observers found that haemolytic action, when brought about by such bodies as the fatty acids, occurred only in cases where the acid employed was one _ of an unsaturated variety containing in its molecule one or more double bonds, while the corresponding saturated bodies produced no effect. In view of this observation, it is likely that the state of combination of the carbon atom constitutes an important factor in many cases of haemolysis produced by chemical agents. The strong probability, on chemical grounds, of certain bile products being constituted by bodies of an unsaturated nature suggested the present ‘investigation; the curious ‘results obtained do not seem capable of explanation by any of the theories put forward at present. SUBSTANCES USED The present paper deals with the results of haemolysis by three = bile derivatives—the sodium salt of glycocholic acid, cholalic acid and ____ tholeic acid. These were prepared as follows : — Soprum GLycocnoLaTe Fresh ox bile was freed from pseudo-mucin by treatment with "4 alcohol; after the evaporation of the aleohol, neutral acetate of lead was aa added, the resulting precipitate separated off and decomposed by heat in ee "a. a a et ee ae ee ee ae ee ee ey eer ee eee Be ee = a Mange , : ; 870 BIO-CHEMICAL JOURNAL the presence of a sodium carbonate solution. The mixture was evaporated to dryness and the residue extracted with alcohol; the alcoholic extract was filtered, the filtrate evaporated to dryness and the residue dissolved in water. This watery solution of sodium glycocholate was now decolorised by animal charcoal, and the free glycocholic acid thrown out by means of a dilute solution of hydrochloric acid. The acid was thoroughly washed with water, then dried and again dissolved in alcohol. To this alcoholic solution sodium carbonate was added, and the whole again evaporated to dryness. Residue was dissolved in a little cold alcohol, and the mixture filtered. This process of purification by dissolving in alcohol was repeated; the alcoholic solution was then evaporated to dryness and the residue dissolved in a little water, filtered, evaporated to dryness over the water bath, and ultimately dissolved in normal saline solution, and in this solution utilised for the experiments. CHoLanic Acip Ox bile was boiled for thirty hours with one-fifth of its volume of 30 per cent. caustic soda, the total volume of the mixture being kept constant by the addition of water from time to time. The solution was then saturated with carbon dioxide and evaporated almost to dryness. Residue was extracted with 96 per cent. aleohol, and the extract diluted with water so as to contain not more than about 20 per cent. of alcohol; it was then treated with a solution of barium chloride, the precipitate filtered off, the filtrate treated with weak hydrochloric acid, and the precipitated cholalic acid separated off and thoroughly washed with water. This acid was now changed into the sodium salt, and the above process of treatment with barium chloride, etc., repeated twice. The free acid ultimately obtained was dissolved in alcohol, and by the addition of sodium carbonate again changed into the sodium salt; the alcoholic solution was filtered, evaporated to dryness, the residue dissolved in a little cold aleohol and filtered; filtrate was evaporated to dryness, residue dissolved in water, filtered, again evaporated to dryness and final residue utilised for experiments. oe CuoLteic Acip Choleic acid was prepared from the precipitate obtained by barium chloride in the preparation of cholalic acid: this precipitate, which consisted of an impure salt of barium choleate, was purified on the lines described above; it was also utilised in the form of the sodium salt. HAEMOLYTIC ACTION OF BILE DERIVATIVES = 3871 EXPERIMENTS _ The first set of experiments was carried out with the sodium salt of - cholalic acid. In all cases red blood corpuscles from the sheep were used ; these were carefully washed four times in the ordinary way with normal galt solution, and then made into a 5 per cent. emulsion with isotonic saline. : + + a For convenience in preparing the somewhat varied strengths of -__ gholalic acid utilised in the experiments, two solutions were made—one a * + deci-molecular and the other a centi-molecular; in each case the molecular : t of the salt was taken as corresponding to the formula C,,H,,0;Na, = all solutions being made in normal saline. Sie! For each experiment a series of tubes, each containing 1 ¢.c. of the above emulsion of sheep’s red blood corpuscles, was taken, and to this was added a measured amount of sodium cholalate. By the addition of the - necessary amount of normal saline, the final volume of each tube was a tained constant, and in all our experiments amounted to 5 c.c. The + ie were then placed in the incubator at 37°C., and observations . ‘constantly taken at short intervals. As the result of repeated experiments, it was found that this salt _ displayed well-marked haemolytic properties, but at the same time * ¥ exhibited some peculiarities so striking as to amount to what may be die peageectively termed a haemolytic paradox. In tubes containing a moderately strong dose of sodium cholalate, the corpuscles were completely laked in intervals of from one to two hours; ___ when the substance was present in somewhat smaller quantities, haemolysis was delayed for three or four hours; on still further reduction of the a amount of substance, however, the peculiar fact was observed that haemolysis took place in a shorter and shorter space of time, until a point __-was reached at which a minimum dose gave a maximum effect. With _ such an amount this minimal-optimum dose often caused complete laking of the corpuscles in six minutes, or even less; while on the other E.: hand, a dose several times as great might take hours to produce a similar 2H effect. When exhibited in amounts smaller than this minimal-optimum dose, the effect became gradually less, until ultimately a strength was used beyond which no further haemolysis could be obtained. The accompanying chart gives a good idea of the results obtained; in a long series of experiments the laking always took place as indicated. '% =} 7 a. a i : - 872 BIO-CHEMICAL JOURNAL | Cuart No. [ Soptum CHOLALATE al - eT ee Shs TO Rie TR we The eae - can —- : ~~ io) Time during which sodium cholalate had been acting 2/2 )8 8 ¢ 8 a -—— + aus Ban ae | _ . Masti, 1 I co. gpien's 8. Bi that oh secsbecvsiees} cavetnqos tense +4 ¢.c. sodium cholalate — >> > >>} ¥ at oy Fy ¥ OS saline...... +3 ss Re ~~ eS q ‘ eT 2 + 8.0.1 sodium cholalate | —|—|-|—|—/ | -\4 , reece se j pat ” ” +2 4, ‘ ” +2 Fe oe —|-|— | ae es 5 4 ae ei t ” + 3 ” ” + 1 os ” -—l—| ei rie 7 J ++ PPT aia : +09 Tost tee -|-|-}-|-|-|-|=|=} ” ” + 3-2 ” ? + 0-8 ” %9 gm Bek fiom. = = int = = + ” ” + 3-4 ” ” + 0-6 ” 7 ‘be: i "ex -| -|-|- +44 + ae ed ” 7 35 ” ” + O05 ” ” A ms fy. eS ‘Lee uy a +++ ”? ” + 36 9 "9 = 0-4 ” ” | Tiel abe —+> ‘ ; »” ” + 3:7 oh . ote O-3 . ” >) | = +> 4 ” ” + 2 a ary + 0-2 99 ” - $444 J if ” ” + 3 ” ? 2 x 0-1 *9 ” $+44444 ¥. + » +35,, 4 +005 ,, s |_| —| —| —jedledled . 7” ” + 36,, ” + 0-04 ” ” | -|-|-|-|-}- ’ Bs ere ; ” ” oa 37 ” ” + 0-03 ” ” \— ons aed Gees loa Dee ‘ as ” ” + 38,, ” + 0-02 ” ” bi Si —} =} a) eae ss Fe -- - i ” ” + 3-9 ” ” + 0-01 os + — -|-|-/='- = ERLE _— a, ” ” + 4 ” ” (control) > - -| -| a — _- ae én _— — fe Eee i x Norr.—The dct of red seein i was rayidly added last of all, the difference in time between the first and last tube being barely 4 minutes. Immediately after the corpuscles were added, the tubes were placed in a thermostat [37° C.] where they were observed. The times were calculated from the moment when the tubes were — in the thermostat. “2 ss In measuring amounts of sodium cholalate less than 0-2 ¢.c., an 100 solution was used. * es % + Denotes point at which laking became complete. wn The results are also shown graphically plotted in fig. 1, in which the ordinates represent time of complete haemolysis in minutes, and the abscissae, the concentrations of the sodium cholalate, in ¢c., as indicated, a) i of 0°1M solution added to a total volume of 5 c.c. when completely — 5 made up. HAEMOLYTIC TION OF BILE sodium cholalate M 10 tres of lec centime b fe, In-cu lume of 5 c.c. ted abov ica CHOLALATE to maintain a constant emulsion) + amounts, as ind ts A ) + sufficient normal saline Each test tube contained 1 c.c. sheep's r. b. 3 es & 8 —) ssApouteuy oyopdutoo soy poaynbea seynuyut uy eur, | a an _ ——— a 874 BLO-CHEMICAL JOURNAL The following reproduction also shows these results; the tubes were left to stand for one hour and then centrifuged; by this means a more suitable condition for photographic purposes was obtained. Haemolysis is distinctly seen in tubes 1, 6, 7 and 8, the remainder being quite free from laking, with the exception of Nos. 2 and 9, where a slight action has taken place. } From these results, it is obvious that the haemolytic power of the so-called minimal-optimum dose is much more marked than that obtained when much larger amounts are used, and that between these two points there lies a sort of neutral ground where the haemolytic power is very much reduced. In the experiment recorded in the chart it is seen that hhh O OR aig PHOTOGRAPH SHOWING HagEMotytTic PAarRApox Tubes with black appearance are those in which laking has occurred. The following table indicates the amount of sodium cholalate in each tube in the photograph :— No. 1. 8c.c. = Sodium cholalate | No. 6. 08 c.c. a Sodium cholalate 9 M | a hs ee Oe ae rv 99 9 ire. “i 7 . M Me. ae Pe Pe ea Ee Se TE RC. Se mts " » & O8c.c. ,, eS i SL OR gles Sh 7 r » 8 O5cec ; | sie. OS: 8. Fr: 0'1 c.c. of O'1 M. sodium cholalate lakes completely in five minutes, whereas it takes thirteen minutes for 4 c.c. of 0°2 M solution to produce the same effect; in other words, a certain minimum dose gives in five minutes an effect equivalent to that produced by a dose about eighty times as great in thirteen minutes. HAEMOLYTIC ACTION OF BILE DERIVATIVES — 375 In another series of experiments performed under the same conditions, but using the sodium salt of choleic acid, similar results were obtained. When a strength of 4 c.c. 0°1 M sodium choleate was used, haemolysis was complete in about siz minutes; in this strength laking was quickly followed by an action on the liberated haemoglobin, as indicated by a _ dark brown coloration of the liquid. With 0°8 c.c. 0'1 M solution the full effect was in evidence in about eleven minutes; when, however, only 1 c.c. ~ 001 M was used, haemolysis was almost instantaneous, the corpuscles being - completely laked within one minute. This salt is a much more powerful agent than the corresponding ~ compound with cholalic acid. Whereas with sodium cholalate a solution sof 03. c.c. 0°01 M required about eighty minutes for complete laking, and a or oun strength corresponding to 02 c.c. 0°01 M gave only the merest indication of -_ a positive reaction after twenty hours’ observation, with sodium choleate _ in strength of 0°7 c.c. 0°001 M complete haemolysis was obtained in one hundred and sixty minutes, and with 0°6 c.c. 0°001 M in twenty hours; even ~ with 0°5 c.c. 0°001 M, traces of haemolysis were evident after this time. The full results of one experiment are indicated in Chart 2. Similar experiments, giving in the main the same kind of result, were performed with sodium glycocholate. Here, however, there was not the same marked difference between the haemolytic action of large and small amounts as was seen in the case of the above described salts; this is due to the fact that the action of the minimal-optimum dose is in this case not nearly sorapid. In other respects, as indicated by the chart, the same general result is in evidence. The results are shown also in fig. 3. On the other hand, haemolysis was ultimately obtained with smaller amounts than in the case of sodium cholalate, though, even in this respect, it did not appear to be so powerful as the choleic salt. A few observations were also made with free cholalic acid; consider- able diffieulty was experienced in procuring a suitable method of application owing to its lack of solubility in inert substances. We succeeded, however, in emulsifying a small amount with normal saline, and with this emulsion similar results to those ébtained with the sodium compound were noticed; that is to say, with a certain small amount of emulsion a much more marked haemolysis was produced in a given period than was in evidence after the exhibition of perhaps nine or ten times as much in a much longer space of time. 876 BIO-CHEMICAL JOURNAL Cuart No. Il Soprum CHOLEATE Minutes Tine panes which sodium choleate had been acting vised bac’ hick Ga 7 |e S18 | 1 c.c. sheep's r. b. ©. 4+ .ceeeeeeereeeeeeee + 40.0. F sodium choleate | — —| — ++ t++4444 . el of lee ee, : ~~ $$444444444 = - ae. eee oe, Rae - pike lak: $+ +t44+444+44 LS Fe Se eee ri Ber fa fi Fae ft Pic Dic icc DD DDD : =, Ole ae eee w — colo - | o> + 32 + 0-8 ' Peete et Gt fo Gt cc cd ad 0 . +3, +07 lati lati nshandnsasncncnancne ey sy. SBeetial = ws sgt! —|—|—|—| “so > 9 > TGs a +35, Bat ea Sade ~|-|-|- foe $+ooto++s 6 ES __ [oo | ol] oH +4 4444444 » +87, +03 , " —\~|—| “>> t+ +4494 44 aa + 3-8 +02 ,, ge 444444444444 + 3-9 Seo TB os $$$ oott++4+4+4+44+44 $35, 4 FOTO > het + thee » +36, » +04 ,, $$$ 444444444444 J es ee e — + $444444444444 : EC ee oe ; ~ = -|-|- -|- boo , 5 " » +30, » + lO ce.on -|-|-|-|-|-|-|-|-| ere 3 » BBR io ROBoI od -|-|-1-|-|-|-|-|-|-|-|- ee , +33 +07 -|-|-|-|-|-|-|-|-|-|-]- =e ‘ » +34 +06, ; -|-|-\-|-|-|-|4-|-|-]444 a ; , +35 + 05 n _|-|-'-|-|-|-|+|-|-/-/4) |e » +36 +04 ,, -|-|-!-]-[-|-|-|4-) =] [ey StS 4 es. = SE eee” Ye . -|-|-'-|-[-|-|-[-|-|-|4-|-[-- F ” oe +. o » (control) -|-|-'-l-}-1- aed ford ee tee bow os ae be + Indicates complete ‘haemolysis. Norr.—The same conditions existed during this experiment as in the preceding Chart a except that the tubes were not placed in the thermostat for 8 minutes after adding the corpuscles. The corpuscles were added during 2} minutes, and the times are calculated from the time when the last tube received its corpuscle. O Trace of haemolysis. These results are shown graphically in Fig. 2, where ordinates show as in Fig. 1. 33 $ tH 5333333: Si Sahss iss $535} fe . Hania 20 10 5 6 : " —! s1sAjourewq o7o;duroo 10; pearmbes seynurut oy eur, 0-8 L-O 90-0 . ’ Sopium CHOLEATE 2. Fia. choleate lum sod M 10 icated above, of i es, as inc Each test tube contained 1 c.c. sheep's r. b. c. (5% emulsion) + amounts in cubic centimetr HAEMOLYTIC ACTION OF BILE DERIVATIVES t normal saline to maintain a constant volume of 5 c.c. cien + suffi 377 than a larger amount. 1e1ent more eft yser is . The curve shows well the paradoxical result that a small amount of the haemo! ‘o°O G JO OUTN[OA JULISMOD B ULEZULEUT OF OUTTYS [VUTIOU yuOIOWjNS + é toy aqujoqr004]3 tunpos JO ‘@A0G® pozwoIpUT su ‘soaqouNyUVD oIqno Ut SsyUNOUTY + (MOIsTNUTe %g) *o “q ‘a s,dooys ‘oo T pouTE_UO 0qN}3-3804 org, ALYIOHOOOATH) WAIGOG “Eg DIY wo ¢ os ts RA Oey eee © So co o ie) bt _ 8 c za ea a * ee zl “FT : ® & # = =] - | = os =I pal S = 3 — ee a — 3 - : Fy = $3 333; & — € 3 = on + le) S is g o : oF B — : iS = $ eH ; 2 + ber] 5 : =a + ttt + » : : Hay 6009) |B ; HEA 2, $ Bt i] tf ; $333 seeesss ¢. Hit 3 : 5 otal Seti $ ¢ SH + : sess Hh STITT : a SHH $ 333 $3 3333 tt ; 3 HE . o Sesss3¢ $3353 $8333 $33 $3 tf $3 : 878 HAEMOLYTIC ACTION OF BILE DERIVATIVES — 879 Cuart No. Ill Soprom GLYCOCHOLATE Times in minutes 25,3085 4044 80! 20 hours 1 o.e, sheep's r. b. c. + 1.0. saline + 3 cc. 3 sodium glycocholate 444444444 Pa ae PS ne cw HT ” : —|-|-|- o> + ree, .. +0, » |--1-|-|-e + a OO: gs ieee Ds ge = a -|-|- -|-|- +> ist Det OO ne Ot a i —\—|—|—|—| lp 2nd Rema ge oo yee, ay ARE RARRAR 100 Bel Set Dl By “a See eee - ” - - -+oo44 Shes che bike Domeitt HED RE Ue ol ood + ete PEGS OO; z al - +44444 + 7 pay vee TF oboe + eee ROR SC —$4444444 Se ee oo tteee + 6 F385 o $05 Se etait tata ances i 9B iin ORG on » | ee + ee, 487, . 408, » |= —|-|-|- bi > em F2 w w +2 iggy eo fHl|-|-|-|-Hee tie eee a iy er, é » |=|-|-|-|-|-|-|-1@ os wel, +00, Cl » |——-|-|-|-|-|-]-| partially laked ” » +32, » +08,, a me a A pa ad os Trace of laking . Se a eek » |—-|-|-|-[=]-]—l reece of laking a 5. eee als OE si ¥ Oe Se 2 ed a » +4 ww (Control) 0 | A ys, Bo terds 1b + = Complete laking. _ Times calculated from time at which tubes were placed in thermostat. 380 BIO-CHEMICAL JOURNAL Of late years, it has been repeatedly demonstrated that the serum of an animal is able to exert a well-marked protective action against haemolytic agents on behalf of its own red blood corpuséles. We therefore tried the effect of substituting 1 ¢.c. of fresh sheep’s serum in place of 1 c.c. normal saline in a series of tubes in which sodium cholalate in various strengths was used as a haemolytic agent; here it was found that the serum exercised a very marked inhibitory action on the haemolytic power of this substance. The results of such an experiment are seen in Chart 4. It was found, for instance, that 1 c.c. of 0°1 M sodium cholalate, which when used alone was able to completely haemolyse the 1 c.c. of sheep’s red corpuscles within five minutes, was, when used in conjunction with 1 c.c. of sheep’s serum, absolutely devoid of haemolytic action. Such a result indicates very marked power indeed in the inhibition and prevention of haemolysis by the protective action of normal serum. In examining the chart, one apparent contradiction will be noticed ; in the case of the tube containing 3 c.c. 0°1 M solution of sodium cholalate, haemolysis was complete in twenty-two minutes. Such an amount, however, when used alone, is capable of completing haemolysis only after eighty minutes, so that in this particular case the serum has increased instead of diminished the haemolytic action. This result is easily explained in the light of our former observations; — if we assume that the amount of sheep’s serum used was insufficient to neutralise the effect of the total amount of cholalic acid salt present, but — had neutralised its equivalent amount, it is obvious that in this case a reduced amount of sodium cholalate would how be present in an active form, and since a weak dose (within certain limits) is more powerful than a stronger one, an increased action might naturally be expected. Some sheep’s serum was then inactivated by heating it at 56°C. for one hour; it was then tested to make sure that the process was complete; this was done by means of several controls against inactivated pig’s serum which normally lakes sheep’s red corpuscles. Ina series of tubes similar to those used in the preceding experiment, 1 c.c. of the inactivated serum was substituted for the fresh material; it was found that the protective action was in no way diminished as the result of inactivation; indeed, if anything, it seemed more marked. It may, therefore, be assumed that in these cases the presence or absence of complement is immaterial in so far as the protective action of the serum is concerned. A similar series of experiments were carried out using the sodium salt of choleic acid, and similar results obtained. Chart 4 shows these results epitomized. > > . a ‘ 7 ‘DO oL€ JO oanqesod io} v 4¥ WwysoUrIeY) v UT poovd o10M soqN7 OY) peppT o1eM *o “q “1 & .daoys oN) IY ‘SINOY PZ 19AO POpUd}Xe soqN} ase} UO SUOTPWAIOSqO OY — ALON stsAjourovy ON eee Ben... - eee 5 Oee wee wee eee age eee ee “ee ee oor +e soa} 00R sts{jourovy ON eee eee “ “ “ CO + “ “ GZ + “ “ “ + “ “ stsf{jourovy ON eee eee “ “ “ I+ “ “ 4 + “ “ “ be “ “ 881 ‘ Soul OPT ul s~sAjowovy oj0;du10g | ** @yve]OYo UINTpos — ‘oo «3 + ouNUs‘o’o =| + uInsos s.dooys “youu! ‘oo | +0 “q ‘1 8.dooys oo] sisAjourevy on see wee aoe “ “ “ CO + “ “ CZ + “ “ + . . stsAjowovy ON eee eee eee “ « “ I + “ “ z + “ “ ae bys = 4 sopnuu ETE ul sisAjowovy oyojdui0g “* @yReTOYo wmIpos _ ‘oo «Z + oUuNYs ‘oo §«6| + unsas s.dooys 0°90 [+ 0 “q “4 s,dooqs oo] stsAjourovy ON | **- ese “ “ “ oO + “ “ OZ + “ “ “* a os pa qsfjouewyon | -" “ a os + 9,4 “25 ee a ee OF BILE DERIVATIVES stsAjoutory oN eee eee “ “ “ £-0 + -< “ LZ 4 “«< “ “ + “ “- , | ‘ganoy gy 497J¥ s{sAjouoeY Jo 90va3 Ody eee ee “oe “ “ee co + - “ee 3% + * “- “ + “ “ “say P34 aoqye sisfjowoey 030;dwW09 qsowly eos eee “ “ ss i + euyes ‘oo Z + “ “ “ + “ “ sopynuyu gz ul sysAjourevy o30;duI0g *** oyupepoqo wmtpos ND eS htteecrsnceese + uinsas s,dooys “jouut ‘ovo 1+ “0 °"q “1 8.deoqs wo] stsAjowory On | °° eee eee “ “ “ Ol +}. “ “ t+ “ “ + “ “- ' stsf{jouory on | °° ose eee “ ~—* OS + “ “ i + “ “ + \ sis{jouovy On wee wee +e. “ “ “oe £0 + “ “ L3+ - “ + -“ “ | SanoY SF 10938 sysKjowoLY I43Is Aso, eee ove sos “ “ “99+ “ “og+ “ “ + “ “ sanoy g 49zj¥ S{sXjouroeYy 230;duI09 eee ose eee “ “ *9°9 1+ ouryes oo g + “ “ {s “ “ HAEMOLYTIC ACTION “syuouTtedxe ‘= Yqog AOj *o"0 G FV JUBYSUOD Bureq eqny qowe jo eurNnjoA a OYJ, “2qng Yous oF poppe SA LOIS|NUTO UII94SOTOYD 3 4 ‘oo % Wey oAIno orp fjouory oy} SMOYS OUL] SNONUIZUOD OT, a : “ONT 03 : soqng oaoge oy} Aq UOYe} ONT OY} SozVOKPUL OUT] Po}IOP ONL, < i : Te TS egeee eas rs T i : a : 46 ‘ re F-O+ ‘ 7 9-E+ ‘“ “e =) :. =) = - “ec 66 06...08 ¢-0+ “cc G-g+ “ “ = & 3 ee “oL.0+ a ‘ ou-g+ a3 “cc = = reed jen) 7 +t as ee se see O-T+ ae ae O-g+ he ae o i esece © — a OOT se se sé se se : Est O.8+ b aa} ses : N 0-3 06+ + ja ehsassen ; OPBLVLOYH "pos 7? P-O+ OULLBS "9°90 9-§+'0°q'a s,dgoys OOT . eee ee Pe 6 a ee Oe SS So gue NOILIGIHN] NIUALSAIOHO—'G “ON LUVHO “Ff DLT 382 HAEMOLYTIC ACTION OF BILE DERIVATIVES 383 According to Liebermann, this inhibitory power of the blood serum is dependent on the serum-albumin. Iscovesco,! on the other hand, _ attributes it to the cholesterin normally present in blood serum. Gerard and Lemoine? have studied the anti-toxic action of cholesterin as applied to tubercular poisons. Ranson’ has also demonstrated the anti-haemolytic power of cholesterin on certain chemical substances. ; In view of these results, we have tried the effect of cholesterin on the % haemolytic action of sodium cholalate. Using an 0°002M emulsion of cholesterin, it was found that even in strengths of 0°0008 M solution it ce exercised a considerable anti-haemolytic action, and in the presence of weak doses of sodium cholalate was sufficient to entirely prevent a laking These results are demonstrated in Fig. 4, Chart 5. When using 04 cc. 0-1 M sodium cholalate, the amount of cholesterin used (2 c.c. 002 M) was sufficient to delay the usual laking effect for about thirty minutes; with 0-5 c.c. 0°01 M there was a delay of seventy minutes, and in the case of 0°4 c.c. 0-01 M haemolytic action was totally abolished. - In all these experiments, the cholesterin emulsion and the sodium ___ cholalate were incubated together at 37°C. for one hour previous to the addition of the red blood corpuscles. — The above are some of the results obtained with the substances mentioned ; it is hoped to extend them at a later period. No theoretical considerations with regard to them have been advanced, since much more work must be done before any satisfactory explanation can be forth- coming; they are merely given as facts obtained as the result of repeated experiments. ConcLuUsIONS Such bile derivatives as the sodium salts of cholalic, choleic, and at glycocholie acids are capable of producing haemolysis in the ordinary - way when strong doses are used, but exhibit marked peculiarities when ‘ present in considerably weaker amounts :— 1. It was found that under similar conditions the same haemolytic effect can be produced in a given time by widely divergent amounts of the salts. Between these two points lies what may be termed a more or less - 1. ‘Les Lipoides,’ 1908. 2. Congris de Médecine, 1907. Soc, Médic. des Hospitaux, Paris, 1907. 3. Deutsche med. Wochenachr., 1901. a 384 BIO-CHEMICAL JOURNAL neutral zone, in which haemolysis is very considerably delayed depending on the relative amounts of haemolytic agent employed. The minimum dose giving the maximum effect in the shortest time we have designated the ‘ minimal-optimum haemolytic dose.’ For instance, it was found that this minimal-optimum dose in the case of sodium cholalate was 0°1 c.c. 01 M solution; this quantity, mixed with 1 ¢.c. of 5 per cent. sheep’s red corpuscles (the total volume being made up to 5 e.c.), gave complete haemolysis within five minutes. Stronger solutions gave a much less marked effect, a solution eight times as strong requiring one hundred and fifty-five minutes for a similar result, while a dose eighty times as large as the minimal-optimum dose required thirteen minutes to complete haemolysis. 2. In all these cases the addition of cholesterin produced a marked anti-haemolytic effect. 3. Ina similar manner, the addition of fresh sheep’s serum exercises a markedly anti-haemolytic action; in some cases, however, an apparent augmentation is in evidence. This is obviously due to the fact that the amount of serum used was unable to completely neutralise the laking action of the bile salts; this would result in a smaller relative amount of active haemolytic agent being present, and hence a more powerful effect would be seen (within certain limits), in accordance with the above observations, . 4. This inhibitory action of serum does not depend on the presence of complement; an inactivated serum acts equally well. | We wish to thank Professor Benjamin Moore for advice and assistance in the above investigation. | THE PHARMACOLOGY OF APOCYNUM CANNABINUM* By J. C. W. GRAHAM, M.A., M.D., B.C. (Cantab.). Communicated by Prov. W. E. Dixon. From the Pharmacological Laboratory, Cambridge (Received August 25th, 1909) ) L INTRODUCTION. Action on 1. Vascular System, (a) Heart. (i) Frog. (ii) Mammal. (iti) Relative Toxicity of Apocynum. (b) Blood Vessels. (c) Blood Pressure. 2. Toxic effects. 3. Muscular System. . Comparison or APocyNUM CANNABINUM WITH OTHER MEMBERS OF THE CARDIAC Group or Drugs. I Apocynum is a member of the group of cardiac tonics, and is official in the United States. It has been credited with being the most powerful indirect diuretic known. Descriptions of the drug are given by Paine! and two Russians, Gliski? and Gvozdinsky.* In this country records* of we patients suffering from pleuritic effusion have been given, under the influence of Apocynum it was thought that the fluid subsided ‘ rather quickly.” Hauseman® first suggested that Apocynum might contain a cardiac poison allied to the digitalin group, and in 188% Schmeideberg isolated two so-called active principles, apocynin and apocynéin. Rose Bradford showed that its principal action was upon the heart. Sokoloft® showed that the drug caused slowing of the heart’s action, * enlargement of the pulse’ wave and marked rise of blood pressure. Petteruti and Somma’ found different results were obtained according as to whether they used the decoction or the tincture, the decoction acting chiefly on the stomach and intestines causing catharsis and emesis, when this action was *A t was made towards the expenses of the research by the Scientific Grants Committee of the “British Medical Association. 386 BIO-CHEMICAL JOURNAL absent there was diuresis and acceleration of the heart beat. The tincture was stated to be unirritating to the gastro-intestinal tract even in large doses. The emetic and cathartic action of the decoction was attributed to the admixture of the bitter fibre of the wood with the bark of the root.’ Further experimental work was carried out by Dortschewski,® Klopotovitch! and Lapshin. Since my experiments have been completed, I find that Laidlaw and Dale have been working with a crystalline active principle isolated by H. Finnemore.'! Some apology is, therefore, necessary for the late publication of this paper. : The preparations used in my experiments were (a) Tinctura Apocyni Cannabini. One part of the root to ten parts of 60 per cent. alcohol. (6) Apocynin. The animals employed were frogs, rabbits, dogs and cats. Frogs were used for simple injections into the dorsal lymph sac, and for demonstrating the action of the drug on striated and unstriated muscle, the gastrocnemius muscles and a sectional ring of the stomach being used respectively. Perfusion experiments were also performed by tying a cannula into the hepatic vein. The effect of the drug on the heart in situ, and also in connection with recording apparatus, was also observed. The frogs were always pithed, except those used for simple injections. Observations were made with rabbits on the effects of injecting both large and small quantities of the drug; the action on the vascular system was also determined. Dogs and cats were employed for obtaining cardiometer and oncometer records; experiments on the urinary flow were performed on dogs. Chloroform, A.C.E. mixture and urethane were used as anaesthetics. In a few cases no anaesthetic was used, the animals being pithed. II. Action Fe Vascular System. (a) Heart. (i) Frog. The most important action of Apocynum is seen in its effects on this organ. In a general way Apocynum acts on cardiac muscle in the same manner as it acts on both striated and unstriated muscle. The tonus is always increased, the systole and diastole are greater in amplitude. Cardiac slowing is a marked feature. On placing on the heart a few drops of a crude extract of the root of Apocynum a marked effect is immediately apparent. The beat is slowed and a clear rise. of tonus occurs. 3 THE PHARMACOLOGY OF APOCYNUM CANNABINUM 387 Such experiments are of little value, since the effect of a drug applied to the outside of the heart is not necessarily the same as when the drug is given by the circulation, but they suffice to indicate the probable effect. Fig. | shows the movements of a frog’s heart recorded by the suspension _method,'so! that the systole is represented by the upstroke. A saturated alcoholic solution of Apocynin was used for injection into the subcutaneous tissues. Section I shows the normal beat. In IT is seen an increase of systole and diastole with some slight slowing after a subcutaneous injection of 10 minims of the Apocynin solution. An increased effect is noticed in III and IV after applying the solution directly to the heart. Section V gives the results of placing crystals of Apocynin on the heart. The greatest amplitude and most marked slowing of the beat is seen in the first part of VI, after this, the beat gradually quickens until it becomes very quick and irregular (delirium cordis) and the heart dies in systole. The experiment illustrated in fig. 1 is particularly interesting, as it shows that the substance Apocynin is active, and that it causes an effect like the tincture. It is also of some interest to observe the delirium cordis, as this is a condition particularly difficult to obtain in the frog. After a subcutaneous injection of 14 minims of tincture of Apocynum in the frog, a similar series of events occurred, and death ultimately - occurred in systole. These simple experiments show that the crude extract of Apocynum, the glucoside Apocynin, and the ordinary standard tincture, all give rise to the same series of effects in the frog. The heart beat is slowed and increased in amplitude, later in the action the beat becomes quicker and irregular and death occurs in systole. The development of these effects was also seen in tracings of perfused frog’s hearts. (ii) Mammalian heart. Experiments were undertaken to demonstrate the effect of small doses of Apocynum on the isolated mammalian heart. Fig. 2 represents the records of an experiment in which the drug was introduced (5 c.c. Tinct. Apocyni, | in 6,000) into the side tube of the perfusion apparatus so that after considerable dilution with the Ringer’s solution in the apparatus all the drug would rapidly pass through the isolated heart. In other words, five minutes after the injection, no Apocynum would be in the ciroulating fluid. Section I shows the heart beating normally. II shows the effect five minutes after injection; the rate of the beat is the same, but the amplitude of the systole is increasing. Tracings LII and IV show still later effects, 20 and 25 minutes after injection the rate of beat still remains about the same, 42 to the minute. In V, a record taken 30 minutes after injection, the heart is beating much more slowly and ventricular beats are being lost, so that two auricular beats are shown to each ventricular beat, and in the later portion of the tracing the ventricular beats entirely cease for a time. This stage is rapidly succeeded by that of delirium cordis in which the heart beats very rapidly and the tonus of the cardiac muscle rises (VI, VIL, and VIII). The heart ultimately stops in systole (IX and X) fifty minutes after the injection. It may be noted here that the death of this heart, which is typical of many experiments, results from a very small dose of the drug, in spite of BIO-CHEMICAL JOURNAL ‘IA 88 ‘ c ‘qQvep PUL sTPOO UINWTEG “A JO WowNUTyUOD— "TA *y2vey 043 04 APoearp urafoody jo speysdio BayA[dde soypy— A ‘(eu pug) “ “a “e a“ —Al ‘(ourty 487) Javoq O43 04 ApooIrp urafoody jo uorjnyos Buy 08 ¢.c. 0-5 c.c. (1 in 2 sol.) . Tinct. Apocynum injected ode) 0-6 e.c. gee 0-4 e.c. : 0-9 o.¢. ll e.c. 13 c.c. 1-2 ¢.c. 1-3 cc. 0-8 c.c. 0-8 c.c. urine blood-stained ‘y ¥ “5 S * eu sun 88s gas ane ! 2 ec, “ee 3-2 e.c. 3-2 c.c. 43 cc. 55 o.0, 1-5 c.c. l-2c.c. Delirium cordis 2 ec. = 4 . : oe 0 o.c, my : Be ya bk ie 3 THE PHARMACOLOGY OF APOCYNUM CANNABINUM 399. ie 400 BIO-CHEMICAL JOURNAL It is to be noticed that the increase of urinary flow is coincident with the rise of blood pressure. Apocynum is sufficiently irritant in character to cause the appearance of blood in the urine. The administration of a saturated solution of Sodium Sulphate by the jugular vein causes a very much greater diuresis than Tinct. Apocynum; the latter is very feeble as a direct diuretic. From this it appears that Apocynum causes a feeble inerease of urinary flow in the healthy animal; if the action is continued long enough and the dose is sufficiently large, haematuria may result. The slight diuresis is not due to any direct action on the kidney, but is due to the increase of blood pressure and passage of a greater quantity of blood through the kidney; because the flow of urine in normal animals always bears a direct relationship to the condition of the renal vessels and blood pressure. If the renal vessels are much contracted the flow of urine diminishes, but if they are only slightly contracted and the blood pressure is considerably raised, the flow of urine is slightly increased. In patients with cardiac disease and a failing heart, when there is back pressure and venous congestion and oedema of the kidneys as well as in other organs, little urine is secreted. The kidney is particularly sensitive to venous blood, and ceases to excrete as soon as the oxygen reaches a certain point. Now it is just in these cases that Apocynum produces so great a diuretic effect. It acts, then, not by any direct influence on the kidney, but by improving the condition of the circulation and so sending arterial blood once more to the kidneys. The kidney vessels are at the time of diuresis actually constricted, but this constriction must not be very complete, otherwise the diuresis will lessen. As in the case of Digitalis, the imerease in urinary flow is due neither to the ris? of blood pressure nor to any action on the excreting arrangements of the kidney, but to the increased quantity and quality of the blood which is driven through the kidney — owing to the greater force of the heart beat. The diuretic effect of Apocynum is a purely dynamical one, and is quite different to the action of such a diuretic drug as Caffeine, which produces dilation of the kidney vessels as well as a rise of blood pressure; the very marked flow of urine which follows an injection of a solution of Sodium Sulphate (see table) is an example of a third method of diuretic action, and occurs probably because the salt solution has some kind of direct action on the renal epithelium. aa - cen iid es ( . e a 4 | THE PHARMACOLOGY OF APOCYNUM CANNABINUM 401 2 Toxie effects. - ‘The drug bes a penetrating bitter Gist; which gradually increases until a feeling of nausea supervenes. Large doses have a very toxic effect on the mucous membrane of the alimentary canal. Experiment. A rabbit weighing 1,350 grammes was taken, and for eight consecutive days 5 minims of Tinct. Apocynum with 10 minims of normal saline solution were injected into the marginal vein of the left ear. “3 On the sixth and seventh days the animal was noticeably ill and exhausted ; it was constantly vomiting and had lost some weight. Death occurred suddenly on the eighth day. On examination the most obvious changes had occurred in the _ alimentary canal. The whole of the stomach and intestines were closely - ~ marked with haemorrhagic spots and areas, chiefly situated on the side of ‘the gut distal to the attachment of the mesentery. On opening the | fomach and portions of the intestines these haemorrhagic areas were found : 2 “te correspond with ulcers of various shapes and sizes in the mucous membrane. The ulceration was general throughout the whole length of aa the alimentary canal, from the lower end of the oesophagus to the rectum. The ulcers varied in size from 2 cm. square, with complete denudation of ‘epithelium, to mere punctures. These small punctate ulcers easily perforated on washing portions of the gut through from a pressure tap, when the water sprayed out in many small streams. The main blood vessels appeared to be normal, but some small white __. selerotic patches were noticed in the aorta a short distance above the semi- __Iwnar valves. The heart was larger than would be expected. Another _ small sclerotic patch was noticed in the wall of the left ventricle just below ee ‘the semi-lunar valves. The remainder of the organs appeared healthy. ih Experiment. A second rabbit was used weighing 1,200 grammes. “Two minims of Tinct. Apocynum were injected into the ear veins at intervals of one, two or three days over a period of seventy days altogether. At the end of this time the animal was killed. The left ear was oedematous and swollen to three or four times its ordinary size. The left eye was very prominent, and the conjunctiva extremely swollen and oedematous. On opening the thorax, some slight amount of fluid was found in the pleural sacs, both lungs were oedematous. The pericardial sac was distended with fluid. There was no increase of fluid in the abdominal cavity. 402 BIO-CHEMICAL JOURNAL One other effect of Apocynum deserves mention here. The mucous membrane of the alimentary canal, like all other plain muscle in the body, is stimulated; peristalsis is increased, and may give rise to nausea, vomiting, and diarrhoea. This effect is, no doubt, partly local; — stimulation of the mucous membrane causing local reflexes through Auerbach’s plexus, with resulting peristalsis. But this is not the whole explanation, since Apocynum still causes some increased movements of the alimentary canal even after absorption, or the same effect can be induced by injecting the drug directly into the circulation of an animal. The results of the experiments serve to illustrate the extremely irritant action of Apocynum on the tissues of the body. In the first rabbit to which the larger dose was given, the intestinal ulceration may be regarded as a secondary lesion occurring at the points of elimination of the drug into the intestine. As far as the naked eye appearances go, the frequent haemorrhagic areas point to profound changes in the blood vessels of the intestine, and these changes are certainly correlated with the appearance of the patches of sclerosis seen in the aorta. Further evidence of the influence of Apocynum on the vascular system is afforded by the lesions found in the case of the second rabbit; these were localised oedema, pulmonary oedema and slight hydrothorax, and distinct hydropericardium. 3. Muscular System. ; (a) Striated muscle. The action of Apocynum on muscle is comparable to that of Veratrine and Barium. The most characteristic ~ action of Apocynum on striped muscle is the delayed relaxation and constantly increasing rise of tonus; in this respect Apocynum exactly resembles Digitalis and Squill. It can be shown that this drug has the same type of action on all forms of muscle, but that the effect on the more delicate cardiac muscle overshadows the others. | (6) Unstriated muscle. This is thrown into a state of prolonged contraction, and the tonus is also raised so that in isolated preparations the death of the muscle occurs in contraction. Plain muscle throughout the body is affected in this way. The typical effect on this tissue may be simply observed in ring preparations of the frog’s stomach, in which the movements are recorded by the suspension method, so that the down stroke of the tracing indicates the contraction (systole) of the preparation. . Apocynum applied directly to the preparation causes a decided contraction, and the tonus of the muscle is permanently raised; and this THE PHARMACOLOGY OF APOCYNUM CANNABINUM 403 effect is obtained in spite of the fact that the tincture which was here employed-contains alcohol, the action of which is to reduce tonus. This effect on plain muscle may be regarded as representing the type of action occurring in all plain muscle throughout the body. I would observe here, however, that the alimentary canal shows irregular colicky contractions, the spleen and bronchioles are induced to contract, there is marked vaso-constriction and the tonus of the bladder and uterus is increased. The effect of Apocynum on the uterus was determined on the isolated uterus of a cat. Experiment. Fig. 5. The uterus was suspended with its lower end fixed in an oxygenated saline solution and the movements recorded by suitable levers. The introduction into the saline solution of a trace of tincture of Apocynum causes a great increase in the muscular tonus. The peristaltic movements become less and less and ultimately cease, but the muscle remains in a state of tonic contraction. The uterus in this case was a pregnant one, and 0-2 c.c. of the tincture was administered at the arrow mark. The type of action shown in the above experiment is very similar to that of digitalis, except that the effect of Apocynum on the uterine muscle is of a much more intense character. Pregnant uterus of Cat. 0°2 c.c. Tincture gp bor given at arrow produces rise of tonus and decrease in size of con- tractions. Death in systole. Full Scale. II]. Comparison or Apocynum CANNABINUM WITH OTHER MEMBERS or THe Carpiac Group or Drves Apocynum will be compared only with Digitalis, Strophanthus and Squill. The experiments show that, of the four drugs, Apocynum stands sécond in relative toxicity on frogs’ hearts. Strophanthus has the greatest toxic action, Digitalis and Squill, which are about equal in toxicity, have a less toxic action than Apocynum. of the other three drugs; in several experiments delirium cordis set he a almost immediately after the administration of the drug, and in others no- ie —o so-called therapeutical effects were obtained, also no tracings show obtained from Apocynum. ae ey generally such wide variations of blood pressure at ae stage as ‘those’ . This drug is the most irritant of the four towards mucous metal Be as shown by the intense ulceration produced in the alimentary canal after iy the intravenous injection of moderate doses of the drug. Strophanthus has been shown to be the least irritating to mucous membranes, and is i ine consequence the most readily absorbed. Apocynum is the most powerful vaso-constrictor of this group of drugs, and its property of constricting mee the blood vessels stands in direct relationship to what is really the specific action of the drug, that is its ability to increase the tonus of muscular tissue of every kind. In connection with this action, a greater rise of blood pressure is produced by Apocynum than by either Digitalis, Strophanthus or Squill; it is known that Strophanthus produces a comparatively small effect on the blood pressure, Apocynum causes a more immediate and sudden rise of blood pressure than Digitalis or Squill. There is no special difference in the action of these drugs on the kidney, which in each case is an indirect one, but Apocynum appears more likely — to produce haematuria owing to its pronounced irritant properties. REFERENCES 1. Gould, Year Book of Med. and Surg., p. 472, 1904. 2. Lancet, 1894, Vol. I, 841. 3. B.M.J. Epitome I, paragraph 447, 1896. 4. Murray, Physiolog. Act. and Therapeut. Val. Apoc. bined. M.B. Thesis, Therapeutic Gazette, 1890. 5. Hale White, Txt. Bk. Pharmacol. and Therapeut. 6. Dabney, Therapeutic Gazelte, Nov. 15, 1898. . 7. Il Policlinico, Nos. 10-14, May-July, 1894. , 8 9. .S B.M.J., Vol. I, p. 1714, 1897. Lancet, 1896, Vol. I, paragraph 447. 10. B.M.J., Epitome 1, paragraph 447, 1896. 11. Journ, Physiol., March 27, 1909. 12. Brit. Pharm. Conf., p. 387, 1905, the Heart, Bio-Chem. Journ., Vol. I, No. 2, 1905. a Ss “* ? * on | ee < 4 a eat Li 13. Haynes,»G. §., The Pharmacological Action of Digitalis, Strophanthus and Squill on 4 THE PHYSIOLOGICAL EFFECTS OF SELENIUM COM- - POUNDS WITH RELATION TO THEIR ACTION ON GLYCOGEN AND SUGAR DERIVATIVES IN THE TISSUES By CHARLES 0. JONES, M.D. (Liverpool). “3 Communicated by Prof. Benjamin Moore % ‘ From the Bio-Chemical and Physiological Departments, University of Liverpool (Received September 6th, 1909) “ . Historicat ae Dibditis were ins by id in 1985. He found that on adding traces of _ selenium and tellurium salts to the water in which plants grew, that although no influence on the growth of the plants took place, yet selenium ey : was absorbed. The same was found true of algae and infusoria by __ Bokorny in 1893. Scheurlen, in 1900, seeking a substance which -__ contained loosely-bound oxygen, to grow bacteria in absence of atmospheric oxygen, tried sodium selenite, and found that though the bacteria were unaffected, yet they were coloured with the reduced selenium. This selenium was found entirely in the cell, none being found in the media. ’ A careful study of these effects was conducted by Klett, who found that _ bacteria and moulds were not as a rule hindered in their development by traces of selenite of sodium, but a few, such as the bacillus of malignant i oedema and symptomatic anthrax, were arrested in growth. He also found the bacteria coloured with the reduced selenium, the surrounding media being colourless, and that as the amount of selenite was increased growth was inhibited. He concluded that the reduction of selenite to selenium took place in the protoplasm of the bacterial cell, and not outside the cell by secondary action of metabolic products. Action on animals.—Gmelin appears to be the first who investigated the effects of selenium and tellurium salts on animals. He found that they were poisonous, that they produced a deposit of the reduced element on the intestinal walls, and that the animal gave off a garlic-like odour. This odour was also noticed in the animal’s breath by Hansen, who attributed it to ethyl selenide; this observer found also that after a large —_ f 406 BIO-CHEMICAL JOURNAL dose the animal vomited, and that the vomit contained selenium. On — making sections of the animal’s organs he noticed that they all contained selenium deposited in granules. The work was continued by Rabuteau, who observed that after a large dose vomiting, profound dyspnoea, anasthesia, opesthotonos, and death from asphyxia took place. The — post-mortem findings were intense congestion and ecchymosis of the whole intestinal tract, also of the liver, spleen, lungs, and kidney. The right side of the heart and large blood vessels held a multitude of small prismatic crystals of unknown chemical composition. Rabuteau concluded that these crystals acted as a mechanical obstruction and caused death. These results were not confirmed by Czapek and Weil, who could not find any crystals or mechanical obstruction, and concluded that selenium was very similar in its action to tellurium, arsenic and antimony, and that death was due to paralysis of the so-called excito-motor ganglia. They further noticed marked distension of the abdominal capillaries. The blood was normal, but it gave off a marked garlic-like odour. This odour was noticed by Wohler to be similar to methyl] selenide, which he was then preparing. Hofmeister confirmed this, proving by analysis that they were the same, and further showed that all the organs gave off the odour, but that it was most pronounced in the testes and lungs, and marked in the blood, liver and kidney. If the organs were placed in an ineubator the smell was intensified, but blood loses the smell. Hofmeister concluded that all the organs could absorb selenium and form methyl selenide from it; lastly he discovered that the reduction to selenium and the formation om methyl selenide were independent of one another. On heating an . organ to 55° C. the formation of methyl selenide ceases, but the organ will still reduce selenite to selenium. The explanation suggested was that the reduced selenium was slowly built up into a soluble compound in the alkaline blood and was changed in the lungs to methyl selenide. The meth] groups he supposed to be derived from cholin, creatinin, and other methyl-bearing substances. he effects of selenium and tellurium salts on metabolism were investigated by Mead and Gies, and Woodruff and Gies, who found that selenium salts had little or no effect on metabolism, but that the ether-soluble substance in the faeces was increased. This they attributed to diminished absorption. They also examined the vomit resulting from selenium salts, and found that there was complete absence of free hydrochloric acid, the pepsin was unaltered, and on addition of hydrochloric acid digestion proceeded at a normal rate. Ptyalin, on the other hand, was markedly affected by selenium salts, PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 407 r The compounds of selenium are thus extremely toxic; but if the _ amount present is very small, the cells are able to reduce it, forming an inert substance, and can then continue their metabolic changes. This research was undertaken to endeavour to find out how this reduction is accomplished and also to find out more exactly the cause of death in selenium poisoning. Two compounds of selenium were used in this reasearch, viz., sodium _ selenite and sodium selenate, both obtained from Kahlbaum. _ Sodium selenate is a comparatively stable salt, easily soluble in water and neutral in reaction. ~~ he lethal dose of selenate for a moderate sized rat (about 80 —— was found to be 0°6 c.c. of 0°125 per cent. solution. Selenite of sodium is an extremely unstable salt. It is stated by “Mead and Gies to be reduced by all protoplasm, and they have seen _ reduction take place in contact with fresh meat. It is even said that reduction takes place in contact with all organic matter. If this is so, __ it is difficult to see how any can be absorbed if given by the mouth, so _ that in this research all doses were given hypodermically. i. The preparation used was found to be acid in reaction, the acidity . of 1 gramme being equal to 2°75 c.c. normal sulphuric acid. It is distinguished from selenate most easily by the insolubility of the selenites of copper, cobalt and nickel. Cobalt salts give a mauve precipitate visible 1 in 800 of water; copper salts give an apple-green precipitate visible 1 in 1,200 water; nickel salts give a green precipitate visible 1 in 1,600 _-__— water. Owing to its instability the selenite solution was always made fresh as required. _--_—__._- The lethal dose for a moderate sized rat was 04 c.c. of 0-125 per cent. solution. The lethal dose for a moderate sized rabbit was 0:5 c.c. of 2 per cent. solution. The lethal dose for a moderate sized cat was lc.c. of 2 per cent. solution. From these results it appears that selenate of sodium is only two- thirds as toxic as selenite. After a small dose no symptoms were observed, and even the appetite was unaffected. As the dose was increased and was just sub- lethal, it was observed that, after about ten minutes, the animal became ‘restless; this was followed by movements of the mouth, tongue and nose. As there were now present a peculiar garlic-like odour in the breath, it is probable that these movements are due to stimulation of the nerves Pree a ee ee Seer fae 408 BIO-CHEMICAL JOURNAL of taste and smell. Very shortly afterwards retching and vouitiiaaia : commenced. If the dose has not been too great, recovery soon takes place, no after effects being noticeable. If the dose be too great, the vomiting and retching continue and somnolence passes on to unconscious ness and death. ee . It may be mentioned, as death has been aseribed by previous vocal bane to dyspnoea, that laboured breathing was seen in one case only. It was in this case due to excessive reduction of the salt to selenium and consequent embolism of the pulmonary vessels. The paralysis, con- — vulsions and other symptoms noticed by former investigators were — probably due to the same cause, and are in no way connected with death — from chemical poisoning with selenium salts. Macroscopic AND Microscorrc CHANGES IN THE TISSUES The macroscopic post-mortem changes were very few. The liver was usually soft and friable. All the organs gave off the garlic-like odour noticed in the breath. The right side of the heart was distended and full of clot. The splanchnic vessels were enormously dilated. The microscopic changes were more pronounced, and were investi- _ gated as follows:—The tissues were immediately placed in formol, dehydrated with acetone, embedded in paraffin and stained with eosin and haematoxylin. 7 The most noticeable feature of all the sections was a golden-brown er amorphous deposit found in almost every organ. It is chiefly found = around the blood vesséls and between the cells, but some is also to be SS seen inside the cells. It was suggested that this substance might be iron, but it gave none of the staining reactions for iron. On grinding up the organs with sand this substance could be extracted, and formed a brick- red deposit. This was found to be identical in every way with the - amorphous form of selenium produced on reducing sodium selenite in the test-tube. It volatilized with heat, burning with a blue flame, and gave _ | off the well-known horseradish smell of selenium. a a This golden-brown deposit was found, if the dose was laripesit in almost | 7 q every organ, the whole of the tissues being flooded with it, but this is han not the cause of death, for if a just lethal dose is given there is no such flooding and death still takes place. Physiologically this deposit is inert, for if a small dose of a selenium salt is given and the animal killed some time afterwards, this deposit will be seen in the cells, which are evidently — still capable in its presence of performing their metabolic changes without noticeable change. PILYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 409 E The action of selenium salts on different isolated physiological ____ systems prepared by the usual methods was next investigated, and they were found to be without action on (1) a muscle-nerve preparation, (2) isolated heart muscle, (8) nervous mechanism of heart, (4) higher nerve centres, (5) blood pressure, (6) intestinal movements. The urine was also normal, traces of selenium salts were found present, but never any solution that reduced Fehling’s solution. The blood was occasionally _ found altered; the most frequent change was a slight lymphocytosis - followed by a more marked increase in the polymorphonuclear leucocytes. The red blood cells were fpund normal both in their number and in _ their haemoglobin contents, and spectroscopically the blood was found normal. In small amounts, selenate had no effect on gastric digestion, aor had it on pancreatic, while selenite had an inhibiting effect on “pancreatic action, this being in part at least due to its acid reaction. The reduction which takes place in the tissues was next investigated. _ After heating an organ to 60°C., as Hofmeister showed, it will still yeduce selenite to selenium. This was repeated, and it was further seen that higher temperatures do not stop this reduction. . It therefore seemed Sale probable that this effect was not due to an enzyme, but was some direct _ chemical effect. A fresh solution of sodium selenite was therefore made, and 5e.c. added to 0-5 gramme of each of the following carbohydrates with aseptic precautions. The mixtures were then placed in an incubator at 35°C. for twenty-four hours and again examined. Reduction was shown by the solution becoming pale brown, while if the reduction were intense a fine brick-red powder became deposited. Glycogen io No action Tnulin hs Profuse reduction POLYSACCHARIDES ... ; ea Starch rye No’action Dextrin Fe No action H é ‘ Mannite ry No action ee eee oe | Duleite he No action Arabinose Ae Reduction PeNTOSES . 4 Rhamnose sx No action a Xylose on No action 4 Maltose ie Faint reduction Glucose al Reduction HEXOSES ... cee cee +e" Galactone vse Slight reduction Lactose es Slight reduction Levulose can Profuse reduction Ammo Hexose ...._—_... 1 Glucosamine is Profuse reduction 4 ; 8 | Saccharose ose No action 4 ComPouND BOGARS... ++ | pom ce ae 410 BIO-CHEMICAL JOURNAL The reduction of inulin is due to the formation of levulose. Tt will be seen that reduction takes place with arabinose, levulose, glucose, and the sugars yielding glucose. The reduction by glucose and levulose was then tried at a low temperature, viz., 30°C., and it was’ found that glucose caused no reduction at this temperature even after several weeks, while levulose caused a profuse reduction. When the two sugars were mixed it was found that no reduction occurred at 30° C., and both came down at 38°C. It was thought that perhaps this reaction with more rapid heating might serve to differentiate the sugars, but on heating a solution of the sugars with sodiym selenite solution in a test- tube, it was found that levulose commenced to reduce at 58°C., while glucose, lactose, galactose and maltose all reduced at about the same temperature, viz., 72°C. The derivatives of glucose and its compound sugars were next tested as reducing agents for selenite, with the following results :— Glucose ica Reduction Gluconic Acid oa No action Glucuronic acid ins No“action Saccharic acid aey Reduction Mucie acid oT No action Furfurol Ele No action. The above reductions at first sight would appear due to the aldehyde or ketone groups in the sugars, but here we are met with the fact that while rhamnose and xylose, which contain aldehyde groups, have no action, yet saccharic acid, which has neither aldehyde nor ketone group, acts very strongly. We tried the action of benzaldehyde, formaldehyde and acetone, but found they all gave negative results. Other possible degradation products of the sugars were tried, for example, lactic acid and acetic acid were without action, but formic acid had a powerful reducing action. It is interesting to note here that selenite is reduced by arabinose, levulose, glucose, maltose and lactose, all of which are found in, or are excreted from, the human body. The only exception is xylose, which is a necessary constituent of the nucleoproteids, but in such a position an energetic sugar would be a source of danger to the animal. These results cannot be explained on the structural formulae at present ascribed to the different sugars, so that the reduction rests more on a physiological basis than a purely chemical one. It was next ascertained whether proteins, fats, or other substances of animal origin, would perform the same PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 411 reduction. Although treated in the same way as the carbohydrate or heated together directly in the test-tube, no action was found associated with any one of them. The following substances were all tried, viz. :— Olein, oleic acid, palmitin, palmitic acid, potassium palmitate, erucic acid, lecithin, cholesterin, glycerine, uric acid, hippuric acid, urea, gelatin, glycocoll, tyrosin, casein and creatinin. From this it appears probable that this reduction can be performed by ; ae glucose and a few allied sugars, and cannot be produced by organic matter from which carbohydrates are absent. To ascertain whether glucose is the agent which accomplishes this _ reduction in animal organs, the following method was adopted. Ten - Sma of finely minced liver were taken with aseptic precautions, and to this were added 5 grammes of yeast, 50c.c. of distilled water and a . - few drops of toluol. A control was prepared in the same way, but without any yeast. Both vessels were placed in an incubator for twenty- four hours, they were then heated to 70°C. to destroy the yeast and _ glycolytic enzyme, and 1 gramme of sodium selenite added to each. After ___ being in an incubator at 38° C. for a few hours, both were examined. The control which contained glucose from the glycogen was of a deep red —__ eolour, showing that quantities of selenium were present. The flask which had its sugar destroyed by the yeast showed no red colour, so that no selenite had been reduced. This experiment makes it probable that in the absence of glucose selenite is not reduced, and, accordingly, that reduction of selenite to selenium in a cell indicates the presence of glucose. a _ Since selenite of sodium is reduced in the cells by glucose, it appeared as necessary to ascertain where, especially in the body, this reduction takes place. As has been already seen, if a large dose is given the whole organism is flooded with the selenium. A rat was therefore given several small doses, and was then killed, and the organs quickly removed and examined histologically. The spleen was found to contain abundance of selenium, as well as an excessive number of leucocytes. The portal vein was examined and found to also contain selenium and leucocytes, and the same was found true of the liver; while the “vessels leaving the liver, the lungs, kidney and intestine, were found to be quite free from selenium. ‘: In some cases the liver cells show destructive changes. The nucleus 412 BIO-CHEMICAL JOURNAL stains less deeply while in others the nucleus has disappeared, the cells being mere shadows. It therefore seems probable that reduction takes place firstly in the spleen. The reduced selenium is brought by the blood stream, only a small amount by leucocytes to the liver. The liver also reduces any selenite that has escaped the spleen. If the dose is not excessive no selenium is allowed to pass the liver. DiIsAPPEARANCE oF GLYCOGEN FROM THE LivER Finding that selenite is reduced by glucose in the liver and spleen suggests that the glucose must be derived from the liver glycogen, and this was found to be the case. ‘Two well-fed rats were taken, and to one was given an injection of sodium selenite just sublethal. As soon as it began to recover, which happened in a few hours, another injection was given. ‘Treated in this way, in a time varying from three to seven days, the animal dies. The control rat was then killed, and the livers from the two animals contrasted as to their glycogen content by the following method: —The livers from both rats were quickly removed, cut in pieces in each case, and placed separately in boiling water acidified with acetic acid. The pieces were then ground up in each case in a mortar with hot distilled water. In the rat which had been injected with selenite, there resulted a perfectly clear pale orange coloured solution, which gave no coloration with iodine and no precipitate with alcohol or basic lead acetate. The fluid from the normal rat’s liver gave an opalescent solution, which on addition of iodine gave a dense brown coloration. This experiment, on account of its importance, was repeated several times, with the same result. It may be stated that the dose given must be carefully regulated so as to be just sublethal. If an over-dose is given, the animal will die, due probably to not sufficient glucose being available to reduce the selenite; glycogen will then not have disappeared entirely. This disappearance of glycogen was also found to occur in both frogs and rabbits. This gradual using up of glycogen and glucose made it interesting to find out if any other metabolic changes took place at the same time. A well-nourished cat was used for the experiment. The normal excretions were examined and estimated daily for a week, during which period the urine was invariably found to be acid, A small injection was 4 a PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 413 then made of ('5 e.c. of 0°25 per cent. solution of selenite; the only change following this small dose that took place was that the urine next day had become alkaline; this continued so throughout the experiment. The dose was gradually increased, and it was noticed that the amount of urea excreted on the day following the injection had fallen considerably, but had returned to normal on the following day. As the dase increased, the urea decrement became greater, and an increased number of days were required _ for the return to the normal. When the dose became excessive the animal yomited. No urine was excreted the following day. The next day the excretion of urea was exceedingly small, and continued small for some days, only very slowly returning to the normal. The total amount of nitrogen excreted showed no relation to the urea; it kept steady all through the periods when the urea fell. When an excessive dose was reached the total nitrogen fell to about one-third the normal amount and ~ gradually returned to normal. ~ It would thus appear that while the wrea was excreted in less amount the nitrogen was got rid of in some other form. That it was not excreted as uric acid nor as ammonia seems probable as these showed little or no change throughout the experiment. Sulphates and phosphates showed no change. | The most striking effect was noticed in the excretion of chlorides. Until the dose had become excessive the amount of chlorides thrown out had continued steady. After the animal vomited the excretion of chlorides suddenly dropped, so that the daily amount excreted became half or less pe than half the normal amount; thus, in the first cat the average daily a output was 1°28 grammes; this fell to 0°061 grammes. 4 In the seeond cat, as will be seen in the following table, the average daily excretion of chlorides for fifteen days during which it was having - increasing amounts of selenite was 0°0749 grammes. After 0°75 c.c. of 2 per cent. solution had been injected the animal vomited. No urine was passed the following day. The next day the amount was only 00305 grammes, that is, less than half the normal amount. The following three days there was very little increase. The following day, as will be seen in the table, the whole of the retained chlorides were thrown out, the amount excreted afterwards returning to normal again. Vomited 00805 0°0835 00881 OO 0° 1820 o-os1li No urine Injection 414 BIO-CHEMICAL JOURNAL The vomiting took place about ten minutes after injection. The if 2 ae vomit consisted of the stomach contents in a state which showed that there = had been no interference with digestion until the injection was given, = THe animal during this period was under the influence of the drug, rr: and it is only when it reaches an excessive amount that vomiting occurs. = | This vomit was found, as already pointed out by Mead and Gies, to be free from any trace of free hydrochloric acid. The reaction was acid, and quantities of organie acid were present. It therefore seems probable that the hydrochloric acid is suddenly withdrawn from the stomach to serve some other necessary purpose, and if 02 per cent. hydrochloric acid be added to the stomach om digestion will proceed normally. This withdrawal of hydrochloric acid is accompanied by a iieiilly diminished excretion of chlorides in the urine. This observation coincides with the withdrawal of hydrochloric acid from the stomach. While the chlorides are retained, appetite and digestion are in abeyance; after a certain period, varying from one day to five days, during which time the excretion of chlorides is only about half the normal amount, the whole of the chlorides retained are thrown out, their purpose having been fulfilled. Then the animal regains its appetite and the exeretion of chlorides becomes normal. Still another interesting fact bearing on the subject was noted: when the animal had lost its appetite, although it refused fresh meat it would still eat salt meat. Possibly the excess of chloride helped the return of hydrochloric acid. : Lastly, with these changes there was also a remarkable loss of weight. One cat lost 38 per cent. of its total weight, the average daily loss being 26 grammes. A second cat lost 335 grammes during the first week, or 16 per cent. of its weight, and in one day it even lost as much as 65 grammes. This loss of weight is too great to be accounted for by diminished consumption of food alone, for until the dosage became large the animal still retained its appetite, and even after a large dose it only | refused its food for a day or two, the appetite gradually. returning. It was observed by Mead and Gies that after a dose of a selenium compound the amount of ether-soluble substance in the faeces was increased. This we found true for small doses, but on investigating the effect of large doses we found that the amount of ether-soluble matter in the faeces was very much diminished, and that the relative amounts of neutral fat, fatty acid and soap (reckoned as oleic acid) were altered. = a Te ee eS id pit a |) OU! * = a hy ent ie, om? — a = ae sia hlY aeaiala a ae PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 415 _ The faeces were dried and extracted with ether in a Soxhlet apparatus, the acidity of the fat being titrated with 01 N alcoholic potash, using phenol phthalein as indicator. After an injection of selenite of sodium the total amount of fat in the faeces became very much reduced and was at its minimum the second day after injection. The excretion of fatty z acid was less affected or increased in amount, so that the normal proportion _ of neutral fat to fatty acid being 2 to 1 on the second day, the fatty acid ~ became equal to, and sometimes greater than, the amount of neutral fat. This may be seen from the following table, where the amount of fat has been worked out to a constant: —- Fat Free fatty acid 0-5 0-284 0-5 0-292 0-5 sie 0-290 Injection of selenite 0-5 a 0-350 0-5 0-530 0-5 0-170 0-5 0-280 - from then onward. - These observations seem to show that glucose, or a derivative, is the means by which the body protects itself from the toxic effects of selenium salts, and death may even in certain cases be due indirectly to the using up of the glucose; but this cannot be the cause of death after a single large dose, therefore the effect of selenite on the living cells of the liver was next investigated. A fresh liver was finely minced, using aseptic pre- -__-vautions, and 10 grammes were weighed out in every case for the purposes of the experiment. To this were added 50 c.c. of sterile distilled water and a few drops of toluol. The substance to be tested was added to one, and the control and the one containing selenite were placed in an incubator at 38° C. for varying periods of time. They were then boiled and filtered. The filtrate and solid matter were each separately estimated by Kjeldahl’s method. SeLenate or Soprem Soluble Nitrogen Insoluble Nitrogen Normal cas nde 0-0883 ce 02125 Selenate 0-5 gramme ue 0-1076 < 0-1704 Normal soe ae 0-1674 set 1618 Selenate °° wed ais 01268 = 02047 Selenate 1%) an v3 01570 0-1751 We see that there is no definite effect on autolysis caused by selenate of sodium. oo : ¥ ¢ i te 416 BIO-CHEMICAL JOURNAL Seiexttr or Soprum oft. Third day ; inte a Soluble Nitrogen Insoluble Nitrogen — Normal boiled ee ii 0-0192 Sis OS7LL |. nite Selenite 0-5 gramme ae 0-0211 sing 0-2708 Normal liver ont ous 0-0883 = 02123 Seventh day , pie ta Soluble Nitrogen _ Insoluble Nitrogen mr Normal liver sae “a 0-1911 Pie 0-1497 © ‘ Selenite 0-5 grammes aire 0-0762 nee 0-2636— Selenite 1 gramme ... Be 0-0566 de 0-2763 Tenth day Roy +. Soluble Nitrogen Insoluble Nitrogen Normal liver das oe 0-1674 ons 0-1616 i i Selenite 0-5 grammes Sc 0-0636 a OTL © Selenite 2 grammes ... 0-0745 oat 0- 1047 It is evident here that selenite of sodium has a very marked inhibitory effect on autolysis. The presence of selenite in a cell in sufficient amount — would seem to inhibit all metabolic changes and destroy the cell. As the action of selenate of sodium in the body is similar to selenite in its ulterior — effects, and as selenate is not poisonous to the cells, it seems evident as its toxic effects are present only when it has been reduced in the body ated to selenite. ate The ease with which the glucose molecule is broken up by soit a selenite and selenate in the body suggested that if diabetes mellitus were — due to the inability of the animal cells-to break up the glucose molecule, as the believers in the oxidation theory hold, then the presence of selenite or selenate which effects this splitting up should lessen the amount of 3 sugar in the urine, for it is well known that the degradation products of glucose can be easily dealt with by the diabetic. Selenate of sodium was the salt used, being less easily reduced in the intestine than the selenite. — The patient was under the care of Dr. J. Hill Abram, whom I have to — thank for trying the drug, and also Dr. A. F. Jackson for the care with — er which he performed the sugar estimations. The patient was a case of ae severe diabetes with twelve months’ history. an : Per diem ~~ ots eT aay Average sugar, Urea, in grains ‘in grains Common diet as yi 5232 so 538 Special diet ba as 2212 3 307 Selenate ee Gen 3087 Ra 455 PHYSIOLOGICAL EFFECTS OF SELENIUM COMPOUNDS 417 The dose of selenate given was 5 minims of 1 per cent. solution, gs gradually increased until 25 minims was given. As will be seen, there was an actual increase in excreted sugar while taking selenate, which seems to show that diabetes is not due to any lack of oxidation power nor to any difficulty in breaking up the glucose molecule. ConcLUSIONS These observations make it probable that selenate is reduced in the body to selenite, so that the action of selenite only need be considered. When an injection of selenite is given, it is quickly taken up by the blood stream; only a small quantity is excreted by the kidney, the oa ‘remainder is carried to the spleen and liver, where it is reduced by glucose to selenium. According to Zsigmondy, this reduction with glucose can, _ with the aid of the ultra microscope, be seen to take place fairly easily outside the body, particles of the reduced selenium being visible in about two minutes, so that the living cells would have no difficulty in effecting This reduction does not appear due to the aldehyde group of the ‘sugar, but more probably is due to some special configuration of the glucose molecule, in which it is closely resembled by arabinose and levolose. This glucose, as required, is furnished from the glycogen of the liver, but when this is becoming exhausted fat is called upon. Whether the fat is used up as such or 1s transferred into sugar, it is ___, diffieult to say. The excretion of excess of fatty acid after an injection ay when sugar is urgently required, would point to the glycerine being ___ possibly required for transference to sugar, but it is clear that there is no effort on the part of the organism to form sugar from proteins. If it were possible an effort would be made by the cells to manufacture glucose from protein to save themselves from destruction, but that no such action occurs is shown by the excretion of nitrogen remaining low, even until death. One must conclude that the organism cannot under such conditions transfer protein into sugar. The evidence as to the transference or at least equivalence of fat and sugar is better. There is complete disappearance of fat as well as glycogen and sugar, which points to their transference or utilization. The loss of weight also suggests the same. In a moderately fat cat the loss of weight was about 38 per cent. This would just about represent the weight of fat, glycogen and glucose. Méchel’s estimate of fat in a 418 BIO-CHEMICAL JOURNAL — moderately fat dog being 26 per cent., the remaining 12 per cont: would ‘ account for the glycogen and glucose. bees It is necessary to consider at this stage the suddeu disepppaha | of hydrochloric acid, the holding back of the chlorides by the organism, and the extraordinary relish for sodium chloride. These factors are all present when selenite has just been given, and when there is a sudden demand for glucose on the part of the cells. The most likely explanation — is to be found in an observation by Eckhard that a one per cent. solution _ of common salt introduced into the blood caused glycosuria. Fisher showed that other sodium salts had the same effect, and that the stronger the salt solution the more glycosuria resulted, even up to 73 per cent. of sugar was found. volts Excess of sugar in the urine represents excess of sugar in the blood, and therefore it would be possible for an animal holding back its chlorides by using its hydrochloric acid to fix some sodium salt, and taking sodium chloride in its food to raise its blood sugar content. If this sugar is picked out by the spleen and liver, and also possibly by leucocytes, it must result in a wonderfully increased power of reducing selenite to selenium, and so saving the cells from the poisoning effect. If the organism is unable to neutralize the selenite, either because the selenite is in too great excess or because the available stores of glucose are used up, then selenite will act on and destroy the cells. The action is on the ferments of the cells, causing all metabolic changes to cease.. It is curious to note here what a slight effect it has on bacteria and a powerful — effect on ferments, quite a contrary effect to toluol, chloroform and similar _ antiseptics. It seems to show that the defences in single cells are much more highly developed than is the case in cells which rely on others for protection. It is interesting to consider here whether glucose may not poise , be the means by which all reduction processes take place in the body. The well-known reductions taking place in the organism, such as methy- lene blue and Prussian blue, can be accomplished with ease by glucose in faintly alkaline solution. Considering the universality of occurrence of glucose in the body cells, and its well-known power of reduction outside the body, which is immensely multiplied within by cell activity, it would hardly seem necessary for the cells to require any other means of reduction. I take this opportunity of expressing my indebtedness to Punta Benjamin Moore and Professor C. §. Sherrington for their kind assistance and advice. LITERATURE , Verauche tiber die Wirhetigen des liaryib es: Miubicas che. au} den thierischen Organismus 1824, p. 43. Ann, de Chemie wnd Pharm., UXXXVI, p. 208. Gazette hebd. de Med. et de Chem., XVL, pp. 194-241. Botanisches Centralblatt, XXII, p. 35. k and Weil, Arch, fiir exp, Path. und Pharm., XXXII, p. 438, , Chem. Zeit., XVII, 2, p. 1098. Tbid., XVIII, 2, p. 1739. r, Arch. fiir exp. Path. und Pharm., XXXII, p. 198. Arch. f. Physiol, 1895, p. 225. Jen, Zeit. j. Hyg. u. Inj. Krank., XXXII, 135. 420 THE EFFECT OF WORK ON THE CREATINE CONTENT OF MUSCLE By T. GRAHAM BROWN anp E. P. CATHCART. From the Physiological Laboratory of the University of Glasgow (Received September 30th, 1909) Previous Work The question—Does work influence the amount of creatine in muscle, and lead to a change in the output of creatinine in the urine, has long been a debated one. Much of the older work on this point is of little value owing to the defective methods then available for the estimation of creatine and creatinine. The papers of Weber (1) and van Hoogenhuyze and Verploegh (2) give good accounts of these earlier investigations. ; Of the older workers, Liebig (3) found an increase of creatine in muscle after work; as did also Sarokow (4), Sczelkow (5), and Monari (6). Nawrocki (7), and Voit (8) thought that there was a slight decrease. Of modern workers Weber (loc. cit.), using Folin’s method, found that work caused a slight decrease in the creatine content of muscle. He also found that traces of creatine and creatinine could be demonstrated in the Ringer’s fluid with which, using the method of Langendorff, he had perfused an isolated heart; while Mellanby (9), also using Folin’s method, obtained results from which he concluded that work had no influence on the creatine content of muscle. As regards the effect of work on the excretion of creatinine in the urine there is also a difference of opinion. Meissner (10), employing very defective methods, found an increase of creatinine in the urine on the day of exercise, followed by a decrease on the succeeding day. Grocco (11), and Moitessier (12) also found an increased output on the day of exercise. Gregor (13) concluded from personal experiment, using a creatinine free diet, that exercise increased the output of creatinine in the urine. Voit (loc. cit.), Hoffmann (14), Oddi and Tarulli (15), using the older methods found no increase. Van Hoogenhuyze and Verploegh (loc. cit.), using Folin’s method, found that, with an ample diet—creatinine free, work left the excretion of creatinine unaffected, but that when the diet was defective, as during the complete fast of their subject, La Tosca, exercise resulted in a slight increase of creatinine in the urine. Weber _ EFFECT OF WORK ON CREATINE CONTENT OF MUSCLE 421 (loc. cit.) also came to the conclusion that, if work were performed during a fast, a rise in the output of the creatinine followed. Shaffer (16) agreed with the results of van Hoogenhuyze and Verploegh. One of us (E. P. C.), although not investigating this particular point, working in conjunction with Drs. Kennaway and Leathes (17), was unable to detect any definite rise in the output of creatinine even after severe work under very different conditions, in all of which, however, the diet was ample. We may therefore accept that, provided the supply of food is sufficient, work does not bring about any increase of creatinine in the urine. PRESENT INVESTIGATION As regards the results on stimulating frog muscle we have already made a communication (18), in which we showed that there is an increase in the amount of total creatinine in stimulated isolated frog muscle (ordinary nerve muscle preparation), whereas, when the circulation is left intact as in the decerebrated frog, there is always a slight decrease in the amount of the total creatinine present. The following table gives the results with frog muscle :— Taste I Serres A. (Isolated nerve muscle preparation.) Experiment Per cent. of total Per cent. of total Difference creatinine in creatinine in normal (controls) stimulated A 0-32 0-36 12 % increase B 0-30 0-32 T% ss c 0-32 0-36 13% on Be D 0-36 0-39 oe ' Serres B. (Muscle in situ, circulation intact.) A 0-37 0-26 lb-6 % decrease B 0-35 0-29 62% ,, 33°G6 Cc 0-30 0-23 9-1 % OT D 0-32 tly 0-24 77% We have now carried out a series of experiments on rabbit muscle under different conditions, and have obtained constant results. The methods we employed were as follows:—The rabbit was deeply anaesthetised with ether and was kept under the influence of the anaesthetic till the end of the experiment, when it was destroyed. The extensor muscles of the right knee were exposed by means of a skin incision and removed, the amount taken being usually 10 to 15 grammes. The 422 BIO-CHEMICAL JOURNAL bleeding points were secured and care was taken not to injure the great vessels of the thigh. All visible fatty and fibrous tissue, as well as blood, was rapidly removed from the excised mass, which was then weighed, transferred to a small mortar, minced fine by means of a pair of sharp scissors, then rubbed up with finely powdered glass, water being gradually added until a fine suspension was obtained. This mixture was next carefully transferred to an Erlenmeyer flask, filled up to 150 e.c. with distilled water, some chloroform and thymol solution added, thoroughly shaken up, and then placed in a hot water oven at 50°C., where it was left, being repeatedly shaken, for eight to ten hours. At the end of this period the flask, after faintly acidifying its contents with acetie acid. was put on the steam bath, or boiling water bath, for thirty minutes and then filtered. The solid residue was extracted with boiling water (as a rule by boiling the residue with water in a porcelain basin) five to seven times and filtered through the original filter. The united filtrates were then concentrated to 40 c.c., and the content of creatine + creatinine estimated by Folin’s colorimetric method. The same procedure was carried out in the case of the stimulated extensor muscles. of the left thigh, care being taken (i) that the corresponding muscles were taken for examination, and (ii) that the amount taken differed by several grammes from that taken from the right (unstimulated) side—sometimes more being taken, sometimes Jess—-so that the readings on the colorimeter were widely apart, thus obviating to a considerable extent any possible personal bias. As an additional precaution one of the observers frequently manipulated the solutions in such a manner that the observer who took the readings was unaware which of the two solutions he was examining. The reason why definite corresponding muscles were chosen was that as the result of several experiments we carried out with different kinds of muscle (red and white) we are inclined to believe that there is some difference in their creatine content. That such might be the case is suggested by such work as that of Bonhéffer (19) and of Paukul (20), — who showed that in muscle structural differences might be associated with functional. So far we have not performed a sufficient number of experiments on this point to be able to draw definite conclusions. —__ As regards our method of stimulation, immediately after the removal of the extensor muscles of the right side those of the left were transfixed by a pair of electrodes, one of which passed through the muscles close — to the patella and the other near their origin. Through these electrodes the muscles were stimulated with rapid alternating faradic shocks, A a _ EFFECT OF WORK ON CREATINE CONTENT OF MUSCLE 428 Berne coil was used, the primary current being about 5 volts and the secondary coil was placed between the 2,000 and 4,000 unit marks. The muscles were stimulated over a period of thirty to forty-five minutes, but the secondary currents were not allowed to pass continuously through them, being short-circuited for five seconds in every ten seconds. We carried out control experiments to determine the accuracy of our chemical methods, and found that, for small amounts of muscle, the average difference between the extensors of the two sides, neither having been stimulated, was a little over 1 per cent. of the total creatinine, and for larger amounts (10 to 15 grammes) was about 2°3 per cent. The general result of the whole series of experiments has been that, with the circulation intact, stimulation of the muscles brings about a constant, although small, decrease in the amount of total creatinine (i.e., creatine + creatinine) extracted from the stimulated muscle. _ Bxperiment II.—Rabbit, weight 1,550 grams. 12 grams muscle removed before stimulation (stimulation 30 minutes) and 11 grams after. Total creatinine before stimulation, 43-42 milli- grams = 0-361 %, and after, 30-84 milligrams = 0-280 %, i.e. a decrease of 22-4 % as a result of stimulation. Experiment I1V.—Rabbit, weight 1,850 grams. 7-5 grems muscle removed before and 17 grams after 40 minutes’ stimulation. During the period of anaesthesia a free supply of pure oxygen was given. Total creatinine before stimulation 28-8 milligrams = 0-334 %, after stimu- lation 54-6 milligrams = 0-321 %, i.e. a decrease of 4-3 % as a result of stimulation. Experiment V.—Rabbit, weight 1,450 grams. 9-2 grams muscle removed before and 16 grams after 30 minutes’ stimulation. Total creatinine obtained before, 37-84 milligrams = 0-411 %, and 52-4 milligrams after = 0-327 %, i.e. a decrease of 20-4 %. Experiment VI.—Rabbit, weight 2,200 grams. 10 grams muscle removed before and 12-5 grams after 30 minutes’ stimulation. A free supply of pure oxygen was allowed during the period of anaesthesia. Total creatinine before stimulation 30-60 milligrams = 0-306 %, and 37-68 milligrams after = 0-301 %, i.e. a decrease of 1-6 %. Experiment IX.—Rabbit, weight 2,250 grams. 9-1 grams muscle removed before and 14-2 grams after 35 minutes’ stimu.ation. Total creatinine in muscle, before stimulation 39-04 milligrams = 0-429 %, and 51-42 milligrams after = 0-361 %, i.e. a decrease of 15-8 %. Experiment X.—Rabbit, weight 1,900 grams. 11-5 grams muscle removed before, and 14-8 grams after 35 minutes’ stimulation. Total creatinine in muscle, before 43-24 milligrams = 0-375 %, and 50 milligrams after = 0-337 %, i.e. a decrease of 10-1 %. oO As an example of two control experiments done, the following may be given, No. 7 with our maximum error, and No. 12 with our minimum. Experiment VII.—Rabbit. * 15 grams of muscle removed from right, and 13-1 grams from left thigh. Total creatinine, in muscle from right side 53-08 milligrams = 0-354 %, and from left side 48-0 milligrams = 0-366 %, i.e. a difference of 3-3 %. Experiment XII.—Rabbit. 8 grams of muscle removed from right thigh and 11 grams from left. Total creatinine, in muscle of right side 36-93 milligrams = 0-461 %, and from left side 50-31 milligrams = 0-457 %, ie. a difference of 0-86 %,. ey 424 BIO-CHEMICAL JOURNAL he. If Mellanby’s figures be examined, although he draws the conclusion. from them that work is without influence on the creatine content of muscle, it will be noted (/oc. cit. pp. 459-460) that his frog’s musele stimulated when isolated always show a slight increase in amount of total creatinine present, perhaps somewhat smaller than ours, and, in the two experiments on rabbits, which he did with their circulation intact, there — is in both cases a slight decrease. As already mentioned, van Hoogenhuyze and Verploegh have come to the conclusion, in which they are supported by other investigators, | that work does not increase the output of creatinine in the urine when the diet is sufficient. It was thought that the state of the nutrition of the animal, or, perhaps, variations in the carbohydrate reserve, might influence the total creatinine content of worked muscle. The experiments given above may be divided into two series, one in which the animals were kept on a very low cabbage diet for four or five days preceding the experiment, and the other in which the animals were allowed to feed freely on bran, turnip, and carrot for the same length of time. In every case there was a decrease in total creatinine, but the decrease was somewhat greta: in the badly fed than in the well fed animals. Taste II. Serres A. Badly fed Experiment Totalcreatinine Percent. present Decrease Mean decrease in milligrams in muscle per cent. " per cent. a) ee ee 5 {8 mk BARB ang FRE» here BAB 0 0 BE Promise 52-0 17-63 Serres B. Well fed cs) 4 {h 546 0-321 “3 ot SA et a(R a0 Hae 21-7 7-23 » b “ r “ 60 ‘UY L EFFECT OF WORK ON CREATINE CONTENT OF MUSCLE 425 Other experiments with frogs were carried out along these same lines, previous to the rabbit experiments. In these the frogs were given large quantities of glucose for two days before stimulation, but the results which we obtained by this method were not very concordant. Another point which appeared in the course of our work was the constant gain in fluid which occurred in the muscle as a result of stimulation. We found that, in the muscles of both frogs and rabbits, the average gain after thirty to forty minutes’ stimulation was about 2 per cent. We discovered subsequently that this figure was almost identical with that given by Ranke (21). In our experiments the fresh muscle was weighed as soon as possible after removal, then dried at 100° C. till constant weight was obtained. In every experiment, although our method is not calculated to elucidate the question, we examined the muscle extract for preformed(?) creatinine, but found that the amounts present varied very largely in quantity, the variation being also found in the control experiments. We are inclined to agree with Mellanby that muscle contains no preformed creatinine, or at most mere traces of this substance. The variation in the amounts found in our experiments were due in all probability to a partial conversion of creatine to creatinine during the process of extraction. There was, however, one interesting fact which may be mentioned in this connection, and that was that the percentage amount of preformed (?) creatinine present in the above experiments with rabbit muscle showed, with the exception of one experiment, a decrease when there was a decrease of total creatinine. The expenses of this research have been defrayed by a grant from the Carnegie Trust. 426: ee ee ee OS ee ae ee Pe , ae oo Se a ee ee wogl sah > a edt Oe ee ee “Weber, Arch. f. exp. Path... Pharm., Vol. LVIIL, p. 93, 1907. 123 tage ~ Liebig, Ann. d. Chem. u. Pharm., Vol. LXII, p. 257, 1847 (cit. in 2). sivas at. . Sezelkow, Centralbl. j. d. med. Wiss., p. 481, 1866 (cit. in 2). ~ Voit, Zeit: f. Biol., Vol. IV, p. 77, 1868. Gregor, Zeit.’ f. physiol. Chem., Vol. XXXI, p. 98, 1900.°° . . . » Hoffmann, Arch. f. path. Anat., Vol. XLVIIL p 908 lade Siete oii ee % Oddi and Tarulli, BoWl. dell. Acad. med. di Roma, Vol. XIX, 1893 (cit. Maly., Vol. _ Shaffer, Amer. Jour. of Physiol., Vol. XXII, p. 445, 1908. ’° BIO-CHEMICAL JOURNAL § 9 = REFERENCES van Hoogenhuyze and Verploegh, Zeitech. f. physiol. Ohem., Vol. XLVI, p. 416, 1905, Sarokow, Arch. f. path. Anat., Vol. XXVIII, p. 544, 1868. sits tig ee Monari, Arch. ital. de Biol., Vol. XIII, p. 1, 1890. Nawrocki, Centralbl. j. d. med. Wiss., p. 416, 1865 (cit. in 2). Mellanby, Jour. of Physiol., Vol. XXXVI, p. 446, 1908. Meissner, Zeit. f. rat. Med., XXXIV, p. 297, 1868. Groeco, Maly. Jahresbericht, XVI, p. 199, 1886. :) Moitessier, Compt. Rend. Soc. Biol., Vol. XLII, p. 573, _ (cit. Maly; aL vie - p. 182, 1891). thot vei p. 522, 1894). Cathcart, Kennaway and Leathes, Quart. Jour. of Medicine, Vol. 1,’p. 416, 1908 Graham Brown and Cathcart, Jour. of Phasiel. (Fyos. Phyotal. See.) Yo). XE YT iam Bonhaffer, Arch. f. d. ges. Physiol. Vol. XLVII, p. 125, 1890. | Paukul, Arch. {. Physiol., p. 100, 1904. Ranke, Tetanus, 1866, p. 69. 427 THE ACTION OF EXTRACTS OF THE PITUITARY BODY By H. H. DALE, M.A., M.D. From the Welleome Physiological Research Laboratories, Herne Hill, London, S.E. (Received October Ist, 1909) I. Iwrropvuctory Though the activity of pituitary extracts was discovered by Oliver and Schafer (1) almost simultaneously with that of suprarenal extracts, the conceptions of the nature of the action of the former are as yet far less precise. A comparison of the two was inevitable, and it has more than once heen suggested that their action, at least as regards vaso-constriction, is of the same kind and produced by stimulation of the same structures. Herring (2) advanced this view as regards the arteries: a more recent Observation by Cramer (3), of the action of pituitary extract on the pupil of the frog’s eye (enucleated), lends support to the same idea: still more recently an account given by Bell and Hick (4) of the action on the uterus emphasised the similarity between the action of extracts from the two organs. I thought it worth while, therefore, to bring together a number of observations, made at different times and in different connexions, which appear to me to indicate that such correspondence as exists is wholly superficial and illusory. In the first place it must be admitted that the actions of pituitary and suprarenal extracts have superficially several points of suggestive similarity. Both raise the blood-pressure, peripheral yaso-constriction being a principal factor in the effect (Oliver and Schiifer) : in both cases the active principle is limited to a small, morphologically independent portion of the gland, developmentally related to the central nervous system in the one case, as to the sympathetic system in the other. Attention is drawn to these points of similarity by Schafer and Herring (5), who state that ‘here the parallelism ends’: but the divergence of which they make specific mention is that the pituitary extract has an additional effect on the kidney. Since they attribute this to a separate active principle, no true divergence is indicated between the pressor principles of the two organs. It has been shown (Langley (6), Brodie and Dixon (7), Elliott (8) ) that the action of adrenaline reproduces with striking accuracy the effects of stimulating nerves of the true 428 BIO-CHEMICAL JONNTRNAL sympathetic or thoracico-lumbar division of the autonomic system. An examination of the action of pituitary extract on various organs and systems containing plain muscle and gland-cells will indieate whether its action has more than a superficial resemblance to that of adrenaline by showing whether its effects, or any group of them, can be similarly summarised by relating them to a particular element of the visceral nervous system. Incidentally evidence will be discussed which throws light on the contention of Schafer and Herring that two active principles exist in the extract, one acting on the circulatory system, the other specifically on the kidney. The extract used in my experiments, except where otherwise stated, was a 5 per cent. decoction of the fresh posterior lobes of ox pituitaries. The posterior lobes were dissected clean from the rest of the gland and from dura water, weighed in the moist condition, pounded with sand, and boiled with water faintly acidulated with acetic acid to produce coagulation. The exfract, filtered from coagulum, is a clear colourless fluid giving a faint biuret reaction. For experiments on isolated organs the extract was prepared with Ringer’s solution and carefully neutralised before use. Il. Tae Errect on THE Crrcv.atory System It has been mentioned that pituitary extract causes a striking rise of blood-pressure, chiefly due to arterial constriction. If the action had any relation to innervation by the sympathetic system we should expect to find that the effect on the arteries was accompanied by an increased frequency and force of the heart-beat, corresponding to the effect of the cardio-accelerator nerves. It was pointed out by Schafer and Oliver that this was not the case: the beat of the heart usually becomes slower, even after exclusion of vagus action, though it may be somewhat augmented. Reference will be made later to the action of the extract on the isolated heart, which enables the effect to be studied in its least complicated form. We should further expect to find, if the action were like that produced by sympathetic nerve-impulses, that the action on the arteries showed irregularities of distribution corresponding to that of sympathetic nerves. It was of special interest, therefore, to examine the action on those arteries which have been shown to be exceptional in their innervation and in their reaction to adrenaline. The pulmonary arteries. Brodie and Dixon showed that the peripheral branches of the pulmonary artery are exceptional in that their THE ACTION OF EXTRACTS OF THE PITUITARY BODY 429 muscular coats are not under the control of sympathetic nerves, and made the interesting parallel observation that adrenaline, perfused through the pulmonary vessels, produces no vaso-constrictor but a small vaso-dilator effect. With segments of the main branches of the pulmonary artery, treated as isolated organs, others have obtained definite constrictor effects with adrenaline (Meyer (9), Langendorff (10) ). It is clear that there is no real discrepancy between the two sets of observations: the only conclusion justified by the evidence is that the sympathetic nerves send motor fibres to the muscular walls of the pulmonary artery and its main branches, but that the innervation stops short of the peripheral arterioles, the calibre of which is alone concerned in determining the rate of ' perfusion under constant pressure, as measured by Brodie and Dixon. In a few experiments with isolated rings of large branches of the pulmonary arteries of large dogs and goats, I observed contraction on adding small quantities of the pituitary extract to the Ringer’s solution in which the rings were suspended. Since these experiments were made similar observations have been published by de Bonis and Susanna (11). Since, however, I obtained even more pronounced constriction of the strips of pulmonary artery on adding adrenaline, these results only add another to the cases already known in which adrenaline and pituitary extract both cause constriction of an artery, and are of no significance for our present enquiry. I owe to Professor Dixon the opportunity of making with him observations on the effect of pituitary extract on the peripheral pulmonary arterioles. The observations were made in connéction with experiments concerning action on these arterioles of certain organic bases. The lungs were perfused with Ringer's solution, or defibrinated blood diluted therewith, according to the method described by Brodie and Dixon. After it had been shown that either adrenaline or p. hydroxyphenyl-ethy- lamine caused only a slight acceleration of the rate of perfusion, 1 ¢.c. of the pituitary extract was introduced into the circulating fluid. As soon as the extract reached the lungs there was a pronounced retardation of the outflow. The observation was repeated several times, in different experiments, with uniform result. Here, then, is a clear case of vaso- constriction produced by pituitary extract on a system in which no such constriction is produced by adrenaline or substances of similar action. The coronary arteries. ‘The innervation of the coronary arteries ‘cannot be regarded yet as definitely settled, even the more recent observations being by no means concordant. Maas (12) found that the vagus supplies vaso-constrictor fibres to this system: Dogiel and 430 BIO-CHEMICAL JOURNAL Archangelsky (13) found that vaso-constrictor fibres are contained in the accelerator nerves: on the other hand Schafer (14) could not find any evidence for vaso-motor nerves to these arteries, and observed no constriction of them under the influence of adrenaline. The last observation was confirmed by Elliott (8), who found the outflow from a perfused segment of ventricle increased by adrenaline. Langendorff observed that adrenaline caused relaxation of an isolated ring of coronary artery, and this has been confirmed by de Bonis and Susanna. Still more recently Wiggers (15) has found evidence of vaso-constriction when adrenaline is added to a fluid perfusing the coronary arteries. From all this conflicting evidence emerge the facts that the coronary arteries are slightly, if at all, controlled by vaso-motor nerves, and that the constrictor effect of adrenaline on the peripheral branches, if it exist at all, is very weak compared with the effect of that principle on other arteries. In this instance | made no experiments with isolated rings of artery, but such have recently been published by Pal (16) and by de Bonis and Susanna. ‘These observers agree in finding that pituitary extract causes a marked constriction of a ring cut from a large coronary artery. De Bonis and Susanna also confirmed Langendorff’s observation that adrenaline causes relaxation of such a ring, so that in this case the action of the two principles is again contrasted. My own experiments were made with the isolated heart of the rabbit, perfused with oxygenated Locke-Ringer solution, by Langendorff’s method as modified by Locke. There are several errors involved in the measurement of the coronary outflow from such a preparation. These have recently been discussed by Wiggers. The outflowing Ringer’s fluid always accumulates to a certain extent in the right auricle and ventricle, and, as Schafer pointed out, a certain amount may pass the semi-lunar valves and so reach the left ventricle. With small hearts I have not found that these defects seriously disturb the average rate of outflow: the principal drawback is that the dripping of the fluid from the heart is rendered irregular by the accumulation of fluid in the right side of the heart during diastole, and its ejection by the systole. With a small, rapidly-beating heart the quick and irregular succession of small drops which results can be averaged and converted into a regular series of large drops by a simple device. I used a large glass funnel, placed immediately beneath the recording lever. A skein of threads, hanging loosely from the heart and lever into the mouth of the funnel, ensured the delivery = eget - _- THE ACTION OF EXTRACTS OF THE PITUITARY BODY 431 into it of all the fluid leaving the heart, without at all interfering with the record of the contractions. The funnel was fixed in an inclined position and over the lower opening of the stem was drawn a short length of rubber tubing, the diameter of which could be reduced by a clip. This device converts an irregular series of drips and splashes into a regular series of large drops, which fall at a constant rate so long as the average rate of the drippings from the heart remains constant. These large drops were recorded on the smoked drum by the ordinary arrangement of receiving and recording tambours. When the beat of the heart and the rate of the coronary outflow, as shown by the drop recorder, had become constant, a small quantity of the filtered and warmed pituitary extract was introduced into the bulb of the heart-cannula by means of a hypodermic syringe, the needle being thrust through the wall of the rubber tube leading to the cannula. Fig. 1 shows a typical effect. It will be seen that the outflow from the coronary sinus becomes very much slower as soon as the extract reaches the heart. The effect shown in the figure is quite typical, and I know of no other drug which, in doses not immediately fatal to the heart-muscle itself, will produce so pronounced a constriction of the coronary arteries. That the effect is genuinely due to constriction, and not to viscosity or mechanical accident, can easily be ascertained from the fact that a second dose, introduced when the effect of the first has subsided, produces a very small change in the rate of outflow. This is quite in accordance with the observation, first made by Howell (25), that a second dose of the extract, given intravenously when the effect of a first large dose has passed off, produces hardly any rise of arterial blood-pressure.' One other point needs mention. It is clear from what has been said above that a weakening or stoppage of systole might lead to an apparent temporary retardation of the coronary outflow by allowing accumulation in the right side of the heart. The phenomenon illustrated is not of that kind. It is a prolonged effect, which persists to some degree for upwards of half an hour after the injection, and its maximum coincides with a phase of increased ventricular activity. There is no room for doubt, therefore, that the coronary arterioles afford another example of an arterial area slightly, if at all affected by adrenaline, stimulated to intense constriction by pituitary extract. The effect on the ventricular beat of the isolated heart 1. It is of interest to note that Dr. W. H. Harvey, to whom I communicated my observa. tion of the constricting effect of pituitary extract on the eoromary arterivs, has produced sclerotic changes in these arteries by repeated injections of the extract. JOURNAL BIO-CHEMICAL 182 ‘rnoul § eTtog ‘uOIgNyos JaBaryy-axoory Dursnyzod ay3 04 4owsjxo Arvjzingid jo suru g Zurppe jo yooyy “qq @ JO J4¥OY PazV[Os! Oy JO SfossoA AzeUOIOD qZnNoIy. MOY PUY yveq IRNOATOA—"| WN urMyAqipunyul Jo yowrpxe jo surrurur ¢ ——y-97 —ty L — fp r—nmwg MOU Prtrrrey rrerrteerererrrrrrrrtetrrrtrtr ter maf Bene) LIVMOIOL ADP LAP DAD ROA RADAR DD RAD DA DAA nnn nnnns a q¥eq qne} , ee ae roy —— sé oT —— ‘me be Sle SS = eo * THE ACTION OF EXTRACTS OF THE PITUITARY BODY 488 can also be studied in fig. 1. It will be seen that, immediately after the injection, it becomes slightly slower and considerably more vigorous: later, with persistent retardation, it becomes weaker than before the injection. Similar effects, in the same order, have been previously described by Hedbom (17) and by Cleghorn ( 18). It is difficult, however, to decide how far these changes in ventricular activity are due to primary action on the cardiac muscle, how far to reduction of the oxygen supply by coronary constriction. Neither effect is modified by previous atropinisation, so that there can be no question of the peripheral vagus-mechanism being concerned. There is further, in the ease of the effect on the heart-beat, as in that of the coronary constriction, no resemblance whatever to the effect of accelerator nerves or of adrenaline. The safest conclusion is to regard the action on the coronary arteries as certainly a primary effect of the extract, that on the heart-beat as probably in part due to direct effect on the heart-muscle, and in part secondary to the altered rate of coronary perfusion. It should be noted, in this connection, that under conditions of natural circulation, in which the effect of coronary constriction would be antagonised by the great rise of systemic pressure, the secondary weakening of the beat is not usually observed. . The renal arteries. Schafer with Magnus (19), and later with Herring (5), found that the kidney expanded when pituitary extract was injected intravenously. It was of interest, therefore, to examine the effect of pituitary extract on the rate of perfusion through the renal vessels. The perfusion was made with oxygenated Ringer’s solution under constant pressure, as for the isolated heart, the outflow from the renal veins being measured by the drop-counter. The kidneys used were those of cats and dogs. Both kidneys of the cat were perfused, the cannulae being inserted into segments of aorta and vena cava. From the dog one kidney was used, with cannulae in the renal artery and vein. The pituitary extract was added by injection into the circulating Ringer’s fluid. The following results were obtained :— Rate or Ovtriow ty Drops rer Ingection or Prrurrany Extnacr 20 seconps Before injection After injection Bxperiment 1.—Cat. 5 minims 34 20 Experiment i.—Cat. ist. 5 minims ay 27 2nd. 10 minims 20 31 Kaperiment [11.——Dog. Lat. 5 minims a4 20 2nd. 10 minima 20 22 434 BIO-CHEMICAL JOURNAL It will be seen that the first injection causes in each case a decided though small constriction. The genuineness of the phenomenon is again shown by the failure of second injections, which even slightly reduce the resistance of the constricted arteries. Similar results were obtained by Houghton and Merrill (24), in the course of experiments made to determine whether the extract locally excites the renal epithelium to secretion. On the other hand Pal states that isolated rings of the proximal portion of the renal artery were constricted, while rings from more peripheral portions were relaxed by the extract. On the whole the evidence obtained with isolated organs suggests that the marked swelling of the kidney in its natural relations must be chiefly due to a relative insensitiveness of the renal arteries towards the vaso-constrictor effect of the extract. It might seem, at first sight, that even this implied, as Pal concludes, an action of the vaso-constrictor principle on some nervous structure, and not on the muscular coats of the arteries themselves. This, however, is by no means the only instance of an exceptional reaction of the renal arteries towards general stimulants of plain-muscle contraction. The various drugs of the digitalis series, for example, injected in small doses, cause expansion of the kidney and diuresis, especially in the rabbit; but the result of most experiments on the artificial perfusion of these drugs through the vessels of the excised kidney, especially of the dog and the cat, has been to demonstrate a marked constrictor action even on the vessels of that organ. There is no reason at all for supposing that these drugs act on nervous structures, and there is as little in the case of the pituitary extract. The anomalous reaction of the kidney vessels in their natural relations is clearly a similar phenomenon to their reaction to the digitalis series; but since the pituitary extract acts more powerfully on the arterioles and less on the heart than digitalis and its allies, the phenomenon is presented by the former in an exaggerated form. The Spleen. The spleen may be regarded, in so far as its contractile activity is concerned, as belonging to the circulatory system. Schafer and Magnus showed that pituitary extract caused contraction of the muscular capsule. I have repeated this observation with a like result. A plethyomographic record of the effect is shown in fig. 2. Oe ee THE ACTION OF EXTRACTS OF THE PITUITARY BODY oer of Rin O0 cc. + a 4 a al on | 7 =< al J + Lepiririri Liprirairr PMacn a ra Assen td ay be ¢ 436 BIO-CHEMICAL JOURNAL Ill. Tue Uterus In a paper on another subject (22) I mentioned incidentally the powerful uterine contraction produced by pituitary extract. I have since extended the observation, finding, as expected, that the action, like that on the arteries, is possessed by extracts of the posterior lobe only. Bell and Hick, working with the extract which I myself used, appear to have obtained a comparatively small effect on the rabbit's uterus in the resting (i.e., non-pregnant and non-oestrous) condition. This is quite contrary to my own experience. They worked exclusively with the rabbit. This animal is not really suitable, however, for our present enquiry, since its uterus responds, under all conditions, to the stimulus of sympathetic nerves or adrenaline, by contraction. In the cat, on the other hand, as was shown independently and almost simultaneously by Cushny (20), by Kehrer (21), and by myself (22), the uterine tone and contractions are inhibited in the non-pregnant, stimulated in the pregnant animal, by sympathetic nerves or supra-renal preparations. I regard it, then, as of great significance that in the uterus of the cat, as well as in that of the dog, the guinea-pig, the rat, and the rabbit, I have always observed, in all functional conditions, powerful tonic contraction as the effect of applying pituitary extract. The results were obtained by intravenous injection into the anaesthetised or brainless animal, and also by Kehrer’s method of adding the extract to a bath of warm oxygenated Ringer’s solution, in which the isolated horn of the uterus was s0 suspended as to pull on a recording lever. The effect, under these conditions of adding a few drops of pituitary extract to the 200 c.c. of Ringer’s solution in the bath, is illustrated in figs. 3 and 4. So little, in my experience, is the effect dependent on the condition of the uterus as regards oestrum or pregnancy, that the uterus of a virgin, half-grown cat responded to the pituitary extract by as marked a tonic contraction as was given by any of the numerous pregnant or multiparous organs examined. The effect of pituitary extract on the uterus, then, shows again the absence of parallelism to the effects of sympathetic nerves, the effect of the extract being always tonic contraction, even when stimulation of the hypogastric nerves produces pure inhibition of tone and rhythm. THE ACTION OF EXTRACTS OF THE PITUITARY BODY 487 . co r nod | x. of _ bath t th I ; t pituitary xtra lated retractor peni I t of adding 0-5 « Scale, 4 linear Eff 4 linea of a pregnant uterus o the bath. Scale, . from the ided of o that 488 BIO-CHEMICAL JOURNAL IV. Ovner Orcans Contarxinc Praty Musee The intestines and the urinary bladder give no such marked response to the pituitary extract as the organs hitherto mentioned. In a dog anaesthetised with A.C.E. mixture I observed, indeed, a distinct inhibition of intestinal movements when the extract was given intravenously, even when the splanchnic nerves were cut. This might be regarded as indicating a similarity of action to sympathetic nerves. An isolated loop of intestine, however, the rhythm and tone of which are immediately inhibited by adrenaline, contracts, though but feebly, when pituitary extract is added to the bath. It is probable, therefore, that the inhibition, seen under normal conditions of circulation, is due to the intense anaemia which the vaso-constrictor action of the extract produces. The bladder of the cat, when the extract is injected intravenously, usually exhibits a temporary weakening, followed by more prolonged increase of tone. Neither is of any great extent. A guinea-pig’s bladder, suspended in the Ringer-bath, contracted feebly when pituitary extract was added. The plain muscular coats of the intestines and the bladder contract, then, like other plain muscle, in response to pituitary extract, but their sensitiveness thereto is small in comparison to that of some organs. The retractor penis of the dog, a convenient sheet of plain muscle for examination in the Ringer bath, contracts, as might be expected, when the extract is added (fig. 5). No effect could be detected on pilo-motor muscles or on the mammalian pupil. ‘VY. Guanp CELLS Schafer and Herring found that the extract caused secretion neither of saliva nor pancreatic juice, which observations I have confirmed. In its failure to evoke salivary secretion the extract is again contrasted to adrenaline. The profuse flow of urine which the extract causes, as first shown by Schafer, in conjunction with Magnus (13) and with Herring (3), can hardly be regarded as a true glandular secretion. VI. Tue Action arrer Ercoroxtne I have shown (23) that the specific ergot alkaloid ergotoxine, when injected intravenously in certain doses, annuls all motor effects of sympathetic nerves and adrenaline, so that the latter produces, in the cat, q q 7 aa \a THE ACTION OF EXTRACTS OF THE PITUITARY BODY 439 a fall of blood-pressure and relaxation of the pregnant uterus in place of the customary rise and contraction. Ergotoxine may be given, however, in any quantity without affecting the contraction of arterial and uterine muscle produced by a subsequent injection of pituitary extract (fig. 6). AcTION OF ENZYMES, ETC., ON THE EXTRACT Schafer and Herring (5) state that peptic digestion reduces the action of the extract on the blood-pressure without affecting the action on the kidney, but that neither action is affected by tryptic digestion. They also obtained results which they regarded as indicating that oxidation by H,O, destroys the pressor action more quickly than the diuretic action. Certain obvious precautions seem to have been omitted: there is no _ indication that they controlled the activity of their enzymes or the response of their animal. A negative result should obviously not be accepted as indicating destruction of the agent unless a positive effect could subsequently be obtained with the untreated extract. Adopting these precautions I have failed to confirm them on all points. Digestion for twenty-four hours with a peptic extract of proved activity and 0:2 per cent. HCl failed to alter in any perceptible degree the pressor or diuretic action of my extract. I can only conclude that the peptic extract used by Schafer and Herring contained some antagonistic depressor substance, or that their animal was for some reason unresponsive to the pressor effect. On the other hand every active preparation of trypsin which I have tried has reduced the action on the blood-pressure and on the urinary flow practically to ni after a few hours’ digestion. Commercial trypsin, ‘liquor pancreaticus,’ pure pancreatic juice obtained by secretin and activated by enterokinase—all gave the same result. In all cases a subsequent injection of the original extract produced the usual rise of blood-pressure and acceleration of the flow of urine (figs. 7 and 8). It may be suggested, in the absence of evidence for control on that point, that Schafer and Herring were using an inactive preparation of trypsin: at least it is clear that the tryptic preparations used by me contained something which was not present in theirs. In my experience oxidation with H,O, failed likewise to discriminate between the pressor and diuretic activities. Both effects ‘ were smaller after oxidation than those produced by a subsequent injection of the original extract; but that either had suffered greater change than the other was not apparent. youryxe Areqingid ‘o-o J—gq 4 ‘qUI[wUOIpe "WSU [-O—V¥ IV :suornoafuy = -Aysnotaoad pojoofur ayeydsoyd ourxojoF10 ‘swisut ¢ -ye0 payyid yo ormssaid-poojq pyoiwg—"g oandiy CUTIE ERTT Cr TEBE Tri tae el Lee ae) U.). ba tod al ad a ROPER Cg abe q FOURNAL ‘AL _— — - = _ —_ — : — ~~ _— ACTION OF EXTRACTS OF THE PITUITARY BODY 441 it injection en hours ol trypsin yp Intravenou ure of pithed ca und ‘ame tu tid blood-pres trypsin d for the ‘ ted with t digests — _ _ a | ~ ~ pm | _ —_ _ = _ — _ ~ -_ —_= ~ ~~ _ _ _ _ ~ =~ _ _ | —_— _ _ _ _~ _ _ —_ i —_— _ — _ ~« ~ _ _ _ _ _ _" _ = _ _ ~~ — _ _ a" _ _ ~ -- _ ~ _ _ _ _ ~ _~ —~ _~ JOURNAL BIO-CHEMICAL *xeoul, § ‘opeos ‘uisd £23 po[log YILA pozyEqnoul 4owIZXO *o°O [—<{{ FV uisd £13 YBLM poyseDIp Jovl}XO *0°O [—V IV 12 ‘Big urse suonooluy *(410q}9) ywo yo (eynuuTO Jaeppr]q) outim yo prooei-doap puv aInssoid-pooyq pryomvwg—'sg WNT * 2939) vrs Gk Os On Ses Sea We Se We fee Ole ea oe as Oe Weave. a mt Oe nee ee TR OO qd Vv u Tee ER NY ten, ee be tt th ’ — f——+ ; aan fo dovy ‘ | ¥"79 ryerr) 4 ul THE ACTION OF EXTRACTS OF THE PITUITARY BODY 448 Excretion. Arremper to Propuce Iuuunitry The fact, discovered by Howell, that second doses are relatively ineffective, suggests that the active principle is not readily destroyed or rendered inactive in the body. I found that the urine of a cat, excreted in response to an injection of the extract, had a pressor action, like a dilution of the extract, when tested on another cat (fig. 9). Probably the active principle, therefore, is at least to some extent excreted unchanged. The refractory state to further injections has nothing to do with a true ‘immune’ reaction. In the serum of a rabbit, treated for a month with increasing injections of the extract, I could distinguish no trace of a body neutralising the physiological activities of the extract. . Discussion OF THE RESULTS It is clear from the foregoing that the characteristic action of extracts of the posterior lobe of the pituitary body is stimulation of plain muscle fibres. Different organs containing plain muscle show a varying sensitiveness of response to the extract, the arteries, the uterus and the spleen being conspicuously affected. This unequal distribution of effect cannot, however, in any way be related to inequalities of innervation by nerves of the true sympathetic or of the autonomic system as a whole. Ergotoxine, which excludes motor effects of true sympathetic nerves, and of drugs acting through those nerves or like them, leaves the action of pituitary extract intact. Neither atropine nor curare affects its direct action in any degree. The muscle of the mammalian heart is possibly affected to some extent by the extract, apart from effects secondary to constriction of the coronary arterioles: Herring’s observations on the frog’s heart render this most probable. No effect could be detected on the response of voluntary muscles, either to direct or indirect stimulation. The active principle is then essentially a stimulant of involuntary, and especially of plain muscle. The question of the diuretic effect needs some further discussion. Houghton and Merrill (24) have recently taken the somewhat extreme view that this is entirely secondary to the rise of blood-pressure. They state that the rise of blood-pressure produced by adrenaline is accompanied by a similar diuresis, This latter observation is directly ‘opposed to the experience of others, and I have never myself been able to confirm it. Further it was shown quite clearly by Schafer and Herring that a second injection of pituitary extract may cause distinct ‘(sanoy Z Buump pozooyjoa je ut ‘oo Og) “yousyxo Axeqymyid oo F JO WOWoefur 19zze pozOOTJOO OULAN Jo ‘o°0 S—'T] I¥ ‘QULIN 8,489 JVULIOU JO *9°0 S—"T IV : suotoefur snousavijUy =«“44y8O peyszid so oinsseid-poojq pyorwp—'6 ound JOURNAL Ay nade ¥**949 ae ry LA) _ a“ — — ss - — _ ~_ " — ~ _ Pd THE ACTION OF EXTRACTS OF THE PITUITARY BODY 445 diuresis without any perceptible rise of blood-pressure. While such an observation, which I have been able repeatedly to confirm, sufficiently disproves the statement that the diuresis is secondary to and runs parallel to the actual rise of systemic pressure, it does not remove the possibility of the dependence of the diuresis on vascular effects. A redistribution of the blood in the system, caused by the comparative irresponsiveness of the renal arterioles, is conceivable without actual rise of general systemic pressure, especially if the arterial constriction is accompanied by weakening of the heart’s action, due to the depressor constituent which the extract always contains, the action of which, moreover, is much more evident in the case of a second injection. The differential action of enzymes and oxidation on the supposed : pressor and diuretic principles, alleged by Schafer and Herring, has not _ been confirmed in my experiments. On the contrary I have found that whatever destroyed one action destroyed both. Their other evidence for the existence of two principles seems to me also inadequate. They lay stress on the difference in the time relations between the two effects and the relatively greater effect of second injections on diuresis. The difference in time-relations of a diuretic and pressor effect is, however, a familiar phenomenon in cases where there can be no question of the presence of more than one active principle. If strophanthin, for example, be injected intravenously into a dog or cat, the immediate effect on the diuresis is usually a distinct retardation: later, as the rise of arterial blood-pressure passes off, there is generally a secondary acceleration which often persists after the blood-pressure has regained its original level. A similar sequence of events was recently observed by P. P. Laidlaw and myself in experiments, in course of publication, on the action of a pure, erystalline active principle from Apocynum. Such a difference in time-relations cannot, therefore, be accepted as necessitating the presence of two principles. The relatively greater efficacy of a second injection in causing diuresis as compared with its pressor effect can also be interpreted in another way, as indicated above. The blood-pressure tracing is complicated by the presence of the heart-depressing principle : it is not a fair index of the degree of vaso-constriction in this instance. An apparently greater relative efficacy of second injections can also be observed in the case of the uterus, when the effect on that organ is ‘compared with that on the arterial pressure. I have frequently seen, as the result of a second injection, marked contraction of the uterus accompanying a very slight or no rise of blood-pressure. 1 tam il ai all | alll 2 ae oat oe eae ae ee a See, Be ‘ 7 sila ' Khe 446 BIO-CHEMICAL JOURNAL | It does not seem justifiable, however, to draw from this catia the — conclusion that the principle acting on the plain muscle of the te rus is different from that which acts on the plain muscle of the arteries. It is, — of course, true that nothing short of the isolation of a single pr principle, producing both pressor and diuretic effects, would 1 ke the view that two principles exist untenable. While Tae fu i evidence, however, the conception of both effects as due to one one | : seems to me adequate and simpler. CoNncLUSIONS 1. The action of extracts of the posterior lobe of ne is a direct stimulation of involuntary muscle, without any innervation. The action is most nearly allied to that of the ¢ series, but the effect on the heart is in this case bl that muscle intense. ig 2. The active principle is excreted in the urine. 3. No true immune reaction is produced by — the extrect. 4. The evidence advanced in proof of the exiatinee of pressor and diuretic principles is inadequate. . SSRPFSERE Serene apeywe . -—- « Re ae hw ee ee ee —_— vr eS eo a ee Sie a : . _ THE ACTION OF EXTRACTS OF THE PITUITARY BODY REFERENCES Oliver and Schiifer, Journ. of Physiol., XVIII, p. 277, 1895. Herring, Journ. of Physiol., XX XI, p. 429, 1904. Cramer, Quart. Journ. of Exper. Physiol., 1, p. 189, 1908. Bell and Hick, B.M.J., 1909 (1), p. 777. Schafer and Herring, Phil. Trans., 1906. Langley, Journal of Physiol., XXVIT, p. 237, 1901. Brodie and Dixon, Jbid., XXX, p. 476, 1904. Elliott, Jbid., XXXII, p. 401, 1905. age mags f. Biol., XLVIIT, 1906. vendorff, Zentralbl. f. Physiol., XX, _p. 551, 1907. s and Susanna, Zentralbl. j. Physiol.. X XI, p. 169, 1909. Pfliiger’s Arch., LXXIV, p. 281, 1899. el and Archangelsky, ibid., CXVL., p. 482, 1906. x, Arch. de Sci. biol. de St. Petersbourg (Pawlow Festschrift), p. 251, 1904. rs, Amer. Journ. of Physiol., XX1V, p. 391, 1909. n, Skand. Arch. {. Physiol., VUIL., 1898. n, Amer. Journ. of Physiol., U1, p. 273, 1899. and Magnus, Journ. of Physiol., XXVIL, p. ix (Proc. Phys. Soe.). Barger and Dale, Bio-Chem. Journal, U1, p. 240, 1907. Houghton and Merrill, Journ. of the Amer. Med. Assoc., LI, p. 1849, 1908. Howell, Journ. of Exper. Med., Lil, p. 2, 1898. 447 ¢ 448 A METHOD FOR THE ESTIMATION OF THE UREA, ALLANTOIN, AND AMINO ACIDS IN THE URINE © & By DOROTHY E. LINDSAY, B.Sc., Cannzom Scuoran. \ Sz Communicated by Prof. D. Néel Paton » From the Physiological Laboratory, University of Glasgow 3 aay (Received October 2nd, 1909) In investigations involving the determination of the distribution of nitrogen in the urine, the separation of the various nitrogenous constituents is a matter of no little difficulty, and involves so much time that it is almost impossible to carry through any prolonged series of observations. The object of the present investigation is to determine how far the distribution of nitrogen can be more rapidly determined indirectly by taking advantage of the differences in solubility and stability to reagents of the various substances. The chief substances in which nitrogen occurs in the urine are: — Urea, purin bodies including uric acid, creatinin (with or without creatin), ammonia, allantoin, amino acids (including hippuric acid), sulphur- containing bodies and bodies of unknown composition. a The present investigation is confined to the determination of urea, ial allantoin and amino acid nitrogen. For the determination of Urea Bohland (1) recommended the precipitation of the other nitrogen-containing substances by phosphotungstic and hydrochloric acids. The urea was then estimated in the filtrate. He claimed that the results got by this method gave only — E as urea nitrogen and ammonia nitrogen, all other nitrogen-containing bodies __ et being precipitated. sg Later Schéndorff (2) showed that the amino acids (glycocoll, leucin, = &c.) are not precipitated by phosphotungstic and hydrochloric acids. He also showed that while creatinin is precipitated, as Bohland had already found, creatin is not. Allantoin also is nof precipitated; a statement later confirmed by Mérner (3). Pfaundler (4) experimented with various samples of phosphotungstic | acid and found that, if Merck’s preparation is used, ammonia is is Ce | precipitated. ig _ ee be ae lt 7 i eas 7 ESTIMATION OF UREA, ETC., IN THE URINE 449 The nitrogen obtained by this method therefore includes urea, amino acids (with hippuric acid), creatin and allantoin nitrogen. Morner and Sjéqvist (5) recommended a method for the estimation of urea in which the urine is precipitated with a saturated solution of barium chloride in which five per cent. of barium hydrate is dissolved, and an aleohol ether mixture containing two parts aleohol to one of ether. After twenty-four hours it is filtered, the filtrate evaporated at 50° to small bulk, after the addition of a pinch of magnesium oxide to drive off the ammonia, and the nitrogen in it estimated by Kjeldahl’s method. Creatinin, hippuric acid, and also some amino acids (leucin, bile acids) are not precipitated, and their nitrogen is thus included in the amount obtained by this method. Folin (6) proposed a method of heating the urine with crystallised aa. ium chloride and hydrochloric acid. At the temperature thus __ employed—150° C.—urea and allantoin alone are decomposed to give off ammonia. Thus the nitrogen obtained by this method includes allantoin nitrogen in additionsto*urea nitrogen and ammonia nitrogen. ______- Mérner, in a moré recent paper (7), develops what may be called the __-Mérner-Folin method by which urea alone yields its nitrogen. The bulk a of the nitrogen-containing substances are precipitated as described above, ____ with barium chloride, barium hydrate and alcohol ether solution, but the evaporated filtrate is heated with magnesium chloride and hydrochloric acid as in Folin’s method. Allantoin is by this procedure almost entirely removed before heating with magnesium chloride, thus avoiding Folin’s _. error. The creatinin, &., which are not precipitated in the Mérner _ method are not decomposed in the Folin method. Thus the Mérner-Folin ___ method gives only the nitrogen which is contained as urea. ee ‘In the present investigation a slight modification of this method _ was adopted, a modification first employed by Underhill and Kleiner (8), viz., the use of an alcohol ether solution containing equal parts of __ aleohol and ether, since as Haskins (9) had previously observed, oa allantoin is more insoluble in such a solution than in the one employed iby Mérner. In addition a few drops of hydrochloric acid were at once _ added before the evaporation of the alcohol ether filtrate, thus preventing a the escape of ammonia, the nitrogen in which is thus included in the results. This addition of acid was also recommended by Folin, instead of i driving off the ammonia by means of magnesium oxide as practised by 1 Méruer, 450 BIO-CHEMICAL JOURNAL The method in detail as employed by me is, 5c.c. of the urine are mixed with 5c.c. barium chloride, barium hydrate solution, and 100 ¢.c. alcohol-ether (50 c.c. absolute alcohol, 50 ¢.c. ether). After twenty-four hours it is filtered, an aliquot portion of the filtrate is taken, and a few drops of hydrochloric acid immediately added. This is then evaporated almost to dryness at a temperature of 50°C. in an apparatus described by Haskin; 20 grs. magnesium chloride are then added and a small piece of paraffin wax to prevent frothing, and the whole is heated for three hours on an electric heater described by Catheart (10). The direct determination of Allantoin has always proved difficult and unsatisfactory. The similarity between its properties and those of urea renders complete separation extremely difficult. Poduschka’s method (11), in which allantoin is precipitated by silver nitrate in alkaline solution, is comparatively simple, though somewhat lengthy, but, as Salkowski says, the precipitation of the silver compound with ammonia is one of the most difficult of chemical procedures. Wiechowski (12) proposed using mercuric acetate, which precipitates allantoin but not urea, instead of the usual mercuric nitrate, by which urea too is precipitated. Allantoin when thus precipitated shows a strong tendency to crystallise. The details of the method are, however, long and troublesome, though giving accurate results, and it is impracticable for a long series of daily observations. For the estimation of the Amino acids various methods have been suggested. Fischer and Bergell (13) proposed a direct method which depends on the power of napthol-sulphonie acid to combine with amino acids. The urine was shaken with an ethereal solution of 8 napthalene sulpho chloride and the amino acid compound subsequently precipitated with hydrochloric acid. This method was later modified by other workers. Glaessner (14), in criticising the method, states that he, as others also, © did not recover on an average more than 60 per cent. of the amino acids. Neuberg and Manasse’s direct method (15) depends on the fact that — on shaking a urine with a strongly alkaline solution of a-napthol isocyanate, hydantoic acid is formed, which is precipitated after acidification. Glaessner found this method not at all constant. Some good results were obtained, but in general they were quite unreliable. Pfaundler (16) endeavoured to devise an indirect method for the estimation of amino acids. His method was to precipitate the urine first with phosphotungstic acid, The precipitate and filtrate were then each . = ESTIMATION OF UREA, ETC., IN THE URINE 451 heated with phosphoric acid. The urine nitrogen was thus divided into four fractions. I. Substances precipitated by phosphotungstic acid. (a) Nitrogen easily removed by heating with phosphoric acid. (b) Nitrogen not thus easily liberated. II. Substances not precipitated by phosphotungstie acid. (a) Nitrogen easily removed by heating with phosphoric acid. (6) Nitrogen not thus easily liberated. Of these four fractions, II (a) consists mainly, if not- entirely, of amino acids. Kruger and Schmid’s method (17), also an indirect one, was based on _ the fact that amino acids do not give off ammonia when heated with concentrated sulphuric acid at 160° to 180°C. They estimated the _ nitrogen in the filtrate from phosphotungstic acid and also the nitrogen _ which was obtained after heating with half the volume of concentrated } sulphuric acid. The difference between these two should correspond to the amino acid nitrogen. e, Glaessner (18) proposed a somewhat similar method for the estimation of amino acids. He precipitated with phosphotungstic acid and _ evaporated the filtrate to dryness at a low temperature. The residue was freed from water and then extracted with alcohol-amyl aleohol for six hours. It was then filtered and a Kjeldahl nitrogen estimation done on the residue. This gave the amino acid nitrogen. The difficulty of this method is to ensure that the urea is completely dissolved. Present Meruops _____ It appeared possible that the combination of certain of these methods _ —-Bohland, Folin, Mérner-Folin—might yield definite results as to the distribution of nitrogen, and the method which I employ is as follows :—- Kstimations of the nitrogen present are made by these three methods, and by the differences between them the nitrogen in urea, allantoin and amino acids is determined. (a2) Bohland nitrogen, using Merck’s phosphotungstic acid, which leaves unprecipitated amino acids, hippuric acid, creatin, allantoin and urea. '(6) Folin nitrogen, which includes urea, allantoin and ammonia nitrogen. 452 BIO-CHEMICAL JOURNAL (ce) Mérner-Folin nitrogen, which includes urea and ammonia . os] nitrogen. The ammonia nitrogen was separately estimated by Folin’ 8 method and subtracted from (6) and (ce). i ~The difference then between the nitrogen of (a) and (6) ideal give the amount of amino acid nitrogen and creatin nitrogen present, hippuric acid being included as an amino acid. The creatin can be determined separately by Folin’s method. The difference between the nitrogen of (4) and (c) should hee the amount of allantoin nitrogen present. To verify the method estimations were made on solutions ovata 3 varying amounts of urea, allantoin and amino acids. The nitrogen in each solution was determined by Kjeldahl’s method. I. A solution was made consisting of 25 c.c. of a 2 per cent. solution of urea, 25c.c. of a 0°5 per cent. solution of allantoin, and 10c.c. of a (5 per cent. solution of alanin. 5c.c. of this solution contained 00016 | grs. nitrogen as allantoin and 0°0022 grs. nitrogen as alanin. | Bohland nitrogen required 9 c.c. ws acid = 0-0126 grs. nitrogen Folin d » Ih 0.0. o = 060-0104 » Mérner-Folin __,, » S5Occ. ,, = 00088 ,, B. — F. = 0-0022 grs. nitrogen = 100 p.c. alanin nitrogen * F -M-F.= 00016 _,, = 100pe.allanton , ¢ II. Solution consisted of 25c.c. of a 05 per cent. solution of © allantoin, 25¢.c. of a 1 per cent. solution of glycocoll, 25 c.c. of a 2 per cent. solution of urea, and contained 0°042 gre. nitrogen as allantoin and 0044 grs. nitrogen as glycocoll. Folin nitrogen required 13-1 ¢.c. jy acid = = 0-275 grs. nitrogen Mérner-Folin _,, a 8-9 o.c. ms = 0234 i F.— M.-F. = 0-041 grs. nitrogen = 97-6 p.c. allantoin nitrogen IIT. Solution consisted of a mixture of a 2 per cent. solution of urea and a 0°5 per cent. solution of allantoin, 5 c.c. of which contained 0°0034 grs. nitrogen as allantoin. Folin nitrogen required 15-260.0. -\-acid = 0-02136 gre. nitrogen Mérner-Folin ,, rm 12-9 0.0. sa > = OORT oF — F, — M.-F, = 0-00825 gra. nitrogen = 95-5 p.c. allantoin nitrogen = 9 | ESTIMATION OF UREA, ETC., IN THE URINE 458 2] IV. Solution consisted of a mixture of a 2 per cent. solution of urea wnd a t per cent. solution of glycocoll, 5 e.c. of which contained 0°0035 grs. nitrogen as glycocoll. | Boland nitrogen = 15-2 ¢.c. —\ acid = 00212 grs. nitrogen Folin - « = 8760. , = 00178 7 fe B. — F. = 00034 grs. nitrogen = 97 p.c. glycocoll nitrogen _ V. Solution consisted of a mixture of a 0°25 per cent. solution of alanin and a 1 per cent. solution of urea, containing 0°45 grs. nitrogen as urea, and 0'037 grs. nitrogen as alanin. eg Bohland nitrogen = 34°15 cc. acid = 0-478 grs. nitrogen Folin oe = 31-6 ec. 7 = 0-442 ” B. — F. = 0-036 grs. nitrogen = 97 p.c. alanin nitrogen : These results are summarised in Tables I and II. TABLE I No, of solution Grs. allantoin F.—M.-F. Pc. allantoin —_Solution contained nitrogen = A 0-0016 0-0016 100 Urea, alanin, allantoin IL. 0-042 0-041 97-6 Urea, allantoin, glycocoll LL. 0-0034 0-00325 95-5 Urea, allantoin No. of solution Grs. aminoacid B. — F. P.c. amino Solution contained nitrogen acid t L 0-0022 0-0022 100 Urea, allantoin, alanin— IV. 0-0035 00034 97 Urea, glycocoll v. 0-037 0-036 97 Urea, alanin _ In order to ascertain whether these methods held when applied to nes, estimations were made on urines to portions of which known 1ounts of allantoin and amino acids had been added. Folin ni = O16 gra. ni trogen grs. nitrogen os oka re ” F. — M.-F. ae aed vols nitrogen was added Folin masta i 8 Be Mamner-Fotin » 0/1036 ,, F.— M.-F. = = 00168 Increase of difference = 0-0168 grs. nitrogen = 105 p.c. allantoin nitrogen P. — M.-F. = 00005 =" 98-0 p.c. allantoin nitrogen as : i. , 454 BIOCHEMICAL JOURNAL I Goose win tlio 10 ¢.c. urine + 10 ¢.c, water. le tacit Folin nitrogen = 0-00582 gre. nitrogen ty al ea 10 ¢.c, urine + 5 ¢.c. water + 5 ¢.0. allantoin solution ontainin 0-0096 grs. nitrogen —— h bani Folin nitrogen = 0-01456 grs, ows 8 . Difeence — 00824 gr. nitrogen. 8548p. al Hante 1V. Goose's urine. ite 15 c.c. oo + We. is water. 4. - =e 0-00798 : Fh ting + Malar yl sation 0 clined: sliveeek a Naiiaeeanes nitrogen | Folin = 0-00798 B. - F, = 0-00546 gra Increase of difference = = enemas RE a sors The results are summarised in Table Til. TABLE IL No. — Grs. ni added) Grs. ni P.c. ni i en arc wel aaowere Se ian L : 0-016 0-0168 105 ye ee IL. 0-0096 . 0-0095 98-9 UL. 0-0096 0-00924 95°6 IV. 0-0091 0-00924 WL * age : ‘ REFERENCES oes . rh 4) Bet Arch. }. d. g. Physiol., XLILL., p. 30, 1888. / 1 Seed 2.. Areh. f. d. g. Physiol., LX, p. 15, 1896. — Ly op, vara. ihe aes 3. Skan, Arch. fur Physiol. XIV, p. 2981908, Hike 4. Ztech. j. physiol. Ohem., XXX, p. 75, 1900. , 5. Skand. Arch. }. Physiol., U1, p. 448, 1891. 6. Ztach. j. physiol. Chem., XX X11, p. 504, 1901. 7. Skand. Arch. }. Physiol., XIV, p. 297, 1903. 8. Journ, of Biol. Chem., IV, p. 166, 1908, 9. Journ. of Biol. Chem., Ul, p. 243, 1906. 10. Proc. Physiol. Soc.. XXXV, 1906. ll. Archiv. j. exp. Pathol. u. Pharmak., XLIV, p. 60, 1900. . 12. Beitr. z. chem. Physiol. u. Pathol., X1, 1907. 13. Ber. d. d. Chem, Gesellsch., XXXV, p. 3779, 1902. 14. Ztach. j. exper. Path. u. Therap., IV, 1907. 15. Chem. Berichte, XXXVIU, p. 2,359. : 16. Ztach. j. physiol. Chem., XXX, p. 75, 1900. 17. Ztsch. f. physiol. Chem., XXX, p. 556, 1900. 18. Ztech, j. éxper. Path. u. Therap., TV, p. 338, 1907. Fon THE NATURE OF THE SO-CALLED FAT OF TISSUES AND ORGANS By HUGH MacLEAN, M.D., Carnegie Fellow, University of Aberdeen, _ axypj OWEN T. WILLIAMS, M.D., B.Sc. (Lonp.), M.R,C.P., _ Hon. Asst. Phys. Hosp. for Consumption; Lecturer in Pharmacology, University of Liverpool. a From the Bio-Chemical Laboratory, University of Liverpool (Received November 5th, 1909) The question of the nature of the fatty substances present in animal research in this direction has elicited some noteworthy facts. On the other hand, the general problem of fat metabolism in almost all its details till awaits solution, and though the processes undergone during fat orption are now fairly well understood, we have absolutely no knowledge of the methods utilised in the body in connection with the _katabolism of these substances. The recent interesting observations of _ Leathes and of Hartley point to the liver being an active agent in the ss preparation of fat prior to its final oxidation in the tissue cells; it is, however, not improbable that other organs may also be capable of _ participating in this preliminary desaturation of the fatty acid radicles. NaTuRe OF so-caLLep Tissur Far S For many years it has been recognised that fatty substances may be _ present in, or at least derived from, an organ which, on ordinary ac pical or microscopical examination, appears to be absolutely fat- 4 and gives no trace of reaction to the specific fat stains. This ‘masked * fat can, however, be rendered visible under particular circumstances, and the question of its origin has given rise to one of the _ greatest controversies in the annals of pathological chemistry; it is now generally accepted as arising from a combination of fat and protein Ms a _ normally present in the tissue, the fat becoming evident only as the result of certain post- or ante-mortem changes, by which the compound is broken up and the fat liberated. The recognition of the presence of this combined fat explained the fact that ordinary solvents, such as ether, are ineapable of extracting all the fat from an organ, and it was found that better yields were obtained by the addition of auxiliary substanees, such as aleohol and chloroform, 456 . BIO-CHEMICAL JOURNAL Various modifications have been suggested, but in most cases the general principle of fat extraction adopted and recommended by the different investigators depends on thorough extraction of the tissue by combinations of the above solvents with the aid of heat. That an organ can be freed from fat-like substances by the above means may be granted, but it is exceedingly doubtful whether the actual substances present in the extract represent with any degree of exactitude the fatty substances as they actually existed in the tissue prior to extraction. Of late, attention has been chiefly focussed on the fatty acids obtained, but the nature of the compounds in which these fatty acids are actually present in the tissues has been to a great extent lost sight of. It seems obvious, however, that a correct knowledge of the nature and disposition of the ‘fat’ in the animal organs is the only way by which a key to the difficult problem of fat metabolism is likely to be found. Experiment shows that in many cases the greater part of the fat obtained is really not fat in the ordinary sense of the word, but to a great extent complex combinations of fatty acids with glycero-phosphorie acid and a nitrogen-containing compound—the so-called phosphatides. Though it has been long known that tissues and organs contain ‘lecithin,’ it is hardly recognised that very much of the ‘fat’ they do contain may be present in this or in an allied form. What ordinary neutral fat can be extracted from an organ seems to be generally interstitial, and is present as stored fat, just as glycogen represents stored carbohydrate. On the other hand, fat which is actually being made use of by the living cells seems to be represented to a very greet extent—if, indeed, not altogether—by phosphatides. These substances, as pointed out by Heffter,! are Pen fe labile, and undergo partial decomposition when heated to a temperature of over 50° C.; even an acid reaction has a similar tendency. Under these circumstances it might be expected that the nature of the material obtained by the different extraction methods would vary in its com- position, and investigation proves that this is actually the case. This fact in itself indicates that the general extraction methods do not suffice for the determination of what appears, after all, to be one of the fundamentally important points in such investigations—the general nature of the fatty substances originally present in the tissue. It is obvious that such methods as that recommended by Dormeyer,? in which 1. Arch. j. exp. Pathologie, XXVIII, p. 97, 1891. 2. Arch. f. d. ges. Physiol., Vol. LXV, p. 90, 1907. NATURE OF FAT OF TISSUES AND ORGANS 457 ___ digestion with pepsin and hydrochloric acid is utilised to separate off the ___ masked- fat, must also result in more or less marked disintegration of the more labile fatty substances. The varying saponification values obtained in the following experiments show that there must be marked differences in the different extracts prepared by various methods. EXPERIMENTS WITH DIFFERENT Extraction METHODS Dog’s liver was taken, and an equal amount used for each experiment. The final extracts obtained were then saponified in the usual way with | alcoholic potash, and the saponification values compared. Bale (1) Noel Paton’s Method.—Liver was cut up into small pieces and placed in methylated “a spirit for a week ; the spirit was then poured off into an evaporating basin and the liver pounded ____ im @ mortar and thrown again into the spirit. The whole was then dried on a water bath, the = temperature being always kept below 80° C. The contents of the basin were then put into a = ‘Soxhlets apparatus and extracted with ether for about 12 hours. The syrupy extract obtained was then dried and saponified. Saponification value = 215. (2) Liver was cut up and extracted for a week with cold alcohol ; it was then treated with ~ hot alcohol for several hours, and with ether as described on page 458. Ether extract was separated and the ether distilled off at a low temperature. Saponification value = 248-2. (3) Liver was thoroughly ground up with calcium sulphate until a fine dry powder was obtained. This was repeatedly extracted with ether in the cold. Ether was then distilled off at a temperature below 50°. Saponification value = 234-84. The essential features of these experiments, given in tabular form, are as follows, and show that both the nature of the extracting substances and the order in which they are used, play a part in determining the nature of the final extract obtained. While the saponification values are given as one instance indicating a difference in the extracts, there are other variations with regard to the amounts of free fatty acids, etc., present :— Saponification Organ used Nature of extraction value of extract Dog's liver Placed in methylated t for 7 days; dried 215 in all at 80°. Total we prt vith ether experiments for 12 hours, ‘ First cold alcohol ; then hot alcohol ; then ether. 248-2 Dried with CaSO, Extracted with cold ether , 23484 458 BIO-CHEMICAL JOURNAL An extension of such experiments showed that not only are different results obtained by different methods of extraction, but that the same method may give variations in the nature of the substances obtained: the — chief explanation seems to be the one that naturally presents itself, se, that during the ordinary process of extraction, in which heat is invariably employed, there is, together with the abstraction of the fat- like substances from the tissues, a more or less well-marked disintegration of the extracted products themselves; the extent to which this destructive — change becomes manifest depends no doubt to a great extent on the — amount of heat utilised during the process. 1 nl Again, the above methods are absolutely useless for determining another most important point-—the relative amounts of free and combined fatty substances in the tissues. It may well be that the extremely labile nature of these substances causes their extraction to be inevitably associated with a certain amount of change in their constitution, but it is obvious that this tendency is exaggerated by the use of hot solvents, and the only rational method would appear to be extraction at as low a temperature as possible. By a careful combination of solvents used in the cold, a fair idea can be obtained of the nature and disposition of these fatty substances in the tissues, and it is hoped to publish shortly a set of experiments giving information with regard to these important details. In all our experiments it was noticed that the saponification values obtained were very high, and the following samples indicate the nature of the figures found in the case of different organs from the dog. oa a The animals were anaesthetised with chloroform, quickly bled, and — transfused with saline. The organs were then removed, transfused separately, and then minced and weighed in the moist condition. Meruop or Extraction Tissues were extracted for several days with cold alcohol, the being changed several times. The residue was then treated for one to two hours with bo‘ling alcohol, a reflux condenser being used. After removal of the hot alcohol, the residue was extracted several times with ether. Both alcoholic extracts were mixed, the alcohol evaporated, and the residue taken up in ether. This was mixed with the ether extract of the tissue, and after the chief part of the ether was driven off, residue was _ dried in vacuo over H,SO,. Saponification was carried out with alcoholic potash. + NATURE OF FAT OF TISSUES AND ORGANS 459 Saponification figures of ‘fat’ from organs of dog are Dog A Dog B Blood = bes a. 153-89 a5 _ Connective tissue... tear? 223-9 Fa 195-2 Liver : 231-6 ‘ne 230-5 Kidney 264-6 ait _ Muscle piss nS me 254-5 =# 264-5 3 a. 190 and 200; Gaicebiedis ae substances as cholesterol are sg to a varying extent, but these and other similar substances would 4 be obtained. The alternative niplapaton seemed to point to the ence of phosphatides as the cause of the high values, and igation showed that this was the case. It occurred to B. Moore sited fat or free fatty acids, and observations made by us at his request - indicates that the same holds good for certain other organs; it is very probable that the free fatty acids found in extracts obtained by the Be _ ordinary methods are to a considerable extent disintegration products of ____ tissue phosphatides. The high saponification values were found to be eaused by combination of part of the sodium with phosphoric acid and . glycero-phosphoric acid liberated during the process of saponification. __ Every organ and tissue naturally contains more or less neutral fat in the interstices of its substance; but though it would seem that the preponderating portion of the ‘ fat’ combined with protein in the bioplasm as masked fat is present as phosphatide, in addition some of the _phosphatide is present in free form, perhaps as a phase in its passage to _ combination; free phosphatides would, on this view, constitute ___ preliminary substances which subsequently pass on to actual combination in the tissues. In a normal organ, therefore, the less microscopical ___ evidence there is of fat, the less neutral fat is actually present; while the -__- eombined fat—a phosphatide—seems to represent one of those steps in that __ gynthetical elaboration of fats which appears to be a necessary prelude to actual assimilation. It is not improbable that phosphatides represent a necessary step in the elaboration of fatty substances destined ultimately to undergo actual assimilation into living matter. That such substances 460 BIO-CHEMICAL JOURNAL are essential for the vital processes seems indicated by their presence in all living cells hitherto investigated; it cannot be doubted that one of the steps which ordinary fat undergoes in the cell is a transformation into phosphatide, and probably in these bodies the desaturation of the fatty acid radicle is brought about. Whether this elaboration is necessary for — the ultimate oxidation of all fats, or whether we have here wholly or in part a process somewhat analagous to the endogenous metabolism of protein, can as yet be but conjecture. The fact, however, that phosphatides contain practically all the constituents (even iron, according to Glikin) of nucleo-proteins, is not without significance, and it is not unlikely, as partly suggested by Hammersten, that they may be the source of the cell nucleo-proteins. The marked amount of phosphatide obtained in two experiments in which pig’s liver was used is shown by the following figures. The phosphatides were separated by means of acetone. As full details of similar experiments will be published later on, the general outlines alone are given here. In the first experiment cold alcohol was followed by hot alcohol, and then by ether. In the second case the more rational method of cold extraction, first with ether and then with alcohol, was carried out: this method, which seems to give the best results, has the advantage of furnishing a good idea of the amount of ‘ free’ and ‘ masked ’ phosphatide and other substances; at the same time the necessary manipulation does not tend to cause a disintegration of the extracted substances. Experiment I Pig (1) Cold alcohol extract = 18-2 grm. { Fhospaatide =17-0084) nized = 2623 (2) Hot alcohol extract = 11-3692 grm. veren eee cane (3) Cold ether extract = 2-9608 grm. \veetae m4 AS Tae mixed = 220 Total extract = 32-5300 grm. | FasPen t= *7 abe, orm. Phosphatide = 84 _— per cent. of total extract. Fate, &. = 16 per cent. of total extract. Experiment II Liver 20 grm. dried substance Ether extract {Phvsshatide = OS416 cox | Proportion of Fat to Phosphatide = 2: 1 Alcoholic extract | Phraphotide ve seen ane Proportion of Fat to Phosphatide = 1:20 NATURE OF FAT OF TISSUES AND ORGANS 461 = This liver showed fat distinctly on examination with the naked eye, __ and the total amount of neutral fat was somewhat large. On the other hand, it will be noticed that the alcoholic extract contained about 95 per cent. of phosphatide; the amount of ether-soluble fat other than _ phosphatide in the liver is probably intimately connected with the digestive processes, and is a much more variable constituent of the liver _ than the more complex phosphatides. This acetone-soluble ‘ fat’ would likely be reduced in amount as the result of a period of starvation. rae Tn short, it would seem that the essential fat of the liver, and probably of —s gertain ~=other organs, is really phosphatide, and under certain circumstances, if care be taken to avoid disintegration during the process ___ of extraction, it may be practically the only one found in any appreciable amount in the combined part of the ‘ fat.’ “dl _ i = ee ee a a —— Se ree Re Pe Re yy a ee ei ie whe te ahs oo ie ee * ee 462 THE OSMOTIC PRESSURE OF LIQUID FOODS By JUDAH L. JONA, B.Sc. (Adel.) bags From the Physiological Laboratory, Melbourne University Communicated by Proressorn W. A. OsBoRNE (Received November 7th, 1909) One of the admitted functions of the stomach is the osmotic equilibration that takes place between the blood and the fluid food swallowed. Hypertonic solutions are diluted and hypotonic solutions have salts, ete., added until isotonicity is attained, though it may be a matter of debate whether the diluting (or concentrating) fluid is physiologically secreted or is due to physical diffusion. That the lining cells of the mucous membrane of stomach and gut would be injured by prolonged contact with a hypertonic fluid may be stated a priori. Even the mucous membrane of the mouth is open to injury in this way—-witness the ‘ roughness’ produced when a piece of confectionery is retained for a few minutes between the teeth and cheek. Also the discomfort which follows the intake of such substances as strong salt solutions, very strong soups, or peptone solutions which ‘irritate’ the stomach, is thus easily explainable. 7 A The object of the present research was to determine the osmotic pressure of the fluids ordinarily admitted to the stomach, and at the same time to discover whether the sense of taste afforded us any guidance in the choice of fluids with reference to their osmotic pressure, more particularly as regards the rejection of the hypertonic. Of the food- stuffs ordinarily eaten, the vast majority are in solid or gelatinous or colloidal form. To such substances the consideration of osmotic pressure cannot apply. The actual fluid foods admitted to the stomach are milk, the ordinary beverages, fruit juices, beef-tea, meat extracts and soups. In tea, coffee, or cocoa there is usually a sugar addition which varies with personal taste, whilst in beef-tea, soups, ete., common salt is invariably = R an added ingredient. The osmotie pressure of milk has been determined | so frequently that I have not thought it necessary to make any confirmatory experiments. In the case of soups the ‘salting to taste’ els was carried out by a laboratory attendant, who was not aware of the sit THE OSMOTIC PRESSURE OF LIQUID FOODS 463 purpose of the research. ‘The Beckmann freezing point method was employed throughout. A mixture of ice and salt water was used to produce the requisite cold, but care was taken to prevent excessive _ supercooling. In none of the recorded readings was the degree of super- cooling more than about 15° C. — Crystallisation was started by i inoculation with a fragment of frozen distiJled water. The stirring was “carried out by a simple elock-work mechanism. Centigrade scale employed throughout. REsvULtTs. __ Coffee.—(2 spoonfuls of sugar in ordinary breakfast cupful.) (1) 4 0-341°C.; (2) A 0-343°C. " Pea Infusion.—(2 teaspoonfuls, about 12 c.c., of tea in 200 ¢.c. boiling water.) Allowed to infuse 5 minutes. (1) A 0-052°C.; (2) A 0-049°C.; (3) A 0-050°C. TT pear 10. 0 Infusion, 50 c.c, water, 25 c.c. milk, 10 grms. of sugar.) Tasted ‘ just nice.’ | (1) A 0-457° C. ; (2) A 0-458°C,; (3) A 0-456° C. nd infusion, made with 200 c.c. more water added to leaves from infusion and allowed : to stand 35 minutes. (1) A 0-026°C.; (2) A 0-025°C. Lemon Juice.—(As used in Melbourne Hospital.) Strained juice of lemon (one lemon) 33 c.c. in 250 c.c. distilled water. (1) A 0-126°C.; (2) A 0-125°C.; (3) A 0-122°C. 100 ¢.c. of diluted juice + 1 teaspoonful (5 grms.) cane sugar added. Tasted ‘ just gight.’ aa (1) A 0-487°C.; (2) A 0-485°C. as “Beer.—{Carlton draught XXX Beer.) (1) A 2-407°C.; (2) A 2-409°C. a : aif sne.—{Cheap Australian claret.) Cooled to —5°C., but ice would not separate out. _ Wine.—Kept between 77°C. and 80°C. for 35 minutes, and then boiled briskly for about : 7 minutes to get rid of alcohol, at end of which time boiling point rose to about 103°C. 75 o.c. of wine subjected to this treatment yielded 45 0.0. (1) A 3-240°O.; (2) A 3-238°C.; (3) A 3-241°C. Foodstuffs ‘Treacle. —Diluted with water to 1 in 7 and this solution gave A 1-730° C. Peptonised Milk.—Benger’s peptonised milk as used at the Melbourne Children’s Hospital, it are Carlton, Melbourne, Victoria. Pat e's Milk ‘ A.’—(Milk 3, water 1, peptonised 20 minutes. Boiled. Sweetened with cane sugar about 1 oz. to 1 pint milk.) One drop gave pink biuret reaction. (1) 4 0-652° 0. ; (2) A 0-656° C. Benger’s Milk ‘ B.—(Milk 2, water 1, peptonised 20 minutes. Sweetened.) (1) 4 0-630° C. (2) A 0-628° C.; (3) 4 0-626°C. Peplonis ed Milk.—(Milk 4, water 1, peptonised 20 minutes.) (1) A 0-548°C.; (2) A 0-546 C. _-—- Sowps.—An ordinary soup which had been served up but rejected as unpalatable on account a of salt taste. A 1-984° C. A Vegetable Soup was made of the following ingredients ;—Carrot, 100 grms. ; parsnip, 110 ‘ grms.; turnip, 55 grms.; spring onion, 47 grms.; celery, 25 grms.; parsley, : 12 grms.; water (distilled), 1500 c.c, Brought to boil and kept simmering for 2} hours. Strained. Salted to different degrees. Vegetable Soup Plain.—(1) & 0-374°C.; (2) 4 0-873" ©. ; (3) A 0-372" C. 464 BIO-CHEMICAL JOURNAL Vegetable Soup Salted.— Soup + salt to 4 per cent. A 2-°757°C. Soup + salt to 2 per cent. A 1-536°C. ee Soup + salt to $ per cent. (1) A 0-851°C.; (2) A 0-856°C. dat Pe opp Soup + salt to } per cent. (1) A 0-586°C.; (2) A 0-584°C.; (3) A 0-582°C. Soup + salt to } per cent. A 0-780°C. ot ae i The verdict of the taster was :—The unsalted vegetable soup possessed a 7A very flat and unsatisfactory taste. A, B, C, and D distinctly too salty. A and B distinctly unpleasant taste. E was about right. Beef Tea.—Made with about 6 c.c. meat extract (Fitzroy Brand, Queensland manufacture) in 1000 c.c. distilled water. Beef Tea Plain.—{1) A 0-141° C.; (2) A 0-139° C.; (3) A 0-140°C. Beef Tea Salted.— Beef tea + salt to 2-5 per cent. (1) A 1-626°C.; (2) A 1-626°C.; (3) A 1-625°C. Beef tea + salt to 1-25 per cent. (1) A 0-882°C.; (2) A 0-887°C.; (3) A 0-886°C. Beef tea + salt to 0-625 per cent. (1) A 0-546°C.; (2) A 0-544°C.; (3) A 0-643°C. Beef tea + salt to 0-416 per cent. (1) A 0-419°€.; (2) A 0-416°C. Sample C was much the tastiest—just salted to taste. A and B having too much salt, and sample D and the original not enough salt. Bee} Tea made with about 6 c.c. meat extract in 1000 c.c. boiling distilled water. A 0-160° C. Beef Tca Salted.—Salt added till the flat and unsatisfying taste of the beef tea was abolished, but still no salty taste perceptible. Taster A. (1) A 0-330°C.; (2) A 0-331°C. Taster B. (1) A 0-329°C.; (2) A 0-330°C. Beej Tea Over-salted.—A 1-922° C. Sap Pr Sugar Solutions Dextrose Solutions. —100 c.c. taken into mouth in sips of 25 c.c., each sip kept in mouth 4 minute, spat out ; in } minute another sip taken, kept in } minute, spat out; and so on for four sips. . 10 per cent. Dextrose Solution. A 1-156°C. 10 per cent. Dextrose Solution Salivated. (1) A 1-068°C.; (2) Al 066° C. 5 per cent. Dextrose Solution. A 0-566°C. 5 per cent. Dextrose Solution Salivated. (1) A 0-536° CG. ; (2) A0-532°C.; (3) 40-534°C, Cane Sugar Solution, 20 grms. in 150 c.c. (13-3 per cent). 100 c.c. treated in similar manner as dextrose solution above, 7 c.c. Saliva were added by this process to the 100 0.0. sugar solution, and there was an after secretion for several minutes. Cane Sugar Solution (13-3 per cent.) A 0-868°C. Cane Sugar Solution Salivated. (1) A 0-788°C.; (2) A 0°792°C. ; Fruit Jwices Lemon.—Weight 140 grms. Peel and connective structure 90 grms. Yielded 40 o.c. strained juice. (1) A 0-937°C.; (2) A 0-940°C.; (3) A 0-939°C. Orange.—Orange 135 grms. Yielded 50 0.0. strained juice. (1) A 1-100°C.; (2) A 1-101°C. ; (3) A 1-100°C. Pineapple Jwice.—From fresh Queensland pineapple. (1) A 1-462°C.; (2) A 1-464° ©. ; (3) 4 1-460° C. Cocoanut * Milk.’—{ About 150 0.0. were yielded by the nut.) (1) A 0-521°C.; (2) 4 0-518°C, ; (3) A 0-518° C. ar THE OSMOTIC PRESSURE OF LIQUID FOODS 465 Saline Aperients Magnesium Sulphate Solution.—(15 grms. in 100 ¢.0.) A 1-136°C. Balanced Saline Aperient,—(The stronger one) as recommended by Professor W. A. Osborne in a paper in the Intercolonial Medical Journal of Australasia, July 20th, 1909. (1) 4 0-864° C. ; (2) A 0-862°C.; (3) 4 0-861°C: Saliva produced from Sucking Confectionery.—Barley-sugar stick (about 12 grms.) sucked for 15 minutes led to prodution of 72 c.c. Saliva. (1) 4 1-008°C.; (2) A L-006°C. Saliva from about 30 grms. boiled cane sugar. Sweetmeat — 100 c.c. (1) A 1-488°C.; (2) A 1-484° 0. GENERAL CoNCLUSIONS It will be seen from the above experiments that of all the fluid foods which are admitted to the stomach, alcoholic beverages and fruit juices alone are hypertonic. Further, it may be safely stated that in no case is ___ a fluid admitted in which hypertonicity is due to the mineral ingredients alone. When, therefore, we find the kidney elaborating a fluid (urine) with sufficient piline ingredients to render it hypertonic, we must regard the high concentration of this fluid as so much external work done and of sufficient moment to be taken into consideration in calorimetric experiments on an animal or on the human subject. These experiments also demonstrate that we must ascribe to the sense of taste a distinct osmotactic character. Not only is this sense potent in testing the food qualitatively, but also from the quantitative standpoint of molecular concentration. Even those hypertonic fruit juices which are admitted to Z __ the stomach are passed, so to say, under protest, for their taste is - recognised as astringent or highly acid, and are apt to be followed by a sense of thirst. The mechanism is faulty, however, when dealing with alcoholic beverages, a fact which we may ascribe to the artificiality of fermented liquors, and their manufacture and consumption being restricted to man only. The great majority of fluid foods are, however, hypotonic, and thus a margin is left for the addition of hydrochloric acid and other constituents of the gastric juice. With regard to alcoholic beverages, it may be stated that a solution of alcohol in pure water, isotonic with the blood, would only be about 15 per cent. As this percentage is almost invariably exceeded in fermented (and, of course, distilled) liquors, and as other substances are present in addition, the high osmotic pressure of the beer and wine tested is not surprising. The association of the raising of osmotic pressure of beverages with 466 - BIO-CHEMICAL JOUR the induction of thirst is made use of in some depar the excessive salting of wines and the over-sugaring 0: In the case of cane sugar, a solution isosmotie with the t about 11 per cent., whereas the fluid which reaches the s of even the slow methods of ingestion of sweetmeats, | process of sucking confectionery, is much higher tha for the disagreeable after-results bet often ex indulgence in such delicacies. lke’ in this work. 4 THE RELATIONSHIP OF DIASTATIC EFFICIENCY TO AVERAGE GLYCOGEN CONTENT IN THE DIFFERENT TISSUES AND ORGANS By HUGH MacLEAN, M.D., Carnegie Research Fellow, University of Aberdeen. From the Bio-chemical Laboratory, University of Liverpool (Received November 17th, 1909) + The first demonstration of an enzyme capable of hydrolysing glycogen is associated with the names of von Wittich (1) and of Claude Bernard (2). 5 The brilliant researches of the latter observer, which ultimately led up to the discovery of glycogen in 1855 (3), formed the basis on which the probability of the presence of such an enzyme rested, but the actual proof of its existence was first furnished by von Wittich; a short time . ipietwards independent evidence was advanced by Bernard, both observers ____ having found the substance in the liver. atti _ These discoveries, however, were not allowed to pass unchallenged, J and to Pavy (4) belongs the credit of having settled beyond dispute the fact that the liver really contains a substance of the nature of an enzyme which is capable of converting glycogen and starch into sugar, and acts quite independently of the vitality of the tissue cells from which it is i derived. 2 ‘yy - __-‘The presence of such an enzyme both in the animal and vegetable a organism is now universally admitted, but its exact function is still in certain quarters a matter of controversy; the fact that glycogen occurs in the body tissues, and that the diastatic enzyme! possesses the power be é transforming this substance into dextrose and intermediate products, suggests that the normal function is directly associated with the conversion of glycogen into the less complex substance—sugar. Pavy (5), however, is unable to accept this view, and suggests that the enzyme is really a product of the dead or dying cell which is generated from an inactive pre-existent zymogen. On the other hand, analogy with other intracellular enzymes points to the probability that this diastatic enzyme actually exerts its influence during life in the same manner as 1 * diastatic enzyme ’ is used in this paper to indicate the substance or substances vilok ah co i transfrmed into red maltose, dextrose, eto. ; it is hy which lycogen si ¥ SSISnOnT Gauiidaedaeti thes farmaiton il detvoee dossnceaeaile or glycogen. 468 BIO-CHEMICAL JOURNAL its activity is evidenced in vitro—as a hydrolyser of glycogen—the difference being solely one of degree, and its activity being called forth in response to some condition not yet understood. That the conversion -f of glycogen is not regulated by the condition of the animal is suggested — by the experiments of Kisch (6) carried out on adult muscle; his results _ seem to indicate that regulation of sugar formation from glycogen is apparently not brought about by the needs of the tissues for sugar. If, then, the function of this ferment is to act on the glycogen present in the organs and change it into sugar in response to some unknown body stimulus, it might be expected that the amount of glycogen normally present in any particular organ would bear some general relationship to the richness of that organ in diastase. It has been known for many years that the chief seat of glycogen in the animal body is the liver; the muscles may also show a very considerable amount, while the other organs contain but traces. Consequently the literature of the subject abounds with data concerning the diastatic activity of the liver and muscles, but comparatively few observations dealing with the other organs are available; it has generally been recognised that many or all the tissues of the body contain traces of a glycogen hydrolysing ferment, but until quite recently no attempt has been made to investigate the relationship of average glycogen content to amylolytic efficiency. A short time ago, however, an interesting series of observations have been undertaken by Mendel and Saiki (7); for their experiments muscle and liver of pig embryos of various sizes was utilised. In the case of the liver it was found that the diastatie activity increased markedly with the growth of the embryos, while in the case of muscle, which showed in general a well-marked initial activity, the increase was not nearly so pronounced. Hence it would seem that the diastatic activity of embryonic liver and muscle varies directly with the normal glycogen content, for it has been shown by Lockhead and Cramer (8) that the liver of foetal rabbits is at first very poor in glycogen, but that after twenty-five days a considerable amount is present; again the estimations of Mendel and Leavenworth (9) indicate that in the very young pig embryos no glycogen at all may be found. Thus, the foetal liver is at first very poor in glycogen, and apparently also in glycogen hydrolysing substances, while on the other hand the embryonic muscles may contain a fair amount of glycogen even at very early age, but tend to show only slightly increased amounts as age advances; in correlation with this slight rise in glycogen content the increase in diastatic power is correspondingly slight. . — -* DIASTATIC EFFICIENCY 469 e So far, therefore, as the subject has been investigated there is, in _ the case of embryonic tissues, undoubted evidence pointing to a _ relationship between diastatic efficiency and glycogen content, but whether varying diastatic activity is directly dependent on the relative glycogen content, or whether the increase of diastatic power constitutes a factor which directly influences the amount of glycogen, is still obscure. An answer to this question might help us in attributing its proper function to the diastatic enzyme, for it is not impossible that its chief sphere of action during life is synthetical rather than destructive. If the view entertained by several authorities with regard to glycogen is correct— that under normal circumstances it does not pass on to any marked extent to form dextrose, but is utilised in other ways—then it might perhaps be expected that the measurement of post-mortem amylolytic activity ___ im an organ would afford some clue to its glycogenetie power during life. It will be shown, however, that the amount of diastatic enzyme actually _ found in different adult tissues bears no apparent relationship to the glycogen content of the same tissues; it is thus obvious that amylolytic efficiency and glycogen storage do not necessarily go together. It might be argued that the comparative absence of glycogen in an organ did not of itself imply the inability of that organ to form glycogen, for the latter substance might be formed and carried away in the blood; or again it might be immediately utilised at the seat of formation by the tissue cells. That the blood does not carry the glycogen as such from an organ is suggested by the very low glycogen content of this fluid, and the - conception of a simultaneous process of formation and destruction of glycogen by which this substance might be kept at a minimum, can hardly be entertained if we accept the theory that it is as dextrose that _ earbohydrate is utilised by the tissues; such a procedure would be quite out of harmony with the ordinary methods of nature, for in that case formation of glycogen would be but a useless step; only on the theory that glycogen is utilised as such by the tissue cells could the latter possibility be entertained. ee This apparent lack of correlation between glycogen content and glycogen hydrolysing capacity makes it much more difficult to understand what the amylolytic activity as evidenced post-mortem really means in connection with the vital processes. It may well be that these enzymes exert their destructive action chiefly as the result of some interference which threatens the life of the organ. It is well known that the kidney, for instance, contains normally 470 BIO-CHEMICAL “JOURNAL but a very small amount of glycogen, and yet it is often found re bem sh richer in diastatie enzyme, per unit weight, than any organ in he body. On the other hand, muscle which generally contains a_ a amount of glycogen is often very weak in diastatie effect. The wil 3 figures, taken from an experiment recorded by Piek (10), are —— with regard to this point; they indicate the results of some conten estimations of amylolytic activity of different adult tissues. + 19a It was found that a a head ee * 100 grms. kidney digested 2°37 grms. glycogen in 3 hours. ROA 100 grms. liver digested 0°69 grms. glycogen in 3 hours. — iat has es ia ee 100 grms. blood digested 0°31 grms. glycogen in 3 hours. Pee re Here it is seen that kidney possessed between three and four times the diastatic power of liver and about eight times that of blood. Experiments . made by the writer gave in general very similar results, and showed that — almost invariably the kidney is the organ which possesses, weight for weight, the greatest amylolytic efficiency. Often other organs were also found to have quite a marked effect. It-is interesting to note that in __ the case of another endoenzyme—erepsin—it has been shown pie 2 ia that the kidney possesses far and away a greater ereptic action than any other tissue, with the exception of small intestine. The richness of the kidney in these and perhaps other enzymes is not easily explained in the light of our present knowledge of vital processes. war s(t neers In order to demonstrate the comparative efficiency of the different sate organs the following procedure was adopted. - Shue: ‘et a Ady i" Estimation oF AmyLonytic AcTIVITY oa ay} The majority of observers conducting research on amylolytic wet have chosen as an index of activity the amount of reducing substances formed in a given time. Hither the dried tissue or the expressed His ue juice was incubated with a starch or glycogen solution of known stret sth, 3 2 and subsequently the extent to which reducing substances had 1 ‘beeitt formed estimated by Fehling’s or some similar alkaline copper solution. 7% This method supplies information as to the activity of the enzyme in terms of its power of forming products which reduce copper solutions, but affords no information as to the relative amounts of these substances” actually present—dextrose, maltose, etc. For general comparative work, — however, such detailed information is not necessary, and this method is: ae quite suitable. i? = i oS ee eee se DIASTATIC EFFICIENCY 471 In other cases the amount of glycogen present before and after =a incubation was—calculated, and the difference taken as an index of amylolytic action. This again merely indicates the power of the enzyme to transform glycogen into substances soluble in alcohol, but gives no -__ indieation as to the extent to which this change has been carried on towards the formation of the final products. As exact estimation of glycogen, especially where many experiments have to be undertaken, is a tedious process, a good indication can be obtained by substituting starch for glycogen and testing with iodine in order to ascertain when the starch ceases to give its characteristic iodine reaction. This method has been used and recommended by Wohlgemuth (11), but in my experiments on dried tissues it was found occasionally to give somewhat indefinite results ; _ only a few experiments, however, were carried out, and so far as they : a went the results were in general similar to those obtained by the reduction ace method. On the whole the most satisfactory method was found to be the estimation of the reducing power, and dried tissue was mostly employed ; in a few experiments moist fresh tissue was also used. Merruop The method utilised is in general the same as that made use of by many observers. The various organs were obtained as soon as possible after death, freed from adherent fat, and quickly cut up into small pieces; these were washed in normal saline solution in order to free them from blood, dried by being pressed gently in a cloth, and finally passed through a mincing machine. The finely divided substance was | _ placed in « large volume of alcohol and left there for 48 hours. The : aleohol was then filtered off and the tissue dried in vacuo over H,SO,, and ground to a fine powder by a coffee mill. To 50 c.c. of a 1-2% solution of soluble starch or glycogen, 1 gm. of the dried powder was added, and the mixture placed in the thermostat at 37° and left there for 18 to 20 hours. Small flasks of about 100 c.c. capacity were found to be most convenient, and the utmost precautions were adopted in order to ensure the sterility of all vessels used; toluol, alone or with sodium fluoride, was always added to the fluid. The flasks were thoroughly shaken up from time to time, and next day the contents were boiled in order to coagulate the albumin; after filtration a measured volume (10-30 ¢.c.) was taken and gradually added to excess of boiling Fehling’s solution. After boiling for about 5 minutes, the cuprous oxide was 472 BIO-CHEMICAL JOURNAL collected on a weighed Gooch crucible containing asbestos; after thorough washing of the precipitate with distilled water, the crucible was heated in order to oxidise the lower oxide, and the precipitate | hen weighed as cupric oxide. Parallel experiments with boiled controls: were also made. In all cases the ordinary Fehling’s solution was employed, x, diluted with an equal volume of water, and in every comparative experiment the same amount of Fehling’s solution was used; this precaution was necessary in order to obtain accurate results, and likely depends on the power of caustic alkali to dissolve small amounts of cuprous oxide. At first some difficulty was occasionally experienced in filtering a too finely divided cuprous oxide precipitate; it was found, however, that this — could be overcome in all cases by boiling both the sugar-containing fluid and the alkali before bringing them together, or by adding the sugar solution very slowly to the boiling Fehling’s fluid; by adopting this small precaution a well defined red precipitate was easily obtained in every case, and no difficulty arose in filtering. Some preliminary experiments showed that this method, when carried out carefully as above described, gave practically identical results in parallel experiments with the same tissue. Wohlgemuth, however, raises the objection to results based on reduction methods, that they do not necessarily indicate the diastatic activity, since there may be present glycolytic enzymes which destroy part of the sugar as it is formed. The evidence for the presence to any appreciable extent of active glycolytic enzymes in the tissues is by no means very convincing, as may be seen by consulting the literature of the Conheim controversy relating to the alleged glycolytic action of muscle juice combined with pancreatic extract. In my experiments in which it is sought to establish the different diastatic powers of the various tissues, it -is obvious that glycolytic effect is of most importance from a comparative point of view — in cases where low results were obtained. Some experiments made on this point showed that mixtures of sugar and tissue, treated exactly as in the diastatic experiments, did not tend to decrease in strength to any : appreciable amount; the results obtained indicate that this factor is of little importance in determining the results of diastatic efficiency caleulated in terms of reducing substance. In one experiment the following figures were obtained :— ; ‘i 10 e.c. sugar sol. = 01126 grm. CuO. DIASTATIC|ANNIchincy 178 50 c.c. of Bare sugar séletion digested with 1 grm. of following gans for 20 hours; other 50 ¢.c. + tissue boiled and used as control :—- (10 e.c. used) (10 c.c. used) solution ad doe Liver 0-1130 grm. ek 0-1109 grm. ~ + 0-0004 grm. = Bent. Olli 5: ellie eNO 5 id ~ 00005... ‘% ; z Lung 00-1046 - ae 0-1090 - aes we 0-0080 - Muscle 06-1102 ,, | isa Os ual ~ 00024 ., a at any rate, any glycolytic. action that may exist is much too all and indefinite to be of any appreciable account in determining r static activity, especially from a general comparative point of view. |Rerative Drastatic Errvictency or Dirrerenr Orcans Nx Terms or CuO given below. In almost all cases where sufficient material was available, two sets of parallel experiments were carried out; by this means the is possibility of accidental results due to contamination by micro-organisms was excluded. In a it cases in which the was too small to vield : Tae Amount of digest used = 20 c.c. Result in grms. CuO Kidney = 0-3708 Langs = 0-3008 Liver = 0-2720 Heart a 02566 Stomach = 2210 Bladder na 01984 Masele = 1448 Here it is seen that kidney shows the greatest diastatic action, while slung comes second and liver third; muscle is apparently weaker than any of the tissues examined. Almost identical comparative results were found in another cat examined, except that the lung was less active. 474 BIO-CHEMICAL JOURNAL Experiment II.—Rabbit 1 germ. dried tissue + 50 c.c, ie cent, starch solution + toluol. ted 19 hours, — 20 c.c. digest u Result in grms. Kidney = 02694 Liver me 0-2028 Stomach = 0-1572 L = 01546 Bladder = 0-1348 Muscle ad 0-0621 In the case of another rabbit, experiments were made with the fresh tissue in order to test the relative effects immediately after death. The animal was killed by bleeding, and the organs removed as quickly as possible and cut up into small pieces. They were then thoroughly minced up and 1 grm. of each quickly weighed and then digested with the above starch solution + toluol. Only kidney, liver and muscle were used for this experiment. - ) Amount of Result in Kidney 5 minutes after deeth; ts: ee | Wen: are Liver 10 7 “ 7 0-0516 Muscle 12 = o 0-0294 In order to ascertain whether there was any difference if the organ was not used quite fresh, it was tested again three hours after death, exactly as mentioned above. ” ? Result—Kidney = 0-1016 grms, CuO Liver = 0-0584 ” Muscle = 0-0318 9 In another rabbit the following figures were obtained; same starch solution as above + 1 grm. tissue were used, and digestion went on for 20 hours 30 minutes :—- 20 c.c. digest used. Kidney 10 minutes after death 0-1872 CuO 4 hours ” => 0-2000 ” Liver 5 minutes after death = 0-0640 _,, 4 hours ” = 0-0599 ” Lung 14 minutes after death = 00576 _,, 4 hours hm = 00521 .,, From these results it appears that tissue does not lose any of its: activity on standing, at least for a few hours; other experiments show that no change is apparent after standing for a very much longer time. Here, again, it is seen that kidney possesses the most marked amylo- lytic efficiency, while liver has a very much less marked effect and musele comparatively little. In one rabbit, however, it will be noticed that liver tissue was twice as active as in the next case, while muscle was about equal in both. In the last case, lung is as effective as liver, though lung at most contains only traces of glycogen under normal circumstances. In another case it was found that muscle was somewhat richer in diastatic ferment than liver. eee Reg: ih a ae i os DIASTATIC EFFICIENCY ees... Here, when tzeated a as other dried tissues for 18 hours, it was Kidney = 0-2264 grms. CuO Liver = 01136 pm Muscle = 0-1200 ” Lung = 0-0716 These results indicate sufficiently well the marked fluctuations that 2 “may exist in the corresponding tissues of the same species of animal. Experiment ITT _ Ptc In this case only one experiment was made, and the lung gave higher results than any of the other tissues examined. Liver was also somewhat more active than kidney, while skeletal muscle was lowest of all. In this In many experiments carried out on animals of other species, it was often found that lung had a relatively high value. ‘Eaperiment IV Doe ; The figures obtained from two dogs examined showed that kidney _ was again most effective, but the difference was not so marked as in some other animals, It is likely that an increase in the number of experiments would have shown greater variations than are here indicated. 1 grm. dried tissue + 50 ¢.c. 2 per cent. starch solution + toluol. Digested 20 hours. 10 ¢.c. digest used. Result in grms. CuO = 0-1612 es $1 roe = 0-1051 Bladder - O1214 — = 1333 uscle - 01051 Small intestine > 01850! 1. Be Gee Meare etter of tathatey yuanooaaie face 4 is ory comaga Pe ee eee ee, juice, this tissue has ees ee oe 476 BIO-CHEMICAL JOURNAL Experiment v Sueep ‘vt a were phone: both in the solpele of corresponding organs of Z arn animals and in the relative efficiency of the different tissues d the same animal. Sometimes kidney was found to be very aia active than liver, or any other organ; in other cases liver and k displayed practically an equal effect. In a few cases the heart exerted but very slight diastatie action. Some of the ° \ obtained are indicated by the following figures from two animals :—- 1 grm. dried tissue + 50c.c. 14 per cent. starch solution + toluol + NaF. Digested 20 ¢.c. digest used. Result in grms. CuO (1) ae: Liver = 0-1876 Kidney = 0-1796 Muscle = O44 Heart = O- 1384 Lungs i 01204 Kidney ¥ ieetikes 0-2576 Liver = ~ 01846 Lungs = 0-1642 Stomach = 0-1401 Muscle = ~ 00920 f. 0-074) i Se A few investigations were carried out with sheep's antiaar in orde ascertain whether there was any change in diastatic activity after t fresh moist tissue had stood for a considerable time at room a e in a cold chamber. 2 grms. liver were taken at different times atter death ona dig with starch solution in the usual way, under exactly similar co: The results indicate that liver does not lone. its full diastatic sti very considerable time: : Result in CuO given by 10 ¢.c. digest } hour after death = 0-0852 24 hours ie ' = 0-0754 ¢ 86 ” ” = 0-0835 og DIASTATIC. EPFICIENCY 477 E In another case liver was left for several days in aleohol, part being takenout-at different times and dried in the usual way. No difference could be detected between liver treated for 24 hours with alcohol and the same liver treated for four days; the figures given are merely comparative, _the same amount of dried tissue being used in each case :— Result in grms, CuO =: 0-0876 48.» ” = 0-0851 , Sa = 0-0836 Samples of powdered teens kept in a dry state did not seem to have changed in the slightest degree after several months. No indication of any difference in the action of the liver when used as soon as possible after death, and a very considerable time afterwards, was observed, though it is possible that this may not obtain for the glycogen actually deposited in __ the liver itself when acted upon by the cells immediately after the death of cs the animal. ‘That diastatic enzymes in general are exceedingly stable bodies, _ especially when protected from moisture, is proved by many researches. Thus Sehrt found active diastatie ferments in the tissue of mummies several thousand years old, and lately it has been shown by White (12) that the seeds of such cereals as wheat, maize and barley contain diastatic enzymes, which, if stored dry, retain their activity for twenty years or more; that these substances are necessarily comparatively stable bodies is obvious from the observation that barley diastase remains unaffected in efficiency after being subjected to temperatures varying from — 200° C. to + 138° C. These facts indicate that observations on human tissues which can generally be obtained not sooner than 18 to 24 hours after death, probably give quite a fair picture of what would be produced by fresh tissues. In the cases of normal organs examined, the results were generally of the same order as those obtained for other animal tissues. Kidney was generally much more active than any other part, liver being second and lung third. Muscle was again about the bottom of the list; great variations were, however, in evidence, and only a few cases were investigated. Tissues were also obtained from two patients who had died from diabetes; it has been shown by Bainbridge and Beddard and by others that diastatic activity is present in diabetes, but the relative power of the ‘different organs has not been investigated. In these two cases, however, no appreciable variation from the normal was observed; in No. 1 the marked effect of the kidney over all the other organs is well brought out, 24 hours in alcohol 2 a= Sa rere, oa ee rt i a a a er me 478 BIO-CHEMICAL JOURNAL Experiment VI Drapetic Tissues 1 erm. tissue + 50 e.c. 1 per cent. starch + toluol + NaF. Digested 20 hours. 30 c.c. digest used. No. 1 Result in grms, CuO Liver = 0-1191 Kidney = 0-2642 Muscle = 0-0959 Heart = 0-0820 Lungs = 02214 No. 2 ’ Kidney = 0-1498 Liver | 0-1089 Lungs a= 0-1186 Muscle oa 0-0707 In these two eases, at any rate, it would seem as if the diabetic condition had at least no tendency to cause diminished diastatic activity ; obviously many more cases would be required before any definite statement could be made. GENERAL SUMMARY Just as the present investigation was almost finished, several papers — by Wohlgemuth (15) on tissue diastases appeared. One contribution dealing with the relative diastatie power of certain organs of the rabbit— liver, kidney, muscle—states that kidney was most powerful, followed by muscle and liver in the order mentioned. The figures given show fairly marked variations, and in some cases liver and muscle appeared to be about equal; sometimes muscle was more powerful than liver. From these investigations, and those given in this paper, it is quite obvious that there are considerable variations in the relative diastatic activity displayed by different animals; at the same time there is no very definite relationship between the results obtained for the different tissues of two animals of the same species. The experiments given above are intended to indicate chiefly the comparative results obtained with animals of the same species, for sometimes different samples of soluble starch were made use of. At first it was intended to make comparative observations on many different species, but the extent to which variations manifested themselves in animals of the same species indicated that such an investigation would lead to no definite results unless a very great number of animals were used, DIASTATIC. EFFICIENCY 479 = The results obtained, however, give sufficient data for an answer to ep question of the existence of correlation of diastatie efficiency to recogen content. — ; Tust as the liver normally contains the most glycogen, and the scles a considerable quantity, while such organs as the kidney, bladder id lungs contain but traces, it might be expected that these same organs a 1 show diastatic power in the order of their glycogen storing power. s, however, i is not the case, and sometimes an organ containing at most mut vis mere trace of glycogen-—e.g. lung—shows more marked amylolytic ower than liver; again, muscle may contain less ferment than any other Tt is obvious from these results that there exists no definite correlation ven glycogen content and diastatie efficiency in the case of adult tissues. REFERENCES Pfliiger’s Archiv., VU, p. 28, 1873. Comples Rendus, LXXXV, p. 519, 1877. Bernard, Legons (Cours d’Hiver, 1854-55). Journ. of Physiology, XX, p. 391. The Physiology of the Carbohydrates, 1895. Beitrage z. chem. Physiol., VIII, p. 210, 1906. . American Journ, of Physiology, XXI, p. 64, 1908. 8. Journ. of Physiol., XXV, p. 11, 1906; and Pror. Roy. Soc., B. LXXX, p. 263, 1908. 9. Amer. Journ. of Physiol., XX, p. 117, 1907. 10. Hofmeister’s Beitr., U1, p. 174 (1903). 1. See Noel Paton, Journ. of Physiol., XXII, p. 423, 1898, - Proc. Roy. Soc., B. LXXXI, p. 417 (October, 1909). Biochem. Zeits., Ba, XX1, 8. 380, 484 (October, 1909). 480 THE OSMOTIC PRESSURE OF THE EGG OF THE COMMON FOWL AND ITS CHANGES DURING INCUBATION veri - By W. R. G. ATKINS, M.A. (Trinity College, Dublin). -— (Received November 17th, 1909) 2 Having shown in a previous paper’ that the blood and eggs of birds ure not isotonic, it seemed of interest to study the changes, if any, taking place during incubation, thus tracing the pressure variations from germ cell to chick. This difference in osmotic pressure between the blood and — the egg was quite unexpected; its magnitude may be seen from the following table, in which A denotes the depression of freezing point of the fluid below that of pure water and P stands for the osmotie pressure, which was caleulated from the formula P = 12:06 A — 0°021 A® Number of Bird experiments A P in atmospheres Common fowl (Gallus bankiva) 15 0-607° ©. 7-31 Blood 12 0-454" C. 547 Egg Duck (Anas) 8 0-574° C. 6-92 Blood 9 0-452° C. 5-45 Egg Goose (Anser) 4 0:552° C. 6-65 Blood 1 0-420° ©, 5-06 Egg The above difference in osmotie pressure is accounted for by the diminution in the inorganic salts of the egg as compared with the blood serum. This was ascertained by estimating the chlorides in egg-white % and plasma by Volhard’s method, after incineration, but as only . 0-03 to 0°02 grm. of chlorine was found in the ash owing to the small quantity of material available, the following table is not of great aceuracy : Duck Plasma, per cent. chlorine Egg white, per cent. chlorine 0-278 oom 0-080 0-276 ms 0-088 0-312 ay 0-088 vi 0-104 Mean 0°287 °,, Mean 0-090 °,, As NaCl 0-473 °,, As NaCl 0-148 % To examine the changes during incubation, eggs were placed in a Hearson egg-incubator and maintained at 40° to 41°C. Samples were __ 4 taken out at intervals, and their freezing point determined with a r Beckmann thermometer, care being taken to avoid freezing out of the ' Proe. Royal Dublin Soe,, Vol, X11 (N.S.), May 1909, THE OSMOTIC PRESSURE OF THE EGG 481 nt, water, thus leaving a too concentrated solution. The zero of @ ruometer was re-determined frequently, both in the apparatus sds suai in powdered ice and water. Vigorous and continued stirring is necessary in dealing with viscous liquids such as eggs. It is to be regretted that, possibly owing to the lateness of the season—August and ptember-—most of the embryos had died before the shell was broken. is is not a very serious drawback, however, as owing to the variations the freezing points of fresh eggs of Gallus, from 0-427° C. to 0-480° ©. in a dozen determinations, quantitative results proportional to the time of incubation are not to be expected. The experiments are tabulated maize present Noembryo Musty eggs 0-480° C. - i 0-458°C. -- -—~ 0-605° C. 0-532° C. 0-620° C. 0-565" C. — 0-656° C. embryo present 0-605° C. 0-538° C = 0-565° C. pas as 0-563" C _ — 590°C. 535° C. _ — 0-550" C. _ 0570 C. 05388" C = 0-590" C, — = OGIEC. -_- 0-633°C. embryo present 0-598°C, * 533°C. co ras — 0-658°C. embryo present = ; -- 0-680°C. embryo present Control egg. in room at 15° to 20° C. for twenty days A = 0-480° (, In these determinations the whole of the liquid contents of the egg tube containing the thermometer. It may be seen from the table that _ there is a rise in the numerical value of the depression of freezing point throughout the period of incubation, the final value, 0°590° C. to O-611° C., being about that given by the blood, 0°591° to 0-662° C., mean 0-607° C. On the other hand, eggs containing no embryos show a similar rise, but to a much less extent; apparently, as the figures range from 0:532° to - 0550° C., there is an initial rise due possibly to evaporation, although the egg chamber is nearly saturated with water vapour. Thus the rise in osmotic pressure during incubation may be divided into two parts :— (a) The rise, as shown by unfertile eggs, probably due to evaporation, (b) The rise due to metabolism of the embryo. Oa, 482 BIO-CHEMICAL JOURNAL In connection with the latter it is to be noted that both yolk and white become much less viscous during incubation if there is a developing embryo, probably owing to the presence of an enzyme Maier cd reserve materials. “3 Eggs with or without embryos, but which were musty, gave high - values in every case, 0°680° C. being the maximum obtained, though itis quite reasonable to expect that if bacterial action had been more extensive a much higher figure might have been reached. More attention was paid to this effect of bacteria in the second series of experiments than in the first, so this, combined with the impossibility of detecting putrefaction in its initial stages, may account for some of the high values of A obtained for eggs with embryos in the early stages of incubation, — The difference in osmotic pressure between the blood and eggs of birds, together with the gradual rise in pressure of the egg to approximate isotonicity with the blood, may, perhaps, be accounted for by the following speculation. There is much evidence that the ontogeny of an— organism is a more or less abbreviated repetition of its phylogeny; by extending this view, based on morphological grounds, to physiology, there is reason to believe that birds are descended from ancestors with a lower osmotic pressure, about five or five and a half atmospheres. _ Fossil remains point to the reptilia as the class from which birds developed. It remains to examine the osmotic pressures of the blood of this group. The following determinations are available :— A P Thalassochelys Caretta, 1. = — 0615 ... Bottazzi and Dueceschit = — 0-602 -- Rodier ? Emys europoea = — 0-463 to-- 0-485 ... Bott. and Duce. (freshwater species of above) Thus the osmotic pressure of the blood of the only freshwater reptile which I have been able to see recorded, is not very different from that of the eggs of birds, being, in fact, within the limits of variation, It may seem fanciful to regard the osmotic pressure as a hereditary character transmitted with great regularity, but sueh a possibility seems well worth serious attention in view of the high elaboration of the organs regulating this pressure in all the vertebrates, a regulation not quantitative only, but qualitative. In this connection Loeb’s researches (see ‘ Dynamies of Living Matter’) show how marked is the effect of a qualitative difference in the salts present upon developing 1 Quoted from Rodier. * Station Zoologique d’Arcachon, 1899, a 5 4 ; THE OSMOTIC PRESSURE OF THE EGG 488 embryos. The whole question of the constancy of osmotic pressure is diseussed-at tength by E. H. Starling (see ‘ Fluids of the Body’), where _ Macallum’s interesting views on the origin of the blood plasma ‘salts from the waters of a pre-Cambrian ocean are also considered. So apart 2 altogether from the, possibly misleading, agreement in osmotic pressure ___-with certain reptilia, it seems not unreasonable to suppose that birds are _ descended from a stock which possessed a considerably lower osmotic pressure. Turning now to the egg membranes which are easily observed, the existence of a fair degree of semi-permeability may be demonstrated by the experiment figured by Bergin and Davis (‘ Principles of Botany’), in which a chip having been taken out of the rounded end of an egg, over _ the air space, without piercing the outer membrane, a glass tube of narrow bore is inserted into the pointed end and cemented in position. _ The contents of the egg will slowly rise in the tube to a height of over a metre when the egg is placed upright in a few centimetres depth of distilled water. If the yolk be pierced it will colour the column, which will stay at its upper limit for several days and then slowly sink. That _ this membrane is somewhat permeable to sodium chloride may be seen by adding a few drops of silver nitrate solution to the water in which the egg is standing, when a turbidity or precipitate is produced. If, however, _--—s the outer membrane be pierced, the rise in the tube will only amount to a : few centimetres, for the inner membrane is much more permeable than RE the outer, as seen by the silver nitrate test. It is to be noted that, while the two membranes are in contact throughout the greater part of the egg, at the blunt end they are separated by the air-space. Acetic acid also penetrates the two membranes, for if an egg be placed in the dilute acid till the shell is dissolved, and then washed and placed in distilled water, it will be found that the egg swells greatly—to ; nearly twice its former volume, in fact. The water surrounding it a becomes acid, even after numerous changes. If now the egg be placed first in a strong solution of sodium chloride, and then in water, it will swell again, and in this condition may be freely handled without risk of uncture. If the membrane be cut after the shell is dissolved, the egg will be found to be coagulated by the acid, having the appearance of having been boiled. The yolk is also enclosed in a delicate membrane with some degree of semi-permeability, for on carefully breaking a fresh egg into water, and rinsing to remove the white, the yolk swells considerably and becomes a 184 BIO-CHEMICAL JOURNAL paler yellow with a turbid appearance. The germinal dise soon disappears " from sight, apparently sinking into the yolk, which by the dilution is now . of a lower specifie gravity, in which the dise can no longer float. This is evidence that the dise is surrounded by a membrane either impermeable, _ or more probably less permeable, to water than the yolk membrane. In _ this distended condition the yolk membrane is very delicate, being ruptured by the weight of its own contents if the water be drawn oft from around it. In spite of this slighter permeability of the germinal dise as compared with the yolk membrane, there seems to be no doubt that the dise is normally approximately isotonic with the egg as a whole. In the above experiment I have been unable to observe whether the dise really sinks into the yolk; it becomes lost to view, but it is just possible that in the imbibed condition it may not be practicable to distinguish it from its surroundings. That the germinal dise is not necessarily absolutely isotonic with the fluid surrounding it, but is rather in a state of osmotic equilibrium with it, seems extremely likely from the researches of Moore and Roat (Bio-Chemical Journ., p. 55, Jan. 1908) on the equilibrium between the cell and its environment. These authors showed that there was normally a difference of 0°02° to 0-03° C. between the freezing point of the serum and red blood corpuscles of the pig, and that diluting the blood affected serum and corpuscles unequally. ‘Che work of Dakin on the variations in the osmotic pressure of marine vertebrates by change in the external medium (Bio-Chemical Journ., p. 475, Dee. 1908), shows that in these cases also the systems are in equilibrium rather than isotonic, SUMMARY The osmotic pressure of the egg of Gallus bankiva, as calculated from freezing point depressions, rises during incubation from about 55 atmospheres to about 73 atmospheres, the latter value being approxi-_ mately that of the osmotic pressure of the blood of the same bird. Bacterial action during incubation may cause the pressure to rise to over eight atmospheres. The view is put forward that birds are descended from organisms with an osmotic pressure of five atmospheres or less. I have much pleasure in thanking Professor A. F. Dixon for his advice and the loan of apparatus, and also Professor H. H. Dixon for permission to carry out the work in the School of Botany. > + Pa % J “ ae | Catan / he, SIAL Ry i Pt a . 7 ¢ ¥ x + * ‘ . ? oh. * $ . 6) 68 : é 3 ’ d = - : : . . : d o a Se ; ‘ ‘ wl - * 4 a az) - We. : - a] o, 4 , * : ° Sr. . ‘ \ : ‘ ‘ . * : 5 * = ‘ 7 re ‘ ‘ ‘ 4 ‘ « eA . ? » & ‘ 3 . a ¥ cre . - et Oy s . . 4} te 4 b os P ‘ i ‘ '