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_ BENJAMIN MOORE, M.A., D.Sc. “EDWARD WHITLEY, M. A..
VOLUME IV 1909 Pte $5.4 ee. g 7 |
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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 "<<a from its own intrinsic interest in the whole wide field of mn ge One of the most obvious lines of attack in-inv pagakeas the reactions
ae . change in mS scien. or orientation, in fixed or sessile organisms. Tt must, however, be clearly borne in mind that this movement is an __ index of other things, that the underlying problem is ultimately and __ essentially a chemical one, or, better expressed, one of chemical transformation of light energy.! The organisms move because of an action of light upon chemical constituents in the cells, that is to say, there is a _ change in the metabolism of the cell stimulated, giving rise to the movement of the organism. Also, according to the nature and condition _ of both cell and light-stimulis, which form the two inter-acting factors, the character and sense of the movement of the organism will vary. Thus, we shall see that with the same condition and previous treatment of the organism, the reaction varies and becomes positive or negative with varying intensities of the light-stimulus, and, secondly, with the same constant intensity of light-stimulus, the reaction varies when the _-_——s previous history and condition of the reacting organism have been P ms artificially varied. That is to say, the light induces chemical alterations in the cell, and the nature and amount of the chemical changes vary with ____ the two factors, the condition of the cell at the time, and the intensity of the incident light. ‘Tt has been clearly pointed out by Loeb that the orientations or _ tropisms of sessile organisms, and the movements of free organisms __ towards or away from light, are essentially the same in character, the free organism being first orientated and then, by the action of its locomotor organs, carried in either direction according to the sense of the previous orientation. This is a fundamental observation which to a certain extent unifies the problem, but there still remain the questions of why the light induces
1. This view has been also put forward by Loeb, Dynamics of Living Matter, 1906, pp. 112 seq.
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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. <p Vm 1-84 %
2 0-4194 & = 55 % = 1-86 % r mn | Average = 1°85 %
by PHOSPHORUS © ; ' 1. 0-872 grm. Substance = 27:22c.c.5 NaOH = 39% 2 60-2918 a = 21-37 me = 4-05 %
Average = 3-975 N:P =1: 103 %
C ann H ee 1. 01747 ¢grm. == _. 0-4234 grm. CO. = 612%C dt pate and 0-164] grm. HyO = 10-44% H gm. = 0-4688 CO. = 66-41%C and 0-1784 erm. HO Fe
he fale combined with its physical qualities show that this > 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 <p tube, it melted at 42°5° to 43° C., and solidified sharply at 40°5° C. When warmed with pyridine it dissolved, but on cooling, the solution set to an opaque gelatine-like mass. It did not give Salkowski’s test and did not absorb ine. This substance, therefore, is very probably a hydrocarbon ‘similar to those found in the pupae of the silkworm moth. ____B. The part easily soluble in alcohol. The alcohol was diluted to out 90 per cent. strength, and on allowing a drop of the solution to 7 stallise under the microscope, thin plates closely resembling those of terol were observed. As the substance was somewhat soluble, even in the diluted alcohol, methyl alcohol was used as a solvent, and from this after repeated crystallisation, about 0-1 gram of crystals, with a constant melting point of 139° to 140°. was obtained. That these were not cholesterol was shown by the following tests:—-(i) When moistened with lgpkibentrated sulphuric acid the edges of the crystals did not turn red. (ii) On benzoylation in pyridine solution no action took place, as the substance easily soluble in alcohol, crystallisimg in oblong plates and melting sharply at 139° to 140° C., was recovered unaltered. Pe The body gave Salkowski’s test with chloroform and sulphuric acid exactly as cholesterol, but owing to the small quantity of the pure substance that could be isolated, it was not possible to characterise it _ further. It would, however, appear to be similar in its general behaviour __ to other members of the cholesterol group. ANNULATA. Cueroropa. Lumbricus terrestris, the earth-worm. ee! -worms,! weighing 286 grams, were killed and ground up with ___ sand and plaster of Paris. The dry mass was extracted for five days with i ether, and the extract, on saponification, gave a considerable quantity of _ a pale brown, slimy soap. The filtrate from this yielded 0-95 gram of 4 unsaponifiable residue, which was practically solid at L00°C. It was soluble in 90 per cent. alcohol, with the exception of a trace of brownish x matter. A microscopic examination showed very badly-formed choles- s terol plates, together with a few long, blunt-pointed, needle-shaped __erystals. Crops weighing 0'1 gram and 0°22 gram, respectively, were . separated, and, after re-crystallisation from acetic ester, melted at 142° to 148° C., and consisted largely of plates. The 0-31 gram of substance ___ thus obtained was benzoylated in pyridine solution in the usual manner.
AL The worms used were the lob or dew worms of the freshwater fisherman.
86 BIO-CHEMICAL JOURNAL
‘The benzoate obtained was very insoluble in alcohol, and after re-crystal- lisation from acetic ester, melted at 144° to 145°C. to a turbid liquid, which became clear at 180° C., and on cooling showed a brilliant colour play. ‘he filtrate from this was examined under the microscope, when, beside a few crystals of the benzoate, a number of minute spherular crystals were observed. .
All the residues, consisting of a dark brown, sticky resin, were put together, dried, and treated with benzoyl chloride in pyridine solution. No crystalline matter, however, could be obtained from them. The total yield of cholesterol was thus about 0°3 gram, or 0'1 per cent. The tissues of the worm thus contain cholesterol itself as their most important cholesterol constituent, although some indication of the presence of — another crystalline body in small amount was observed.
ECHINODERMATA. AsreromeEa. Asterias rubens, the starfish. ~The starfish, which were still living when received, weighed 1,658 grams. ‘They were minced (tlie juices being collected in plaster of Paris), ground up with sand and plaster of Paris, the mass allowed to dry, and again reduced to powder. After extraction for twenty-one days with ether a pale red-brown extract was obtained, which, on saponification gave a large quantity of a firm porridge-like soap. The red-brown colour of the filtrate was removed on washing with water, leaving the ether solution almost colourless. The crude residue, which was yery fluid at 100°, weighed 3°5 grams, and dissolved almost completely in 90 per cent. alcohol. A microscopic examination showed that a mixture of substances was present. Plates, closely resembling those of cholesterol, were seen, together with masses of minute curved needles. After many attempts it was found possible to effect a partial separation in the following way. ‘he crude substance was treated with dry pyridine. A portion proved very