UNIV. oF TORONTO lire papy THE BIO-CHEMICAL JOURNAL EDITED BY BENJAMIN MOORE, M.A., D.Sc. AND ERPWARKRD:: WHITLEY, “MA. VOLUME Iii 1908 t COPYRIGHT BIO-CHEMICAL DEPARTMENT, JOHNSTON LABORATORIES UNIVERSITY OF LIVERPOOL JAUAUO ta aa ee es oe aby tM AROO AINE ae sae ; ae aia: = Gime eh ee | bE eee \ + . MS G3)? sean res OT ALOU LR Ke A TO] ARI Ce ai LOTTA S C1 Te? Ae ae eins . e CONTENTS OF VOLUME III The Action of Muscarin and Pilocarpin on the Hearts of certain Vahobs, with Observations on Seasonal Changes. By Hugh MacLean, M.D., Lecturer on Chemical Physiology, University of Aberdeen Observations on the Action of BocrrNavia Diffusa. By Lal Mokke Ghoshal, IMS. _ A Contribution to att Study of Calcium Metabolism. By S. W. Pueciiian _M~.B., B.S. On the Eqilibrium Pee the Call Pia its may Ses in ee to Soluble Constituents, with special reference to the Osmotic Equilibrium of the Red Blood Corpuscle. By Benjamin Moore, M.A., D.Sc., Johnston Professor of Bio-Chemistry, and Herbert E. Roaf, M.D. , Lecturer on Physiology Univ sg - of Liverpool Prosecretin in nine to Diabetes Mellitus. By F. A. Bainbridge, M. A., -M.D., Gillson Scholar in Pathology, Guy’s Hospital The Presence of a Nitrate Reducing Enzyme in Green yi? Em Annie ’ A, Irving, B.Sc. (Lond.), and Rita Hankinson, B.Sc. (Lond.) Observations on the Significance of the Haemosozic Value of ie Blood Serum. By Captain D. McCay, M.B. ite U. he I.M.S., Professor of Phy: sii Medical College, Calcutta : On Variations Observed in the Cidmentine of "Gilivm Ge catia Prepared by Different Methods. By Wm. C. M. Lewis, M.A. Notes on the Action of Atropine, Hyoscyamine, Hyoscine, Scopolamine, -Duboisine, and Daturine. By W. Webster, M.D., C.M., Anaesthetist to the Winnipeg General Homies Lecturer in Anaesthetics in the Manitoba Medical College a ; ; A Note on the Dagibeann of the Salts in Hacmolyis, iy ‘Mbak Woelfel, M.D. On the Action of eras Onpilishag Agerits upon Blood Re aisae By J. A. MacWilliam, M.D., Professor of Physiology in the University of Aberdeen On the Presence of Oxydases in India~Rubber, with a Theory in Regard to their Function in the Latex. By D. Spence, Ph. D., A.IL.C., Research Chemist to the Liverpool Institute of Commercial Research in the Tropics On the Application of Barfoed’s Reagent to show the Hydrolysis of Disaccharides by Enzymes. By Herbert E. Roaf, M.D., Lecturer on ee: University of Liverpool ; A Rapid Method for Separating Flaca Acid rae tides By Bates E. Roaf, M.D., Lecturer on Physiology, University of Liverpool , A New a eaceic Method to Show the Activity of either ‘ Peptic’ or ‘Tryptic’ Enzymes. By Herbert E. Roaf, M.D., Lecturer on patents University of Liverpool On the Formation of Lactic Acid aid Carbonic “Add dithe Masiclae Contraction and Rigor Mortis. By P. W. Latham, M.D., ec Professor of Medicine in the University of Cambridge (1874-1894) On the Complete Hydrolytic Decomposition of Egg-Albumin at 180° C. By P. W. Latham, M.D., Downing Professor of Medicine in the University of Cambridge, (1874-1894) > On the Synthesis of Living Albumin. By P. W. Dathath: M. D.. Dow ning Professor of Medicine in the University of Cambridge (1874-1894) The Osmotic Concentration of the Blood of Fishes Taken from Sea-Water of Naturally Varying Concentration. By W. J. Dakin, M.Sc., *51 Exhibition Scholar, University of Liverpool . : : tie = ‘ . ; PAGE ii CONTENTS The Effects of Variations in the Inorganic Salts and the Reactivity of the External Medium upon the Nutrition, Growth, and Cell-Division in Plants and Animals. _ By Benjamin Moore, M.A., D.Sc. , Johnston Professor of Bio-Chemistry; Herbert E. Roaf, M.D., Demonstrator and Assistant Lecturer in PAYNE 2 ; and Robert E. Riowiles. M. ‘cx University of Liverpool . ‘ An Investigation of the Toxic Actions of Dilute Solutions of the Salts of certain Heavy Metals (viz.: Copper, Iron, Nickel, Cobalt, Manganese, Zinc, Silver, and Lead) upon the Bacillus Typhosus, with a view to Practical Application in the Purification of Shell-fish, By Benjamin Moore, M.A., D.Sc., Johnston Professor of Bio-Chemistry, University of pei and Jame Leonard Hawkes, M.D., (Liverpool) Note on the Chemical Composition and Physical Properties of ree Calculi. By J. Sydney Rowlands, M.D. (Liverpool), M.R.C.S., L.R.C.P., Thelwall Thomas Fellow in Surgical Pathology, University of Liv erpool On the Presence of an Oxidising Enzyme in the Latex of Hevea Braziliensis. By D. Spence, Ph.D., A.I.C. Experimental Pekdenes of the Local Production of * ee By H. Leith Murray, M.D., Pathologist, David Lewis Northern Hospital, Liverpool ; R. Stenhouse Williams, M.B., D.P.H., Assistant Lecturer on Public Health Bacteriology, University of Liverpool ; and J. Orr, L.R.C.P. and S.E., D.P.H. Experimental Inoculation of Meningo-Coccic Vaccine. By R. Stenhouse Williams, M.B., D.P.H., Assistant Lecturer on Public Health Bacteriology, University of Liverpool; H. Leith Murray, M.D., Pathologist, David Lewis Northern Hospital, Liverpool ; and J. Orr, L.R.C.P. and S.E., D.P.H. : Changes in the Chemical Composition of the Herring during the Repro- ductive Period. By T. H. shag Professor of ere Queen’s it Belfast ; The Microchemical Chaat Bee in Apebaliais ‘wife a Noe! on the Incidence of the Disease in Various Countries. By Owen ‘T. Williams, M.D., B.Sc. (Lond.), M.R.C.P., Hon. Assistant Physician, Hospital for Consumption ; Pathologist to the Children’s Nee Demonstrator of Bio-Chemistry, University of Liverpool The Action of Pilocarpine on de selec By James M. McQueen, M.A., B.Sc., M.B., Ch.B., Senior Assistant in Physiology, University of Aberdeen The Action of ~—_ on Mammalian Uterus. By Harold J. Fardon, M.A., M.D. ; : The Effect of Acid ids Alkali on fine Samii: Saciaeta of Si sou Pidstéine. By L. Adamson, M.D., University of Liverpool, and Herbert E. Roaf, M.D., Lecturer in Physiology, ‘University, of Liverpool Urobilin Excretion in Diseased Condition. By N. F. Suidvedbe, M. A., M. D. (Bombay), M.R.C.P. (Lond.), Professor of Bacteriology, Grant Medical College Honorary Physician, Jamshedji Jijibhoy Hospital : Variations in the Free Hydrochloric ‘Acid of the Ginssle a stctois in Cancer and the So-called ‘ Physiologically Active’ Hydrochloric Acid. By Benjamin Moore, M.A., D.Sc. fii Professor of BIE hE: said of Liverpool ‘ : The Hydrolytic Sacha of Savakabiatic By H. EB. Roaf, M. D., ‘cane in Physiology, University of Liverpool : Variations in the Osmotic Concentration of a Blood nd Guickornie Fluids of Aquatic Animals, Caused by Changes in the External Medium. By W. J Dakin, M.Sc., 1851 Exhibitioner, University of Liverpool - PAGE 279 sid 346 S5t 353 359 366 391 402 405 422 439 “449 462 473 THE ACTION OF MUSCARIN AND PILOCARPIN ON THE HEARTS OF CERTAIN VERTEBRATES, WITH OBSERVATIONS ON SEASONAL CHANGES By HUGH MacLEAN, ML.D., Lecturer on Chemical Physiology, University of Aberdeen. From the Physiological Laboratory, Aberdeen University (Received October 14th, 1907) Whether muscarin and pilocarpin, when applied to the heart, act directly in virtue of their depressing influence on the inherent. motor activity, or indirectly as excitants of the cardio-inhibitory mechanism, is still an open question. : Schmiedeberg and Koppe’ attributed the action of muscarin to its influence as a stimulant of inhibitory ganglia. Kobert® sub- sequently emphasised the same idea. Later on Schmiedeberg’s general results were confirmed by Prevost and Monnier*®. Since. that time many investigators have published results favouring the theory of vagal stimulation, and many authorities. of the: present day accept the view that these drugs act by stimulating the vagus endings. On the other side, the direct action of a comparatively weak solution of muscarin or pilocarpin as a paralyser of the cardia¢ muscle substance, has been emphasised by various authorities. One of the strongest advocates of the theory of direct depression of the cardiac motor mechanism is Gaskell*. After the application of muscarin he found a general depression of rhythm, strength, ‘tone and conductivity. He holds that the drugs act on the muscular tissue atonically. 1. Schmiedeberg u. Koppe, Das Muscarin, Leipzig, F. C. W. Vogel, 1869, S. 28; Studien. uber Herzgifte, Wiirzburg, 1871. 2. Archiv. }. exper. Pathol. u. Pharmakol., Bd. 20, S. 92. 3. Gaz. méd. de Paris, 1874, p. 243. . 4. Philosophical Transactions, 1882. ‘fournal of Physiology, Vols. II, IV, and VIII. Schéfer’s Text-book of Physiology, (1900) p. 223. 2 BIO-CHEMICAL JOURNAL DescrIPTION OF SAMPLES OF MuscarIN AND PILOCARPIN USED IN THIS INVESTIGATION In all investigations bearing on the action of muscarin with regard to cardiac inhibition, it is obviously of primary importance that the drug should be pure. Schmiedeberg has pointed out that the samples obtained from dealers are never sufficiently purified, and are liable to contain, amongst other impurities, traces of an atropin- like base. The solution used in these experiments has been kindly supplied by Professor Schmiedeberg himself, and a similar solution has been used by Gaskell in his experiments on muscarin action. The latter observer found that the strength was such that one or two drops placed on the sinus of the frog’s heart were sufficient to cause immediate standstill; placed on the sinus of the tortoise, the beat ceased immediately’. With this solution of muscarin the majority of my experiments were made; in suitable cases one drop or even less applied to the sinus gave the characteristic effect; if one or two drops were not sufficient, an increase in the quantity never gave an immediate effect, the result of the exhibition of a large quantity being essentially different in nature from that of a small amount. | For some experiments pilocarpin was used, and in order to insure the elimination of any possible errors arising from impurities or _ defects in the salts, five different specimens were used—three nitrates and two chlorides—two of these being specially certified to contain no impurities. ‘The solutions were made up in Ringer’s circulating fluid, so that 1 drop as obtained from a special pipette contained exactly 2 mgrs. of the salt ; by dilution it was thus easy to apply to the heart any fraction of a mgr. of the pilocarpin. The activity and purity of the samples used, I have often demonstrated on the normal heart of the frog and eel, when a small quantity applied to the sinus produced the usual results. | 1. Fournal of Physiology, Vol. VIII, p. 407. ACTION OF MUSCARIN AND PILOCARPIN 3 Heart or Froc On Seasonal Changes in the Frog-Heart, and their Influence on Inhibition —When making observations on the heart of the British frog in the months of January, February and March, | was surprised to find that very considerable changes were in evidence with regard to the manner in which the heart responded to vagus stimulation. In some cases the inhibitory function of the vagus seemed to be quite inactive, while in others it was manifested only to an exceedingly slight degree. In January, certain hearts responded to faradisation applied to the ‘ posterior white crescent,’ but in many the vagus influence seemed to be very slight indeed, the heart quickly escaping from the effects of stimulation and regaining its former rhythm ; on the application of a stronger faradic stimulus there was occasionally a slight inhibitory effect, but it was always of very short duration, and soon no strength of stimulus had any effect. Stimulation of the vagus trunk or medulla did not, as a rule, alter in any way the rate of cardiac action, though in a few cases there was a rather ambiguous result produced probably dependent on the intermixture of accelerator _and inhibitory fibres in the frog’s vagus (Rana temporaria). In February and March the same features were in evidence, only the elimination of vagus influence became gradually more pronounced, till towards the end of March it often happened that no inhibitory effect could be demonstrated. In April, also, there was little response to vagal stimulation, but in May the heart was found. to be gradually assuming its normal condition, for faradisation of the posterior white crescent often resulted in a. marked slowing or tem- porary stoppage of the heart ; even in May, however, normal con- ditions did not by any means prevail. In the autumn months the result of vagus stimulation on British frogs was quite different, the majority giving unmistakable evidence of an active inhibitory mechanism; some German frogs examined in October, however, gave no result, and it is interesting to note that these were distended with ova (Rana esculenta). Many observers have noted certain changes in the ordinary 4 BIO-CHEMICAL JOURNAL frog-heart during the winter and spring months, but there does not seem to be any definite account of the very marked phenomenon in regard to this diminution of the inhibitory power, and the exact cause of it is difficult to establish. A striking fact is the observation that in a general way this change coincides more or less with the season of sexual activity in the frog, while such factors as temperature and the diminished activity of the animal in the cold season may also have some influence. Ringer’ noticed that certain antagonisms were slight during the breed- ing season. Pandelejeff? also found that the antagonism of atropin for quinine on the frog-heart was affected by the time of year. In summer frogs, quinine arrested the heart in diastole and atropin caused the heart at once to resume its pulsations. In winter frogs, quinine arrested the heart much more slowly, while atropin, instead of obviating actually increased the arrest. Low temperature would not entirely account for the changes observed in regard to inhibition, for while the mean temperature in January was fully 6° F. lower than that in the beginning of April, yet these changes were much more pronounced in April. Sexual activity in the frog at any rate seems to be the dominant factor in the production of this change. Jordan found the action of muscarin difficult to demonstrate in - winter frogs.® The action of Muscarin and Pilocarpin on the Frog-Heart affected by Seasonal Changes.—The diminution, or in some cases, absence of any apparent inhibitory action in response to stimulation of the cardio-inhibitory apparatus in frogs affected by the seasonal changes discussed above suggested the idea that advantage might be taken of the condition in order to test the mode of action of muscarin and pilocarpin. Here was a case where in certain frogs there was a comparative elimination of the function of inhibition, while in others it was markedly less than normal, probably an expression of some profound general change. 1. Fournal of Physiology, Vol. III, p. 115. 2. Lancet, July 31st, 1880, p. 176. 3- Arch. f. exp. Pathol. u. Pharmakol., VIII, 1878. ACTION OF MUSCARIN AND PILOCARPIN 5 If muscarin stopped or slowed the heart in these frogs, then this drug could not be acting through a medium which had been proved inactive by other means. In other words, if muscarin acted as an excitant of the cardio-inhibitory nerves, its effect on the cardiac rhythm should bear some comparatively constant ratio to the effect of electrical stimulation of the inhibitory apparatus. If, on the other hand, muscarin acted as a direct depressant of the inherent motor mechanism of the cardiac muscle, then the fact of the inhibitory apparatus being inert should not influence its action when applied to the heart substance. During a long series of experiments carried out at different seasons during the last three years, it was found invariably that when electrical stimulation of the inhibitory mechanism through the posterior white crescent failed to affect the rhythm of the heart, then no effect was produced by the application of weak solutions of muscarin or pilocarpin. When electrical stimulation gave a positive result (z.¢. slowed the heart) muscarin also gave a like result in many cases; as a general rule, faradisation quickly resulted in tiring out the already attenuated inhibitory apparatus so that subsequent increased stimulation had no effect, and in such a case muscarin likewise proved ineffective; if the faradisation was immediately - stopped after an effect was observed, the subsequent application of muscarin generally resulted in slowing the heart for a time; an escape from the inhibition, however, generally occurred in a com- paratively short time ; this setting free of the heart could be brought about by faradisation and by muscarin or pilocarpin. Even in late summer and autumn when normal conditions apparently prevail, many variations were met with in the course of this investigation, but no instance was observed in which a heart unaffected by faradisation of the posterior white crescent gave any result in the direction of slowing or stoppage after the exhibition of muscarin. : Fig. I’ represents a tracing from a ‘normal’ frog heart, showing result of a local application of a few drops 2 per cent. pilocarpin 1, All the tracings réad from left to right. 6 BIO-CHEMICAL JOURNAL solution; heart is seen to be considerably slowed. ‘The result of atropin is also well seen. Drum was stopped for a few seconds on each application to give drugs time to act. ee a Nowch Cect ° seeks 28/e PLscofp | Whhhbhanh>AhhnhaAnhnnnhhjnhhnlhly Anh hhh hha Fic. I. Effect of pilocarpin on frog’s heart. (Autumn). Fig. II is from an ‘abnormal’ heart in which faradisation failed to cause inhibition, showing result of pilocarpin application. Here such a strong solution as 4 per cent. pilocarpin failed to influence the beat even after ten minutes. | | ue | Rlncop ret, Erk 4h Sat 86% RE Rip for 10 me lic Fic. I]. Effect of pilocarpin on frog’s heart. (Spring). With large doses of muscarin or pilocarpin, direct depression of the properties of cardiac muscle became evident; the time and amount of drug required for this varies greatly in different frogs according to the vitality of the heart and animal in general. In nearly all my experiments the depression of these special functions of cardiac muscle followed a well marked rule. The first effect of a large dose seemed to be on the muscular tone. Very soon after the application of a strong solution of pilocarpin the whole heart became somewhat flabby, both the auricle and ventricle exhibiting well marked distension during systole. ‘This did not seem to influence ACTION OF MUSCARIN AND PILOCARPIN 7 immediately the rate or force. Some time afterwards the rate became less, due probably to a diminution of excitability. A weakness of the cardiac contractile force was next manifested, followed often by a condition in which a considerable interval elapsed before the conduction of the auricular beat to the ventricle was affected. The order, therefore, in which pilocarpin seems to depress the properties of cardiac muscle is— Diminution in (1) Tone (z) Rate (3) Contraction force (4) Conduction. A heart slowed by direct depression by a large dose of pilocarpin acting for a comparatively long time, differs from a heart quickly arrested or slowed by a small dose in the fact that the rate of the former heart is not increased by atropin, whereas digitalin immediately caused a marked acceleration. On a heart quickly slowed by a small dose, digitalin in weak solution has little or no effect when the in- hibition is strong, but atropin-gives a most marked and immediate result. Several of the above points are seen in Fig. III. In A fara- disation was applied at the point marked, but without effect. B was taken half a minute after the application of a few drops 2 per cent. pilocarpin; no change is apparent. The heart was then covered with § per cent. pilocarpin nitrate solution and left for 25 minutes (see C, D, E); it was then again covered with pilocarpin solution every 5 minutes until 65 minutes had passed; gradual slowing due to direct muscular depression is seen, but this is not marked till after one hour or so. After 65 minutes, atropin was applied (E) : heart still slow. Four minutes after first dose of atropin more was applied, the result being to cause more marked slowing. Digitalin in weak solution had a marked effect, the rate being instantly quickened (f). | In a strong frog the heart may often be soaked with pilocarpin nitrate, from § to 10 per cent. solution, and yet be able to beat quite strongly after an hour or more, though in direct contact with the 8 BIO-CHEMICAL JOURNAL AN i) Ah Sa anit W Faradisation Pilocarp. sol. A B 40° 45 50 D (4 bs be X t+ ; ve eabaatatatstieet taal t ratte ANIARAR TATU EATER NAR ‘ 55 60° 65’ Atropin’ applied E VV Yona hanunannannnnannnsss AAU AU Un AL 4' after atropin Digitalin applied F Fic, III. Direct weakening action of pilocarpin after long period, with effect of atropin and of digitalin. ed ACTION OF MUSCARIN AND PILOCARPIN 9 drug the whole time; in general a diminution in rate is observed in much less than an hour. | Weaker frogs vary considerably in regard to the time and amount of drug required to cause depression, but they never give an indi- cation of depression unless the dose is much greater than is usually necessary to produce a result in a normal frog-heart—also the time necessary for the occurrence of appreciable direct depression is much greater than is generally required for the causation of slowing or stoppage in a normal heart. . The following experiments serve as an indication of the condition obtaining in certain frogs during the months when sexual activity is most manifest ; it will be noticed that in the cases quoted, in- hibition was often found to be practically absent ; such cases serve best to bring out a certain phase of the parallel action of muscarin and faradic stimulation. In many experiments there was, of course, a certain amount of inhibitory power present, but as a general rule, as the result of a great number of experiments repeated during several years, it was found to be markedly diminished during the spring season. In all experiments mentioned in this paper, faradisation was applied to the ‘ posterior white crescent.’ EXPERIMENTS _ Experiment I.—January. Rate Rate Remarks on before after Condition of heart Drug Dose Faradisation (per (per heart minute) minute) Exposed in Muscarin — Negative 22. oa — in situ ; 1 drop ous a 22 Strong beat fairly vigorous yee Fi bod 22 na a 2 drops ee ie 20 -— 4 » Pas is 21 Heart quite strong after 8 drops After 10 minutes rate 18 per minute and fairly strong ; gradually became weaker Atropin 2 drops oe cas 14 Weaker still I % sol. Digitalin Few drops ab Ae: 23 Slightly stronger of weak beat solution BIO-CHEMICAL JOURNAL | fe) Experiment II —February. Remarks on heart Drug Dose Exposed in Pilocarpin — situ; strong nitrate beat (3% I drop I 5; I 5 I ” I» 2 drops 3 bb) 4 Leen Experiment II1I.—March. Excised ; Muscarin — feeble heart 4 drop I ” I ”> I Direct weakening ac Rate after (per Condition of heart minute) minute) Rate before Faradisation (per Stopped heart 30 for Io seconds ; afterwards failed to affect it Negative 18 30 31 27 30 29 25 26 26 25 Quite strong Quite strong After } hour beating strongly 18 per minute Digitalin increased rate to 25 per minute Weaker Still getting weaker Very. weak; soon only faint con- tractions noticed passing over heart; then stopped Digitalin no effect Se Aon ACTION OF MUSCARIN AND PILOCARPIN II Experiment IV —April. Rate Rate Remarks on 2 before after Condition of heart... Drug Dose Faradisation (per = (per heart og minute) minute) Excised ; Muscarin _ Negative 16 — — fairly vigorous + drop bine es 16 — heart ee ree a 14 — a“ on: Bot I4 — oy iy: fi 16 — 5 ae sai a 16 — tie a ti 13 — Lois. ee vas 12 Beat rather weak ae vis oats 15 — Atropin 3 drops es re 12 Beat still weaker; (1 % sol.) heart placed in muscarin solution, continued to beat though weakly for several minutes; washed and treat- ed with Atropin —no change Helped slightly by digitalin Experiment V.—May Exposed in Muscarin — Slowed to 30 — — situ ; average of strongly IO per beating minute 4 drop “a oak 24 Strong I ys 13 hm ry dee bed 21 Strong | ae Som wae 25 _ ee in he 20 Slightly weaker 2 drops se ee 20 oo 1 drop Bad ae 18 Somewhat weaker but beating after other 6 drops Atropin—no effect Digitalin—beat quicker and stronger 12 BIO-CHEMICAL JOURNAL On tHE ANTAGONISM OF MuscarIN AND PILOCARPIN Many years ago Ringer’ concluded that pilocarpin antagonizes the action of extract of muscarin on the frog-heart, a curious result in regard to two drugs exhibiting such closely allied pharmacological action as pilocarpin and muscarin. Judging from the general action of these two drugs it would be expected that a heart slowed by a: certain amount of muscarin would be further slowed by an additional dose of pilocarpin. Instead of this, however, Ringer has found that in certain cases, a heart slowed by muscarin is accelerated by the application of pilocarpin, and he therefore concludes that pilo- carpin acts as an antagonist to muscarin. This curious phenomenon I have observed on several occasions, and the idea suggested itself that if muscarin acts on the cardio- inhibitory nerves, and not on the muscle directly, an explanation of this action might be afforded by the fact that the primary dose of muscarin resulted in a stimulation of the nervous mechanism, and thus brought about a slowing of the heart, whilst the subsequent acceleration of cardiac action by pilocarpin might be due to the increased dose paralysing the inhibitory mechanism to a greater or less extent and thus increasing the rhythm. In a heart where the inhibitory function was but little marked, this would probably happen. If this was the case the same result should be brought about by the application of more muscarin after the preliminary slowing by that drug. In the course of my experiments it was very obvious at certain seasons that a heart slowed by muscarin in small dose was, on the application of a little more of the drug with a view to increasing its inhibitory action, accelerated instead of slowed, and the same result was even more frequently obtained with pilo- . carpin. ‘This was not so much in evidence with muscarin, at any rate in the normal frog-heart, and here it would seem that a dose of the drug sufficient to paralyse the inhibitory apparatus is so great as to directly depress the muscle. In this case no result could be expected. Again, at the season of increased sexual activity, when 1. Fournal of Physiology, Vol. Il, p. 135. ACTION OF MUSCARIN AND PILOCARPIN 13 the inhibitory mechanism was practically functionless, this effect was not obtained; any depression of rhythm in this case, however, must have been due-to the effect of a large dose in depressing the cardiac muscle, and so no subsequent acceleration could be expected ; the phenomenon was best seen in a heart where inhibition was feeble but still active. The above observations do not account for many of the results obtained by Ringer, and are not inconsistent with the idea of the existence of a real antagonism between the two drugs ; on the other hand, it is obvious that the phenomenon described above would probably be accepted as a true antagonism, whereas it really is but an apparent one. Here the escape of the heart from the effects of muscarin while the drug is still in contact with the cardiac tissue admits of practically the same explanation, and the fact that within certain limits this escape can often be brought about by the application of an increased amount of the original drug, seems to be inconsistent with the idea of direct muscular depression; if, however, too much muscarin be added, depression of the muscle may of course be evident, and so prevent increase in the rhythm, while if the increased dose is too small to paralyse the mechanism, the inhibitory effect is more marked, as generally occurs in the normal frog-heart. In view of these observations, it would seem that in certain hearts at any rate the apparent antagonism of muscarin and pilocarpin is due to the condition of the cardio-inhibitory apparatus. Again, if muscarin produces its effects by its direct action on the cardiac muscle, it is difficult to understand how it should sometimes have its apparent effects considerably lessened by an additional dose. On a TENDENCY FOR THE EsTABLISHMENT OF IMMUNISATION TOWARDs MuscARIN AND PILOCARPIN IN THE FRoG-HEART A very important factor exhibited in the frog-heart is a tendency for the production of a certain amount of immunisation to muscarin or pilocarpin after the exhibition of these drugs. When the heart recovers from the effect of a small dose of muscarin, as often happens 14 BIO-CHEMICAL JOURNAL in certain hearts, it was found that a very much greater dose of the drug was required to produce slowing or stoppage a second time ; in many cases, as previously stated, it was not possible to produce a second effect, but in a fairly normal heart this could sometimes be done. I have never seen an instance of any heart escaping from the effect of a second dose being slowed by the application of more of the drug, except in cases where, after a comparatively long time, direct muscle weakening set in. It is interesting to note that many of the above results agree with observations made by Marshall on the mammalian heart’. In view of what we know of the behaviour of muscle towards depressant drugs, such immunisation would be difficult to explain if muscarin (or pilocarpin) is in small doses a direct muscle depressant, but is perfectly intelligible if the drug is a stimulant to nerve endings’—repeated doses leading to exhaustion of their function. That the action of pilocarpin is on nerve-endings, however, has been comparatively recently disputed by Matthews’, who still advocates direct action on the animal cell, at least in .the case of gland-cells. Heart or EE The heart of the eel, as shown by MacWilliam‘ many years ago, possesses a very marked susceptibility to vagus inhibition, and differs from the frog-heart both as regards the state of the cardiac tissue while. under the influence of vagal stimulation and in the peculiar manner in which spontaneous cardiac rhythm becomes re-established. On Seasonal Variations in the Eel-Heart.—Here, as in the heart of the frog, great variations in susceptibility are met with at different seasons. In some experiments done on eels 1. Fournal of Phystology, Vol. XXXI, p. 127. The above observations were presented as part of a thesis to the Senatus of the University of Aberdeen before I knew of Marshall’s paper. 2. In this paper the term ‘nerve endings’ is used to indicate the peripheral terminations of the cardiac inhibitory apparatus. The present investigation shows that the drugs mentioned act ‘indirectly by stimulation of a peripheral inhibitory mechanism and not directly by simple depression of the contractile mechanism ; whether the exact portion of this inhibitory apparatus is of nervous or muscular origin is not within the scope of the investigation. It is very possibly intra-cellular. 3. American Fourn. of Physiol, Vol. VI, p. 207, 1901. 4. Fournal of Physiology, Vol. VI, p. 218. ACTION OF MUSCARIN AND PILOCARPIN 15 obtained about the beginning of March, it was found that in every case faradisation of the sinus or vagus nerve had little or no effect on the cardiac rhythm or contractile force ; in twelve eels experi- mented upon not one was definitely influenced by faradisation ; here again muscarin and pilocarpin proved equally ineffective, and comparatively large doses not only failed to influence the rhythm, but a heart covered with such strong solutions as 4 per cent. pilocarpin nitrate or chloride for several minutes, showed no appreciable diminution in the force of the cardiac contraction. In eels obtained early in April the same phenomenon was in evidence in some of them; in a certain number of these, however, fairly definite results could be obtained by faradisation as well as by the application of muscarin and pilocarpin, but here the heart could not be always brought to a standstill by faradisation, though a con- siderable slowing was often observed ; if the faradisation was applied for even a comparatively short time, the heart generally succeeded in escaping from the inhibitory influence and regained its normal rate; the application of a stronger stimulus sometimes gave a result, but it was always less than that obtained from the primary stimulation, and soon the heart escaped and could not be again influenced by vagal excitation. After this faradisation the heart was quite unin- fluenced by muscarin or pilocarpin even in large amounts, and the fact that in these hearts the usual effects of the drugs can be prevented by previous faradisation of the vagus seems to prove that muscarin and pilocarpin are directly dependent on a functionally intact in- hibitory mechanism for their action in slowing or stopping the heart. Again, the effect of muscarin and pilocarpin always ran parallel with that of faradisation ; in several cases a heart which had escaped from fairly weak faradisation was slowed by pilocarpin, but the effect very soon passed over, the heart regaining its former rhythm ; when this happened, pilocarpin, even in large doses, was quite ineffective. Fig. IV shows a tracing from sucha heart. In A is seen a stoppage from weak faradisation, which was applied at the point marked on the left. After a short initial stoppage, beat escaped though current still applied. B is a continuation of the same tracing showing 16 BIO-CHEMICAL JOURNAL result of application of pilocarpin. (The difference in level is due to a reflex movement of the eel). In C the pilocarpin is almost recovered from; atropin has been applied at part marked. Fic. IV. Effect of faradisation and pilocarpin on a heart in which inhibitory action was present to a slight degree. In these ‘hearts it was obvious that vagal inhibitory power was present only tova slight extent, and this inhibition seemed to be still less in evidence if the heart was injured in the process of exposure ; an excised heart at this season seldom gave any definite indication of inhibition either as the result of faradisation or drugs, and it would seem that even in hearts in which inhibition is very pronounced, mechanical injury tends to diminish the phenomenon. More eels were obtained at different seasons later on, and it was found that normal conditions became gradually established. In May, however, many hearts were obtained that seemed to possess no very marked inhibitory mechanism; here the same relationship as above stated obtained as regards faradisation and the application of muscarin and pilocarpin. In July, some eels were examined and found to be almost normal, though inhibition was not so marked in all as it appeared to be later. In September and October, inhibition was found to be much more active, and both faradisation and muscarin or pilocarpin instantly stopped the heart for a long period; the application of atropin immediately restored cardiac action. Fig. V shows a tracing taken from a normal eel-heart showing effect of pilocarpin : drug was applied at part marked on left and drum ACTION OF MUSCARIN AND PILOCARPIN 17 stopped for a few seconds; single ventricular beat in middle shows result of mechanical stimulation. On the right is seen the effect of atropin; here drum was also stopped for a few seconds. k (deh 2Fe Pbscn nek nw suuintouhhhhhhhhuhanninnhhhnhhhhhhinhnhhhnhhannnnnnahnnankannnnanhnnn ws UU PUA PRA Fic. V. Effect of pilocarpin on normal heart: At all seasons of the year a heart may occasionally be found in which faradisation of the vagus gives no result, and in such a case muscarin and pilocarpin are likewise incapable of slowing or arresting the rhythm. This close relationship between the effects of faradi- sation and the application of muscarin or pilocarpin can only be rationally explained by assuming that these drugs act by stimulation of the cardiac inhibitory nerves. As in the case of the frog-heart this difference in regard to inhibition at different seasons is difficult to account for, but it is likely that the same general causes may be present as factors in both cases. On the Condition of the Eel-Heart when brought to a standstill by Faradtsation of the Vagus and by Muscarin or Pilocarpin—tIn the normal eel-heart arrested by faradisation of the vagus, the ventricle generally remains quite responsive to stimulation, while the auricle remains inexcitable: the same thing commonly obtains in the eel-heart arrested by moderate doses of muscarin or pilocarpin; the ventricle responds readily to stimulation while the auricle remains quiescent. In many other points also, the eel- heart behaves in a peculiar manner while under the influence of vagus inhibition, and the same conditions seem to obtain under the 18° BiO-CHEMICAL JOURNAL influence of muscarin and pilocarpin; these points are at present being investigated. , The following are notes of a few of the experiments done at different seasons of the year :— Eret I.—March— 12.0 Heart exposed in situ: rate 28 per minute. 12.5 Faradisation applied to sinus: no result even when strong. 12.8 1 drop muscarin applied to sinus: no result ; 21 per minute. 12.12 Another drop applied: rate 20 per minute. 12.20 4 drops applied: heart beating fairly strong; 18 per minute. 12.25 4 drops applied: slightly weaker; 19 per minute. 12.30 Atropin applied, but no change in rate or force; 19 per minute. 4.0 Heart still beating: very weak; 17 per minute. Eext [J.—March— 11.10 Heart exposed in situ: rate 25 per minute. 11.15 Faradisation slowed heart slightly, but only for about 1 minute. 11.20 Pilocarpin nitrate (20 %)—1 drop applied: no result; 25 per minute. 1.24 ss a 2 drops applied: no result ; 23 per minute. 11.33 a 5, several drops applied: no result; heart quite strong. 11.37 Heart excised. 11.40 Pilocarp. applied, many drops: no result; 25 per minute. 11.45 Heart much weaker: 22 per minute. 11.55 Heart getting weaker: 19 per minute; after little time 16 per minute. 11.55 Heart getting weaker: 19 per minute; after little time 16 per minute. 12.5 Atropin applied: no result; 18 per minute and weak. 12.8 Digitalin solution applied: beat stronger; 16 per minute. , 32.30 Heart beating, but very feeble: 13 per minute. Eex I.—April— 10.30 Heart exposed: 19 per minute. 10.33 Faradisation effective ; only tried for } minute. 10.38 Pilocarpin applied: beat slowed; rate 14 per minute. 10.41 strong beat; rate Io per minute. 10.46 More pilocarpin applied: strong beat ; rate 12 per minute. 10.55 strong beat ; rate 16 per minute. 11.0 beat rather weaker ; rate 16 per minute. 11.3 Atropin applied: beat 22 per minute. Heart continued to beat for over 6 hours.” ACTION OF MUSCARIN AND ‘PILOCARPIN ag" Err I.—July— 11.15 Heart exposed: reflex inhibition from gill easily obtained ; heart stopped for 65 11.27 11.29 11.37 12.5 12.30 12.35 12.42 12.46 10.16 10.19 10.25 10.29 10.30 10.31 10.35 10.40 10.41 10.43 10.47 10.50 11.0 11.5 11.6 ___seconds»” Another arrest for 65 seconds occurred when gill again pressed. Faradisation of sinus : ready arrest of whole heart for 1 minute. Heart excised. Faradisation : arrest of whole heart for 2 minutes 10 seconds; then one beat occurred. followed after an interval of 40 seconds by another. Faradisation : inhibition for over 3 minutes. Faradisation of auricle: arrest of whole heart for 3 minutes (not tried long): Went on all right when current stopped. Heart dull: Ringer’s fluid applied. Beating very slowly ; 44 per minute. Ringer’s fluid removed to try faradisation : heart did not beat. > 93 Ye-applied: heart again gave auricular beat. Ventricle not beating. Faradisation of auricle stopped it for 1 minute (not tried longer) : sinus went on beating. Faradisation of sinus readily arrested sinus and auricle. Pilocarpin readily arrested and atropin restored the beats. Ee I1.—July— Heart exposed. Reflex inhibition from pressure on gill (1 minute) easily got. Faradisation of sinus: arrest of whole heart (for I minute). Piece of auricle ligatured off with some blood on it. Heart excised. Inhibited from faradisation of sinus. Separated piece of auricle began to beat some little time ago—now 6 per second. Inhibitory effect of faradisation of excised heart is absent even with fairly strong current. Faradisation of separated (autom. beating) auricle does not inhibit. Some ocioag effect at beginning. Pilocarpine chlor. (1 %) applied to (a) excised heart and (6) isolated piece of auricle: many drops poured on. Pilocarpine has not arrested or apparently depressed (a) or (b). Pilocarpine still no effect. . Heart still beating at good rate though weaker; separated piece of auricle still beating. Fresh blood from another eel (very large) has been applied to separated piece of auricle; now beating rapidly 23 per minute and vigorously: pilocarpine again applied—no arrest. Atropin applied to excised heart : no improvement. 20 BIO-CHEMICAL JOURNAL 11.20 (a) and (6) still beating; (a) weaker and ventricle responds only to every second auricle beat. | 12.45 (a) and () still beating though weakly, Pilocarpine no effect. Eret [.—October— 11.30 Heart exposed: rate 26 per minute. 11.33. Faradisation gave marked result. 11.35 Pilocarpin applied: heart stopped after 4 minute. 11.38 Heart beating slowly: 5 per minute; strong. 11.39 More pilocarpin applied: 3 per minute ; strong. 11.42 Atropin applied : 23 per minute (after 4 minute). 11.48 Faradisation negative after atropin. 11.55 Heart at 26 per minute. After 20 hours— Heart examined. Sinus 16 per minute (weak) ; ventricle 6 per minute (also weak). Pilocarp. nitrate applied (3 drops 2 % sol.). Heart: 3 per minute (ventricle) ; 9 per minute (sinus). Atropin sulphate sol. applied: 3 per minute; sinus 9 per minute. Heart very much weaker, and only shows feeble wave of contraction. Eet I].—October. 11.15 Heart exposed: 28 per minute. 11.16 Faradisation gave marked result—(tried for 1 minute only). _ 11.20 Applied 1 drop (2 % sol.) pilocarp. nitrate: 12 per minute: strong beat: 4 per minute. | : 11.23 Applied another drop pilocarp. sol.: 4 per minute. , strong beat: 3 per minute. fairly strong beat: 3 per minute. 11.41 Applied 2 drops pilocarpin. sol.: 3 per minute. 11.46 Applied 3 drops es 3 per minute. 11.48 Heart covered up with blotting paper saturated with pilocarp. After 2 hours. Solution (2 %). Also many drops poured-on heart. 1.48 Beat not very strong: rate 26 per minute. On tHe Depressant Action or MuscarIN AND PrLOCARaat ON THE Eet-Hearr Muscarin.—The sample of muscarin obtained from Profamm Schmiedeberg was used, and it was found that the direct depressant effect of the drug on the cardiac muscle, though much greater than pilocarpin, was not very marked, at least for some considerable time, ACTION OF MUSCARIN AND PILOCARPIN 21 unless a comparatively large amount had been applied. A normal heart could be arrested by the application of one drop to the sinus region, but hearts-in which inhibition was absent were often not affected by many drops, either as regards rhythm or contractile force— in fact it generally required a considerable time (from five to ten minutes or more) for any appreciable diminution to become apparent in the cardiac rate or force. ‘The contrast between the comparatively sudden stoppage or slowing of the normal heart caused by say one drop of muscarin applied to the sinus region, and the very gradual diminution of force and rate noticed only after a considerable time in a heart in which the inhibitory apparatus is functionless, is very marked and suggestive; this gradual weakening is not helped by atropin, but the application of digitalin in weak solution increases the force. On the other hand, digitalin applied to a heart stopped by a drop or two of muscarin in the ordinary way has little effect. These points indicate that the drug must necessarily possess two different modes of action. As the supply of muscarin was limited the majority of eel-heart experiments were performed with pilocarpin. Pilocarpin.—As the result of very many experiments with pilocarpin, it was found that this drug in doses sufficient to produce profound cardiac inhibition, proved to have only very feeble muscle depressant power ; different hearts require different strengths within certain limits, but generally a few drops of a I per cent. or 2 per cent. solution of the salt was quite sufficient to produce a marked result in the normal heart. In a heart possessed of but feeble inhibitory power the initial slowing consequent on the application of a few drops of pilocarpin solution was quickly recovered from, and in these cases the application of the drug in quantities sufficient to cover up the heart so that it was beating in a solution of pilocarpin, did not seem to depress it, at least for a very considerable time: the same held true when as much as 30 minims was injected into the circulation. In some hearts covered with 2 per cent. pilocarpin nitrate, the heart was wrapped up with blotting paper saturated with the same solution and left over night ; next day it was sometimes found to be beating fairly strongly, though naturally very much weaker than. at first ; 22 BIO-CHEMICAL. JOURNAL at other times there was no spontaneous contraction, but there was quite a ready response to mechanical stimulation of the ventricle. In this case, as with muscarin, atropin had no effect, but digitalin in Ringer’s solution often revived it. In short, pilocarpin, though undoubtedly possessing muscle depressant action in large doses, does not seem by any means to be very active in this respect, especially when used in moderate amount. Were the primary stoppage the result of direct cardiac muscle depression, it is impossible to conceive how the muscle could so often overcome the result of this depression while still in actual contact with a strong solution of the drug. On GaskKELL’s ELECTRICAL ExPERIMENT ON THE TORTOISE HEART Gaskell showed that in the heart of the tortoise, when the auricle was cut away from the sinus and the coronary nerve left intact, the isolated auricle and ventricle preparation would, after a time, beat with its own rhythm, and then stimulation of the right vagus nerve in the neck would diminish the size of the beats of the auricle’, this showed that the vagus must be active during the period of quiescence, the non-manifestation of that activity being merely due to the want of suitable means of making it visible. Later on, he describes a method by means of which he ascertained that stimulation of the vagus in the neck caused some alteration in the non-beating muscle of the auricle, which was manifested by an electrical change of an opposite sign to that which accompanied contraction of the muscle’, this change in the quiescent auricle he did not get on stopping the sinus by muscarin directly applied, and from this it is argued that muscarin does not act through the inhibitory nerves. — Under normal conditions the sinus acts as the leader of the heart, the auricular and ventricular beat following in direct sequence to the lead of the sinus. When the heart is inhibited by the application of muscarin to the sinus, standstill is observed on account of the fact 1. Fournal of Physiology, Vol. IV, p. 85. 2. Journal of Physiology, Vol. VIL, p. 406-407. ACTION OF MUSCARIN AND PILOCARPIN 23 that the leader is stopped and no active inhibitory action is set up in the auricular or ventricular tissue as long as the drug does not come directly into contact with these parts. If muscarin acts on the nerve endings, then in Gaskell’s experiment the sinus would of course stop as the result of local stimulation of the nerve endings in the sinus, but there is no reason why its influence should be apparent by the production of an electrical change in the separated auricle with which it did not come into contact; it merely acted locally at the seat of application, and no change, electrical or otherwise, could be expected in any other part of the heart. On the other hand, this experiment affords evidence that muscarin does not indirectly influence the auricle by acting on the sinus ganglia or on the vagus fibres passing through the sinus (pre-ganglionic or post-ganglionic). ‘The experiment, however, is quite in accord with the view that muscarin acts on nerve endings. Heart or Newr Newts were examined in April and June. In both cases ova were in evidence, and more particularly in the June newts. In April, several hearts were found to respond to faradisation and to- pilocarpin, but as a rule it was not possible to prolong inhibition for any considerable period ; in general the heart gradually escaped from the effects of stimulation and seemed unaffected by a stronger application of the faradic current or pilocarpin. Some hearts gave no response when acted on by an interrupted current, and in these no pilocarpin effect was obtained. In the June newts it was practically impossible to elicit any trace of inhibition. Under normal conditions, as proved by the researches of Professor MacWilliam, the heart of the newt is most susceptible to vagus stimulation, very weak excitation causing arrest of whole or part of the heart according to the nerve acted upon; local inhibition is also easily procured as the result of direct local application of the faradic current, the part arrested being inexcitable to direct stimu- lation. Several features of vagal stimulation peculiar to the newt’s _ heart seem to be reproduced when the heart is arrested by pilocarpin. 24 BIO-CHEMICAL JOURNAL Owing to the general unsatisfactory condition of the newt-hearts examined it was difficult to arrive at definite conclusions about some of the points examined, but there was a certain amount of evidence showing the possibility of obtaining local inhibition by careful circumscribed application of the drug; again, during strong pilocarpin inhibition, there was a distinct diminution in the tendency of the heart to respond to direct stimuli, a response being readily elicited only as the drug effects began to wear off. It is hoped that a fuller investigation of these points may be made later, when the newt-heart becomes more normal with regard to inhibition. Heart oF SALAMANDER A few experiments performed on the salamander-heart in June elicited the fact that inhibition was but little in evidence at this season. In this heart there was practically a reproduction of what was noted as occurring in the frog-heart during the spring and summer months. In several hearts stimulation of the vagus nerve seemed to cause no change, or if any change was in evidence it generally consisted of slight acceleration of the rhythm. In one heart electrical stimulation had a very slight effect, and this heart was brought to a standstill for about thirty seconds by one drop of a 2 per cent. pilocarpin solution : after this time it began to beat and very quickly regained its former rhythm. Subsequent soaking with 2 per cent. pilocarpin failed to affect it for a considerable time. After fifteen minutes it gradually became weaker and slower, but was beating weakly after one hour : the application of atropin did not help to restore the beat. In another heart faradisation of the vagus trunk gave no result, but direct stimulation of the ventricle caused’a temporary stoppage ; this, however, soon passed over, and though stimulation of increased ' strength was now applied, no change took place in-thefrate-or-strength. In this heart pilocarpin was without effect. . A heart in which temporary slowing occurred as the result of direct stimulation to the sinus region, gave no result on-the exhibition of pilocarpin. It is interesting to note that all the specimens utilised were distended with ova and obviously approaching the spawning ACTION OF MUSCARIN AND PILOCARPIN 25 season. Some similar experiments were done in September and October. In the September experiments the amount of inhibition present was not great, but in general much more distinct than was found in the above hearts in June. In the October experiments inhibition was more in evidence. In the salamander it is likely that inhibition becomes still more marked towards the winter and early spring months. 7 __ As in other hearts, it was very apparent that injury to the cardiac walls or cutting of the bigger vessels tended to destroy any little inhibition that might be present, for in no case was inhibition obtained where any serious injury occurred in exposing the heart. ConcLusIons I. In the early months of the year, and especially in the season of increased sexual activity, there is a general diminution of inhibitory power in the frog-heart ; when faradisation of the inhibitory apparatus has little effect, muscarin and pilocarpin also produce but very slight effects ; when vagus stimulation slows the heart, muscarin or pilo- carpin will do so also: in short, the effect produced on the heart by small doses of muscarin and pilocarpin is in direct proportion to the influence exercised on the heart by the vagus nerve. 2. Faradisation applied for some time to a heart not possessed of much inhibitory power will tire out the mechanism and the heart will escape ; muscarin or pilocarpin subsequently applied has, in this case, no effect; consequently, after a heart escapes from the action of pilocarpin, it cannot be arrested by vagal stimulution. 3. Mechanical injury resulting in loss of blood tends to diminish the inhibitory power in even a normal heart, and often destroys it in a case where it was not originally present to any marked extent. 4. Muscarin and pilocarpin in small doses, when dropped on the heart, have no appreciable immediate depressant effect on the cardiac muscle. In very weak hearts, a comparatively small dose may cause weakening, but this takes place gradually and differs from true inhibition in not being helped by atropin ; digitalin tends to increase the force. Many hearts will beat in a strong solution 26 BIO-CHEMICAL JOURNAL (e.g. 2 per cent to 5 per cent.) of pilocarpin for a long period without much apparent lessening of strength or rate. | 5. In the eel-heart there were found great differences in the inhibitory power of the vagus, these differences being most marked in the spring months, when, in some cases, faradisation of the vagus or sinus gives no result. As noted for the frog-heart, in cases where vagus faradisation is ineffective, no result is obtained by the application of muscarin or pilocarpin; the converse of this holds true, | 6. Contractile force is similarly affected by pilocarpin and muscarin and by vagus stimulation—diminished or annulled in the auricle while not directly influenced in the ventricle: the mode of recommencement after standstill also corresponds in the two cases— the tendency of the ‘ interjugular’ part of the sinus to initiate the renewal of rhythm being evident in the eel-heart. | 7. A condition corresponding somewhat to the antagonism of muscarin and pilocarpin described by Ringer, is sometimes probably the result of a primary excitation of inhibitory nerves by muscarin followed by paralysis of these same nerves on the addition of a pretty large dose of pilocarpin; the same effect in certain hearts can be obtained by using muscarin or pilocarpin alone (frog). 8, Gaskell’s muscarin experiment on the tortoise-heart does not exclude the possibility of the drug acting through the nerve endings. . g. In the heart of the newt and salamander, inhibition is absent or markedly diminished at certain periods corresponding roughly to the seasons of sexual activity. Here the same general conditions obtain as were noticed in the frog and eel heart. 10. All the available evidence shows that in different types of ° the vertebrate heart the local application of muscarin and of pilocarpin reproduces the special results obtained by vagal stimulation in each heart, widely different as they are in different types as regards the parts of the heart acted on, the characters of the action, etc. 11. The above observations strongly support the view that muscarin and pilocarpin in small doses cause arrest of cardiac action , Ds. Bowe ‘not 1 Saietil in virtue of their ou. the contractile mechanism. ors a0. MQITOA . ) BROITAVS nere ne adele Gesnio ct AAC } < ; a i ‘ Srey ae Hamar VEN ETT RTs TUE ‘; ¥ i ‘ ae t=. y ake tei ‘: ! * a Rae | ie ‘barat ibis qihest ws awe Ray { g , ¥ Ce : 7 ? Si Scores yi ; fen as ¢ sbnak spas | t : t pe pe TEED i a hey ro t r « eH 4 : : iw : ¥ 4 = «1 . ie 4 Vi PISA m3 . Hy; Poros “aed iT boo fh : ‘av . 4 t> wt Lory y } 3 i es i, * ie 4 a vo yinaypis #3 osteo WARES bros wRag Bsn [tO seh ivhbeuey too Sauls ee: yey Ssronrai beoseeal eb ors ic» Ses b? Dir ieee: ee Beanie Way i reo ber DRA! See eis oid ig w T 4 ‘ + Ra Wy >) tae > i ivy VFA - 3 7 - 7" : : : ¥ = i See le FO Se Oy ath a , . Os Did eas era nd bE) t.35 L Gr ae ‘us Teynel dguete 5 > GAB FFA asi 28 ¢, OBSERVATIONS ON THE ACTION OF BOERRHAVIA DIFFUSA By LAL MOHAN GHOSHAL, I.M.S. Communicated by Carrain D. McKay, I.M.S., Professor of Physiology, Medical College, Calcutta (Received October 14th, 1907) Natural Order Nyctaginecea, popularly known as Punarnava in Bengal and Gadha Purna (Hind.). Distribution—Widely through Bengal and Chota Nagpur, extensively found during the rainy season. Description of the plant.—It is a large diffusely-branched herb, with opposite leaves which are faintly undulated at the margin, small and panicled, flowers capitate; stamens one or two to five ; anther didymous ; ovary oblique and slender; ovule erect; stigma peltate ; fruit enclosed in the ovoid ; seed adherent with testa. There is another plant closely allied to this—it is a red variety, stronger and larger, more branched, lasting longer and bearing red flowers. It resembles the white variety in every particular. The roots of the red plant are bigger, thicker and longer than the white variety. Popular uses.—It is extensively used for food purposes, specially by the Bengalee ; the whole plant is used as fodder for cattle in the: Central Provinces under the idea that it increases the quantity of milk. Illiterate persons use a decoction of the root regularly as a sedative to the urinary passages, specially if there is lessened urinary secretion. [ts antiquity as a drug.—From a very early period its medicinal value was recognised, as may be inferred from its mention in Raj Nirghanta and Bhabaprokash (two very old Sanskrit works on medicine). Composition.—The whole root and half of the stem were first — ACTION OF BOERRHAVIA DIFFUSA 29 turned into a pulpy mass with the help of pestle and mortar. This’ mass was then macerated with water (twenty times its volume) for about a week and then filtered through a fine rag. The filtered extract was then evaporated to dryness; the dried mass was treated with dilute sulphuric acid (equal parts of water and H,SO,) for a week. The acidulated extract was then filtered, and the filtrate was neutralised with ammonia until it gave a distinct alkaline reaction and until no more precipitate began to fall. The precipitate was allowed to settle, the upper liquid was decanted off and the contents evaporated to dryness. A portion of this was treated with petroleum ether, giving no change. Another portion was treated with absolute alcohol, which dissolved it entirely in the course of three days ; the alcoholic liquid was concentrated on a vapour heater until only a little of the fluid remained. On the surface of the fluid floated a very minute quantity of black scum, suet-like in appearance and sticking on the hand like gum on touching. The black scum was removed by decanting and treated separately with ether, in which it was perfectly soluble ; on evaporation of the ether, a greenish-black sticky substance was left. Under the microscope it looked like small oil globules with green colouring matter. This substance is, therefore, probably fatty in nature. Finally the alcoholic extract was evaporated in an air chamber, when a dry brown powder was left. This powder was soluble in alcohol and satisfied all the tests of alkaloid except that ferric chloride gave only a slight brown precipitate. Under the microscope it appeared as dark amorphous granules. The brown powder was then treated with dilute sulphuric acid and water in equal parts and kept for twenty-four hours so that the whole of the granular mass was dissolved—the amount of dilute sulphuric acid added being about twenty times. This fluid was then evaporated to one-tenth of its volume and allowed to cool, when small needle-shaped crystals began to settle at the bottom; the whole was then kept in hot air chamber so that extra fluid evaporated and crystals only were left behind. 30 BIO-CHEMICAL JOURNAL Physical character of these crystals—They were small, needle- shaped, brownish-white in appearance when in mass; taste nearly bland or very faintly bitter ; they were light and voluminous. Under the microscope they appear as long or short needle-shaped crystals, about one-fifth to one-tenth mm. in length. This is evidently the sulphate of the granular body obtained after evaporation of the alcoholic extract. It resembles impure quinine sulphate. The amount extracted was very small, so that the plants were very poor in the amount of the alkaloid. Thus 20 oz. of the original plant treated yielded only 300 mgrms. of the sulphate (about 5 grains) or about ‘ooosth part of the original plant taken. The ash of the dried plant yielded only about 15 per cent. of the solid matter ; it was partially soluble in water, and the solution gave tests for chlorides and sulphates of the alkali metals (specially of sodium and potassium),and minute traces of nitrates, and a very faint trace of chlorate. Thus we see the composition to be (1) a sulphate of a body, alkaloidal in nature; (2) an oily amorphous mass of the nature of fat (probably) ; (3) sulphates and chlorides and traces of nitrates and chlorates from the ash. | The amount of the alkaloidal body is very small. PuystioLocicaL EFFEcts A guinea-pig;- weighing about 500 grammes, was given 200 mgrs. of the sulphate with milk; it took the drug well and developed no poisonous symptoms. Only change noticed was that the animal passed a very large amount of urine for two days. As the quantity of the alkaloid obtained was very small in amount, all the experiments were henceforth carried out with a liquid extract of the plant (1 in Io). The same animal was injected with 4 drachms of the extract at I2 noon, the only symptom it developed was that there was a little excess of urine passed by 4 o’clock ; now another injection of 2 drachms ACTION OF BOERRHAVIA DIFFUSA 31 was given, which was repeated at 6 o’clock; poisonous symptoms began to appear late at night, about seven hours after the last injection ; the animal became stupid, took no food that was given to it at night, ' passed-neither “stool nor urine, had occasional convulsions and a difficulty in standing. Next day it became perfectly stupid although consciousness was retained ; later in the day it became more or less comatose, with frequent convulsions. All the time there was no ‘urine nor any stool. In this state it remained till § o’clock in the evening, and died at 6 o’clock in deep coma. _ The post mortem examination madé next morning showed the following appeararices :— = (r) Stoppage of the heart in ventricular systole. (z) Acute inflammation of the glomeruli of the kidneys, which were acutely congested and almost flesh coloured—more so at the cortical region ;. on section of the kidneys the glomer- ular portions stood out prominently as so many reddish dots. (3) Congestion of the portal area, possibly due to the wi gk vs el of the auricles and right side of the heart. Microscopical examination of kidney.—Section of kidney showed excessive cell infiltration in and around the glomeruli; the glomeruli were large and standing out prominently amongst other tissues ; around the glomeruli there appeared to be a kind of thin fibrinous deposit. The tubules showed practically no change except a little hazy material occupying the lumen of the tubules. Other organs _ showed practically no change. Death was probably due to uraemia caused by the stoppage of the excretions from the kidneys. Effects on isolated heart.—Direct application of the drug on the heart increases the force and frequency of the heart beat ; the duration of the systole is increased along with the force of the beat so that all the blood is squeezed out of the ventricle during the contraction and the heart appears paler for a time. In its increasing the force and fre- quency of the heart beat the drug resembles strychnine; while © in increasing the force of systole it resembles digitalis. The following 32 BIO-CHEMICAL JOURNAL is a tracing of the frog’s heart before and after the application of the drug while it was beating forcibly :— Cardiographic tracing of frog’s heart before and after the application of the drug ; the upper one represents the normal tracing before the application of the drug; the lower one represents the tracing after the application of the drug; all the while the heart was strong and beating forcibly. The upper tracing represents the normal beats, while the lower tracing is taken after the application of the drug. In this lower curve the systolic tracing is sharper and more prolonged, indicating the force of the systole, while the downstroke, representing the pause, is about the normal or very slightly longer. The following tracing was taken while the heart was weak and beating slowly ; various doses of the drug were applied—first two drops, then four drops and then treble the dose. Here the upper curve represents the normal, while the lower curve after the application of the drug. Double dose Treble dose the slowing of the heart due to weakness. OBSERVATIONS ON THE ACTION OF BOERRHAVIA DIFFUSA 33 Here, too, we find the systole more prolonged and sharper, systolic plateau being distinctly longer after the application of the drug. The tracing-after the third dose is quite remarkable. In both cases the tracing represented by the systole is more prolonged and sustained. This action of the drug on the heart is probably muscular and not nervous, as may be deduced from the following experiments. Stimulation of the vagus at the crescent after the application of the drug causes the heart to cease beating, while the removal of the stimulus and re-application of the drug restores the former action of the heart. First, Stannius’s ligature after the application of the drug causes the inhibition of the heart, while removal of the ligature and re-application of the drug brings about the contraction of the heart after a little while. Again, application of the drug after double Stannius’s ligature brings about one or two contractions of the apex. Thus we see that the nerve (vagus) is unaffected by the drug, and the action exerted by the drug on the heart is probably muscular. The number of heart beats was markedly increased, and it rose from 76 to go in case of one frog; in another from 68 to 81; ina third from 70 to 80, and so on. These phenomena were more marked while the heart was in its wane, and then the frequency in the number of beats was well marked. Effects on pulse beat in man.—These facts were also borne out by the condition of the pulse as experimented on human beings. The following tracings indicate the condition of the pulse before and after the application of the drug. Cnet Sphygmographic tracing before the administration of the drug. Pulse beat = 83 per minute; sphygmographic pressure = 92. 34 BIO-CHEMICAL JOURNAL Sphygmographic tracing after administration of the drug. Pulse beat = 92 per minute. sphygmographic pressure = 128. Here the upstroke is longer and sharper, while the downstroke is more prolonged and the dicrotic wave is well marked. Sphygmographic tracing before the administration of the drug. Pulse beat = 82 per minute ; sphygmographic pressure = 95. Sphygmographic tracing after the administration of the drug. Pulse beat = 108 per minute ; sphygmographic pressure = 140. Here, too, the dicrotic wave is very well marked, the upstroke longer and sharper. Similarly, other pulse tracings were taken, and all gave similar results. In all the cases we see that the upstroke due to the expansion of the artery is sharp and long, consequent on the increased force and duration of the systole of the left ventricle. The dicrotic wave is more prominent in all the cases, and that is probably due to the larger amount of blood flowing into the aorta and thereby closing the semi-lunar valves more completely and tightly. OBSERVATIONS ON THE ACTION OF BOERRHAVIA DIFFUSA 35 The pulse rate is also quickened—thus :— — Before After eee Roghy’........... 82 Pa 108 Sipe) f2.2 i 78 a 82 Gokal is.i.se FS 83 as 92 The blood pressure is also raised, as experimented by sphygmo- meter ; thus we have :— ; Before After Roghu\.......:.0-. - 9§ a5: 140 CRON Fo inn» 42 BIO-CHEMICAL JOURNAL the carotid artery of the anaesthetised animal, placing this in a weighed crucible, evaporating and burning over a low bunsen flame. The residue was then extracted with water, the ashing continued over a blow-pipe flame, and the watery extract added in the usual manner. For determining the total ash of bone, a weighed amount of dried bone was placed in a tared crucible, burnt, and ignited to constant weight over a blow-pipe flame. All reagents employed were tested for impurity but with negative results. EXPERIMENTAL I.--Experiments with Rabbits The animals used for the following experiments were the ordinary brown wild rabbits which had been netted, and brought to the laboratory. Young, healthy animals of both sexes were used, care being taken to exclude lactating females, in order that the secretion of milk, which is rich in calcium, might not vitiate the results. The control animals were fed on a green vegetable diet. This consisted mainly of grass, but lettuces and cabbages were used as well. The experiment animals were fed on oatmeal, as it has been found that animals on this diet soon suffer from calcium insufficiency. At first the oatmeal was made into a thick paste with distilled water ; but it was found that this caked into a hard mass which was refused by the animals. So the oatmeal was given in the dry form, and a dish of distilled water for drinking was placed in the cage. During the first days of each experiment, a certain amount of green food was also given, until the animal became accustomed to the dry diet. During the later experiments, maize meal was added to the animal’s diet of oatmeal and distilled water. ‘To carry out the analyses the animals were anaesthetised with ether. A cannula was put into the carotid artery in the neck, and the animal was bled as long as the blood ran freely. Care was taken not to continue the bleeding so long that fluid would be withdrawn from the tissues and cause an error in the composition of the blood. The blood was collected in a measured vessel ; a certain amount was measured off and used to estimate the CALCIUM METABOLISM 43 ash of the blood, while the remainder was dried and its calcium- content was determined. After getting the blood in this way, the animal was killed by breaking the neck. The femora and humeri were then cut-out; cleaned of muscles and tendinous attachments, pounded up and dried. Their total ash and calcium content were then estimated. An examination of the internal organs was made, but no definite micro- scopic changes were observed. In some of the animals,'a hyperaemia of some of the joints was observed, the synovial membrane and cancellous tissue were engorged, but there was no effusion into the joint-cavity, nor any sign of adhesions in the joint. These changes affected mainly the larger joints, which were always more markedly altered than the smaller joints. ‘These changes were noted in three of the animals fed on calcium-poor diet (one of which died) and in one animal fed on green vegetable diet. In these cases no endocar- ditis was observed, and the significance of the condition is not known. In order to obtain a greater amount of calcium in blood, and so, if possible, to diminish the experimental error, in one of the series of experiments two animals were used at the same time in some of the later experiments. ‘Ten c.c. of blood from each were ashed, and the rest of the blood was measured and mixed, dried, and the calcium- content determined. Rabbit I.—Buck: was anaesthetised and killed on November 19th, 1906, after being brought from the country, where it had been feeding on the natural grasses. Under ether, carotid artery in the neck cannulised and 48 c.c. of blood withdrawn ; of these 8 c.c. were dried and ashed, and 40 c.c. dried and the calcium-content estimated. The femora and humeri were removed, cleaned, smashed up, dried ; and the total ash and calcium-content determined. Results: Bone, ash r1’osg6g. in 1°8545 g. dried bone, = 57°08 per cent. Ca 0.5191 g. in 1°6047 g. dried bone, = 32°35 per cent. Blood, ash o°0502 g. in 8c.c. = 0°627 g. in 100 c.c. Ca 0'0026 g. in 40 c.c. = 0°0065 g. in 100 C.c. Ca 56°6 Blood, Ca ae stuees Totalash ... 100 Total ash 100 44 BIO-CHEMICAL JOURNAL Rabbit I1—Doe: died on November 26th, 1906, after one week on oatmeal and distilled water diet. At the post-mortem examination there was synovial engorgement affecting the large joints (hips, knees and shoulders). The femora and humeri were smashed up and dried. Results: Bone, ash 1°1243 g. in 1°8339 g. dried bone, = 61°31 per cent. Ca 02178 g. in 0'8679 g. dried bone, = 25°09 per cent. Ca _ 41°08 Total ash 100 Bone, Rabbit III.—Doe: killed on November 29th, 1906, after being kept in a hutch since November 19th, 1906, on green vegetable diet. 45 c.c. of blood obtained from carotid artery; of these 10 c.c. dried and ashed, and 35 c.c. dried and their calcium-content estimated. The femora and humeri were removed, smashed up and dried. Results: Bone, ash 1°8572 g. in 2°8760 g. dried bone, = 64°24 per cent. Ca 0°3131 g. in 0°8788 g. dried bone, = 35°61 per cent. Blood, ash 0°0476 g. in 10 c.c., = 0°476 g. in 100 c.c. Ca 0°0035 g. in 35 c.c., = O°OI g. in 100 c.c. Ca 55°4 Blood, Ca oa PONE Total ash... 100 Total ash —«S*T00 Rabbit IV.—Doe : killed December 7th, 1906, after being fed since November 19th, 1906, on oatmeal and distilled water. 30 c.c. of blood obtained from carotid artery ; of these, 10 c.c. were dried and ashed, and 20 c.c. dried and their calcium-content estimated. The femora and humeri were smashed up and dried. Post-mortem: hyperaemia of the cancellous tissue and synovial membrane was observed ; most marked in the knees and shoulders, less marked in the elbows and hips, and still less marked in the small joints. No endocarditis. Results: Bone, ash o°9619 g. in 2°0749 g. dried bone, = 46°36 per cent. Ca o'1810 g. in o'gors g. dried bone, = 20°09 per cent. Blood, ash 0°0663 g. in 10 c.c. blood, = 0°663! g. in 100 c.c. Ca o'oog! g. in 20 c.c. blood, = 0°0455' g. in 100 c.c. Ca 43°3 Ca 6'8 Bonese se os pe ages hla Oe rer Total ash 100 mere Total ash 100 Rabbit V.—Doe: killed December zoth, 1906; fed since December 8th, 1906, on oatmeal and maize meal and distilled water. 22 c.c. of blood obtained from carotid artery ; of these, 10 c.c. were ashed, and 12 c.c. were dried. ‘The femora and humeri were smashed and dried. 1. Some error in the method of analysis undoubtedly occurred in this case. CALCIUM METABOLISM 45 Results: Bone, ash 1°1771 g. in 2°5805 g. dried bone, = 45°23 per cent. Ca 0°3396 g. in 1'2874 g. dried bone, = 26°39 per cent. Blood, ash 0°0525 g. in 10 c.c. blood, = 0°§25 g. in 100 c.c. Ca Seer ih —— page Total ash 100 Rabbits VI and V II.—Killed January 29th, 1907, fed on green grass. 10 c.c. of blood from each was ashed; and 60 c.c. of mixed blood was dried and their calcium-content determined. The femora and humeri were cleaned, smashed up and dried. Results: Bone, ash 1°5231 g. in 2°2982 g. dried bone, = 66°31 per cent. Ca 0°3580 g. in 1°2510 g. dried bone, = 28°62 per cent. Blood, Ca 0°0054 g. in 60 c.c. = 0°0090 g. in 100 c.c. ash O°1451 g. in 20 c.c. = 0°7255 g. in 100 c.c. Ca _ 431 Ca a Bera ak” 7 ~ 100 Bod, omer ian 7 Too Rabbits VIII and IX.—Killed March 27th,1907 ; fed since January 30th, 1907, on oatmeal and maize meal and distilled water. 10¢.c. of blood from each ashed ; 53 c.c. (26-27) of blood dried, and their calcium-content estimated. The femora and humeri were smashed and dried. One of these animals showed hyperaemia of the synovial membrane of the larger joints. Results : Bone, ash 170485 g. in 2°1306 g. dried bone, = 49°18 per cent. Ca 03059 g. in 1°3966 g. dried bone, 21°gO per cent. Blood, Ca 070087 g. in 53 c.c., = O°O164 g. in 100 c.c. ash O*ISQI g. in 20 c.c., = 0°7955 g. in I00 C.c. Ca 44°5 Ca 2°06 Pope, Total ash ~ 100 Fined. Total ash ~ 100 Rabbit X.—Doe : killed March 28th, 1907, after being fed since January 30th, 1907, on oatmeal, maize meal and distilled water. 10 c.c. of blood were ashed ; 30 c.c. dried and the calcium-content estimated. The femora and humeri were cleaned, smashed up and dried. Post-mortem showed engorgement of shoulders, hips and knees ; no other joints affected ; no endocarditis. Results: Bone, ash 0°8196 g. in 1°6392 g. dry bone, = 50 per cent. Ca 0°3196 g. in 1°6392 g. dry bone, = 19°49 per cent. Blood, Ca 070046 g. in 30 c.c., = O’0155 g. in 100 c.c. ash 0°0833 g. in 10 c.c., = 0°8330 g. in I00 c.c. Cc 38°98 Ca 1°86 NOR eg ae ie we cal ak “ie 46 BIO-CHEMICAL JOURNAL Taste I.—Raspit EXPERIMENTS Rabbit, Feeding Bone Ca - Blood Ca Bone ash Blood ash I ie Green grass 56°6 he Be. 100 100 II pn Oatmeal 41°08 Tat 100 III rep Green grass 554 ad 100 100 e “Ql IV £9; Oatmeal ... iB ane % ¢ 43°3 68 100 100 Vv oie Oatmeal and maize meal 58 = 100 Viand VII ... Green grass... a i +35 tas Be: 100 100 Vill and IX ... Oatmeal and maize meal ie 44°5 tee 2°06 100 100 x ix Oatmeal and maize meal J, 38°98 vee 1°86 100 100 It will be seen from the above table that the amount of calcium in the blood varied only between 1 and 2 per cent. of the total ash. On the other hand, considerable variations took place in the calcium content of the bones. Though the results are not quite uniform, it will be scen that the average calcium percentage of the bones of animals fed on oatmeal, and oatmeal with maize meal, is lower than that of the normal control animals fed on green stuff. I1.—Metabolism in Man The experiment was continued for five days. All the estimations were made with undried substances. Diet.—For lunch weighed amounts of biscuit (thin Captain) and butter were taken, and after the first day a measured quantity of milk was added. For tea and breakfast weighed amounts of oatmeal 1. See previous note. CALCIUM METABOLISM 47 ‘with a small quantity of salt, were boiled for hours in a double saucepan and a measured quantity of milk was taken with the oatmeal. The body-weight.was taken daily at a regular hour, due pre- cautions being taken to ensure the same clothes being worn : the same weighing machine was used throughout. The exercise taken consisted of a four mile walk each day. The work done during the day was the ordinary routine work of the Physiological laboratory. About eight hours sleep was obtained each night. The total urine passed in the twenty-four hours was collected, mixed and measured ; the specific gravity was taken and the amount then diluted to the nearest convenient whole number to avoid fractions in multiplication. Samples of this diluted urine were then used for analysis ; a few drops of chloroform were added to prevent decompo- sition. The total faeces were collected, weighed, and a sample kept for analysis; a few drops of formalin being added to prevent the growth of moulds. As nearly as possible a full analysis of the urine was carried out, but owing to the lack of a suitable colorimeter and the expense of absolute alcohol, the estimation of the creatinin excreted could not be made. It was intended at the outset to attempt to estimate the complete inorganic metabolism; but it was found that no suitable method of reasonable accuracy could be obtained to estimate the sodium and potassium when present in small quantities mixed with large quantities of organic matter and phosphates. ‘Therefore, the estimations of sodium and potassium in food and faeces were not made. The calcium was estimated in food, urine and faeces; since accurate methods can be used for isolating and determining the calcium even when mixed with organic matter. BIO-CHEMICAL\ JOURNAL 48 Taste II Date Weight Foop Urine Faeces July, 1906 kilos. Lunch Tea Breakfast C.C. gm. 23 71°8 Biscuit 100g. Oatmeal 230g. Oatmeal 230g. 1,200 248 Butter 20g. Salt 20°6 g. Salt 20°6 g. — Milk 290 c.c. Milk 290 ¢.c. 24 72'3 Biscuit 121°5 g. Oatmeal 230g. Oatmeal 230g. 1,620 166 Butter 20g. Salt, 16°5 g. Salt 16°5 g. Milk 400c.c. Milk 348 c.c. Milk 232 C.c. 25 727 + Biscuit 119g. Oatmeal 230g. Oatmeal 230g. 1,705 115°5 Butter 195g. Salt 16°5 g. Salt 16°5 g. Milk 375 c.c.. Milk § 348c.c. Milk 238 c.c. 26 72°8 Biscuit 123g. Oatmeal 230g. Oatmeal 230g. 1,815 163°5 Butter 18°5 g. Salt 16°5 g. Salt 16°5 g. . Milk 325 .c.c.. Milk 348-c.c. Milk 232 C.c. 27 72°7_ _~—~‘Biscuit 122°5 g. Oatmeal 230g. Oatmeal 230g. 1,975 98 Butter 21g. Salt 16°5.g. Salt 16°5 g. Milk 315 c.c. Milk 348 c.c. Milk 232 C.c. Taste III et at 3 Date CaLcruM NITROGEN July, 1906 Food Urine Faeces Food Urine Faeces 23 1°2640 g. 0°1083 g. — 1837 g. 214g. 77238. oh 24. ~=—«1'7373. g. O'1687-g. 149568. 2108 g. 13°55 g. 285g. © 25 1'6gi2g. O1651g. 1°2532g. 20°91 g. " 13°60 g. 19485 26 16428 g. o'1665g. 1°3420g. . 20°63 g: Pca 46g. 2°95-g. 27 16311 g. O'1680g. o'79276g. 2058. 141 18 8: 1°76 g. From the above tables it will be seen that during thes course of the: experiment the body was not.in nitrogenous. equilibrium, but was The weight of the body, it will be Bos iuar HHO os retaining nitrogen. had increased. Intake... I0I°47 g. N ERE Output 84°66 g. N yeesa Balance 16°81 g. NU : dsiw This would represent.a storing of about 109 grammes of proteid during the five days. a 2%. Ue y ae z CALCIUM METABOLISM . 49 The calcium also shews a gain to the body. Retake IO 2hO YOM, AGN ... 6°7024 g. Ca Output Ws Se roe da me 5°6867 g. Ca MeeGaltnce 8... cee 7 a OUT g The excretion of Ca by the urine shows a remarkable constancy of about 0°166 gramme during the last four days of the observations, while the faecal excretion shows a gradual diminution. 2 bs Taste 1V—Urine Date Amount Total N Purin N NH, Urea Chlorides P.O; and sp. gr. in grs. in grs. in in grs. as Cl, in grs. at _N nu, = : Io 23 voit 12°13 O1I7 - 450 I5"12 10°3 1°79 1620 ez: ee 2 eae ae 24 1031 13755 07156 288°" - — Togo 14°9 2°58" ; 1705 i nies Heise $s : : 3 25 1024 13°6 O°312 i #2 18°78 16°5 2°34 o7166 484 24°35 Ce 19'S. 2°60. "300 424 17°57 216 2°83 . ‘Tastt 1V—Continued Date Total SOs Ethereal SO, Ca ; Mg Na . K thas in grs. in grs. in grs. _ im gts. + in grs. in grs. 23 2°03 _ 0.20 01083. _,_-—-«0"1093 5°3181 3°351 24 a ae 01687 =: "1216 8°7483 3°283 : 25. 2°42 ct ae” ernbes o71083—is«8"5568 371003 meet +-*- dee or18 01665" o°1126 10°690 3°168 27 2°40 - O12 0° 1680 O'1215 12°2848 2°912 é | This table shows a gradual but marked increase from day to day of the chlorides and sodium, with.a less marked rise in the excretion of phosphates, and magnesium. On the other hand the calcium * remained almost constant... 50 BIO-CHEMICAL JOURNAL Il1.—Metabolism in Rectal Alimentation This series of observations was carried out on a patient at the Melbourne Hospital, and I am indebted to Dr. Howard for permission to investigate the case. M. G., aet. 26, was admitted to the Melbourne Hospital on the 28th August, 1906, suffering from the symptoms of gastric ulcer. Rectal feeding was carried out from the 26th August until the 18th September, the only thing allowed by the mouth was plain filtered water. The food administered, by four-hourly nutrient enemata, con- sisted in solutions of plasmon in water, starch in 0°7 per cent. salt solution, and white of egg inwater. Thesewere used in varying amounts during the greater part of the experiment. It was found that the amount of plasmon calculated as necessary to keep up the nitrogenous equilibrium could not be got into solution even in the total amount of water used in the twenty-four hours, so that it became necessary to reduce the amount of plasmon injected. During the last three days various nutrient enemata were tried, with the object of increasing the calcium intake. The patient’s rectum and sigmoid colon were washed out each morning at 8 a.m., and the return, together with the faeces, if any, was mixed, measured, and a sample reserved for analysis. The total twenty-four hours’ urine was collected, measured, and samples taken for analysis. The weight of the patient was taken at 11 a.m. on the first two days, but removal to the scales caused such abdominal discomfort that weighing had to be discontinued. On the 1st September, the patient commenced to menstruate,. and this function continued for four days, during which the nutrients were continued but the urine and faeces were not analysed. In the samples of urine and faeces obtained the total nitrogen and total calcium were estimated, and the urea of the urine was approximately determined by the hypobromite method. CALCIUM. METABOLISM 51 Taste V—RecraL Freepinc Dare = Wetcut Foon Water Urine Bower Return Aug: ___—-Plasmion Starch Egg: 5 per . + Mie 6: ead See a Le tO Ge. 10 c.c coke ase TV ... cee 20 SO ‘ Li) Saree. IO C.c. Bat a ee eS. > os — 1°6 c.c. ret, » » Non-diabetic case 120 fell to 60 mm. Hg Sy ES Ce I am indebted to Professor Starling for permitting me to publish, in a footnote, the particulars of two other cases of diabetes ; in each case an acid extract of the duodenal mucous membrane contained secretin. Notre.—Case I.—Diabetes and phthisis. Post mortem: The pancreas was micro- scopically normal. An acid extract of the mucous membrane contained abundance of secretin. Case II.—A diabetic patient, operated on for an ischio-rectal abscess, died three days later from diabetic coma. Post mortem: The pancreas was large and fibrous; sections showed degeneration of the alveoli—the cells being small and free from granules—rather like a pancreas after ligature of the duct. The duodenal extract contained secretin. 86 BIO-CHEMICAL JOURNAL CoNCLUSIONS In all the cases of diabetes described above, a boiled acid extract of the duodenal mucous membrane contained secretin; with one exception, the activity of the extracts was almost or quite as great as that of extracts from the duodenum of non-diabetic patients. These cases, taken together with those previously recorded by Beddard and myself, show that prosecretin is more often present than absent in diabetes, and it may be doubted whether the absence of prosecretin has any causal relation to diabetes. It is quite possible, of course, that, as Moore suggests, prosecretin is deficient in some diabetic patients during life. But this suggestion is not supported by the clinical observations of Dakin, of Foster, and of Beddard and myself ; and the rapid post mortem degeneration which diabetic tissues often undergo might well account for the failure to find prosecretin after death in certain cases. I wish to express my indebtedness to Dr. Beddard, Dr. Andrewes, and Dr. Langdon Brown for their kindness in supplying me with post mortem materials and with notes of the cases. The expenses of this investigation were defrayed by a grant from the Royal Society. REFERENCES (1) Bainbridge and Beddard, this Fournal, Vol. I, p. 429, 1906. (2) Bayliss and Starling, Proc. Roy. Soc., Vol. LXIX, p. 352, 1902. (3) Dakin, Yourn. Biol. Chemistry, Vol. II, p. 305, 1907. (4) Foster, Ibid, p. 297. (5) Moore, Edie and Abram, this Fournal, Vol. I, p. 28, 1906. 87 THE PRESENCE OF A NITRATE REDUCING ENZYME IN GREEN PLANTS By ANNIE A. IRVING, B.Sc. (Lond.), anp RITA HANKINSON, B.Sc. (Lond.). From the Botanical Laboratories, University College, Bristol (Communicated by J. H. Priestiey, B.Sc., Lecturer in Botany, University College, Bristol) (Received December 12th, 1907) INTRODUCTION The question as to the form in which nitrogen is most easily assimilated by the green plant has long been under debate, and very — conflicting statements have been made by various investigators :— Thus Boussingault’s' observations showed that nitrates seemed the most suitable form for the absorption of nitrogen by the plant. Treboux’ found that— | 1. Nitrites are probably useful to the plant.in alkaline solution, but poisonous in acid solution. 2. Nitrates have the same, if not a greater value than nitrites. 3. Ammonium salts are still better than nitrates or nitrites. 4. Amino acids and amides can be used but their nutritive value is much less. He suggests that amino acids are decomposed by enzymes with liberation of ammonia. Mazé® thinks that nitrates and ammonium salts are of equal value in metabolism. 1. Boussingault, Agron., Tome I, pp. 69, 130, 1860. 2. Treboux, Chem. Centr., p. 1619, 1905, from Ber. deut. bot. Ges. XXII, p. 570-572. 3- Mazé, Compt. rend., 1899, pp. 128, 185-187. 88 BIO-CHEMICAL JOURNAL Godlewski' found that higher plants when kept in darkness could produce proteids from nitrates and from the decomposition products of proteids, but in the case of the higher plants the assimilation of these substances is restricted in the absence of light. The necessary energy for nitrate assimilation is supplied by metabolism and respiration. Laurent, Marchal and Carpiaux’ found that plants kept in distilled water containing ammonium sulphate and _ saccharose respectively were able to assimilate these substances when placed in the light. Hansteen® showed that nitrates were assimilated to a small extent in darkness. Laurent’ maintains that neither ammonium salts nor nitrates are assimilated in the dark. Zuleski’s’ results show the possibility of proteid formation in the dark. Suzuki® found that if plants were fed on I to Io per cent. sugar solution, assimilation of nitrate and subsequent formation of proteid took place in the dark, as well as in the light. Plants containing a large amount of sugar were able even in the dark to form proteids from nitrates without the addition of sugar solution. The observations of Suzuki would seem to suggest that in the case of Godlewski’s experiments, and in those of Laurent, the plants had not contained any reserve carbohydrate, and were, therefore, dependent for their sugar upon the supplies formed during their exposure to the light. The same explanation might hold for the other conflicting statements upon this point, as for example, those of Hansteen. From our point of view it is interesting to note that there are statements 1. Godlewski, Bul. Acad. Cracow, Vol. VI, p. 313, 1903- 2. Laurent, Marchal and Carpiaux, Bied. Centr., Vol. XXVII, 1898, pp. 821-823; from Bul. Acad. Belg., Vol. XXXI, pp. 815-865, 1896, and Bot. Centr., Vol. LXX, p. 232, 1897. 3- Hansteen, Ber. deut. bot. Ges., Vol. XIV; p. 368, 1896. Laurent, Bul. Acad. rag. Belg., #. C. 8. Abstracts, Vol. Il, p. 323. Zuleski, Ber. deut. bot. Ges., Vol. XV, p. 336, 1897: Suzuki, Bul. Coll. Agr., Imp. Univ., Tokyo, Vol: III, pp. 488-507, 1898. nv NITRATE REDUCING ENZYME IN GREEN PLANTS 89 by various investigators pointing to nitrates as the best. source of nitrogenous food for the green plant, and there are indications to show that this may-be correlated with the presence of carbohydrates formed in photo-synthesis. As the nitrogen is usually regarded as present in the proteid molecule chiefly in the form of NH, groups, there must obviously be a very efficient reducing apparatus in the green plant capable of converting the received grouping NO, into the necessary NH, form. There are very few statements in the literature of plant physiology suggesting that such is the case, but one or two cases of the existence of a nitrate reducing enzyme have been recorded :— Abelous and Aloy’ showed that an enzyme capable of reducing nitrates to nitrites and nitrobenzene to aniline, is found in animal structures. They also demonstrated the presence of a similar enzyme in potato tubers. Kastle and Elvolve® confirmed its presence in the potato, and showed that it was also present in the fruit of the egg plant (Solanum melongina). Weehuizen*® found that nitrous acid was present in the leaves of Erythrina, and concluded it was set free from a glucoside by the action of an enzyme; because if the leaf were killed by immersion in boiling water for thirty seconds, no nitrous acid was formed. _ It seems very probable that Weehuizen’s enzyme was the nitrate reducing enzyme which has formed the subject of this paper. There are also records of nitrate reducing bacteria. Thus Burri and Stutzer* found that certain bacteria decomposed nitrate with liberation of free nitrogen, and that the action was increased in absence of air. Also very recently Mattio Spica’ has found that under anaerobic conditions yeast was able to reduce nitrates. 1. Abelous and Aloy, Compt. rend. Soc. Biol., Vol. LV, p. 1080, 1903. 2. Kastle and Elvolve, American Chemical Journal, Vol. XXXI, pp. 606-641, 1904. 3- Weehuizen, Pharm. Weekblad, Vol. XLIV, pp. 1229-1232, 1907- 4. Burri and Stutzer, Ann. Agron., Vol. XXII, pp. 491-494, 1896, from Centr. Bact., Par. 1895, Vol. I, p- 2, Abt. 257, 350., pp. 392-422. 5- Mattio Spica, ¥. C. S. Abstracts, October, 1907. go BIO-CHEMICAL JOURNAL It is clear that a more general distribution of such an enzyme is to be expected if nitrates are utilised in the formation of proteids. The present paper is the outcome of work carried out upon this hypothesis. EXPERIMENTAL Water plants (¢.g., Elodea, Vallisneria, etc.) were used in the first experiments on account of the greater facilities which these plants offered for collecting and examining any gases which might be liberated. The following experiments were set up :— A.—Elodea was placed in boiled tap water containing 0°5 gramme of asparagin and I gramme of potassium nitrate per litre. The plant was placed under an inverted funnel in the solution, and a test tube filled with water was put over the stem of the funnel to collect any gases which might be given off during the experiment. ‘Thymol was added for antiseptic purposes to E and F. This was left in the light for two days. B, C, D, E and F were set up in the same way. B.—Elodea in a solution containing asparagin and potassium nitrate in the same proportion as in A but placed in the dark. C.—Elodea boiled for some time, before putting it into a solution containing asparagin and potassium nitrate, and then placed in the light. D.—Boiled Elodea put into a solution of asparagin and potassium nitrate and then placed in the dark. E.—Chloroformed Elodea in a solution of asparagin and KNO, in the dark. In this case the protoplasm would be killed, but many of the operative ferments present in the plant might remain. F.—Chloroformed Elodea in a solution of asparagin and potassium nitrate placed in the light. No gas was evolved on the first day, but on the second it was found that gas had: been given off from the Elodea in A, B, Eand F, but notinC and D. ‘The gas was collected in a Winkler Hempel apparatus and analysed. A.—Total volume = 24°6 c.c. 1. Passed through KOH, bulbs = 24°6 c.c. 2. Treated with KOH and pyrogallol, vol. = 24°6. 3. Sparked with oxygen and treated again with pyrogallol to absorb the oxygen = 24°6. .. Gas = nitrogen. NITRATE REDUCING ENZYME IN GREEN PLANTS gi B.—Total volume = 25°6 c.c. This, treated in a similar manner, also proved to consist solely of nitrogen. E.—Total volume = 37°5 c.c., which proved on analysis to consist of 37°5 c.c. nitrogen, 1'2 c.c. carbon dioxide. F.—Total volume = 19 c.c. On analysis, only nitrogen present. All analyses were carried out as described for A. ‘These experi- ments were carefully repeated, and in all cases nitrogen was found to be given out by normal and chloroformed plants, but not by the boiled ones. The solutions of asparagin in which the Elodea had been placed were examined: A, B, E and F were found to contain nitrites by the starch and potassium iodide test. Before the experiment they contained only nitrates. Therefore, during the experiment the nitrates were reduced to nitrites. Substitution of Ammonium Salts for Nitrates.—Experiments were set up with an equivalent amount of ammonium sulphate in the place of nitrate, but no gas was given off, either in the light or in the dark, when Elodea and asparagin were placed in the solution. The following Explanation is suggested for the Evolution of the Nitrogen :— | The nitrite formed by the reducticn of the nitrate is converted into nitrous acid by the slightly acid cell sap. This in turn acts upon the asparagin giving malic acid and nitrogen. (i) 2KNO, > 2KNO, + 0, Owing to the nature of the reducing action the oxygen is not liberated as a gas, but retained in some form. (ii) KNO, -> HNO, CH - NH, - COOH CHOH * COOH (iii) 2HNO, + | —> | ‘+ 2N, + 2H,O CH, - CONH, CH, * COOH The reaction came to an end after a few days in the dark, and the Elodea was then found to contain no starch. 92 BIO-CHEMICAL JOURNAL The necessary acid medium for the reaction is probably provided by the cell sap. The oxidation of the carbohydrates may provide the energy necessary for the reaction, and its cessation may be due to the exhaustion of the supply of carbohydrates in the plant. This is suggested by the fact that the reaction is accelerated, or restarted when it has stopped, by the addition of cane sugar, or glucose, to the solution. Support is given to this theory by the fact that very little sugar, if any, was present in the plant which had been kept in the dark, whereas in the ordinary plant there was an appreciable supply of carbohydrates. An estimation of total sugars and starches in normal Elodea gave as a result 1°268 per cent. of dry weight as sugars and starch, while some of the same crop of Elodea, after placing in a solution of nitrate and asparagin in the dark, until no gas was liberated, yielded upon estimation merely a trace of carbohydrates. From these experiments it thus seems probable that malic acid is formed in the plant in proportion to the nitrogen evolved as gas. It ought, therefore, to be possible to detect the malic acid in the plant itself. Elodea was treated as described for A in the previous series of experiments. The plant was then finely ground, and shaken with a little water for two hours, and left overnight to extract. ‘The solution was then filtered and treated with lead nitrate. A precipitate, presumably lead malate, was thrown down. ‘The solution and pre- cipitate were then heated, when the precipitate partially re-dissolved. The solution was then boiled to coagulate the proteids, and after- wards filtered, a clear solution being thus obtained. On concen-. trating and cooling small colourless crystals separated out. These were examined microscopically and found to have a similar structure to those of lead malate. They were re-dissolved in water, treated with a solution of calcium nitrate, which brought down a precipitate of calcium malate. ‘This was filtered off, and the filtrate concentrated. No crystals came out, so it was concluded that all the malic acid had been removed as calcium malate. NITRATE REDUCING ENZYME IN GREEN PLANTS 93 This process involves the formation of nitrate as an intermediate product in metabolism, and nitrite is supposed to be poisonous to the plant. Experiment seems to suggest that in dilute solutions the protoplasm is not killed, and in stronger solutions ferment action is not arrested by the presence of nitrite. Sprigs of Elodea were placed in solutions of nitrites of different strengths ranging from ‘oor per cent. to Io per cent. It was found that an enzyme present in all of them catalysed hydrogen peroxide, even after an immersion of three days in the nitrite solution. ‘Trials made to plasmolyse the leaf cells after this immersion in solutions of potassium nitrite, indicated that in all the solutions used that were above o°I per cent. in concentration, the protoplasm was killed even after twenty-four hours. But the amount of nitrite formed by the plant under normal conditions would probably be very minute at any moment, and would be removed almost immediately. Extraction of an Enzyme capable of Reducing Nitrate to Nitrite.— Grass was dried at the temperature of the air, powdered, treated with water and left overnight to extract at 30° C. Chloroform was added for antiseptic purposes. The solution so obtained was filtered, and treated with alcohol to precipitate the enzyme. ‘This was filtered off, washed and dried. The following experiments were then set up :— 1.—Enzyme in a solution of glucose, asparagin and potassium nitrate in water. 2.—Enzyme in a solution of cane sugar, asparagin and potassium nitrate. 3 and 4.—Controls to 1 and 2 in which the enzyme had been boiled for some time. 5.—Enzyme in a solution of asparagin and glucose but with no potassium nitrate present. 6.—Control to 5 ; containing the enzyme after prolonged boiling. After twenty-four hours gas was being liberated by 1 and 2, but not by 3, 4, § or 6. The glucose and cane sugar experiments had approximately the same volume of gas in each, but only about 1°5 c.c. was obtained in 94 | BIO-CHEMICAL JOURNAL either case. No analysis was made, but from its small solubility it was concluded that it was not carbon dioxide. The enzyme obtained in a similar way from Elodea gave the same results. Ata later date this experiment was repeated upon a larger scale, and after two unsuccessful attempts, sufficient gas was obtained to make quantitative analysis quite possible. . By extraction from avery large bulk of the dried plants o°5 gramme of the dried and powdered enzyme was obtained, though, of course, still in a very impure state. This was placed in water containing respectively 2 per cent. of potassium nitrate, asparagin and dextrose. The enzyme, which only partially re-dissolved after drying, was placed in a test tube containing the solution ; over this was inverted a slightly larger test-tube, and both these were placed in a larger dish of the solution. In this way owing to the slowness of the outward diffusion of the enzyme, very little gas was lost as a result of its liberation taking place outside the walls of the larger test-tube. The reaction proceeded in an incubator kept at 30° C. from November 28th until December 11th; thymol was used as an antiseptic, and on the later date when the experiment ceased, there was not the slightest indication of bacterial activity in the solution. During the first week the liberation of gas was very slow, but latterly it collected more rapidly, and at the end of the experiment 6:2 c.c. were available for analysis. This gas underwent no change in volume, either over strong caustic potash or upon treatment with pyrogallol and potash (solutions | made up according to Clowes” formula) ; it was therefore concluded that the only gas present was nitrogen. A nitrate reducing enzyme has also been found to be present in the following plants :—Potamogeton, Vallisneria, Iris, Vicia faba, various Gramineae. 1. Clowes, Brit. Association Report, 1896, p. 74. NITRATE REDUCING ENZYME IN GREEN PLANTS 95 In the case of Vicia faba, it was found in all parts of the plant, in root, stem, and leaves; but the reaction was longer in starting, and slower in progress in the case of the roots when placed in the nitrate and asparagin solution. As far as our experiments go, there seems no reason to doubt its very general distribution in plants. CoNCLUSIONS The presence of a reducing ferment in green plants seems to have been established by means of this reaction with asparagin. It is not intended to suggest that this actual reaction occurs normally to any great extent in green plants, as the asparagin occurring in such plants is presumably to be regarded as an upgrade stage in the synthesis of proteids. Further, as asparagin occurs to a considerable extent in such plants, it seems essential that the centres of nitrate reduction and of proteid formation must be quite distinct. The reaction is to be regarded as abnormally wasteful in the plant economy, and not occurring in nature to any appreciable extent. Its occurrence under the experimental conditions has to be regarded as being due to the excess of both nitrate and asparagin in the solutions in which the plants were placed. Possibly, under the conditions existing in ensilage, and in similar cases, the loss of nitrogen that takes place in the slowly decomposing heaps of grass may be due in part to the evolution of gaseous nitrogen, owing to the distribution of the enzyme becoming, as it naturally would, less localised. In the normal plant the only conditions necessary for nitrate reduction seem to be the presence of ‘the enzyme, found in roots, stems, and leaves, and a suitable carbohydrate. The latter condition suggests the green leaf as the centre of reduction, and this agrees with the distribution of nitrates in the plant. Our results seem to show that any hexose or polysaccharide is suitable for the supply of energy for nitrate reduction ; not as in 1. Evolution of gaseous ammonia takes place at the same time, and probably accounts to a large extent for the loss of nitrogen that occurs. 96 BIO-CHEMICAL JOURNAL later stages of proteid synthesis where, according to Borodin’ and Hansteen,’ glucose is the only carbohydrate which, together with asparagin, can provide the necessary basis for construction of these bodies. In conclusion we wish to thank Mr. J. H. Priestley for his valuable advice and helpful criticism throughout the progress of the work, and writing of the paper. Our thanks are also due to Dr. F. F. Blackman and Mr. F. L. Usher for kindly criticism and suggestions. 1. Borodin, F. C. S., Abstracts, Vol. II, p. 323, 1899. z. Hansteen, Chem. Centr., Vol. 1, p. 295, 1897, from Ber. deut. Ges, Vol. XIV, p. 362-371 97 OBSERVATIONS ON THE _ SIGNIFICANCE OF THE HAEMOSOZIC VALUE OF THE BLOOD SERUM By Caprain D. McCAY, M.B. (R-U.I.), LM.S., Professor of Physiology, Medical College, Calcutta. From the Physiological Laboratories, Medical College, Calcutta (Received December 23rd, 1907) * The term haemosozic value of the serum has been made use of to indicate those constituents present in the serum which preserve the red blood corpuscles from solution.” It is well known that on a gradual dilution of the plasma of the blood with distilled water we arrive at a point when the red blood corpuscles are broken up and the haemoglobin goes into solution—the blood is then spoken of as -*laked blood.’ Based on this reaction Wright and Kilner® and later Wright and Ross,* described a new method of testing the blood and urine, and shewed that by means of the relationship existing between the haemosozic value of the serum and the haemosozic value of the urine we had a means of discriminating between physiological albuminuria and the albuminuria of renal disease. _ Working on the lines laid down by the ‘above-mentioned authors, I found’ that the excretory quotient test, 1.e.— the haemosozic value of the urine the haemosozic value of serum anaemia, and particularly in the anaemia of ankylostomiasis, very similar to those found to be the case in organic disease of the kidneys; viz., the value is always below that observed in health. gave values in the different forms of Dated Calcutta, December 5th, 1907. Armand Ruffer, British Medical Fournal, 1903, 1904. Wright and Kilner, Lancet, April, 1904. * Wright and Ross, Lancet, October, 1905. McCay, Lancet, June, 1907. eee vo 98 BIO-CHEMICAL JOURNAL Reference to the literature of the subject will show that in healthy Europeans the excretory quotient is always represented by the figure 2 or over, and in Bengalis by 1 or over; while in nephritis or anaemia with oedema, as the case may be, it falls much below 2 in Europeans, and usually well under 1 in Bengalis. A full description of the technique of the method of carrying out the test will be found in the numbers of the Lancet referred to, and need not detain us. The points with which the present paper will deal are :— (i) On what does haemolysis caused by dilution really depend. (ii) What 1s measured by an estimation of the haemosozic value— (1) In health, (2) In disease. (iii) The significance of the modification of the haemosozic value of the serum by drugs and the bearing of a lowering on the incidence of blackwater fever. I.—Tue Cause or THE SOLUTION oF THE RED Bioop CorpuscLEs - on DiILuTION oF THE PLASMA The blood may be looked upon, so far as the erythrocytes and plasma are concerned, as a mass of impermeable or slightly permeable globules floating ina fluid medium of the same density. Very careful measurements by Krénig and v. Fiirth’ have shewn that the freezing- point of the blood-plasma, or of the blood serum and blood-corpuscle pulp is the same ; and the same osmotic equilibrium has been shewn to exist between the various body fluids, with the exception of the urine. : It may, therefore, be accepted that the osmotic tension of the’ plasma and of the corpuscles is identical; any modification of the density of the plasma, as, for instance, its dilution by distilled water, will upset the osmotic equilibrium existing between the corpuscles and the plasma, and by osmosis water will tend to pass into the red blood corpuscles and the salts to pass out. 1. Monatschr. fiir Geburtsh. u. Gynak., 1901. -HAEMOSOZIC VALUE OF SERUM 99 In practice it has been found that a dilution of the plasma by a weaker and weaker saline solution (two volumes of the diluted saline being added-to one volume of blood) gives a very definite point when haemolysis takes place—the haemoglobin going into solution in the plasma. This point may be termed the haemolytic point. To make the reaction clear it might be expressed in-the form of an equation :— If x equals the total salt concentration of the plasma of the blood examined, and it is found that— I vol. of blood + 2 vols. of a < NaCl = haemolysis, normally « = about 0°85 per cent., and Bois 0°130 per cent 45 +. 3 P <2 substituting these values in the equation— = I vol. of 0°85 per cent. solution + 2 vols. of 0°130 per cent. solution, _ 0°85 per cent. + 2 (0°30 per cent.) 3 that is, the salt concentration of the plasma in the example at which haemolysis takes place is a concentration of 0°370 per cent.'; in other words, the plasma of the blood will bear dilution until it becomes reduced from a 0°85 per cent. solution to a 0°37 per cent. solution before any general breaking up of the red blood corpuscles takes place. ‘The fact that haemolysis does take place on a dilution of the plasma shows that the envelope of the red blood cells is impermeable to contained salts and haemoglobin ; if this were not the case, these would wander out until the osmotic tension of the plasma and inside the corpuscles reached equilibrium and no haemolysis would occur. Instead of this, the salts and haemoglobin of the corpuscles not being able to pass out, water finds its way in by endosmosis, the corpuscles swell up and, at the degree of dilution corresponding to the haemolytic point, sudden and complete disruption of the erythrocytes takes place and the blood is laked. = 0°370 per cent." 1. Ihave urposely taken the haemolytic point very low. In the blood of man haemoglobin is extruded from the red at a concentration of a sodium chloride solution of 0°47 per cent. 100 BIO-CHEMICAL JOURNAL We have, therefore, in this dilution method a very definite means by which the degree of dilution of the plasma or serum necessary to cause haemolysis can be estimated, and by using diluted normal saline solutions, as introduced by Wright, its value can be expressed in terms of NaCl. So far as our theoretical knowledge of the physical chemistry of the blood goes, it would appear that the solution of the red blood corpuscles brought about by a dilution of the plasma with distilled water or a weak saline solution, as the case may be, depends on a difference of osmotic tension inside and outside the red blood corpuscles —haemolysis taking place when the osmotic pressure within the corpuscles is able to overcome the resisting power of the corpuscles to disruption. I].—_Wuart 1s MerasureD IN AN EsTIMATION OF THE HAEMOSOZIC VALUE OF THE SERUM 1. In health.—As indicated above the haemosozic value of the plasma or serum should vary distinctly with its osmotic pressure, which in its turn depends on the total number of molecules in solution. Neglecting the large proteid molecules, which have very little effect on the osmotic pressure, it would appear that the haemosozic value is really a measure of the total salt concentration or total salinity of the plasma or serum, 7.¢., the total number of inorganic molecules in solution. Like The evidence that this is the case :— (i) The specific electrical conductivity of fluids tested by Dr. Waller was found to be roughly proportional to the salt content. as estimated by this dilution method.’ (ii) Working mainly on Bengalis over a series of eighty-four observations, I have found? the salt content as estimated by the dilution method to be 1°054 per cent. expressed in terms of NaCl. - This is a slightly higher figure than that given for the percentage of salts in the serum in Europeans but, by actual quantitative analyses, I found 1. Lancet, quoted in Wright and Kilner’s paper. 2. Sctentific Memoirs ; * Metabolism of Bengalis ’ ; in press. HAEMOSOZIC VALUE OF SERUM 101 that in healthy Bengalis the average percentage of total salts present in the blood was 1°06 per cent.—a figure practically identical with the average salt concentration obtained by the dilution method. While it may be admitted that the electrical conductivity is a more accurate measure of the number of inorganic molecules in the serum than the amount of ash it yields, still quantitative estima- tions give a fairly close approximation to the total salts present. It may, therefore, be accepted that for all practical purposes in estimating the haemosozic value of the serum in healthy individuals, what is actually measured is the total salt concentration or total - salinity. | 2. In disease—In the observations recorded by Wright and Kilner, and by myself, on the salt concentration of the serumin anaemic conditions, it will be seen that the serum is modified in the same characteristic manner as found in estimations of the haemosozic value in nephritis with oedema—the serum giving in these cases a very high figure for its haemosozic value. Some examples of the results are shewn in Table I. Tasie | Haemosozic Reference Authority Disease value of serum in Remarks terms of NaCl Lancet, 1904. Wright and Kilner Pernicious 24% 1,700,000 Red anaemia blood corpuscles “Do. do. Chlorosis 156% Do. do. do. 3°00% Oedema of legs Do. do. Anaemia 2"40% Gastric ulcer Do. do. do. 2°44% Thrombosis of veins of legs Lancet, 1907 McCay Anaemia 2°12% Ankylostomiasis ; oedema and ascites Do. do. do. 609% Do. Death Do. do do 3°995% Oedema and ascites Do. do. do. 2°32% Ankylostomiasis, marked oedema Do. do. ~ do. 1°72% 102 BIO-CHEMICAL JOURNAL. From these results and from other evidence brought forward it appeared probable that in anaemia and in oedema from ‘whatever cause there was an increase in the total salts of the blood, and par- ticularly an actual retention of chlorides by the tissues and blood plasma. | . While not denying that in oedema and nephritis there isretention of chlorides so that the system and blood contains an absolutely larger amount of total salts, it appeared most improbable that the blood plasma should possess such a relatively high percentage as that obtained for the haemosozic value in Table I. In order to clear up the mystery of these high percentages of salt of the serum—in one instance as high as 6°0 per cent.—a further investigation was undertaken, several different lines of research being resorted to. As the results obtained would appear to be of some importance, I make no excuse for giving them in detail. A.—lInvestications To ExcLupE THE PossIBILITY OF A CoMBINATION OF SALTs wiTH CoLtorip MATERIAL It seemed absurd to suppose that in the series of cases shewn in Table I there could be anything like the large percentage of free salt present in the serum which the estimation of the haemosozic value indicated ; further, it appeared probable that some of the salt might be combined with the colloids of the serum and that, on manifold dilution with distilled water—as actually takes place in the estimation of the haemosozic value—the combined salt gradually becomes dissociated, and coming off pari passu with the degree of dilution, maintains for some time the isotonicity of the diluted serum and the red blood corpuscles. Eventually, however, as the serum becomes | more and more diluted its salt concentration falls below the point necessary for the maintenance of the integrity of the red blood corpuscles, and haemolysis takes place. To put this view to the test a number of cases, in which the haemosozic value was high, were examined with regard to the per- centage of chlorides in the serum before and after a twenty-fold dilution with distilled water. The results obtained are shewn in Table II. HAEMOSOZIC VALUE OF SERUM 163 Taste IT Haemosozic Percentage of Percentage of Case Date value of serum in chlorides of serum chlorides of serum A nee terms of NaCl before dilution after dilution I 14/7/07 1°64.% 105% 1706 % 2 17/7/07 3°24% 0°70% 0°75. % ss 29/7/07 1°358% 0°724.% 0°73 % np 28/8/07 5°44% 0°692% 0°692% ngs 14/9/07 318% - 0°668 %, 0688 %, 6 19/11/07 346% 9°719% 0°734% a 21/11/07 2°26% 0°485%' “+ 0°498% 8 22/11/07 0°97 % 0691 % 0691 %, zs The table shews no definitely large increase in the percentage of chlorides of the serum either before or after a twenty-fold dilution with distilled water. We may, therefore, safely conclude that what- ever is the cause of the high percentage of the haemosozic value of the serum in anaemia and oedema it cannot be due to a retention of the chlorides in combination with a colloid as Forster and Marie are inclined to believe. The percentage of NaCl in the serum is, of course, no guide in determining to what extent retention of chlorides has taken place, for the hydraemic condition of the serum may conceal a great increase in NaCl and, further, the tissues may take up a large quantity of NaCl. This is probably the case in oedema. B.—Investications UNDERTAKEN WITH THE VIEW OF DETERMINING WHEREIN LAY THE AcTuAL Causr or HicH Harmosozic VALUE So far in these investigations I had firmly believed that the high haemosozic values were due to something—most probably inorganic salts—present in the serum in much greater proportion than in health and, therefore, requiring a larger number of dilutions to lower the osmotic pressure of the serum sufficiently to cause disruption of 1. O0cdema disappearing may explain the low percentage of chlorides. 104 BIO-CHEMICAL JOURNAL the red blood corpuscles. Having failed to find any marked increase in the chlorides of the serum, I began to suspect some change in the resisting power of the erythrocytes as a likely explanation. In order to test this view, the following method was devised nd carried out in a series of cases presenting a high haemosozic value :— : The haemosozic value of the patient’s serum was estimated in the usual way—his own red blood corpuscles serving as the indicator of haemolysis. Having obtained the figure representing the haemosozic value of the serum in terms of NaCl, a sample of the same serum was now tested for its haemosozic value, not against his own red blood corpuscles, but using the red blood corpuscles of a normal individual as the indicator of haemolysis. ‘The results obtained in this way are very striking and throw considerable light on the question at issue; they are shewn in Table III. Taste III Haemosozic value of | _Haemosozic value of Case Date serum versus own red serum versus normal Remarks blood corpuscles red blood corpuscles I 17/7/07 . 3°24% 0°984.% Anaemia and oedema 2 29/7/07 1°358% 089% Bright’s disease, etc. 3 30/7/07 169% 1'06% Do. 4 16/8/07 5°44% 1°06 % Ankylostomiasis anaemia and oedema 5 19/11/07 3°46% 102% Do. It is evident from the results obtained in this way that in using the patient’s own red blood corpuscles as an indicator a far larger number of dilutions with distilled water of his serum are necessary to effect haemolysis than when the red blood corpuscles from a healthy individual are acting as the indicator. In both cases it is the serum from the same patient that is diluted, the indicator—red blood cells—alone changing, yet it has been found that the serum may require up to a thirty- or forty- fold dilution to effect haemolysis of his own erythrocytes, whereas a six- or eight- fold dilution will be sufficient to cause disruption of normal red blood cells. These HAEMOSOZIC VALUE OF SERUM 105 figures would, therefore, appear to point toa greatly increased resisting power of the erythrocytes in those conditions in which the haemosozic value of the serum is high, and not toa change in the total salinity of the serum whereby its osmotic pressure is greatly enhanced. We may, therefore, conclude that the increased resisting power of the red blood corpuscles to haemolysis in the several conditions examined does not depend on an increased salt concentration of the plasma—as the results obtained in healthy individuals would lead us to believe—and it cannot be explained by physical reasons alone. Some other factor is present which in some way is able to increase the resisting power of the red blood cells to disruption. What that factor is, it is impossible to say at present. It may be that in some way the permeability of the erythrocytes is greatly increased—as for instance by the presence of ammonium salts—thus allowing of a free exchange of inorganic matter between the corpuscles and the diluted plasma, or, what would appear to be more likely, a new substance is present which is able to prevent a breaking down of the ted blood corpuscles in the diseased conditions in which a high haemosozic value of the serum is found. ‘The evidence that something capable of doing this does exist will be discussed below. C.—InvesticATIons UNDERTAKEN TO SHEW THAT IN CERTAIN DisgasEp ConpiTiIons THE HicH Haemosozic VALUE Is NO CRITERION OF THE ACTUAL QuanTiTy oF SALts PRESENT IN THE BLoop Having failed to find evidence of any great increase in the chlorides or of the salt concentration when measured against normal red blood cells, I next turned to actual estimations—quantitatively— of the percentage of salts in the blood of individuals shewing a high haemosozic value. It will be fairly obvious that, if the blood in the particular conditions shewing such high values for the haemosozic power does really contain a higher—much higher—percentage of salts; and if, due to this, the haemosozic value is greatly increased, then by quantitative analysis this increased salinity should be demonstrated. 106 BIO-CHEMICAL JOURNAL The following table—Table IV—gives the facts obtained by analysis :— Tasie IV Haemosozic value Total salts Total solids Case Date Disease of serum in of blood of the blood terms of NaCl I 28/8/07 Marked anaemia and 5°444% 1'20% 10°579% oedema general 2 14/9/07 Anaemia and oedema 3°18% 1°103% 16°176% 3 19/11/07 Do. 3°46% 0'987% 12°862% 4 21/11/07 Do. 2°26% 0°988%, 15°563% 5 Do. Heart oedema 0°848% 0°926% 14°646% These results would appear to be absolute proof that while the haemosozic value of the serum in health corresponds very closely with its total salts, in certain diseased conditions characterised by a high haemosozic value—notably nephritis, anaemia and oedema generally—the haemosozic value is no measure whatsoever and in no way. corresponds to the actual percentage of salts in the blood or serum. Again, by this method of investigation the conclusion is forced on us that some unknown factor, other than an increase in the percentage of salts in the blood, is present able to increase the resisting power of the red blood corpuscles. Further, from the investigations carried out in cases of marked oedema it would appear that there is very little increase in the per- centage of salt present—the blood being able in some way or other to maintain its chemical composition at a fairly uniform level, at least so far as its inorganic salts are concerned. 111.—Tue Sicniricance or THE Mopirication or THE HageMosozic VALUE OF THE SERUM BY Drucs, AND THE BEARING OF A LoweRING ON THE INcIDENCE oF BLACKWATER FEVER As it was evident from the results recorded above that the haemosozic value of the serum or, more accurately, perhaps, the resisting power of the red blood corpuscles varied within very wide limits in disease, it appeared to be a matter of some importance to determine the action of certain commonly used drugs, and more particularly what effect ‘quinine’ had on the resistance of the erythrocytes. HAEMOSOZIC VALUE OF SERUM 107 In blackwater fever there is undoubtedly a great and wide-spread breaking down of the red cells of the blood ; it was, therefore, evident that any drug, the absorption of which caused a lowering of the resisting power of the red cell, would, other things being equal, tend to bring those cells within the danger zone and precipitate an attack of blackwater fever. In blackwater fever it is generally accepted that there is a virulent malarial infection primarily—the infection probably being repeatedly effected daily—so that many of the red cells have been already broken up, many others on the point of breaking up, and a large number of those remaining injured by the malarial parasite ; if now, for any reason the salt concentration of the plasma becomes seriously lowered the effect will be to bring the red corpuscles— and particularly the innumerable injured corpuscles—nearer and nearer to their haemolytic point according to the greater and greater lowering of the total salinity of the plasma. That it is those corpuscles, injured by the presence of the malarial parasite, that do break up in blackwater fever is evident from the fact that no parasites can be found, once the attack is precipitated, the corpuscles hitherto containing them having disappeared on disruption. In order to obtain information of the effects of malaria and its treatment by ‘quinine’ on the salt concentration of the blood, a series of observations was begun. However, as malaria during the period was rare in Calcutta, I was forced to carry out most of the investigation on healthy individuals, but the results in the few cases of malaria treated with quinine sulphate shew exactly the same modification of the plasma as met with in health on administration of the drug. The following table makes clear this modification due to the particular drugs administered. In order to check the results, a chemical estimation of the chlorides of the blood serum was made before and after the drug was given, and, as will be seen, the results are in harmony with the changes observed in the salt concentration. 108 BIO-CHEMICAL JOURNAL Taste V Haemosozic : Haemosozic Chlorides of Chlorides of Case value of Drug and value of serum serum No. Date Disease serum in dose serum in before drug after drug terms of terms of NaCl (before) NaCl (after) I 20/7/07. ~— Healthy 0°984% Quinine Sulph. 0°754% -— -- gr. 30 2 23/7/07 Do. 085% » gr. 20 o808% —- — 2 25/7/07 Do. 0°85 % » . Bt. 90 O'7585, a — 2 26/7/07 Do. 085% > te ZO “OGZY — — 2 27/7/07 Do. 0°85 % » gt. 20 0'806% — eee 3 29/7/07 Do. o°888% ,, gr. 30 0'742% — — 3. 31/7/07 Do. 0'888% 4, gr. 30 0°707% — — 3 1/8/07 Do. o'888%- ,, gr. 30 0'707% — -— 4 3/8/07 Do. 0°965% ._.;. . gt. 20, 0°762% -- — + 5/8/07 Do. 0°965 % ” gr. 10 0°736% ey Te 5 7/8/07 Do. 107% =, BF. 5 0°965% 0°753% 0°724% 5 9/8/07 Do. 107% 45, BE. S$ O'914% —«*0°753%_~——-0°723% 5 10/8/o7_—Ss«dso. 107% «BT. § 0°863% ~—»-0°753% ~—-0°705% 5 12/8/07 Do. 1:07% » gt. 10 o'701% 0°753% 0612% 5 13/8/07, ~—Ss«zDo. 107% BT. 10 0'876% +. 0°753% 0°720% 5 14/8/07 Do. 107% 5». gt. 10 1'016% 0°753% 0°758% 5 15/8/07 Do. 1':07% 9» Bt..20 104% 0°753% 0°754% 6 23/9/07 Do. 0'928% (Qui.Sulph. 0°779% 068% 0°676% gr. XV, Mag. Sulph. gr. 30, Ac. Sulph. : dil. Mi. 15) 6 25/9/07 Do. 0°928% Do. o'861% a 23/9/07 Do. 1'168% Magnes. . Sulph. gr. 30 —0°928% o'701% 0'684% 7 25/9/07 Do. 1'168% . Do. o'812% _ 0°679% 7 26/9/07 Do. "168% Do. 0'719% — 0°675% 7 27/9/07 Do. 1'168% Do. o'719% — —- ° 7 28/9/07 Do. 1'168% Do. 1°04.% (watery stools) 0°695% The results shewn in above table are of very great interest ; in every instance in which a sulphate was given by the mouth there was a well-marked fall in the haemosozic value of the serum measured in terms of NaCl by the dilution method. Further, it will be seen that the rapidity of the fall depended more or less on the strength of the dose. HAEMOSOZIC VALUE OF SERUM 109 Another important point, brought out in Nos. 2, 5, 6 and 7, is the recovery or rise to the normal of the haemosozic power when daily doses are administered on consecutive days. We might look on the early effects of sulphates on the haemosozic value as a negative phase which gradually passes off on regular administration of the drug. . The following table shews the results obtained in cases of ‘malaria treated by quinine sulphate. Taste VI Haemosozic value - Haemosozic value of serum interms Drugand _ of serum in terms No. Date Disease of NaCl versus dose of NaCl versus Remarks normal red cells normal red cells I 22/7/07 ~—+éBenign 0°928% Quin. Sulph. 0°776% Blood examined on Tertian gr. 20 4th day, 7.¢., after Quin: Sulph. gr. 80 had been given 2 1/8/07 Malig. 1'004% Quin. Sulph. 097% : Tertian gr. 30 3/8/07 Do. Do. Do. 0°848% — 3 24/8/07 Malig. 1:027% Quin. Sulph. 0976% 10 ae given for Tertian gr. 10 two days 29/8/07 Do. Do. Quin. Sulph. 012% § grains for 5 days " be gt. 5 4 26/8/07 Malig. 0°928% Quin. Sulph. 0°849% — Tertian gr. 30 5 26/8/07 Malig. o928% Quin. Sulph. 0°742% 45 grains daily for Tertian daily 45 grs. two days The same fall in the haemosozic value is seen as occurs in health when sulphates are administered. These cases were all examined by using the red blood corpuscles of a healthy individual as the indicator—the patients were in hospital, and a few drops of blood were drawn off and sent to the laboratory in exactly the same way as for Widal’s test. It is important to note that by using normal red corpuscles as the indicator I eliminated any change in the resisting power of the patient’s red cells due to causes other than changes in the total salt concentration of the blood. 110 BIO-CHEMICAL JOURNAL Tue SIGNIFICANCE OF THE MODIFICATION OF THE HAEMOSOZzIC VALUE EFFECTED BY SULPHATES AS SHEWN IN TABLES V AND VI In an earlier part of this paper I brought forward evidence to shew that the haemosozic value of the serum in health expressed. in terms of NaCl gave results almost identical with those obtained by a quantitative estimation of the total salts of the blood. In the few cases of malaria examined—using normal red blood corpuscles for an indicator—it may be accepted that the percentages obtained for the haemosozic value are an actual measure of the total salinity of the blood, both before and after the administration of sulphate of quinine. Therefore, both in healthy individuals (Table V) and in malaria (Table VI) the lowering of the haemosozic value of the serum is really due to an actual diminution of the total salts of the blood, and not to some other cause such as a lessening of the resisting power of the red blood corpuscles. ‘That this is the case we have a certain amount of corroborative evidence in the accompanying depression - of the chlorides of the serum (wide Table V, Nos. 5, 6 and 7). It is, therefore, evident that—other things being equal—this depression of the haemosozic value of the serum or, what would appear to be the same thing—so far, at least, as healthy individuals or those suffering from malaria are concerned—the lessened salt concentration of the blood, produced by the administration of sulphates, does upset the osmotic equilibrium existing between the red blood corpuscles and the plasma in which they float. The diminution in the number of inorganic molecules present in the plasma lessens its osmotic pressure, and the osmotic pressure inside the red blood cells remaining as before, the effect will be to tend to burst the corpuscles open. ‘This effect will be the greater the more the inorganic molecules of the plasma are diminished in number ; and disruption from this cause will, of course, be the more easily effected the more the corpuscle is injured by the presence of the malarial parasite. : As already stated, the haemolytic point of normal blood is about HAEMOSOZIC VALUE OF SERUM III 0°37 per cent., i.¢., the red blood corpuscles will bear a dilution of the plasma until it becomes about equal to a 0°37 per cent." solution before actual general haemolysis takes place. ‘This fact can be easily verified by the simple experiment of mixing an equal volume of blood and distilled water together when, it will be seen, no general laking of the blood has occurred ; but, on the further addition of a fraction of a volume of distilled water quite suddenly, on the proper dilution being attained, general haemolysis takes place. It might be argued from this that if the erythrocytes will bear a dilution of the plasma until its density is reduced from about a 0°85 per cent. solution to ‘one of about 0°37 per cent., the lowering produced by sulphates— such as quinine sulphate, magnesium sulphate or acid sulph. dil.— would be quite insufficient to cause haemolysis. (The greatest reduction observed after the administration of sulphates in any of the persons examined only lowered the density of the plasma from a 1°07 per cent. solution to a o°701 per cent. solution.) This is quite true so long as we are dealing with normal blood or even in con- ditions of ordinary malarial infection, otherwise haemolysis and black- water fever would follow after the administration of sulphates in every case of malaria. In order, therefore, to explain the onset of an attack of blackwater fever, it is necessary to bridge the interval between 0°63 per cent., the lowest salt concentration above recorded, and 0°37 per cent.,’ the lowest depression of the density of the plasma normal corpuscles will bear. A purely physical explanation might be advanced which would cover much of the ground. In blackwater fever we are dealing with a condition in which many of the red blood cells are invaded by the malarial parasite, resulting in injury to the cohesive power of the stroma and haemoglobin so that the resisting power of the cells is greatly lessened ; it is, therefore, quite probable that a sudden lowering of the osmotic tension of the plasma due to a decrease in the number of inorganic molecules contained—brought about by the administration of sulphates—becomes the determining factor 1. Probably 0°47 per cent. NaCl is more correct. 112 BIO-CHEMICAL JOURNAL in the breaking up of those injured cells. In other words, a difference — of osmotic tension between the red cells and the plasma, which in health would have no haemolytic effect, in blackwater fever becomes a very important factor in the causation of the attack. ! The question therefore arises :—What other factor is there in blackwater fever the presence of which permits of haemolysis taking place under circumstances that would not occur in health or in ordinary malaria ? 3 . Two explanations are evident from the results above recorded. — The first of these is the actual injury done to the red corpuscles by the presence of the malarial parasite ; that this in itself is not sufficient would appear probable from the fact that even severe malarial fever treated with heroic doses of quinine sulphate will not develop into blackwater fever, except in certain well-defined areas. The second explanation would rest on the probable formation within the system of an haemolysin or haemolytic ferment by the action of which the resisting power of the red cells becomes diminished so that, in extreme cases, solution takes place without any great change in the composition of the plasma; but, in the majority of recorded cases, the power of resistance of the erythrocytes being diminished by the haemolysin, the lowering of the osmotic pressure of the plasma—due to the action of sulphates—becomes the determining factor, and sets up an attack. The presence of a substance such as this would completely explain the onset of haemolysis long before any such dilution of the plasma. was attained as has been found necessary in health. ‘The degree of dilution necessary for haemolysis would form a most interesting and important subject for research in patients just before the onset - and during an attack of blackwater fever. ! Once recovery has begun to set in, a condition such as found in ankylostomiasis would be expected, viz., a very greatly increased resisting power of the remaining red blood cells due, in all probability, to the formation within the system of an antihaemolysin. That something in the nature of an haemolysin is present and is the cause of the great anaemia of ankylostomiasis seems now to be generally accepted. The anaemia is not due to a mere sucking of HAEMOSOZIC VALUE OF SERUM 113 blood by the ankylostoma—in fact, it is very doubtful if they suck blood at all. The great increase in the haemosozoic value, which I have found to hold true in these conditions, and particularly when oedema is well marked, would appear from the evidence brought forward above, to depend on a greatly enhanced resisting power of the patient’s red blood corpuscles. This high haemosozic value is made up of two factors: one, the ordinary haemosozic value, depending on the number of inorganic molecules present in the plasma; the other, something most probably of the nature of an antihaemolysin, by virtue of which the erythrocytes of patients—suffering from ankylos- tomiasis for some time, or recovering therefrom—are endowed with a very great power of resistance compared with normal red corpuscles. A similar explanation must hold also for certain other forms of anaemia and for the anaemia of nephritis, as, even in renal disease, I have found a high haemosozic value is no measure of the actual total salts of the blood. A body possessing haemolytic properties has been isolated from the tissues of the ankylostoma, and the formation of an antibody within the system would be expected ; indeed, the presence of such a substance forms the only reasonable explanation of the high resisting power of the erythrocytes in ankylostomiasis, at least in its later stages, the only stages I have had a chance of investigating. GENERAL SUMMARY OF CAUSATION OF OnseT oF BLACKWATER FEVER Practically everyone agrees that the malarial parasite is the real cause underlying the condition, and that by prevention of malaria blackwater fever would become non-existent. The great majority of clinical observers consider ‘ quinine’ the actual exciting cause precipitating an attack. This action of quinine is so widely believed in by those of great experience in the disease as to appear worthy of general acceptance. The word ‘quinine’ has been used very loosely in connection with malaria; in probably 99 per cent. of instances of administration of the drug by the mouth, ‘quinine’ 114 BIO-CHEMICAL JOURNAL means the sulphate. From the investigations and results recorded in an earlier part of this paper on the action of sulphates on the inorganic salts of the blood, it seems very probable that the lowering of the total salt concentration from the administration of sulphates, such as quinine sulphate, magnesium sulphate, etc., in a patient saturated with malaria, is quite sufficient to precipitate an attack. In all probability, besides the actual weakening of the erythrocytes from malarial infection, another factor, causing a lessened power of resistance of the red cells, comes into force, and there is reason to believe this is of the nature of a haemolytic toxin or haemolysin. How this haemolysin is formed; the conditions necessary for its formation; why its formation is limited to malaria occurring within certain well-defined areas, etc., are questions which, for the present, cannot be answered. It is not improbable that the relative virulency of the parasites under different climatic conditions may be found to explain the mystery. For the present, it would appear, from the effects of sulphates on the salts of the blood, that the adminis- tration of quinine sulphate even in small doses may just make all the difference between a malarial fever of a special type and a malarial fever that developes into blackwater fever. How tue Haremosozic VALUE OF THE BLOOD MAY BE INCREASED— A PropaBLe Rationat INDICATION FOR THE TREATMENT AND PrRopHYLAXIS OF BLACKWATER FEVER As it was possible, by the administration of sulphates—probably also by giving potassium salts, alkaline carbonates and compounds of alkalis with vegetable acids—to decrease the total number of inorganic molecules in the blood plasma, and thus, in malaria, — tend to produce haemolysis of the red blood corpuscles and perhaps precipitate an attack of blackwater fever, it seemed a rational indi- cation to discover some means of increasing the total number of inorganic molecules of the plasma; perhaps, in that way, lessening the tendency of the red blood corpuscles to haemolysis and thus attain a position of being able to prevent the development of blackwater fever entirely. HAEMOSOZIC VALUE OF SERUM 115 The SO, of sulphates, probably on absorption, combines with the Na, Ca, etc., of the blood plasma, forming sulphates which are eliminated at once on-arrival at the kidneys, thus decreasing the number of inorganic molecules in the plasma; it, therefore, seemed possible by giving a salt that need not necessarily be eliminated at once—i.¢., one not absolutely foreign to the system—to be able to increase the total salts of the blood and thus raise the haemosozoic value of the plasma. ‘The salts most likely to cause this increase were the chlorides. I therefore began a series of observations on the effects of the administration of chlorides by the mouth on normal individuals. As it was very necessary, in view of the malarial origin of blackwater fever, that quinine should be given, the form of chloride administrated was quinine hydrochloride. The results obtained are shewn in Table VII. Taste VII Haemosozic Haemosozic Chlorides Chlorides Case Date value of serum Drug value of serum ofserum of serum No. in terms of given in terms of before after NaCl (before) NaCl (after) drug drug I 17/7/07 0°903% Quinine Hydrochloride gr. 25 1°044% — -- 2 16/8/07 1:°09% Do. do. 20 109% . 0'712%. 0°723% 2 18/8/07 1:09% Do. do. 20 109% — 0'°732% 2 19/8/07 1°09% Do. do. 20 109% -- o'719% 2 20/8/07 1709% Do. do. 10° 1°170% — 0°745 % 2 21/8/07 109% "Nil. 0°899% a SOS 3. 21/8/07 0°865% Quinine Hydrochloride gr. 10 104% 073% — 3. 23/8/07 + 0'865% Do. do. 10 0°865% — o719% 3 24/8/07 0°865% Nil. 0°865 % = — 3 25/8/07 0'865% Nil. 9°959% sr —- 3. 26/8/07 0°865% Quinine Hydrochloride aa 1°392% — 0877% gr. 3 27/8/07 0°865% Do. do. gr. XXX 1°866% — 0740% 3. «28/8/07 0°865% Do. do. gr. XXX 1°856% — o718% 21/11/07 0'865% me eo gr. “et 1'076% 0°723% 0°736% : or. gr. » Ae. Hydrochlor. SL Mo XV 4 23/11/07 0°865% Do. do. 1'I57% — = 4 24/11/07 0°865% Do. do. 143% —- 0'964 — 6 5/11/07 0'814% Quin Hydrochlor. gr. X, Ac. 1'016% ams — : Hydrochlor. Mi. X 116 BIO-CHEMICAL JOURNAL These results present a very different picture to that obtained from an administration of the sulphate of quinine or other sulphates. No lowering of the haemosozic value of the serum was observed in any one instance, and, in the majority of cases, the value was increased. That this increase is in part, at least, due to an increase in the number of inorganic molecules of the serum would appear probable from the accompanying increase in the chlorides of the serum. ‘The increase is very marked when full doses of quinine hydrochloride are given, and this is further enhanced by an addition of sodium chloride and hydrochloric acid to the prescription. The importance of the bearing of these results on the incidence of blackwater fever is obvious; the raising of the haemosozic value of the plasma in the treatment of malaria, especially in districts where blackwater fever is prevalent, should, other things being equal, be of great service in lessening the liability to its onset. With the knowledge of the action of sulphates on the salinity of the blood gained from the above-recorded investigations, and what the significance of that action really is, it appears to me quite time that the treatment of malaria by quinine sulphate should cease, and that a fair trial should be given to its substitute, quinine hydro- chloride or the acid hydrochloride. If this were done I think there is a reasonable hope that the number of cases of. blackwater fever following almost immediately on the ingestion of sulphates in some form—and this covers the very large majority of the cases—would © soon shew a rapid diminution, with a saving of many valuable lives yearly. 3 In connection with the views here put forward it would be interesting to obtain reliable information with regard to the relative liability to blackwater fever of those infected with the Ankylostoma Duodenale, and those—mostly Europeans—who are non-infected, From the arguments made use of above I should expect that the high resisting power of the red blood corpuscles, characteristic of those suffering from Ankylostomiasis, would probably prevent altogether the occurrence of blackwater fever in such cases or, at least, lessen the likelihood of its onset. HAEMOSOZIC VALUE OF SERUM i17 CoNCLUSIONS 1. The haemosozic value of the serum expressed in terms of NaCl corresponds with the total salt concentration. 2. In conditions of an increase in the haemosozic value above the normal, the haemosozic value is a measure of (1) The total salt concentration. (2) A factor other than the salt concentration increasing the resisting power of the red blood corpuscles ; probably an antihaemolysin. 3. The lowering of the haemosozic power of the serum by the administration of certain drugs—sulphates, potassium salts, etc.— would appear to be a most important factor, under certain circum- stances, in the precipitation of an attack of blackwater fever. 4. The raising of the haemosozic value of the serum by the administration of chlorides, such as quinine hydrochloride, sodium chloride, etc., would appear to be a rational indication for the prophy- laxis and treatment of blackwater fever. In conclusion, I wish to express my thanks to those who assisted me in the investigation by providing the material for examination. I am specially indebted to Dr. Upendra Nath Brahmachari, Campbell ’ Medical School, Calcutta, for the great majority of the pathological cases investigated. I desire also to thank Lieut.-Col. Drury, I.M.S., Major Chatterton, I.M.S., and Captain Mackelvie, I.M.S., physicians of the Medical College Hospital, Calcutta, for permission to examine the patients under their care. Since writing the above paper I have had the opportunity, due to the kindness of Major L. Rogers, I.M.S., Professor of Pathology, Medical College, Calcutta—of putting the views advanced with regard to the causation of blackwater fever to some extent to the test. The patient was recovering from a typical attack when the blood for examination was taken. ‘The haemoglobinuria had disappeared, and the ordinary symptoms of the disease were rapidly subsiding. 118 BIO-CHEMICAL JOURNAL At this period it was evident that the active cause of the haemolysis had disappeared and that in all probability the erythrocytes that had escaped disruption would be found highly resistant. Such, indeed proved to be the case. The blood at our disposal was very small in amount and had been mixed with a small amount of citrate of sodium to prevent coagula- tion. | From the examination of this citrated blood it was found that one volume of the blood required dilution with two volumes of, at N least, a $e NaCl solution before haemolysis took place. ‘This is a very remarkable result, and would most distinctly point to the presence of some substance conferring a very high degree of immunity to haemolysis on the red blood corpuscles of a patient recovering from blackwater fever. (Normal red blood corpuscles break up when one volume T of blood is mixed with two volumes of about a pores, pul solution). ‘This substance must be of the nature of an antihaemolysin, the presence of which would certainly mean the earlier presence of a haemolysin. ee 11g ON VARIATIONS OBSERVED IN THE COMPOSITION OF SODIUM GLYCOCHOLATE PREPARED BY DIFFERENT METHODS By WM. C. M. LEWIS, M.A. From the Muspratt Laboratory of Physical and Electro Chemistry, University of Liverpool (Received February 6th, 1908) Having had occasion to determine the molecular weight of sodium glycocholate in connection with other work, the results obtained were of so unexpected a character that it was thought worth while to investigate the matter more closely. The material employed (and referred to above) was Merck’s ‘sodium glycocholeate.? The method of preparation as carried out by the makers is as follows :— _ Ox bile is treated with a solution of lead acetate. The pre- cipitated lead salts are decomposed by sodium carbonate and the residue extracted with alcohol. The alcohol is evaporated off and ‘sodium glycocholeate’ results. It is evident that ‘sodium glycocholeate’ is at least a mixture of sodium glycocholate and sodium taurocholate. The makers themselves state that ‘ the substance is used for medicinal purposes only and is no chemically pure body.’ From the generally accepted view, however, of the relative proportions of the constituents of ox bile, one would expect this preparation to be at least go per cent. pure sodium glycocholate. An examination of the substance, however, showed this to be by no means the case. 120 BIO-CHEMICAL JOURNAL The empiric formulae and percentage composition of sodium glycocholate and sodium taurocholate usually stated are due to Strecker,’ and are as follows :— Formula Molecular weight %N % Na Sodium glycocholate - C,H,NO,Na 487 2°87 4°72 Sodium taurocholate C,H,,NSO,Na 537 2:60 4:25 In a preparation of the mixed salts, therefore, one would expect values for the percentage composition to lie between those given in the table, but approximating more closely to those for sodium glycocholate since this is present in much greater pigportiam than sodium taurocholate. The ‘ sodium glycocholeate ’ having been first tested for absorbed moisture (found 2-6 per cent.) and for the presence of inorganic impurities (¢.g. sodium chloride and sodium carbonate—which were found to be absent), the following systematic examination was carried out :— I. Determination of the molecular weight in aqueous solution— (a) By the lowering of the freezing point. (b) By the rise of boiling point. II. Determination of the molecular weight in alcoholic solution by the rise of boiling point. III. Determination of the percentage of sodium. IV. Determination of the percentage of nitrogen. I.—DeETERMINATION OF THE Mo.tecuLar WEIGHT IN AQUEOUS SOLUTION (a) By the lowering of freezing point of water :— Mass of the solvent = 22-5 grams Mass of * sodium Freezing point Molecular glycocholeate ’ (Beckmann therm.) A weight in grams foxe) 2°78 — —. Or1125 2-71 0:07 134 0°5332 2°46 0°32 139 0°8664 2°26 0°52 139 The mean molecular weight in water is therefore 137. 1. Strecker, Liebig Ann, d. Chem. a oe DEA VARIATIONS IN SODIUM OF GLYCOCHOLATE 121 (6) Molecular weight by the elevation of boiling point :-— Mass of solvent = 26-30 grams it Mass of Boiling point Rise of Molecular ~ * sodium glycocholeate ’ (Beckmann therm.) boiling point weight in grams foe) 1-g18 — _- 0°1765 1-940 0-022 158 0°5265 2-009 o-0gI 114? 0°9205 2°049 O'131 140 First and last determination, mean, 149 The values obtained for a molecular weight by the elevation of boiling point when water is the solvent can scarcely be taken as conclusive in themselves, owing to the comparative difficulty of carrying out a determination with this solvent. The above results must, therefore, be regarded as surprisingly good corroboration of the more trustworthy determinations obtained by the freezing point method. Since we are dealing with an alkali salt in aqueous solution it is evident that dissociation has taken place, and the above solutions are sufficiently dilute to cause approximately complete dissociation. Assuming the substance to be a mono-sodio derivative we obtain the result that the undissociated molecular weight is 278. ‘To check this, a determination was carried out in alcohol.' This requires great care in order to get rid of traces of moisture both in the alcohol and the bile salt—for this latter is exceedingly hygroscopic. Il.—Motecutar WEIGHT oF THE ‘ SopIum GLYCOCHOLEATE’ BY THE ELEVATION OF THE BorLING Point or ALCOHOL * Sodium glycocholeate ’ Rise of Molecular in grams boiling point weight 0°2208 0°037 282 02820 0°047 283 Mean, 283 1." Solutions of soaps in alcohol give molecular weights corresponding to non-dissociation. It was therefore thought (and the results justify the assumption) that no dissociation of the bile salt would take place in this solvent. 122 BIO-CHEMICAL JOURNAL The good agreement between the results obtained for the sub- stance in the undissociated state in alcohol and that calculated from the determination in aqueous solution, points apparently to the possibility of error in the usually accepted empirical formula and molecular weight. II].—PercentTAcE oF Sopium 1N ‘ SopruM GLYCOCHOLEATE ’ The sodium was estimated in the ordinary manner by first incinerating the substance and finally estimating as sulphate. The following results were obtained :— ‘ Sodium glycocholeate ’ Sodium sulphate Percentage of in grams in grams sodium [ 0°7585 01894 8-09 | 10491 0:2813 8-6 1-O125 0°2702 8-6 Mean, 8-6 Assuming the substance to be a mono-sodium derivative— A molecular weight of 283 requires 8-12 % sodium ” ” 487 ” 4°72 ” ” ” 537 ” 4°28 et The above results seem to afford fairly conclusive evidence in favour of the smaller value for the molecular weight. An alternative — hypothesis, however, might be possible. The formulae of the bile acids may be mainly according to those of Strecker, but the acids © may be dibasic and hence their salts may contain two sodium atoms, viz., CygH,NO,Na, and C,H,NSO,Na, This would almost double the sodium percentage, which would be in agreement with that found. Also a disodium salt in dilute aqueous solution would give rise to three ions approximately ; thus yielding a molecular weight—3 x 140 = 420. This, although a much higher value, differs considerably from the accepted value. The fact that this is at complete variance with the actual value observed in alcohol renders the hypothesis practically untenable. To further test the values obtained for the molecular weight the percentage of nitrogen was determined by Kjeldahl’s method. Po al ee eS y = pa ae 7 ay vi VARIATIONS IN SODIUM OF GLYCOCHOLATE 123 IV.—NItrrRocEen PERCENTAGE * Sodium glycocholeate ’ Percentage ar grams nitrogen 0-832 es 1-03 1-087 oh o-go Mean, 0°965 Assuming one nitrogen atom in the molecule of salt— ‘The nitrogen percentage required for molecular weight 283 is 4-94 ” ” ” ” 487 ” 2:87 ” > eS > 537 9 2-60 The experimental values for the nitrogen are therefore at utter variance with all the molecular weights, the discrepancy being most marked in the case of the molecular weight actually determined. All this conflicting evidence can be explained only by assuming that either Strecker’s formula is quite wrong, or that there are other substances present whose existence has been overlooked.’ In fact if we assume that over 50 per cent. “of the * sodium glyco- choleate’ consists of sodium salts of nitrogen-free acids of molecular weight much smaller than sodium glycocholate, the anomalies observed would be accounted for. To form an approximate idea of the proportion of true sodium glycocholate in ‘ sodium glycocholeate’’ a quantity of the latter was hydrolysed with alkali, and the cholic acid produced was estimated by Lassar-Cohn’s* quantitative method. ‘The yield was 3 grammes of cholic acid from 8-5 grammes of ‘ sodium glycocholeate,’ the theoretical yield being 6-4 grammes of cholic acid. This points to the percentage ~ of true sodium glycocholeate being about 50 per cent. of the material. To settle the question, a quantity of ox bile was obtained and from it (1) the bile salts were prepared by Plattner’s method, and (2) ‘sodium glycocholeate’ prepared according to Merck’s method. The results of the analyses of the substances prepared by these two methods are given below. ‘x. Nore: It may be mentioned that the presence of sodium oleate was tested for by formation of the lead salt and extraction with ether—negative result. 2. Lassar-Cohn, Ber. d. deut. chem. Gesell., 26, Part 1, p. 146 (1893). os = ke . bi, a 124 BIO-CHEMICAL JOURNAL (1) Tue Brite Sarr Preparep Accorpinc to Prarrner’s MerHop This method, as is well known, simply consists in evaporation of the bile on the water bath and extraction of the residue with alcohol, thereby removing the sodium glycocholate and sodium taurocholate. (a) Determination of the molecular weight of the bile salt thus obtained. Mass of solvent Mass of solute Molecular Weight (water) in grams in grams A weight 27°70 0:5890 Or15° 262 31°55 0:5830 0°13° 263 Mean, 262°5 Assuming that the substance is about 80 per cent. dissociated at this dilution in water, we find for the undissociated molecular weight the value 472. ‘This is somewhat low but is in fair agreement with the value 487 required for the sodium glycocholate according to Strecker. : rat ie (b) Determination of Nitrogen (Kjeldahl). Grams. of N in grams Nitrogen substance percentage 1*123 0:02856 2°55 _ The value required for Strecker’s sodium glycocholate is 2:8 per cent. (c) Determination of the sodium content. Substance in grams Weight of sodium Sodium sulphate percentage I°1908 0°1931 2 5°2 The value required for sodium glycocholate is 4:7 per cent. This examination of the product obtained by Plattner’s method confirms the usually accepted formulae and constitution of sodium glycocholate when this body is obtained pure. ‘The small discrepancies noted above can again be explained by assuming the presence in | small quantities of that substance (or substances) which appear in large quantities in Merck’s preparation. — = - as 2 . ae : a VARIATIONS IN SODIUM OF GLYCOCHOLATE 125 (2) Anatysts or Propucr Oxstainep By Mercx’s Metuop oF PREPARATION —————— ———— ee A quantity of fresh ox bile was treated according to Merck’s method, the product yielding the following results on analysis :— Percentage of Sodium : Substance in grams Sodium sulphate in % Na grams I-1412 0-2152 6-11 1-0097 0-1902 6-10 . Mean, 6-1 ~~ Percentage of Nitrogen : Substance in grams Percentage nitrogen 0°4426 ¥ 2-66 0°8979 is 2-40 Mean, 2°53 We have here again in the case of the sodium content the discrepancies already noted. ‘The values for nitrogen, however, as will be seen, lie not far from those for pure sodium glycocholate. There can be little doubt but that we are in all cases dealing with the same contaminating substance, though the amount of this substance appearing in the final product depends on the conditions of manipu- lation. The most unexpected thing about this substance is the exceedingly large amount of it evidently present in ox bile ; especially when one remembers that the exhaustive work of Lassar-Cohn* and others point to the fact that in ox bile the sodium glycocholate and sodium tauro- cholate are present in much greater proportion than all the other salts together. For example, after hydrolysis of the glycocoll and taurine derivatives, Lassar-Cohn’ found that ‘ 100 litres of ox bile yield 4,790 grammes of cholic acid and 405 grammes of other acids.’ 1. Lassar-Cohn, Ber. d. deut. chem. Gesell., 26, Part I, p. 146, (1893). 126 BIO-CHEMICAIL, JOURNAL The important question is, of course—what is this hitherto undetected substance or substances? In this connection it may be mentioned that an attempt was made to ascertain whether there was any formation of double salt between the pure sodium glyco- cholate and some of the precipitants or products of precipitation. Thus, a quantity of the material obtained by Plattner’s method was dissolved in water and treated with excess sodium acetate. Lead acetate was then added, and the product decomposed with sodium carbonate and evaporated to dryness. In the presence of sodium acetate it was thought that possibly a double salt of the form (Cys Hy NO,Na + C;H;O,Na) might have been produced and removed by ’ the final extraction with alcohol. ‘The substance obtained, however, proved to be simply sodium glycocholate, an analysis of the material yielding 5-0 per cent of sodium. ie Referring to Merck’s preparation of ‘sodium glycocholeate,’ the analyses already given allow one to calculate the mean molecular weight of the substance (or substances) whose presence has been suspected. Thus assuming that the unknown body is nitrogen free, the deter- mination of nitrogen recorded leads at once to the conclusion that Merck’s preparation consists of 35-7 per cent. sodium glycocholate and 64:3 per cent. unknown substance}j(or substances). Now if M = the undissociated molecular weight of pure sodium glyco- cholate = 487 | M’ = the mean undissociated molecular weight of the mixture = 282 ' 3 m = the mean undissociated molecular weight of the unknown substance (or substances) it follows that : SOD ries f-Shi et 5 gS M’ M m whence m = 227, and as this is a sodium salt or salts (presumably a mono sodium derivative) the mean molecular weight of the acid (or acids) is 205. ’ VARIATIONS IN SODIUM OF GLYCOCHOLATE 127 In the saturated fatty series of acids the following members occur— Saeed 3g SS acia ~C,,H,,0, molecular weight 200 Tridecylic oy) C.3.HsO, ” ” vA 4 Myristic ,, CyH»O, we ee If we are dealing with a single substance in the case of the unknown salt the value obtained for the molecular weight points strongly to the presence of sodium laurate. It is much more probable, however, that we are really dealing with more than one body. In this con- nection one may mention the acids whose salts are present in ox bile according to Lassar-Cohn,’ viz., glycocholic, taurocholic, stearic, _ palmitic, oleic, myristic and some ‘amorphous acids.’ According to Lassar-Cohn, as already stated, the first two acids are present in amount about 10 times that of the remaining acids added together. It is very suggestive, however, that myristic acid should have been found as a constituent of ox bile, although Lassar-Cohn’s analyses point to its being present in extremely small quantity. Myristic acid, as already stated, has a molecular weight of 228, ‘which is not so very far removed from the mean value 205 of the unknown constituent. And indeed this mean value would be readily realised by myristic acid containing a small proportion of some acid of much lower molecular weight, as for example valeric acid (molecu- lar weight 102), or caproic acid (molecular weight 116), both of which are known to be produced in animal metabolism. SUMMARY The result of the foregoing investigation has been to confirm Strecker’s formula for sodium glycocholate when this is obtained in the pure state. At the same time, evidence is adduced to show that in the ordinary methods of extraction of mixed bile salts (7.e. sodium glycocholate + sodium taurocholate) one does mot always obtain a mixture consisting entirely (or almost entirely) of these two salts. 1. Lassar-Cohn, Joc. cit. BIO-CHEMICAL JOURNAL 128 The resulting product appears to contain as well, varying amounts of the sodium salts of nitrogen-free fatty acids of very much smaller molecular weight which may, in certain cases, be present to the extent of over 50 per cent. of the ‘ mixed bile salt’ preparation. It is suggested that sodium myristate is the chief representative of these lower fatty salts, this body being probably present in ox-bile to a very much greater extent than the analyses of Lassar-Cohn seem to show. bp od NOTES ON THE ACTION OF ATROPINE, HYOSCYAMINE, HYOSCINE, SCOPOLAMINE, DUBOISINE, AND DATU- __ RINE By W. WEBSTER, M.D., C.M., Anaesthetist to the W innipeg General Hospital, Lecturer in Anaesthetics in the Manitoba Medical College. From the Physiological Laboratory of the University of Manitoba Communicated by Professor Swate Vincent, M.B., D.Sc. (Received March 8th, 1908) The primary object of the present research was to test the efficiency of atropine as a restorative in poisoning by chloroform or other anaesthetic, or as a precautionary measure before its adminis- tration. ‘The use of atropine in one or other of these ways has been frequently advocated.1 : _ At the outset of my series of experiments a somewhat striking discrepancy was noticed between the statements in the text-books and the results actually obtained, even as regards the most readily observable effects upon the heart and blood vessels; thus it was deemed advisable to perform an extensive series of experiments devoted to the physiological effects of atropine and its allies upon the heart, respiration and circulation, apart altogether from the question of benefit or otherwise in the administration of anaesthetics. Atropine and the allied drugs mentioned in the title are generally supposed to be isomeric with each other, or very closely allied. There is a close relationship between these substances as regards not only their chemical structure but also their physiological action, and as far as my experiments are concerned, it is impossible to detect any difference between them. In regard both to their general physio- logical effects and to the question of the rapidly induced tolerance or immunity described later, all these drugs may be considered as 1. Dixon, Manual of Pharmacology, p. 79, London, 1906. 130 BIO-CHEMICAL JOURNAL identical.1 My experiments, however, have been restricted to what we may call gross effects upon the heart, circulation and respiration. The effects on the central nervous system, nerve endings, secretion, and excretion have not been specially examined in the present investigation, and it is, of course, possible that in some of these spheres of action there may be individual differences between the different drugs. The question of the value of adrenalin as a restorative to the circulatory system will be incidentally discussed in the body of the Soup ‘gecesi? + I have performed in all more than fifty experiments—two on cats, the rest on dogs. Chloroform, ether, or the A.C.E. mixture were the anaesthetics used. In some few cases curari has been used in addition. The blood pressure has been taken from the carotid artery, and the injections made into the saphenous or femoral vein. A glass plethysmograph was used for recording changes in the volume of the limb, and for similar changes in the intestina! wall an air onco- meter was used, each of these being connected with a piston recorder. The method described by Oliver and Schaefer? was employed for recording the effects upon the heart. A hook is caught in the epicardium of the auricle, and another in that of the ventricle. From these threads pass over pulleys moving on a horizontal axis; the threads then pass vertically downwards to be attached to long elastic levers of steel. ‘To the ends of the levers writing points are attached. PuystoLocicaL EFFEcts One of the most familiar, and at the same time most striking, actions of atropine is paralysis of the peripheral terminations of the vagus in the heart. It would naturally be expected that this effect, like section of the vagi, since it cuts off the tonic inhibitory influence 1. For information on the chemistry of these substances see Schmiedeberg, Pharmakologi be . . . 1é I Tomaial, Atti dell. R. Accad. dell. Scienz. Med., Palermo, Ann, 1896; O. es Leibig’s Aubalows tl ey 304, 308; O. Hesse, in Apotheker Zeitung, 1895; Raehlmann, Semaine Medicale, 1893 ; Jan ioc, ee an P 597 jbo ; % R. Pooley, Can. Lancet, Jan., 1895; Sharp, Practitioner, *y ; . sber ed. . Diss., 18 ; 188%, 1892) 1893, “Asa patio Srourn., res #ss., 1889-1890; Year Book of Pharmacy, 1880, 1881, 1882, 2. Fourn. of Physiol., Vol. XVIII, Pp. 256, 1895, ACTION OF ATROPINE AND ALLIED DRUGS 131 of the nerve centre, would exercise an augmentor effect upon the heart, and raise the blood pressure. This is, in fact, usually stated to be the case. Thus Dixon! says: ‘In mammals small injections of atropine produce the same result: this paralysis of the peripheral vagal terminals, like section of the vagi, cuts off the tonic inhibitory influence of the centre, and the heart is quickened. The increased rate will naturally only occur in those animals in which there is some tonic central effect. Thus the quickening is decided in dogs, and little in cats, whilst in man it varies with the age and disposition, but is usually greatest between the ages of twenty-five and forty. In children under two months atropine causes no quickening, and it has also little effect in old age.” This author also suggests that atropine may directly stimulate cardiac muscle, and refers to a vaso constriction as a result of the action of the drug on the medulla. ‘ Blood pressure rises mainly as a result of this vaso constriction . the pressure also tends to rise on account of the quickened heart . + « + « constriction of vessels is pronounced only in the splanchnic area.’ Schmiedeberg? likewise states that atropine, in men and dogs, quickens the heart and raises blood pressure, but admits that a subsequent effect is paralysis of the heart. Sollmann* states that the blood pressure is scarcely altered, but there may be a slight rise from stimulation of the vasomotor centre ; this stimulation is always slight, and may be entirely absent. It is replaced by vaso- motor depression rather early. Large doses depress the vasomotor centre profoundly, so that the pressure falls very low while the heart is still beating. Still larger doses paralyse the heart muscle as well. Sollmann is the only author I am acquainted with who mentions this lowering of blood pressure at all. Thus Dixon states that there is a rise of blood pressure owing to vaso constriction ; Sollmann that there is at first-a slight rise owing to vaso constriction, and later a pronounced fall owing to vaso dilatation. But my own experiments 1. Op. cit. “2. Op. cit. 3. This secondary effect is also mentioned by Pouchet, Lecons de Pharmacodynamie, Paris, 1901. 4. Sollmann, Text-book of Pharmacology, Philadelphia, 1906. 132 BIO-CHEMICAL- JOURNAL farnish no evidence of any action whatever on the vasomotor system. I have been unable to find any original papers giving details of experi- ments upon animals, with tracings of the blood pressure.’ In my series of more than fifty experiments on dogs I have never observed any rise of blood pressure upon the injection of atropine into the circulation.” GODT TP rm nr pete My cme g meio | | AMARA Andaaal oll Respitation K \ ancient Ai "t arotid. "ii bo vai ieee ATT | Aydt yl i neta } Ficure 1.—Dog, 10°5 kg.. Chloroform. No ‘anaesthetic had been given for half an hour before the injection, at A, of 0-4 of a gram of atropine. . ‘The animal had previously received, in increasing doses, commencing with 0°7 of a mg. of hyoscine, 50 mg. of hyoscine and a o's of a gram of atropines ° The‘ blood pressure: falls and: returns ‘to ‘normal im 7°5 minutes. — At B, the kymograph was stopped for 5 minutes. Respiration was first quickened, then, as the blood pressuré returris to the normal, it is markedly slower but deep. ‘The limb follows passively the blood pressure. Scale, half. 1. The literature, however, to which I have access is limited. Professor Vincent inforn ; : : : Vincent informs me that so far as his memory serves him he has never seen a risé of blood pressure on the administration of atropine ACTION OF: ATROPINE AND: ALLIED DRUGS 13 >) This applies to the allied drugs mentioned in the introduction. The drugs have been tested under very varying conditions as to dosage, amount of fluid injected, temperature of fluid injected, and rate of injection. In all cases, when any efféct whatever has been produced, this has been in the direction of a fall of blood pressure. In small doses this. is slight and transient ; in large doses marked and long continued—sometimes for an hour. In some cases, however, even after large doses, recovery is fairly rapid. (See Fig. 1.) In many cases, it is true, the tracings show a slight preliminary rise of blood pressure. ‘This, generally followed by a much more pronounced and significant fall, is, | have convinced myself, simply due to the injection of the fluid in which the drug is dissolved, since an injection of an equal quantity of normal saline solution has been always found to induce a rise of pressure similar in character, and of equal magnitude. (Fig. 2.) Aespiration tha ANAL: \ \\ CULL All UT LLL NAKA AM WAN VALLE WY NA % AM AWA ALS "\ iy a \\ WAIN iv . We 1 as « "Whi ty inant Hada, # Uy ee hy Hs ANA Be ndagtans AT REY AAAI nt Aaya i, ROT 4 Tee CLA MBA His mth wit 2k eh he ie a te Ficure 2.—Dog, 5-3 kg. A.C.E. At A, 0°5 of a mg. of atropine was given in I0 c.c. saline solution. At B, 10 c.c. saline solution only was injected. The same initial rise of blood pressure occurs in both instances. Time intervals, 5 secs. Of course, the effect of atropine on the blood pressure has almost always been recorded in animals already under the effects of other 1. A possible criticism of my results would be that doses small enough had not been tried, and that the initial increased rate of heart beat and concomitant rise in blood pressure, whether due to this or to vaso constriction, had been overlooked. Every precaution has been taken to avoid this error. No dose, however small, has in my dogs produced the slightest rise of blood pressure. In many cases so small was the amount of drug injected that no effect whatever was produced other than that due to the fluid adminis- tered, which point has been very frequently tested by control injections of the same quantity of normal saline solution. 134 BIO-CHEMICAL JOURNAL drugs. Thus the animal has been under the effects of chloroform, ether, or A.C.E. mixture when the first dose of atropine, hyoscine, etc., has been injected, and in the experiment in which records of auricle arid ventricle were taken, curari was administered in addition. On the other hand, many of the larger doses have been given when the animal has not had any anaesthetic for the previous half hour, the atropine already used sufficing to maintain an unconscious condi- tion. Effect upon the Heart.—Contrarily to the usually accepted view, it has been found that in dogs atropine has only at most a very slight and temporary effect in the direction of augmentation of the heart beats ; the chief effect is a diminution in the extent of movement as revealed by the heart levers. (See Figs. 3 and 4.) Auricle. uricie Fis MENA ACR O RUMOR loge etc oct ka ventricle Zeto BR Fic. 3 Fic. 4 Ficure 3.—Dog, 5:2 kg. Ether. 1 mg. of atropine has the usual effect on carotid pressure. ‘There is slight diminution in extent of movement of auricle and ventricle. Time intervals, 5 secs. 7: ett 4.—Dog, 11°5 kg. Ether, curari ; artificial respiration. Atropine, I mg. ery Sight temporary increase of frequency of heart beat, followed by diminution in extent of movement, as shown also in Figure 3. ‘Time intervals, 5 secs. en Sh ~ . ier experiment I have reduced this objection to a minimum by employing only just sufficient naesthetic for the carrying out of the preliminary surgical proceedings. The animal was then allowed to recover from anaesthesia, and atropine in a small dose injected into a vein. The effect in this case, as usual, was a fall and not a rise of blood pressure, and the heart was weakened. (See Fig. 10.) ACTION OF ATROPINE AND ALLIED DRUGS 13 Ww" Mode of Action of the Drugs.—The levers of the piston recorders connected with the limb plethysmograph and the intestinal oncometer always fall on the injection of the drug. The limb and intestinal tracings, in fact, passively follow that of the blood pressure, and frequently show the same preliminary rise as does the blood pressure. (Figs. 5 and 6.) > a Cage Hind Let 7m, SEIU AMAL! ue 11 ji Respiration sateen 59 inca Ni they % | Carotid. mu FIGURE 5.—Dog, 10°5 kg. Chloroform, atropine, o°2 of a gram. ‘The animal had previously been given increasing doses of hyoscine and atropine. The blood pressure “was markedly lowered but he animal recovered. The heart was slowed, respiration was increased in frequency and depth and rendered more irregular. The volume of the limb follows the blood pressure. The heart beat is less frequent and more irregular, as indicated by the movements of the mercury in the manometer. The slowing of the heart beat is shown by the increased excursions of the mercury, which are shown most distinctly at the lowest part of the curve. Such increased excursions are frequently misinterpreted as meaning increased force of heart beat. This must be interpreted as meaning that owing to enfeebled «| heart’s action blood is drained from both the somatic and splanchnic 136 BIO-CHEMICAL JOURNAL areas; there cannot be active vaso constriction, or there would be a rise of blood pressure, and we have seen that there is never any evidence of vaso dilatation. Untes ta we. + eaaamavay nan oe x bbaaahes Aw — AAA IS ANY Wi Wih\} \ \ may \ A ‘ hy AAV AT cia ane Ficure 6.—Dog, 7 kg. At A, 8 mg. of atropine is administered in 10 c.c. saline solution. The animal has already had 7°5 mg. of atropine in divided doses, At B 20m of atropine, in Io c.c. saline solution, proved fatal in 10 minutes. Tatestinal son follows the blood pressure after both doses. The slight preliminary rise of blood pressure is usually obtained and is due to the quantity of fluid injected, as it can be invari es roduced by the injection of 10 c.c. saline solution. The short lines of the time markin show seconds, the long lines 5 seconds. : It would appear that rather too far-reaching deductions have been drawn from the action of atropine in cutting out the inhibitory control of the vagus. Since cutting the vagi always raises the blood pressure, while the administration of atropine always lowers it, in the dog at any rate; and since, on injection of atropine, the volume of both limb and intestinal wall always follow passively the blood pressure, we must conclude that atropine acts upon the heart in a manner quite ACTION OF ATROPINE AND ALLIED DRUGS 137 Jf different from that of section of the yagi. (Fig. 7.) It seems, in fact, that with atropine, although the vagus inhibition is removed, there is a-much more powerful effect acting upon the circulation in an opposite sense, namely, a paralytic effect on the heart muscle itself. \ Mas Respiration. iN 1} if, 5 mug = Vii Nagests We ao ' a Carotié. Ficure 7.—Dog, 52 kg. Overdose of chloroform. At A, both vagi were cut simultaneously ; sudden rise of blood pressure occurs. Respiration, which had stopped, was not re-established, and blood pressure gradually falls again. The animal died in about 6 minutes. Tue Errects or Rapipty REepeatep Doses oF THE Drucs It is well known that a very marked tolerance to atropine as well as to other drugs can be established both in animals and man by gradually increasing the dosage over a period of days, weeks, or months. It is, however, somewhat surprising to find that within the limits of time occupied by a single experiment a dog can be brought to withstand, manifesting only a comparatively slight reaction, a dose which, if administered at the beginning of the experiment, would have been certainly and quickly fatal; nay, further, in many cases even a large multiple of this. ‘This rapid immunity or tolerance to the poisonous effects of a drug is not referred to in any of the books or papers to which I have had access, though the phenomenon is so striking that one can scarcely believe it has escaped the notice of pharmacologists. 138 - BIO-CHEMICAL JOURNAL By commencing with small doses and gradually increasing their size, animals of 5 to 12 kg. body weight have, in the course of an experiment lasting one and a half hours, been rendered so immune to the ill effects of the drug as to tolerate as large a quantity as o-4 gramme injected intravenously (Fig. 1) without other apparent ill effects than a lowering of blood pressure, from which recovery gradually takes place.? The commencing dose varied from oI mg. to 0-5 mg. of atropine, hyoscine, etc., an injection being given every three to ten minutes _ afterwards until the animal succumbed. In some cases 0-5 mg. as an initial dose caused death rapidly, but in others I mg., or even 2 mg., could be used as an initial dose without a fatal result, the animals showing the same idiosyncrasy as the human subject in this respect. In all cases where the initial dose failed to kill each successive dose was doubled or trebled until on many occasions 0-4 of a gramme of the alkaloid was given. From this latter dose animals have recovered without the intervention of any restorative measures; double this amount has been given, and the animal kept alive by artificial respira- tion, the blood pressure, which was very low after the injection, rising almost to the normal. But immediately on stoppage of the artificial respiration the pressure would fall and death ensue. It does not make any difference which of the drugs, atropine, hyoscine, hyoscyamine, duboisine, daturine or scopolamine, are used to commence with in the process of obtaining this tolerance; any one can be used in increasing doses ; then, when the dose has become large, an increased dose of any of the others given with no different result than would be obtained were the first drug continued—each drug immunising the animal from all the rest of the series. Further, the serum of an animal which has been rendered immune to large doses of these drugs will, if injected into another animal, confer an immunity on it. Thus a first dose of 0-5 mg. of duboisine injected into a dog of 6 kg. weight produced rapid fall of blood pressure and death. (See 1. It is impossible to make any definite statements a : : s to the permanent effects of such a dose, as in this and all other cases the animal was killed under the spanniane ACTION OF ATROPINE AND ALLIED DRUGS 139 Fig. 8.) Later, blood serum,! obtained from an animal which had previously had large doses of hyoscine and atropine, was injected into another dog 6 kg. weight. The serum, amounting to 80 c.c., was given in eight doses by means of a Io c.c. syringe. A very slight effect was noticed with the first and second dose, given at intervals of three minutes, and between the second and third doses ; the remaining six doses were given as quickly as the instrument could be filled, and no effect was observed. 12-5 mg. (twenty-five times the dose which killed the same weight of dog mentioned in Fig. 8) of duboisine were injected about ten minutes afterwards with practically no effect, as seen in Fig. 9. Carotid. eo we oF my Dubos rahe ‘Figure 8.—Dog, 6 kg. Duboisine, 0°5 mg. caused death in two minutes. This was a primary dose. From this experiment it would be rash to conclude that anything of the nature of an antitoxin had been formed. The first dose (10 c.c.) of serum from the drugged animal had the effect of cutting out the vagus action in the second animal, and it is probable that this serum contained therefore considerable quantities of the drug injected. This has an important bearing upon one theory of toleration, rendering it clear that since the serum contained the drug in an active form the tolerance established in the first dog could not be due to rapid elimination. 1. Blood was drawn from an animal which had had large quantities of hyoscine and atropine, and after standing twenty-four hours the serum was drawn off with a pipette in the usual manner. 140 :BIO-CHEMICAL JOURNAL AcTION ON THE RESPIRATORY SYSTEM It is well known that the first effect of atropine upon the respira- tion is to increase both the frequency and extent of the movements. (Figs. 1, 5, 9.) Subsequently, however, the respiratory centre is paralysed. (Figs. 6and7.) I have been able to confirm the statement made by Reichert,’ and quoted by Sollmann,? that an animal may recover from many times the minimal fatal dose if artificial respiration be maintained. ‘This power of recovery on the part of the respiratory centre is of supreme practical importance in dealing with cases of poisoning by this drug. Hard Lamb Respivation, puvva Peres very erevs rvvwy Fev¥y PPV FYYWN ErieU covey PUWrc Fe¥ty touts vOVWN GEYYR vurvs rVecyVovTN PereNcrTTY CTT Ficure 9.—Dog, 6kg. 80 c.c. of blood serum from a dog (which had had altogether 65 mg. of hyoscine and 1-64 grams of atropine in an experiment extending over nearly two hours) was administered. At A, 12°5 mg. of duboisine was administered with practi- cally no effect. Previously (Fig. 8) o-5 mg. (#¢ the dose) given as a first injection, without a preliminary one of blood serum, killed a dog of the same weight in two minutes. ‘When the blood pressure has been depressed by an overdose of chloroform, section of the two vagi, by cutting off the medullary effect, will release the heart; the beat will once again recover its normal character, and the blood pressure will bound up. A natural 1. Reichert, Phila. Med. Four., Jan. 1gth, 1gor. 2. Sollmann, op. cit. 3. Dixon, op. cit. ACTION OF ATROPINE AND ALLIED DRUGS 141 ‘inference would be that atropine, by cutting off the tonic effects of the vagus, would have a similar effect. This, however, has not been ‘the-case in my experiments. In chloroform poisoning, just as in the normal condition, atropine does not raise the blood pressure, but lowers it. I have not found the slightest benefit to accrue when atropine has been administered to an animal whose circulation is ‘depressed with chloroform or other anaesthetic. The use of atropine prior to the administration of chloroform has been strongly advocated and no less strongly opposed by various writers, ¢.g., Brodie and Crouch,? J. Harley3, Dastre, Pitha®, Fraser, Brown-Sequard’, Dastre and Morat®, Schafer®, Schafer and Schar- lieb, Hewitt", some of whom question the great danger of primary inhibition of the heart through excitation of the vagus, and also the benefit supposed to be derived from a preliminary dose of atropine. The dose required to eliminate vagus action in dogs varies greatly per kg. of body weight in different individuals. ‘This variability, we know, exists to as great an extent in the human subject. In view of the fact that we have no convenient means of testing vagus condi- tion in the human subject, it follows that after administering a dose of atropine previously determined upon we are in the dark as to whether we have obtained the desired effect. Should we invariably give a large dose, disagreeable and possibly fatal results might ensue ; numerous cases have been recorded where small doses have produced ill effects. 1. I have not invariably found benefit from cutting the vagi, although often, as Dixon states, it restores blood pressure. In the case of the animal whose tracing is reproduced in Fig. 7, although a sudden rise of blood pressure took place, respiration was not restored; the blood pressure again fell, and death pets ensued. Trans. Soc. Anaesth., Vol. V1, pp. 70 and 81, wees Brit. Med. Four., Vol. II, p. 320; 1868. Soc. Biol., p. 242, 1883. Pitha, 1861. Quoted by Schafer from Dastre. Brit. Med. Four., Vol. Il, p. 715, 1880. Brown-Sequard (C. 7. Soc. Biol., p. 289, 1883). Dastre and Morat (Lyon Med., 1882, and C. r. Soc. Biol., pp. 242 and 259, 1883). Schafer, Brit. Med. Four., Vol. Il, p. 620, 1880. Trans. Royal Soc. Edin., p. 333, 1904. Anaesthetics and their administration, pp. 230, 259, 503, 1907. ~SS PYareyry — 142 BIO-CHEMICAL JOURNAL Harley advocates 0-01 to 0-025 grain, and Dastre 00015 gramme (0-023 grain). These doses would certainly cause serious symptoms in some subjects, and would probably cause discomfort in nearly all, and in spite of their size we have no guarantee that the quantity is sufficient to abolish vagus action. It may also be argued that after an anaesthetic the sleepiness and depression are such that the dose of atropine would be eliminated before consciousness completely returned. ‘This would only be the case after prolonged anaesthesia. After short operations, where the anaesthetic is skilfully administered, consciousness returns with comparative rapidity, while the effects of atropine last twelve to twenty-four hours; and in these short cases the atropine is quite as necessary as in cases of prolonged anaesthesia if, as stated, the danger to be chiefly averted is that of primary cardiac inhibition. As regards myself, o-o1 grain administered hypodermically to test the effect on blood pressure produced very disagreeable results ; headache, defective vision, dryness of the fauces, and a slight inco- ordination of the muscles, particularly of the lower limbs, being the prominent symptoms. Blood pressure which was taken previously for three consecutive days at the same hour with Janeway’s instru- ment was recorded every five minutes after the injection at first, then later every ten minutes. It showed a gradual fall until it was reduced by 20 mm. of mercury. Dr. , who was treated in the same manner, but only took 0:0067 grain of atropine, had less pro- nounced symptoms, but still sufficient to cause considerable discomfort, and a very slight change of blood pressure, which was lowered by 10 mm. of mercury. Among the more recent davoedeh of the use of atropine, Profene Schafer must be specially mentioned. His method was to inject atropine prior to the administration of the anaesthetic, and hypo- dermically. In my experiments this has not been done. The atropine has been injected into a vein during the actual administration, and at various stages, and with the blood pressure at different levels. So far as my experiments go they do not support the view that the atropine is beneficial. yw ACTION OF ATROPINE AND ALLIED DRUGS 143 So far as I can conclude from my experiments, adrenalin would be of distinctly more benefit, though, since its effect is transitory, as a rule frequent small doses must be administered in order to be of benefit. If the heart has not completely stopped, adrenalin causes more active contractions! and consequent rise of blood pressure, which a few doses at short intervals may make permanent. If the heart has ceased absolutely, I have never obtained any result. This restoration of heart beat may be obtained after the heart movements can no longer be felt through the chest wall, but when, if the chest wall is opened, small movements can still be observed. Ven trnecle Sr Se _ x a ied ie atall Carotad ro Ficure 10.—Dog, 7-2 kg. Ether, atropine. At A, 0-02 gram causes, after a slight preliminary rise, a fall in blood pressure and slowing of heart with diminution of the force of the beats. At B, 0-08 of a gram was given with similar, but more pronounced, results. SUMMARY 1. In dogs atropine, hyoscine, hyoscyamine, scopolamine, and daturine produce, whether in small or large doses, a lowering of the blood pressure. 2. The volume of a limb or portion of intestinal wall always becomes diminished concomitantly with the fall of blood pressure. 1. The rise with adrenalin is due also to stimulation of vasomotor nerve endings in various parts of the body. 144 BIO-CHEMICAL JOURNAL 3. The conclusion seems justified that although these drugs eliminate the tonic inhibitory action of the vagus, they have a simul- taneous action on the heart substance, diminishing the output. This paralytic effect upon the heart is shown also by direct experiment upon this organ. 4. By frequent administration of increasing doses of these drugs an animal may be brought into a condition of tolerance within one or two hours, so that at the end of this time it will withstand (with comparatively slight reaction) very’ many times the dose which would have been fatal at the beginning of the experiment. 5. In small doses the respiration is quickened and rendered deeper. In large doses it is-often paralysed immediately. 6. ‘The present series of experiments has not yielded results which would tend to encourage the use of atropine in chloroform poisoning. Adrenalin seems to be of much more, though limited, utility. Note added February 6th, 1908 In consequence of some criticism of the preceding observations, which have been privately offered, I have performed a further series of experiments. The first point in which my results differ from those of the majority of experimenters is that in the dog certainly, and probably also in other animals, a dose of atropine sufficient to produce any effect at all upon the blood pressure does not raise it, but invariably lowers it. ‘This can only mean that the increased force and frequency of the heart’s action, which must be brought about by the mere fact of eliminating vagus action, is counteracted even in minimal doses by another: effect. of atropine, viz:, a depressant effect.. In other words, the effect of the administration of atropine is something other than a simple cutting out of the vagus. On this point I have performed a series of ten additional experi- ments on dogs, and find myself totally unable, under any circum- stances whatever, to raise the blood pressure of the animal. The ACTION OF ATROPINE AND ALLIED DRUGS 145 attempt has been made on a dog without any anaesthetic as well as under the influence of chloroform, ether, the A.C.E. mixture, morphia and curari, _and-further with the blood pressure at various levels. In all cases when the dose has been sufficient to produce any effect at all there has been a fall and not a rise of blood pressure. It is scarcely necessary to state that account has been taken only of the initial injection in any one animal, since it has never been supposed that atropine would raise the blood pressure after the vagus terminals have once been paralysed. In my experiments 0-5 c.c. of I-10000 solution per kg. of body weight has usually been found to be the smallest dose which will paralyse the vagus terminals,! and this dose “never raises the blood pressure. The second point of interest in the above communication is that during the course of a single experiment lasting two or three hours a tolerance to the action of atropine may be established. It is not pretended that the large doses mentioned in the body of the paper would be completely recovered from, but that they produce effects which are exceedingly small as compared with effects which would be produced at the beginning of the experiment. This general result has been confirmed in ten fresh experiments. 1. The dose varies considerably in different animals, some requiring less, and some a greater dose. This, however, is about the average. A NOTE ON THE DISTRIBUTION OF THE SALTS IN HAEMOLYSIS By ALBERT WOELFEL, M.D. From the Hull Physiological Laboratory, University of Chicago Communicated by Professor G. N. STEwart (Received March 26th, 1908) In his extensive studies! of the comparative electrical conductivities of normal blood laked by various means, Stewart has shown that com- plete liberation of the haemoglobin can be produced in blood and in artificial suspensions of erythrocytes without any change or with only a slight increase in conductivity. ‘This is the case when what he calls the less violent haemolytic agents (freezing and thawing, heat, foreign serum, strictly minimal doses of saponin, etc.) are employed. When the laked blood is subsequently exposed to one of the more energetic haemolytic agencies (water, supraminimal doses of saponin, etc.) a marked increase in the conductivity is produced. The total increase is sensibly the same whether the haemolysis has been effected in two stages, first by a gentle and then by a more violent agent, or in one stage by the original and more energetic action of a body of the second group. ‘The actionof the more violent haemolytics when consecutive to that of the less violent, is an action on the — shadows or ghosts of the corpuscles already deprived of haemoglobin which reduces them to the same condition as that produced directly by the more energetic haemolytics acting alone. It is not due to the ge Four. of Phys., Vol. XXIV, p. 211, 1899; Ibid., Vol. XXVI, p. 470, 1901; Four. of Exp. Med., Vol. VI, p. 257, 1902; Four. of Med. Research, Vol. III, p. 268, 1902; Amer. Four. of Phys., Wol. TX, P- 72, 1903. ; a 2 vie hei Fle DISTRIBUTION OF SALTS IN HAEMOLYSIS 147 breaking up of (hypothetical) compounds of colloids and electrolytes in the extracorpuscular liquid' of the laked blood. This phenomenon is of interest not only in relation to the mechanism of haemolysis but in relation to the physical chemistry of cells in general, since, as Stewart” has pointed out, and as has been more recently insisted on by Overton and others, it is a common property of living cell ‘ envelopes” to be relatively impermeable to the ions with which they are normally in contact. In the interpretation of the alterations of conductivity in laking the following points must, according to Stewart, be taken account of : 1. The exit of haemoglobin from the corpuscles (supposing it to take place without interchange, on the whole, of water or electrolytes between the serum and the stromata, and without change in the permeability of the envelopes or stromata to the ions) would depress the conductivity of the laked blood by diminishing the conductivity of the intercorpuscular liquid to a greater extent than would be compensated by the diminution in volume of the badly conducting corpuscles. 2. Apart from the depressing influence of liberated haemo- globin, the exit of electrolytes from the corpuscles would increase the conductivity of the intercorpuscular liquid and, therefore, of the laked blood, provided that the volume of the corpuscles is not increased by the passage of water into them. 3. The exit of water from the corpuscles unaccompanied, on the whole, by electrolytes would cause an increase in the conductivity - 1. The extra- or intercorpuscular liquid is a convenient term for the liquid in which corpuscles or ghosts are suspended. In unaltered blood it is, of course, the serum. 2. Fournal of Physiology, Vol. XXIV, loc. cit, 1899. 3. Note by G. N. S.—In this paper the question of the precise physico-chemical action produced by the various-haemolytics or their precise point of attack in the corpuscle has not been entered into. Moore and Roaf (this Journal, Vol. III, p. 55, 1907) have recently published an interesting paper in which they argue against the view that the difference in the inorganic constituents of corpuscles and plasma can be accounted for by the existence of a ‘membrane’ variously permeable to different salts. “I have avoided the use of the term ‘membrane’ in this connection, and have preferred to speak of the corpuscles as being bounded by an ‘envelope,’ a word which does not seem to carry with it the same preconceived histological and physico- chemical suggestions as the word ‘membrane.’ I have, however, demonstrated histologically (Amer. Four. of Phys.,loc. cit.) the existence of an envelope in the large nucleated corpuscles of Necturus. I hope soon to have an’ opportunity of returning to the subject. 148 BIO-CHEMICAL JOURNAL of the laked blood by increasing the volume of the intercorpuscular liquid relatively to that of the corpuscles, and perhaps by increasing somewhat the degree of dissociation of the serum electrolytes, although it would diminish the specific conductivity of the inter- corpuscular liquid. 4. An increase in the permeability of the corpuscles or ghosts to the ions, without, on balance, any increase in the electrolytes of the intercorpuscular liquid or in its amount, would increase the conductivity of the laked blood (apart from the depressing influence of the liberated haemoglobin). According to the same writer, the difference in the action of the two groups of haemolytic agents may be explained in two ways :— (a) By the assumption that in the first case a relatively small, and in the second case a relatively large amount of intracorpuscular electrolytes escapes from the corpuscles ; the relation of a considerable part of the electrolytes of the corpuscles to the stroma and envelope being such that an energetic action of the haemolytic on these structures is required for their liberation. (6) By the assumption that the permeability of the envelope (and stroma) to the ions of the intercorpuscular liquid, and perhaps also to the intracorpuscular ions, is decidedly increased by the second group and slightly, if at all, by the first. ‘The consequence of this would be that after the action of, say, a sufficient dose of saponin the laked blood would approximate more closely to the condition of a homogeneous conductor than after a less violent haemolysis, which left the shadows their ‘ semi-permeable ’ character. Stewart arrived at the conclusion that an increase in the conductivity of blood after laking is not due altogether to a surrender of electrolytes by the corpuscles to the fluid, but rather that some alteration in the corpuscles which allows ions to pass through them more freely is caused by the laking process. Foa, whose views as to the structure of corpuscles are quite at DISTRIBUTION OF SALTS IN HAEMOLYSIS 149 variance with those of Rollett and of Stewart, observed! that repeated freezing and thawing of blood with nucleated corpuscles lowered its freezing-point by stages. He, believing that in the laking of non- nucleated corpuscles electrolytes do pass into the surrounding fluid, holds that any indications to the contrary furnished by measurements of the electrical conductivity of the fluid of laked blood are not admissible, in that we do not know what the relations between the albuminous components of the blood and the electrolytes may be, and what effect their relation may have on electrical conductivity. Moreover, by a method which he used, of separating the fluid of laked blood from the ghosts, he found the ash in the former in excess of that found in the serum of an equal quantity of unlaked blood. Since it seems doubtful if a complete separation of serum and corpuscles, or of ghosts and fluid in laked blood, can be effected by simple centrifugalisation, I made, some time ago (at Dr. Stewart’s suggestion), to test the constancy of the results, determinations of the ash in as much serum of measured quantities of blood and in as much intercorpuscular fluid of measured quantities of laked blood as could, aftercentrifuging, be separated as cleanly as possible from the corpuscular elements with a pipette. It was found impracticable—especially with laked blood, always darkened—to discern the exact line of separation ; and there were always, even with the best available centrifugalisa- tion, loose, flocculent particles, barely visible, above the seeming line of separation. This, I think, ought to preclude the possibility of an exact reading of the comparative volume of ghosts and of fluid in laked blood. It is less easy to imagine how a perfect separation of serum and corpuscles or fluid and ghosts can be effected by the method which Foa used of centrifuging in tubes with stop-cocks at the bottom, and then allowing the sediment to flow off through a small opening in the cock. If blood is centrifuged very long and rapidly, the corpuscles or ghosts will be packed together and pressed against the wall of the containing tube so tightly that they will adhere 1, Archivio di Fistologio, Vol. 1, Fasc. 2, 1904. 150 . BIO-CHEMICAL JOURNAL to the wall of the tube, and will continue to do so after the contents of the tube are poured out. The numerous comparative determinations of the ash from serum and from the fluid of laked blood—made by trying to draw off all of the fluid parts carefully with a pipette—were made with measured amounts of chicken blood because it was expected that the corpuscles of such blood, still ballasted, so to speak, by the nuclei, would sediment well. The results are so inconstant that it is held to be wholly impossible to ascertain the ash from the fluid part of blood—especially when laked—by separating all of the fluid from the corpuscular elements. : With horse blood, also, determinations of the ash from the serum and from the fluid of the blood, laked by freezing and thawing, were made, the fluids being separated as completely as possible from the corpuscular elements after centrifuging a long while. ‘The results of determinations in the fluid of the laked blood were far apart. The proportion between the amount of*ash (L) found in the fluid of the laked blood and the ash (S) in the serum were even much greater in each case than Foa found with horse blood by his method of separation. Thus in two experiments I obtained :— Ls8 23.206: : 1°00 L : S :: 1°3558 : 1-00 es which compares with Foa’s result — : 1*1388 : 1-00 The gross irregularities in the increases in the ash of the fluid: of the same blood laked by the same procedure but separated from the ghosts by such doubtful means, make it quite a certainty that some of these increases are due to salts which get into the ash deter- © mination in ghosts or in solid particles which do not sediment — perfectly. | . It seemed to me that it might be ascertained if salts come out of the corpuscles into the fluid on laking, by simply comparing the ash of a measured volume of serum of unlaked blood with a measured volume of fluid of laked blood, both being taken from the upper layers DISTRIBUTION OF SALTS IN HAEMOLYSIS 151 _ only of portions well centrifuged in closed tubes, while at the same time the relative proportion of serum and corpuscles in the unlaked blood and_of-tiquid and ghosts in the laked blood was determined by the haematocrite. But the same difficulty of effecting a complete _ separation of the ghosts presented itself here. I was, therefore, _ forced to leave out of account the possibility that the relative volumes of fluid and corpuscular elements of blood may change on laking without alteration in the proportion of salts. This would be the case if a quantity of water holding the same amount of salt as the same volume of serum, were transferred one way or the other. As, however, I found positive differences in the amount of ash, this objection does not render my observations valueless. In these experiments the blood to be tested was handled in centrifuge tubes fitted with rubber caps, such as bacteriologists use. With these caps put on the tubes as soon as filled, there can be no evaporation and hence no alteration of volume of the contents. Some of the tubes were centrifuged, immediately after filling, to provide the normal serum; others were subjected to the laking process, well stirred up and then centrifuged. Carefully measured volumes of serum and of fluid of laked blood were pipetted off and brought into crucibles in which their ash-content was weighed after incinerating /Jege artis. In this way the relation of ash (S) in the serum of blood to the ash (L) in the fluid of the same blood laked was found in the following cases to be :— : 0-917: 1-00 (chicken blood laked by heat). 7: O-891 =: 1-00 ( * rs saponin). :: 10284 : 1-00 (horse blood laked by heat). 2: 10544 : I-00 ( iy = » saponin). :: 10618 : 1-00 (horse blood laked by freezing and thawing). Pat ae ANHAHRAD ~ 1-048 : 1:00 ( >” >> »” ” yi In all cases where saponin was used as a laking agent the small amount of ash contained in it was deducted from the total ash of the incinerated laked fluid. In all cases where the ash contained iron derived from the haemoglobin of the fluid of laked blood it was included, since it was seen that the whole excess of L over S$ in the mammalian blood could be accounted for by the iron, and that was all I aimed at determining. 152 BIO-CHEMICAL JOURNAL It will be seen from the above that the increase of ash from the fluid of horse blood laked by freezing and thawing is not nearly so large as Foa found by his method, and may be accounted for by the iron of the haemoglobin. If the intercorpuscular fluid contained only ten per cent. of haemoglobin (and it would almost certainly be more, since the whole, or nearly the whole, of the pigment is in the fluid), this would correspond, say, to 0°035 per cent. of Fe or 0°05 per cent. of Fe,O;. When the original serum contains 0°8 per cent. of ash this would make the ratio L:$:: 1:06: 1, if no salts were exchanged between the corpuscles and the liquid. With chicken blood the quantitative relations above recorded between the ash of the serum and the ash from the fluid of laked blood are in accord with Foa’s belief that there is a difference in the mode of laking of nucleated and the mode of laking of non-nucleated corpuscles. Stewart also found that in heat-laking of blood with nucleated corpuscles the conductivity might be markedly diminished. On the other hand the relations found between the amount of the ash from the serum of blood with non-nucleated corpuscles and from the fluid of such blood laked in different ways are not easy to interpret. In blood laked by freezing and thawing we find that the increase of proportion of the ash from the fluid is almost as much in one series, and more in the other series than the increase of proportion of ash from the fluid of blood laked by saponin, whereas in blood laked by heat it is distinctly less. | Yet, as has been said, saponin increases the conductivity of the laked blood much more than heat or freezing and thawing. So far as these determinations go, then, they support the view that in none of these forms of laking is there a notable increase in the percentage ~ of electrolytes in the intercorpuscular liquid, and that the increase in conductivity produced by saponin must be due either to an increase in the total volume of the liquid or to an increased conductivity of the corpuscles, or to both. That a change in the conductivity of the corpuscles, not necessarily associated with the liberation of electrolytes, is caused by us DISTRIBUTION OF SALTS IN HAEMOLYSIS 154 saponin is indicated by determinations of ash in the fluid of suspensions of formaldehyde-hardened corpuscles treated with saponin, which bear out Stewart’s view as to the behaviour of the corpuscles during the laking process. With suspensions of such corpuscles complete separation of the fluid can be made after centrifugalisation since they sediment to form a viscid mass from which the supernatant fluid can even be poured off ; and the corpuscles can be washed and re-washed in M/4 cane sugar solution. Although formaldehyde-hardened corpuscles will not give up haemoglobin when subjected to the influence of laking agents, a suspension of them in isotonic solutions of salt will undergo an increase in its electrical conductivity when treated with saponin as great as if the corpuscles were fresh. It is very easy to determine that the increase in conductivity of the suspension of formaldehyde-hardened corpuscles after treatment with saponin is not due to a passage of electrolytes from the corpuscles into the fluid of the suspension. Such suspensions, having been treated with weighed amounts of dried saponin containing a known amount of ash and allowed to stand from five to forty-eight hours, were centrifuged, and measured volumes of the supernatant fluid then taken for determinations of the ash obtainable from them, and these were compared with determinations of the ash from the supernatant fluid of a like centrifuged suspension, which had not been treated with saponin. It was found that the excess of ash from the supernatant fluid of the saponin-treated suspension could be accounted for by that added with the saponin when the suspension was in salt solution. Or in other series, the suspensions of formaldehyde-hardened corpuscles in salt solution were first washed with M/4 solution of cane sugar and suspended in the latter when treated with saponin. After centrifuging such sugar solution suspensions, the ash from the supernatant fluid of the saponin-treated suspension was found to exceed that from the supernatant fluid of untreated suspensions in the exact proportions in which it was added with the saponin ; or in a case where the whole supernatant fluid was taken for ash determinations, the excess of ash from the fluid of the saponin-treated 154 BIO-CHEMICAL JOURNAL suspension was found to coincide almost exactly with that added with the saponin. ‘The supposition that formaldehyde-hardened corpuscles on being treated with saponin, which raises the conductivity of a sus- pension containing them, do not give up’any of their electrolytes to the surrounding fluid is therefore, by this method, well substantiated. The increase of conductivity must, then, be due to a change in the envelope (or stroma) which permits an easier passage to the ions of the liquid in which the corpuscles are suspended. 155 ON THE ACTION OF CERTAIN OXIDISING AGENTS UPON BLOOD-PIGMENT! By J. A. MacWILLIAM, M.D., Professor of Physiology in the University of Aberdeen. From the Physiological Laboratory, University of Aberdeen (Received April 9th, 1908) __ While the destructive effects of chlorates on red blood corpuscles and the conversion of the haemoglobin into methaemoglobin have long been known the further effects have not been much studied. Formation of haematin, ultimate disappearance of absorption bands, and conversion of the blood into a dark solid mass have been stated to occur.” Jettyinc or Bioop anp Cotour CHANGES _ The jellying effect of chlorate on blood is a remarkable one, and occurs as one of the results of the chlorate influence being prolonged much beyond the phase of methaemoglobin formation. It can be easily produced by mixing blood with crystals or strong solutions of sodium (¢.g., 10 per cent.) potassium or ammonium chlorate in amounts sufficient to give a strength of 2 to 5 percent. chlorate in the mixture. When crystals are used the fluid must, of course, be stirred until solution is complete ; otherwise there may be excessive percentages in certain portions of the fluid—with disturbing effects. With blood that has not been much diluted the jelly formed is very firm, coherent and elastic, and very difficult to reduce to a state of fine subdivision. Its formation is much accelerated by acidulation of the blood and by warmth (30° to 40° C.). When no acidulation is used the jelly 1. The main facts in this’ paper were communicated to the Physiological Society at the Oxford meeting in July, 1904, when illustrative specimens were shewn. Publication was delayed in the hope of carrying the investigation further in certain directions. 2. A list of papers is given at the end of the article on Chlorates in Cushny’s Textbook of Pharmacology. See especially von Mering’s paper in the Berlin klin. Wochenschr., No. 44, 686 (1883). 156 BIO-CHEMICAL JOURNAL is at first always dark red in colour when seen in mass—probably in , twenty-four to forty-eight hours after the chlorate has been added, at room temperature. Ata later stage, much hastened by acidulation and warmth, the red colour gives place to a deep green, as seen by ~ reflected light, though thin slices viewed by transmitted light show a brownish-red tint. At a still later stage under the influence of decided acidulation and warmth the green colour may gradually change to a yellowish tint. Sometimes the jelly contracts to a certain extent and squeezes out a clear fluid or serum. This fluid is rich in protein, etc., but gives no appreciable iron reaction with ammonium sulphide, ferrocyanide, etc., even after prolonged application of various methods for revealing ‘masked’ iron; the iron of the haemoglobin has evidently been retained in the jelly or clot. ‘The squeezing out of fluid is very variable as regards its occurrence and amount; very often there is none. When blood is diluted beyond a certain point jellying under the influence of chlorate fails and is replaced by a coloured precipitate varying in coarseness and tendency to coherence. The results are essentially similar whether the blood has been laked by dilution with water, etc., or diluted with isotonic salt solution. Ox blood which had been kept for many years behaved like fresh blood, and the results got with the blood of different animals (ox, horse, sheep, rabbit, rat, etc.), and with human blood, were essentially similar. The influence of the reaction of the blood on these changes is very marked. Addition of alkaline salts (sodium carbonate, etc.) retards chlorate action very markedly, while acidulation accelerates the changes very strikingly. Samples of blood which were unusually - alkaline shewed considerable resistance to the chlorate influence ; the effect of slight acidulation of portions of the same blood was very notable. Hence the plan of administering alkalies in cases of chlorate poisoning has obviously a sound basis. Acidulation may be done by adding dilute acids or acid salts like acid sodium phosphate. With different samples of ox blood I have often used half to three volumes of 0-2 per cent. HCl, or about one volume of Os per cent. ACTION OF OXIDISING AGENTS UPON BLOOD-PIGMENT 157 HNO,. To make the mixture faintly acid to litmus paper varying amounts of acid are required, care being taken to guard against the acidity being sufficient to cause the formation of acid haematin, etc. ; this was checked by the use of the spectroscope. Amounts of acid insufficient to make the mixture react acid to litmus are able to favour chlorate action markedly. The relations of the development of the colour changes to the jellying vary considerably. With acidulation and warmth the green phase may be arrived at very early, indeed almost as soon as the jellying, while with an alkaline reaction in the cool the jelly may remain red for many days. Similarly, when the blood is diluted so much that a flocculent precipitate occurs instead of jelly formation the particles may become green at a very early stage in presence of pronounced acidulation. Under certain conditions the blood, especially when diluted to a certain extent, may, after the addition of chlorate, present the appear- ance of a deep green fluid with little or no jelly or precipitate. Such conditions are (1) when the reaction is too alkaline or insufficiently acid, (2) when the percentage of chlorate is insufficient, and (3) when the temperature is too low, the reaction and amount of chlorate present being such as to require warmth for complete precipitation. The addition of acid (¢.g., acetic) in such cases usually leads to pre- cipitation or jellying immediately or in a short time; if much acid is added, the development of the yellow phase is brought on. Alcohol also causes precipitation in the green fluid, the coloured substance being thrown down among others. , The effects obtained with bromates were quite similar to those following the use of chlorates. Jodates on the other hand, have not the same action; a red precipitate is thrown down with properties differing from those got with chlorates and bromates, the blood not being acidulated. | Similar results are obtained when washed red blood corpuscles are used instead of blood. Solutions of the stromata were prepared by Halliburton’s! : Fourn. of Physiology, Vol. X, p. 532. 158 BIO-CHEMICAL JOURNAL modification of Wooldridge’s method, and these were tried with chlorate in the same way as blood, but no jellying or precipitation followed. On the other hand the solution of haemoglobin left after removal of the stromata gave the characteristic results with chlorate— very rapidly indeed on account of the previous acidulation with acid sodium sulphate used to precipitate the stromata. Blood plasma and serum were found to give negative effects with chlorate, whether acidulated or not; acid sodium phosphate was used to acidulate. Even when kept in a warm chamber for consider- able periods this was the case; except where the plasma or serum had been contaminated with some haemoglobin—leading to precipi- tation by the chlorates—the serum pigment remained apparently unchanged, at least for long periods. Similarly negative results were obtained with egg-albumin. Errects oN SOLUTIONS OF HAEMOGLOBIN From the foregoing observations it is evident that striking effects of chlorates on the blood are essentially effects upon the haemoglobin, and not upon the ordinary proteins. ‘This conclusion was confirmed by the characteristic effects got with solutions of haemoglobin crystals and of methaemoglobin; the former were prepared by different methods, commonly by some of the modifications of Hoppe- Seyler’s method. ‘The same phenomena as regards colour changes, . jellying, etc., were readily observed. Samples of haemoglobin obtained from Griibler, Merck and others were also used. Solutions of CO-haemoglobin were found to behave in much the same way. As a rule no acidulation was found to be necessary with the haemo- globin solutions ; the chlorate or bromate was simply added to the — usual amount. Cautious acidulation accelerates the changes. Haematin solutions on the other hand quite fail to give these results ; if blood is treated in such a way as to break up its haemoglobin into haematin and globin, treatment with chlorate in the usual way does not produce the effects seen when unchanged blood or haemoglobin is dealt with. | ACTION OF OXIDISING AGENTS UPON BLOOD-PIGMENT 159 SEPARATION AND Properties OF THE Mopiriep HAEMOGLOBIN From haemoglobin solutions as well as from blood the product was separated in conditions of greater or less purity. When blood was used the most successful results were obtained when considerable dilution was employed, ¢.g., ox blood diluted with sixteen volumes of water and cautiously acidulated with o-2 per cent. HCl, or o-1 to o-5 per cent. HNO,, chlorate or bromate being then added to the amount of 2 per cent. Methaemoglobin was rapidly formed and soon a flocculent precipitate appeared—very marked in less than half an_ hour. _ Centrifugalised and subjected to repeated and thorough washing . first with normal saline and then with distilled water. Boiling was tried with one portion, and helped the separation of the precipitated matter without causing any evident change of colour. In other cases the blood was diluted only three or four times with very weak acid and after addition of chlorate or bromate kept constantly stirred to prevent jellying, a coarse flaky precipitate (green) being got in an hour or two; this was washed thoroughly. The substance obtained in the dry state is a dark powder (looking somewhat like haematin) with metallic lustre. Microscopically it presents the appearance of yellow or brownish- yellow polygonal plates, varying much in size and form. With the micro-spectroscope no absorption bands are visible. Slices of the coloured jelly in the moist state shew similarly negative appearances with the spectroscope. The methaemoglobin spectrum resulting from the addition of chlorate to the blood gradually disappears as a result of more intense and prolonged influence. The dried substance is very stable and insoluble in water and most reagents, ¢.g., alcohol, ether, chloroform, benzol, glycerine, etc. Also in acids like HCl and H,SQ,, in solutions of alkaline salts and in weak solutions of caustic alkalies, while it dissolves after a time in strong solutions of NaOH (20 per cent., etc.) and KOH. A certain amount of solution takes place after many hours in weak alkalies in the warm chamber. The freshly precipitated moist substance is more soluble in dilute alkalies. Boiling causes no obvious effect upon the substance ; 160 BIO-CHEMICAL JOURNAL its colour—whether red or green—remains unchanged. When a solution has been made with strong alkali, boiling with lead acetate gives a negative result as regards reduction. In o-2 per cent. HCl the solution swells up but does not dissolve even with the aid of warmth. A solution in strong alkali shows no definite absorption bands, and when treated with ammonium sulphide shows only a very faint haemochromogen spectrum. Treatment of the substance with H,SO, does not give anything resembling haematoporphyrin, or indeed any definite bands. QueEsTION oF ForMATION oF HaLocen ComPpouNnpD In view of the results that have been obtained by Mulder! and various more recent workers on the combination of proteins with halogens, and especially those described by Hopkins and Pinkus? and by Kurajeff* (combination of haemoglobin with iodine), the question suggests itself as to whether there is any combination of such a kind here. Examination of the chlorate and bromate products by Carius’ method does not lend countenance to this suggestion, the halogen found being apparently of the nature of an impurity. The red, green and yellow products obtained by the action of chlorate and bromate seem to be oxidation products of haemoglobin. RETENTION AND LIBERATION OF THE [RON The substance retains all the iron of the haemoglobin from which it was formed, and the iron is in very firm combination. There is no reaction with ferrocyanide and HCl, with Macallum’s* haema- toxylin solution, nor with ammonium sulphide even when kept for long periods in the warm chamber. This is the case if the chlorate action in the preparation of the substance from haemoglobin solution or blood is not carried too far, in the presence of too much acid, ete. Fourn. f. pr. Chem., Vol. XLIV, 8. 487. Ber. d. deutsch chem. Gesellsch., Vol. XXXI, 8. 1311 (1898). Zeitsch. f. physiol. Chemie, Vol. XXXI, 8. 527 (1901). Journ. of Physiology, Vol. XXII, p. 25. Pet oe pte oe ACTION OF OXIDISING AGENTS UPON BLOOD-PIGMENT 161 The presence of Fe in the solution can be demonstrated in the usual way by incineration, etc., or by setting free the iron by prolonged treatment with acid alcohol (Bunge’s fluid, etc.) and warmth, or more easily by chlorate or bromate plus strong acidification and warmth. By using Io per cent. solution of sodium chlorate with zs to $ volume of saturated salicyl-sulphonic acid in the warm chamber the whole of the Fe is usually removed in a day or two; as soon as it is set free the ferric salt strikes a red colour with the salicyl-sulphonic acid and the progress of the extraction is readily gauged by the depth of the coloration. When the Fe has been completely removed in solution an orange-yellow residue is left. A similar method serves for the extraction of Fe from ordinary haemoglobin or from blood. A weaker solution suffices to extract a good deal of the Fe even at room temperature, and with such a solution (¢.g., containing 5 per cent. salicyl-sulphonic acid) testing with ferrocyanide can be readily done (without HCl), and with ammonium sulphide after neutralisation. The iron of haematin may also be extracted by treatment with chlorate whether treated as (1) haematin in the solid state, HCl, etc., being used for strong acidulation, or (2) haematin dissolved in glacial acetic acid or salicyl-sulphonic acid alcohol. Heat accelerates the extraction. When the Fe is completely removed a_ yellow substance is left which is not ordinary haemato-porphyrin. As regards the possibility of demonstrating the iron im situ in the modified haemoglobin or in ordinary haemoglobin by means of ammonium sulphide, ferrocyanide, etc., the difficulty is that when the Fe is set free it is readily dissolved out. In order to prevent this I sought a solution which should liberate the combined Fe but not dissolve it out, and obtained such by dissolving salicyl-sulphonic acid in ether to saturation. ‘Treatment with this fluid liberated the Fe but did not remove it, and it was possible to stain it 7 situ in the crystals and in blood corpuscles. But in the latter case the injurious effects of prolonged action of the fluid constitutes a serious drawback as regards histological applications. In the case of the modified haemoglobin got by means of chlorate or bromate the substance is 162 BIO-CHEMICAL JOURNAL so resistant that it was found quite possible to obtain successful preparations shewing the iron im situ by the black coloration with ammonium sulphide and with haematoxylin, and the blue with ferrocyanide. Errects oF PEprTic AND Tryptic DicEsTIon Digestion with peptic fluid gave results strikingly different from those obtained with ordinary haemoglobin or methaemoglobin. Instead of there being a splitting of the pigment into the protein component and acid haematin which becomes precipitated, the substance in this case gradually dissolves forming a yellow solution in which proteoses and, later, peptone can be recognised. No haematin appears, and in the earlier stages there is little or none of the iron liberated ; though with prolonged digestion an extensive setting free of iron occurs and some yellowish deposit may form in the fluid. The rapidity of digestion varies much with the exact condition of the substance tested; in the moist state and well subdivided (¢.g., a recently-formed flocculent precipitate) it is very rapidly dissolved (two to three minutes, etc.); the dried product on the other hand is much more slowly acted upon. No absorption bands are visible in the yellow solution. The digestibility of the substance is very notable in view of its generally insoluble and resistant character. Whether this material can be turned to useful account as a source of ‘organic’ iron for therapeutic or nutritive purposes remains to be seen. aitibe When a neutralised digest taken at an early stage is dialysed it may be found that a certain amount of combined iron passes through into the dialysate and can be demonstrated there by methods for revealing the presence of ‘masked’ or ‘ organic’ iron, 7.¢., there is a certain amount of iron combined in the form of a diffusible compound. But if peptic digestion is at all prolonged the iron gradually becomes liberated. If treatment with the chlorate has been carried on too long, or too decided acidulation has been used, iron seems to be set free at an earlier stage of digestion. Pancreatic fluid is similarly effective in digesting the substance ACTION OF OXIDISING AGENTS UPON BLOOD-PIGMENT 163 in question ; alkaline haematin is not formed as in the digestion of ordinary haemoglobin. With tryptic digestion the liberation of iron seems to be slower than with peptic fluid. Treatment of blood or haemoglobin with a soluble persulphate (e.g. 2 to 3 per cent. sodium persulphate) also gives a modification of haemoglobin which is completely digested by peptic and pancreatic fluids without splitting off haematin. In the case of blood the modified haemoglobin is mixed with other blood proteins also coagu- lated by the reagent. _ Some Apprications oF THE CHLORATE AND Bromate AcTION UPON HAEMOGLOBIN In virtue of the above described action in coagulating haemo- globin while not similarly affecting ordinary proteins, chlorates and bromates may in certain cases be successfully used to separate haemo- globin from other proteins, etc., a suitable degree of slight acidification being provided by the use of acid sodium phosphate, etc., as in the case of serum, etc. The same may be done in urine containing haemoglobin (using 5 per cent. of the chlorate at room temperature for a day or two) with proper acidulation, but here certain con- stituents often interfere with the separation of the haemoglobin. The chlorate causes no precipitation in normal urine. When it is desired to liberate and demonstrate the iron of the haemoglobin, ¢.g., by the use of chlorate and strong acidulation with. salicyl-sulphonic acid as already described, complications arise from the fact that certain urinary constituents (uric acid, kreatinin, etc.) discharge the red colour struck by the liberated iron with the salicyl- sulphonic acid—unless the amount of iron be large; ammonium sulphide and ferrocyanide may likewise fail to demonstrate the presence of appreciable amounts of iron. Interfering constituents are also present in muscle extract, but not to any marked extent in serum or plasma. In the absence of such disturbing agents it is very easy to liberate and demonstrate the iron of haemoglobin, etc. For class purposes two or three drops of blood may be mixed with sodium chlorate 164 BIO-CHEMICAL JOURNAL solution (5 to 10 per cent.) and acidulated with salicyl-sulphonic acid to the amount of 2 to § per cent. The tube is left to stand for twenty-four hours, and the liberated iron shews by pink coloration of the fluid, which can also be tested by ferrocyanide and (after neutralisation with ammonia) by ammonium sulphide. The pigments of serum, bile and urine are not precipitated or decolourised by treatment with chlorate (after acidulation with acid sodium phosphate) over periods vastly longer than are Neceseany to change haemoglobin in the way described. Chlorate and bromate may be used as fixing agents for haemo- globin for certain histological purposes, etc., ¢.g., in the case of the natural injection of organs and parts with blood. » 165 ON THE PRESENCE OF OXYDASES IN INDIA-RUBBER, WITH A THEORY IN REGARD TO THEIR FUNCTION __IN-THE LATEX By D. SPENCE, Ph.D., A.I.C., Research Chemist to the Liverpool Institute of Commercial Research in the Tropics. From the Bio-Chemical Laboratory, University of Liverpool (Recetved April toth, 1908) In a previous paper! on the distribution of the protein in Para tubber, I have shown that Weber’s insoluble oxygen-addition compound of india-rubber is in reality of the nature of vegetable protein and have attempted to account for the singular behaviour of this insoluble protein towards solvents by a physical theory based on its peculiar distribution throughout the rubber in the form of a fibrous network. In this same paper I drew attention to the rather remarkable striped appearance which moderately dry sections of raw Para rubber exhibit, and as the peculiar shading of colour in the form of dark and light brown layers alternately throughout the rubber seemed to correspond with the distribution of the protein in the latter as evinced by the microscopical examination of stained sections, I suggested that the protein had probably a not unimportant function in the raw product. These incidental observations in regard to the properties of the insoluble constituent of Para rubber led me, however, to investigate still further the nature of the nitrogenous products which I have since found to be present not only in Para rubber, but in greater or less amount in some forty different brands of raw rubber obtained from the various rubber-producing plants throughout the world.? 1. Quart. Fourn. Liverpool Institute of Commercial Research, Vol. III, No. 6, p. 47, 1908. 2. There seems indeed to be little doubt that nitrogenous products of a similar character are to be found in greater or less amount in the latex of all the rubber-producing trees or vines. Furthermore in the processes for the coagulation of the latex, unless special steps be taken, the protein invariably becomes coagulated and bound up to a great extent in the rubber clot, giving rise:to putrefaction and deteriora- tion in badly-cured samples. 166 BIO-CHEMICAL JOURNAL Some time ago my attention was directed to a short note by T’schirch and’ Stevens! on oxidizing enzymes in gums. ‘The singular resemblance in solubility and in general behaviour of these enzymes to the insoluble constituent of Para rubber led me at once to examine this rubber for oxidizing enzymes, and to attempt to determine in how far the rapid darkening in colour and the oxidation of raw rubber in general might not be accounted for by the presence of such an enzyme in the insoluble constituent of india-rubber. Before proceeding, however, to describe the experimental work which has led to the isolation and identification of such an enzyme, let me briefly outline the literature on this subject. As the result of numerous investigations in the field of plant physiology, our knowledge of the nature of oxidizing ferments and of their function in the plant has been considerably increased within the last few years, and it must be looked upon as a great step in advance that experimentation, in particular the work of Bach and Chodat, has shown us that it is necessary to distinguish sharply between the ‘ direct oxydases ’ and the peroxydases (indirect oxydases) which act only in presence of hydrogen peroxide or another peroxide body. Nor can the function of these enzymes in the vital oxidation processes be overlooked, since many oxidation phenomena have been shewn to be due to the intermediate formation of peroxides and the action of oxidizing enzymes, while other important oxidation processes taking place in the plant have been attributed to the action of specific oxidizing enzymes. The colour changes observed in freshly-cut vegetable tissues exposed to the air have been generally ascribed to the action of oxy- dases, in many cases without even experimental proof of the existence 1. Separatabdruck aus der Schweiz. W ochenschrift fir Chemie u. Pharmacie, No. 31, 1905. 2. A review of the present day theories in regard to the nature and the function of oxidizing enzymes in plants is given by, Bach and Chodat (Biochem. Zentralblt., Vol. 1, p. 416, 1903) ; a very complete account of all the literature on this subject is to be found in Czapek, ‘ Biochemie der Pflanzen,’ Vol. II, pp. 464-481, 1905. According to Bach and Chodat the oxydases are a mixture of two enzymes of distinct types; the oxygenases or protein-like bodies, exceedingly unstable towards heat, chemical agents, etc., which take up molecular oxygen from the air with peroxide formation; the peroxydases or enzymes of nitrogenous but non-protein character which are much more stable towards heat, etc., than the oxygenases and activate the oxygenase, causing it to give up oxygen to the auto-oxidisable substrate. That the oxygenase fails in the extracts of some plants is due to its great instability. PRESENCE OF OXYDASES IN INDIA-RUBBER 167 of such enzymes in the tissues in question. It is not, therefore, surprising that the gradual darkening in colour which has long been observed to take place in freshly prepared samples of raw rubber, became associated in the minds of observers at a very early stage with the presence of an oxydase in the raw product. Czapek,1 indeed, states that the latex of rubber-producing plants contains an oxydase which is partially responsible for the darkening in colour of the coagulated product. Weber attributed the intense darkening in colour of Castilla latex to the presence of an oxydase, without, however, investigating this point, while Tschirch® states that the darkening in colour of raw rubber is due to the action of enzymes (oxydases). I have not, however, been able to find any reference to experimental evidence supporting these views, and on the other hand Schidrowitz and Kaye* have reported that they examined a sample of Hevea latex but failed to get any indication of the presence of oxydases. The latex, however, which they examined could not be regarded as a normal one seeing that it contained barely 4 per cent. of dry rubber. That oxydases occur in the latex tubes of certain plants Raciborski® has shewn, and the presence of a similar enzyme (laccase) in the gum of Japanese lacqueur has been shewn by Bertrand® and others. Furthermore, the presence of oxydases in gum arabic and indeed in almost all gums has been determined by Struve,’ Bertrand and Bourquelot,® Wiesner,® ‘T'schirch and Stevens, and important bio- logical functions have been ascribed to these enzymes by certain authorities." Numerous reagents have been suggested and employed successfully 1. Czapek, ‘ Biochemie der Pflanzen, Vol. IL, p. 705, 1905. 2. Weber, Ber. chem. Ges., Vol. XXXVI, p. 3110, 1903. 3. Tschirch, ‘ Die Harxe und die Harzbebilter,’ ind edit., Vol. I, p. 992, 1996. 4. India Rubber Fournal, Vol. XXXIV, No. 1, p. 24, 1907. 5 . Ber. d. deutsch. bot. Ges., p. 52, 1908. 6. Compt. rend., Vol. CXVIII, p. 1215, 1894; Vol. CXX, p. 266, 1895; Vol. CXXI, p. 166, 1895 ; Vol. CXXII, p. 1132, 1896. 7. Lieb. Ann., Vol. CLXIII, p. 160. 8. Compt. rend. soc. biol., Vol. XLIX, 1897. g- Sitzb. d. Wien. Akad., 1885, and Monatsh. d, Chem., Vol VI, p. 592, 1885. “10. Archiv. d. Pharm., p. §35, 1905, and Pharm. Zentralb., Nr. 26, 1905. 11. See Tschirch, ‘ Die Harze und die Harxbebalter, 2nd edit., Vol. I, p. 883, 1907. 168 BIO-CHEMICAL JOURNAL from time to time for the detection of oxydases, and it has been shewn that the behaviour of these enzymes towards reagents may vary with the source of the oxydases (certain oxydases giving a negative reaction to one class of indicator and a positive reaction with another). One cannot, therefore, rely on the tincture of guaiacum reaction alone in studying the action of oxydases. For this reason, in the work of isolating the peroxydase from Para rubber which I now propose to describe, I have been led to study the behaviour of the enzyme towards some ten well known reagents for oxydases which will be described in due course, and have only used the reaction with guaiacum as a rough and ready test in preliminary experiments. Further, my original intention in commencing this work being to clear up certain points in regard to the so-called insoluble constituent of Para rubber which has been surrounded by mystery in the past, my attention has been confined in the following communication to Para rubber only.} | EXPERIMENTAL Preliminary experiments to isolate an oxydase from raw Para rubber were carried out as follows :— A large quantity of unwashed raw Para rubber, cut from the interior layers of a very large block, was cut up into very fine sections by means of a sharp knife. These sections, which were as thin as it was possible to cut them, were allowed to soak ina quantity of distilled water sufficient to cover them, for a period of seven to ten days, after which time the aqueous extract was filtered off. About 100 grammes of the finely minced rubber were treated in this way each time, and the extract was tested from time to time by means of tincture of guaiacum with and without hydrogen peroxide. The first experiment showed that it was possible in this way to extract a ferment which had the property of rapidly turning tincture of guaiacum in presence of hydrogen peroxide a fine deep blue, and which was destroyed by boiling (peroxydase). ‘The watery extract was alkaline to litmus, and appeared to give marked indications of the presence of protein by the Millon’s and other tests. In order, therefore, to purify the extracts as far as possible they were dialysed for twenty-four hours against running water, preliminary experiments having shewn that in this way the peroxydase activity towards guaiacum was not destroyed. 1. Itis hardly to be doubted, however, that, what has been shewn to hold for the insoluble niteogetea body in Para rubber, will also be shewn in time to be equally true for the insoluble nitrogenous product to be found in all varieties of raw india-rubber. Tschirch’s work on the insoluble nitrogenous body in the various gums, and the gradual darkening in colour of all the ordinary varieties of india—rubber on the market only serve to support this view. y 7 A PRESENCE OF OXYDASES IN INDIA-RUBBER 169 The dialysed aqueous extract was found to have all the properties of a powerful peroxydase (indirect oxydase) but to be practically devoid of activity in absence of hydrogen peroxide. It was tested as follows :— _ Three test tubes were taken and into each was pipetted 10 c.c. of the dialysed extract. _ To serve as a control one of these was then heated to boiling for a few minutes and cooled, after which a measured quantity (1 c.c.) of a dilute standard solution of hydrogen peroxide was added to it and also to one of the other two quantities of active extract. The three tubes were then set side by side while the same quantity of a standard solution of the indicator to be used was added to each.' The activity of the dialysed extract was tested in this manner with reference to the following reagents :—Tinct. guaiacum, p-pheny- lenediamine, o-phenylenediamine, a-naphtol, phenol-phthalin, hydrochinon, amidol, pyrogallol, p-phenylenediamine + a-naphtol (indophenol reaction), tyrosin. The ‘results observed are given in the following table :— Ten c.c. of the extract Ten c.c. of the extract Ten c.c. of the extract Reagent boiled, and hydrogen _— without hydrogen with hydrogen peroxide peroxide added peroxide added Tinct. guaiacum? ... Pure white ... Pure white, Deep blue in five mins. unchanged p-phenylene-diamine* Colourless ... Colourless ... Dark brownish red o-phenylene-diamine* Faint yellow ... Colourless ... Yellow, finally red a-naphtol* ... ... Colourless -.. Colourless, after Marked brown : 2 hours Phenol-phthalin® .... Faint pink ... ,, Deep pink Hydrochinon® ..s Colourless ... Hs Intense port red Amidol’ ... ... Faint pink... Faint pink ... Deep cherry red Pyrogallol* ... ... No change after No change ... Yellow coloration and 24 hours brown deposit ‘Indophenol’ Mixt.® Faint colour ... No change ... Deep violet awa. ... .. Noaction ... Noaction ... No action 1. It is hardly necessary to point out that the solutions of these reagents were prepared fresh imme- diately before use. Peroxide formation in the process of auto-oxidation of these reagents may lead to erroneous results. See * Die Peroxydasereaktion der Kubmilch, etc.,) Dr. P. Waentig, Arbeiten aus dem kaiserlichen Gesundbeitsamte, Vol. XXVI, No. 3, 1907. 2. Van den Broek, Fabresber. Chem., p. 455, 1849-503 and Schonbein, Zeit. /. Biol., Vol. IV, p. 367, 1868. An alcoholic extract of the powdered resin. 3. Aqueous solutions of the hydrochlorides of the bases. 4. Solution in 50% alcohol containing trace of alkali. 5. Kastle and Shedd, Amer. Chem. Four., Vol. XXVI, p. 527, 1901. 6. Dilute solution in water. The developer Amidol has not been previously used. As a rapid indicator of oxydase activity it was found to be very useful, giving an intense dark red coloration within a few minutes. An aqueous solution acidified with a few drops of 5 %, acetic acid was used. 8. Asolution in water. The oxidation proceeds slowly, an insoluble brown precipitate of purpurogallin separating out in the course of 24 hours in the presence of oxydase. Réhmann_and Spitzer, Ber. chem. Ges., Vol. XXVIII, p. 567, 1895. The reagent contains 1 mol. a-naphtol, 1 mol. p.-phenylenediamine, and 3 mol. Na,CO, in water. to. A dilute solution in water containing a trace of Na,CO,. 170 BIO-CHEMICAL JOURNAL These tests suffice, I think, to shew beyond all doubt that the dialysed aqueous extract from Para rubber contains a peroxydase enzyme or enzymes of the general type, readily destroyed by boiling. That the extract had no action on tyrosin points to the absence of © a tyrosinase. Extracts from several different samples of Para rubber were examined in this way under varied conditions and always with the same result. The extract had also the property of a catalase, 20 c.c. of this extract in one experiment in an eudiometer tube over mercury liberating 12 c.c. of oxygen from hydrogen peroxide within a few hours; there was also a very slight indication of proteolytic activity.? The dialysed extract was neutral to litmus, and gave neither the xanthoproteic reaction or the familiar coloration for protein on warming with Millon’s reagent. No definite biuret test could be got with the dialysed extract. Boiled with a trace of acetic acid the extract became opalescent, and a small quantity of to matter separated. Very small quantities of acid or alkali did not appear to influence the peroxydase activity to any extent although large quantities of either of these reagents stopped its action completely. When the dialysed extract was concentrated in vacuo over sul- phuric acid it appeared to lose in activity gradually and became finally inert, the sulphuric acid colouring at the same time due to absorption of ammoniacal products. "This method, then, obviously could not be used for the isolation of the peroxydase, nor was the method of saturating the extract with ammonium sulphate of much value in this connection. ) For the isolation and purification of the Bre es the following method was found to be most satisfactory :— sti The finely cut raw Para rubber was digested for about a week with 40 per cent. alcohol, after which the alcoholic extract, which became intensely yellow during the process due to the extraction of resins, etc., from the rubber, was filtered off and precipitated with several times its own volume of absolute alcohol. On standing, a gummy mass settled out which was filtered off, redissolved in 40 per cent. alcohol, and again precipitated with absolute alcohol. When this process was repeated three times a product was obtained which was infinitely more active than the original precipitate, blueing tincture of guaiacum in presence of peroxide practically instantaneously. 1. Metts digestion tubes. PRESENCE OF OXYDASES IN INDIA-RUBBER 171 PROPERTIES OF THE PEROXYDASE FROM PARA RUBBER The product obtained in this way no longer appeared to possess either catalytic or proteolytic activity to any extent, and in the moist state was a gummy looking mass which left a dry vitreous solid on _ evaporation of the alcohol. The amount of this solid extracted from the rubber was very small, so that a kilogramme of Para rubber had to be extracted in order to obtain sufficient material for chemical tests. The peroxydase dissolved slowly in water, and the solution was found to be many times more active in presence of peroxide than the original aqueous extracts,! although it had not the slightest action in absence of hydrogen peroxide. The specific activity of the aqueous extract of the peroxydase was destroyed by the addition of small quantities of potassium cyanide, sodium fluoride, or mercuric chloride to it, while in presence of mineral acids and alkalies, the activity also disappeared. ‘The activity of the peroxydase was found to diminish gradually when the aqueous solution was left exposed to the ait and light for some time.2_ Towards heat the peroxydase was remarkably stable, and it is interesting to note in this connection that when its solution in water was just raised to the boiling point, and then allowed to stand for some time, its activity gradually returned to a slight extent so that it again gave a marked reaction with tincture of guaiacum or with the indophenol reagent.® By a series of rough measurements with tincture of guaiacum colorimetrically and with pyrogallol, the activity of the peroxydase was determined after exposure to different temperatures. It was found that heating the aqueous extract for 5 minutes to 80°C. 1. It is unnecessary to repeat here the various tests which were carried out in order to determine the peroxydase nature of the substance in solution. All the reactions, however, which have been described on page 169, were repeated many times during the course of the work, and always with more or less the same result. 2. Bach (Ber. d. chem. Ges., Vol. XLI, p. 225, 1908) has determined a reduction in the activity of peroxydase due to the action of oxygen and light. 3. These observations agree with those of Woods (United States Dep. of Agr. Bull., No. 18, p. 17) who found that the oxydase from tobacco juice when killed by boiling, gradually regained its activity on standing. Both Woods and Aso (Bull. Coll. Agric., Tokyo, Vol. Il, p. 220) appear to be agreed on the existence of a zymogen, more stable towards heat than the oxydase, which changes slowly into the active enzyme. 172 BIO-CHEMICAL JOURNAL destroyed the peroxydase activity completely, while 24 minutes reduced the activity by one half. Heated to 70° C. for 5 minutes the peroxydase was still active, whereas after 15 minutes the enzymic action was completely destroyed. ‘The peroxydase when heated for 10, 20, 30 or 40 minutes to 60° C. was not destroyed. The thermal optimum of the peroxydase was determined by similar colorimetric methods and it was found to be about 55°C. In regard to the chemical nature of the peroxydase the following points were noted :— Although the peroxydase contained a considerable amount of nitrogen, it gave no reaction either with Millon’s reagent or by the biuret or other tests for protein. When fused with KOH, ammonia was evolved in quantity along with traces of pyrrol which gave the characteristic red coloration to a splint of pine-wood moistened with hydrochloric acid.t| This point will be dealt with again in full when the properties of the oxygenase from the latex are discussed. The peroxydase, however, gave a slight reduction with Fehling’s solution, and further gave the characteristic coloration for pentoses* with phloroglucin and hydrochloric acid, so that the pyrrol ring is probably formed from the pentose and ammonia set free in the process of fusion. | The peroxydase isolated from Para rubber was found to give a very marked reaction for iron. ‘The coloration with potassium ferrocyanide or with ammonium sulphocyanide was only given, however, when the solution of the peroxydase was treated in such a way that the activity of the enzyme was destroyed. Thus for example, although no coloration was given by adding either of these reagents directly to the peroxydase solution when a drop of dilute hydro- chloric acid was first added to the peroxydase or when the peroxydase _ J. Tschirch was the first to observe that pyrrol is evolved when the oxydase from Japanese lacqueur is distilled with KOH (Archiv. der Phar., 243, 7, 504, 1905. Bach has recently confirmed Tschirch’s observations in connection with the peroxydase from the radish, but has pointed out that ammonia as well as pyrrol vapours are evolved by this reaction (Ber. d. deutsch. chem. Ges., Vol. XLI, p- 226, 1908). 2. From a review of the literature on this particular point it appears that a carbohydrate group of a reducing nature is to be found associated with most of the oxidizing enzymes, and that it is extremely difficult _ if indeed not impossible to separate this complex from the oxydase. Whether in reality acompound between carbohydrate and enzyme exists still remains to be shewn. See Bach and Chodat, Ber. chem. Ges., XXXVII, P- 42, 19043; Cazeneuve, Compt. rend., Vol. CXXIV, pp. 406 and 781, 1897; Tschirch u. Stevens, Archiv. der Pharm., 243, 7, 535; 1905. St ee Se ee eee ee Nr PRESENCE OF OXYDASES IN INDIA-RUBBER- | 173 solution was boiled for a few minutes (although less marked in this case) and then treated with these reagents, a fine pink coloration was got with-the ammonium sulphocyanide and a deep blue precipitate with the ferrocyanide reagent. Without going into a discussion of the present day position in regard to the function of the manganese,} which is usually associated with the oxydases or of the iron? which has already been found in an oxidizing-enzyme and is probably present in a colloidal form therein, from the above observations I am inclined to believe that considerable importance must be attached to the iron-content of the peroxydase from Para rubber. RELATIONSHIP OF THE PEROXYDASE TO THE INSOLUBLE PROTEIN OF THE RUBBER Having thus determined the presence of a peroxydase enzyme, it became of interest to study more closely its relation to the so-called insoluble constituent or protein in the rubber. The peculiar striped appearance of Para rubber, which, as I have already pointed out, falls closely in line with the distribution of the protein as revealed by the microscopical examination of stained sections, gave a valuable indication of the oxydase nature of the latter. Indeed, the presence of a peroxydase alone in the insoluble body would not appear to be sufficient to account for the rapid darkening in colour observed when freshly cut rubber is left exposed to the air. According to Chodat and Bach the peroxydase is inactive unless in presence of the peroxide forming enzyme (oxygenase) or preformed peroxide, and this at once led me to examine the insoluble constituent of Para rubber for an oxygenase-complement which had not been extracted in previous experiments. Here, however, almost negative results were obtained, and it was only later when fresh latex from Funtumia elastica was obtainable, that I was able to prove the presence of an oxygenase as well as of a peroxydase in the fresh latex (see page 175). 1. Bertrand, Ann. chim. phys. (7) Vol. XII, p. 115; Compt. rend., CXXIV, pp. 1032, 1355 (1897); Bulk. soc. chim. (3) Vol. XVII, pp. 619, 753 (1897). 2. Sarthou, Yourn. pharm. chim. (6) Vol. XI, p. 583, 1900; Vol. XIII, p. 464, 1902. 174 BIO-CHEMICAL JOURNAL The method by which the Para rubber was tested for the presence of an oxygenase was as follows :— A quantity of the insoluble protein was isolated from Para rubber by dissolving away the rubber in chloroform! in the cold. After three months’ extraction the insoluble product left was washed with alcohol and dried. It was a hard, stringy, but non-elastic mass which gave a slight reaction for protein with Millon’s reagent. This body was allowed to act on peroxydase extracts in test tubes, but when the extracts were tested they shewed not the slightest activity in absence of hydrogen peroxide. As the oxygenase, as is conceivable, might have been destroyed by the long continued action of chloroform, thin sections of the raw rubber were cut and these were placed directly in a peroxydase extract with tincture of guaiacum. Whereas the tube containing the rubber and extract was only very faintly blue in colour and was hard to distinguish from the control with extract alone, the extract in presence of hydrogen peroxide gave a deep blue colour in a very short time. Numerous experiments on these lines were tried in order to deter- mine the presence of an oxygenase along with the peroxydase in the insoluble constituent of Para rubber, but the insuperable difficulties connected with the isolation of the ‘insoluble constituent’ from Para rubber made all attempts in this direction useless. One is led to the conclusion, therefore, that either the oxygenase is killed in extraction experiments with chloroform and toluol, and is not directly got at in the other tests on account of the large mass of the colloid material with which it is associated, or else that the oxygenase fails entirely in the rubber, being destroyed in the coagulation of the Hevea latex.” In view of the activity of the peroxydase in the rubber as evinced by the colour-changes produced, the first explanation seems to me to be the more probable of the two. The subsequent examin- ation of a sample of Funtumia elastica latex and the isolation of an oxydase therefrom, determines with a fair degree of certainty the existence of an oxydase in the raw rubber. In conclusion, further support was given to the theory of a stable zymogen in the raw rubber, by the fact that boiling the rubber with water alone or with water containing small quantities of cyanide, mercuric chloride, or formic acid for some time did not prevent the further darkening in colour of the rubber on prolonged keeping. 1. See ‘Distribution of the Protein in Para Rubber,’ Quart. Fourn. Inst. Comm. Res., Vol. III, No. 6, p- 51, 1908. 2. It would be interesting in this connection to know approximately the temperature at which coagulation of the Hevea latex is brought about. PRESENCE OF OXYDASES IN INDIA-RUBBER 175 OxyYDASES IN THE LATEX It may seem out of place that the examination of the latex emulsion was deferred until this point. Obviously, if any reaction is to be found it ought to be sought for in the fresh latex from which the rubber is formed. But various difficulties have to be overcome in working with latex, there being the question of supply of an emulsion _which is not to be had in this country, and is withal exceedingly unstable and liable to coagulate. Hence it is that what should in the ordinary course of events have been studied first had to be left until late in the research. A quantity of latex from Funtumia elastica, Stapf., preserved according to my directions, arrived here recently from Southern Nigeria.1 The latex, which had been carefully collected and sealed up in well-filled pint bottles, arrived in this country for the most part in perfect condition. As only a couple of the samples of this latex come under consideration here, these only will be dealt with. The others will be considered elsewhere. The two samples submitted to thorough examination for oxidising enzymes were :— ; Sample I. The natural latex undiluted. Sample II. One pint of latex treated with 2 tea-spoonfuls of ammonia (S.G. 0°880) in } oz. of water. Sample I was a clear white milky fluid which had not coagulated at all. On suitable dilution the globules of caoutchouc on examination under the microscope were found to be intact and in rapid brownian movement in the serum. This sample of latex although uncoagulated had developed a marked acidity to litmus? which is perhaps surprising, in view of the generally accepted theory in regard to the process of coagulation.° 1. This latex was collected for me through the kindness of Mr. T. F. Burrowes, the Acting Colonial Secretary for Southern Nigeria, and I should like to take this opportunity of expressing my thanks to him and to those who have assisted him in securing for me an ample supply of latex for further investigations. 2. Fresh Funtumia elastica latex is practically neutral! Experiments are already in progress which appear to show that the acidity developed in the natural latex is due to the oxydase therein. 3. This latex would indeed appear to be exceptionally stable in presence of acids; see Spence, Quart. Fourn. Inst. Comm. Research, Vol. II, No. 4, p. 45, 1907; Schidrowitz and Kaye, India Rubber Fournal, Vol. XXXIV, No. 7, p. 377, 1907. i elie ’ 176 BIO-CHEMICAL JOURNAL Sample II. This sample had not coagulated at all and was of a pale yellow colour in consequence of the presence in it still of a considerable excess of ammonia. Both samples were tested directly with tincture of guaiacum with and without hydrogen peroxide. The first sample was found to give a very marked peroxydase reaction, but no reaction in absence of hydrogen peroxide. | To free the latex, however, from products which would tend to interfere with the peroxydase reactions, both samples of latex were dialysed for twenty-four hours against running water and then examined, The sample of untreated latex from which the free acid had been removed in this way was still found to give a very marked peroxydase reaction but no oxydase. The sample of ammoniacal latex on the other hand now gave an intense peroxydase and a much less marked though positive oxydase reaction. ‘This was confirmed by a positive reaction against boiled controls using the tincture of guaiacum, the indophenol, the p-phenyl- enediamine and the pyrogallol tests as indicators. The oxygenase in Sample I had, therefore, been more or less destroyed by the acidity developed. ‘The question as to whether new oxygenase is formed on removal of the acid from the sphere of action still remains to be proved. Experiments so far have not shewn whether the oxygenase like the peroxydase will recover its activity, although it would be remarkable, were this not so, that. samples of rubber prepared from the two dialysed latices in the ordinary way should darken in colour at the same rate and to the same degree. Experiments were now set on foot in order to isolate the oxydase from the latex. For this purpose several methods of separating the caoutchouc in the latex without at the same bringing down the protein with it were tried, and although the method in the case of the acid latex was simple, it being only necessary to dilute the dialysed latex with several volumes of distilled water in a separating funnel, and to allow the fine flakes of caoutchouc to rise to the surface and settle before drawing off the mother-liquor, the process in the case of the PRESENCE OF OXYDASES IN INDIA-RUBBER 177 ammoniacal product was not so easy, there being a great tendency for the protein to disappear in the rubber clot. The following method for the ammoniacal latex was found to be satisfactory :— =The dialysed latex was‘diluted by its own volume with water and it was then treated with 50 per cent. alcohol until the agglutination of the caoutchouc particles was complete. The fine flakes of caoutchouc were allowed to settle, after which the mother-liquor was filtered off and treated with a large excess of absolute alcohol. On adding excess of alcohol a gummy precipitate formed which was immediately filtered off and dried in vacuo. This precipitate gave a marked oxydase reaction. PROPERTIES OF THE OxyDASES FROM THE LATEX The product separated from the latex in this way was a dark brown vitreous-looking mass which dissolved only slowly in water, giving a somewhat opalescent solution. The aqueous solution was tested with the various reagents for oxydases both with and without hydrogen peroxide, and it was found to give a marked positive reaction in absence of peroxide and an intense coloration with the various reagents in presence of peroxide. It contained, therefore, the full oxydase complement (oxygenase-peroxydase) although the oxygenase appeared to be very feeble. The enzyme was fairly stable towards heat and light in the dry _ state, but was readily destroyed in solution. It gave the xantho- proteic, Millon’s, biuret, coagulation and precipitation tests for protein in a marked degree. It was also found to contain a con- siderable quantity of iron, and on fusion with alkali evolved ammonia and pyrrol, the latter being identified by the pinewood-HCl reaction already described. As in the case of the peroxydase from Para rubber the oxydase was also found to have associated with it a reducing substance which, from the violet red coloration (shewing an absorp- tion band between D and E) given by it on warming in aqueous solution with an equal volume of HCl and a trace of phloroglucin, appears to be partly at least of the nature of a pentose. This being the case, it does not appear to me to be necessary or even correct to assume that a pyrrol ring is there preformed in the protein nucleus of the oxydase.! It seems far more probable that the pyrrol is formed “1. See Tschirch, ‘ Ueber die Gummi-Enzyme,’ Pharm. Zentralb., Nr. 31, 1905. 178 BIO-CHEMICAL JOURNAL only in the process of fusion of the oxydase with the alkali, the ammonia liberated in this process reacting with the pentose or the partially oxidised pentose.t_ That such a reaction will actually result in the formation of a pyrrol derivative sufficient in amount to give a marked pinewood reaction, I have proved to myself to be the case by fusing together a mixture of pure arabinose, powdered potassium hydrate and ammonium sulphate. Although no coloration is given to the pinewood splint when the reaction is carried out in absence of ammonia, in the presence of this reagent I had no difficulty in getting, in re- peated experiments, marked evidence of pyrrol formation. The oxydase product isolated from the latex of Funtumia elastica did not appear to have proteolytic or amylolytic properties. It had a slight catalytic action. In order to determine in how far the darkening in colour in rubber could be prevented practically by the removal of the nitrogenous products which form the ‘insoluble constituent’ of india-rubber, experiments were carried out with the Funtumia latex, the object being to separate the caoutchouc from the emulsion without at the same time coagulating the protein material in the latex. The following method was completely successful :— The acid latex is diluted with many times its own volume of water in a separating funnel. The caoutchouc globules apparently coalesce in this way to form fine spongy flakes. These flakes gradually separate and rise to the surface of the watery liquor, which is then drawn off. The flakes of caoutchouc are again shaken up with a fresh quantity of water, and when this process is repeated three times the caoutchouc is found to be practically nitrogen-free. The flakes are then worked up into a solid rubber clot by washing with alcohol or by pressure. The rubber obtained in this way was snow-white and did not darken in the slightest on keeping. Samples prepared from the same latex by any of the usual methods of coagulation in which the protein is afterwards to be found in the rubber clot, darken rapidly in colour and become almost black in the course of a week. A sample of rubber prepared by the method described was found to be practically free from protein.® | 1. Compare C. Paal, Furfurane, thiophene and pyrrol syntheses, Wirzburg, 1890. 2. Coagulation does not take place. 3. The protein was found uncoagulated in the first washings. The value commercially of such a method of coagulation will be discussed elsewhere. PRESENCE OF OXYDASES IN INDIA-RUBBER 179 These observations prove conclusively that the darkening in colour of raw rubber is due to an oxydase which is associated with the protein or the so-called tmsoluble constituent of the rubber. In how far this oxydase is responsible for certain other changes occurring in raw rubber on keeping (decomposition, oxidation and the like), further experimental work, I hope, will shew. In addition to its practical importance the presence of active oxydase enzymes in the latex of caoutchouc-producing plants appears to me to be of interest biologically when considered in the light of certain well-known facts regarding the chemistry of india-rubber. Up to the present no explanation of the function of the caoutchouc in the latex has been offered. The chemical inertness of india-rubber as we know it, and the fact that this product can be extracted in such “quantities without any apparent injury to the tree, has led to the general belief that it is an excretory product of metabolism serving as a means of protection to the plant against injury! (Wundverschluss). That the latex plays a useful part as a reserve store of water for the plant and as a means of protection against external injury, one need not doubt, but that these are the chief functions of the latex of caoutchouc-producing plants seems to me improbable. The economy and care with which the vital processes in nature are carried out and regulated, point on the very surface to a deeper and more important function for the caoutchouc in the plant, and it is scarcely to be accepted without further proof that a caoutchouc-producing tree continues to produce immense quantities of material in the nature of a hydrocarbon—the richest form of chemical energy that one can well imagine—solely as a means of protection against injury. From the very nature of the product, it seems to me we must far rather believe that it has a more important function in the plant, and there is considerable evidence on all hands to indicate this. I would venture to suggest that the caoutchouc is probably a reserve food stuff for the plant, and to account for its formation and subsequent decomposition would bring the oxidizing enzymes under 1. See Czepek ‘ Biochemie der Pflanzen,’ Vol. II, pp. 698-701, 1905, and Tschirch, ‘ Die Harze und die Harzbebilter, 2nd edit., Vol. II, 1906. 180 BIO-CHEMICAL JOURNAL consideration. Just as the glycogen in the liver is believed to be a reserve store of energy built up from the simpler sugars only to be broken down again into these and other products by the interaction of the glycolytic enzyme, so the caoutchouc may also be regarded as a reserve food stuff which is broken down by the oxydases in the plant as circumstances demand, into the simpler products from which it has been formed. That the oxydases are capable of effecting such changes might appear at first sight hard to believe. But as evidence that such enzymes have already been shewn to bring about even more com- plicated changes, I should like to point out :— The complete oxidation of sugar into CO, and H,O by oxidizing enzymes has been shewn by the work of Sieber,! Palladin,? and others. The oxidation of complex fats into simpler fatty acids and CO, has been illustrated by the experiments of Tolomei? on the oxidation of olive fat by olease—an oxidation enzyme in olives giving a marked tincture of guaiacum reaction. Finally, the oxidation of the protein decomposition product tyrosin by means of an oxidizing enzyme (the tyrosinase of Bertrand*) into homogentisinic acid, carbon dioxide and ammonia with the uptake of oxygen is sufficient evidence, I think, to shew that in complicated oxidation processes in both the animal and the vegetable kingdom oxidizing enzymes play an important role. Nor does the decomposition of caoutchouc by oxidizing enzymes in the plant appear to me to offer insurmountable difficulties in the way of the theory suggested. Certain observations already made on the action of a peroxydase enzyme and hydrogen peroxide on the neutral latex emulsion of Funtumia elastica seem to indicate that the caoutchouc in the latex is not indifferent towards atomic oxygen liberated in this way. Further, the important discovery of Harries,° that caoutchouc, even in the form in which it is known Sieber, Zeit. phys. Chem., Vol. XXXIX, p. 484, 1903. Palladin, Zeit. f. physiol. Chemie, Vol XLVII, p. 407, 1906. Tolomei, Chem. Centr., Vol. I, p. 879, 1896. - Bourquelot and Bertrand, Ydurn. pharm. chim (6), Vol. 3, p. 177, 1896; Bourquelot, Bull. Soc. Mycol., France, p. 65, 1897; v. Furth and Schneider, Hofmeister’s Beitrige, Vol. I, p. 229, Igo! ; v. Furth and Jerusalem, Hofmeister’s Beitrage, Vol. X, p. 131, 1907, and numerous others. 5- Harries, Ber. chem. Ges., Vol. XXXVII, pp. 2708-2711, 1904, and Vol. XXXVIII, pp. 1195-1203, 1905. - PRS PRESENCE OF OXYDASES IN INDIA-RUBBER 181 commercially, is exceedingly sensitive towards small quantities of ozone, and indeed can be broken down by this means into products very nearly related to the pentoses! from which the caoutchouc is most probably formed by the plant, lends support to the view that the caoutchouc in the latex must be regarded as a reserve stuff which can be drawn upon as required and broken down by the oxidases always associated with it into simple carbohydrate products of value as food stuffs.” If we go further, however, and assume that the reaction brought _ about by the oxydase is a reversible one, then by means of it we can account not only for the breakdown, but also for the building up of the caoutchouc from the sugars (from the pentoses probably as Harries suggested) by the plant. In this light, the presence of a pentose group associated with the oxydase from the latex of Funtumia elastica is highly interesting.® The theory suggested to account for the formation and the function of the caoutchouc in the latex is supported further by experimental facts regarding the anatomy and physiology of caout- chouc-producing plants. It would explain, for example, the occurrence of a definite latex system in the embryo of certain caoutchouc- producing plants, and the complete disappearance of the latex from the tree under certain conditions, but these aspects of the question will be considered in full when further experimental evidence on the chemical side has been obtained. In the meantime, I should like to reserve to myself the right to follow up the work on which I have already commenced, of investi- gating the action of oxidizing enzymes on latex emulsions. In conclusion, I beg to express my thanks to Professor Benjamin Moore for the interest which he has taken in the progress of the work. 1. Laevulinic acid and hydrogen peroxide. 2. The peculiar state of division of the caoutchouc in the latex, and the fact that it is there most probably in a less complex form than in the coagulated product must not be overlooked. See Weber, Ber. chem. Ges., Vol. XXXVI, pp. 3108-3115, 1903; Harries, Ber. chem. Ges., Vol. XXXVII, Pp- 3842-3848, 1904. ; 3 Whether the so-called impurity always associated with the various enzymes may not represent mmute quantities of the substrate in loose chemical combination or adsorption with the enzyme, in the rocess of change, is a point worthy of further consideration. If this were so then the nature of the so-called impurity so firmly attached to the enzyme ought to give a valuable indication of the function of the latter. 182 ON THE APPLICATION OF BARFOED’S REAGENT TO SHOW THE HYDROLYSIS OF -DISACCHARIDES BY ENZYMES By HERBERT E. ROAF, M.D., Lecturer on Physiology. From the Physiological Department, University of Liverpool (Received April 7th, 1908) The methods which are used to show the hydrolytic splitting of lactose and maltose, are not so simple as those which demonstrate the hydrolysis of saccharose and polysaccharides. The reason for this is that in the latter case there is a change from a non-reducing carbohydrate to a reducing sugar, whilst in the former the original material will already reduce cupric hydrate in alkaline solution. It is, therefore, necessary to make a quantitative estimation of the reducing power in two solutions, one of which is a control containing the boiled enzyme and the other is a similar mixture containing the unboiled enzyme.! The presence of lactase and maltase can be just as easily proved as the ordinary amyloclastic enzymes, by using copper acetate in acetic solution.” This reagent is reduced by monosacchar- ides, whilst under proper conditions, disaccharides leave it unchanged. Hinkel and Sherman have recently investigated this reaction by comparing the behaviour of the reagent with glucose, maltose, lactose and saccharose.? ‘They found that using five cubic centi- metres of the reagent the presence of 0-0004 gramme glucose can be shown, either alone, or mixed with disaccharides, provided that the total weight of disaccharide does not exceed 0-02 gramme. The period 1. Polarimetric and other tests have also been used, see Aders Plimmer, Fourn. Physiol., Vol. XXXV, P- 20, 1907. 2. Barfoed, Fresenius Zeit. f. anal. Chem., Vol. XII, p- 27, 1873. 3. Fourn. Amer. Chem. Soc., Vol. XXIX, p. 1744, 1907. HYDROLYSIS OF DISACCHARIDES 183 of heating should, however, not be too long or reduction may occur owing to hydrolysis of the disaccharide by the acetic acid in the reagent. ‘They also recommend that each observer should standardise his reagent before using it. Their observations have been confirmed by me, and} it seemed desirable to apply this reagent as a qualitative test,1 when testing for the presence of lactase and maltase. To demonstrate the presence of lactase and maltase, the following method was employed. ‘To five cubic centimetres of a one per cent. solution of the disaccharide, one cubic centimetre of an aqueous (toluol water) extract of the first part of the small intestine was added ; toluol being used as a preservative. After the period of digestion was ended, one cubic centimetre of this mixture was added to five cubic centimetres of the copper acetate reagent, and the resulting solution was placed in a boiling water bath. ‘The tubes were examined at the end of three minutes, and if no reduction was visible they were replaced and re-examined at the end of the fourth and fifth minutes. It was found that one cubic centimetre of a one per cent. solution of either lactose or maltose does not cause reduction until heated for nine or ten minutes. Thus, if the heating does not exceed five minutes any reduction with this strength of sugar must be due to the formation of monosaccharides. ‘There is also a further check on the accuracy of the method, the control should show no reduction and as it is heated under identical conditions, the absence of reduction proves that the reduction caused by the other solution must be due to the hydrolytic action of an enzyme. Experiment I.—Aqueous extract of duodenum and first portion of jejunum of cat. Solution At end of 1 day At end of 3 days 5 c.c. 1% lactose + 1 c.c. unboiled enzyme _No reduction Slight reduction within 5 minutes 5 c.c. 1 % lactose + 1 c.c. boiled enzyme No reduction No reduction 5 c.c. 1 % maltose + 1 c.c. unboiled enzyme Marked reduction -- within 3 minutes 5 c.c. 1% maltose + 1 c.c. boiled enzyme No reduction — 1. This reagent might also be applied quantitatively, as glucose can be completely removed from a solution if the total amount does not exceed o'002 g. per § c.c. of the reagent (Hinkel and Sherman, p. 1747) and the oxide separates in a form which could easily be filtered off and weighed. 184 BIO-CHEMICAL JOURNAL Experiment 1J.—Aqueous extract of first portion of small intestine of a kitten four days old. At end of 1 day Solution 5 c.c. 1% lactose + 1 c.c. unboiled enzyme _..._ Marked reduction within 3 minutes 5 c.c. 1% lactose + 1 c.c. boiled enzyme ... No reduction 5 c.c. 1% maltose + I c.c. unboiled enzyme ... Marked reduction within 3 minutes 5 c.c. 1% maltose + 1 c.c. boiled enzyme ... No reduction These two experiments show that both lactase and maltase were present in the intestine of a kitten whilst the adult cat possessed maltase, but the amount of lactase was much less than in a young animal. This result, agreeing as it does with previous workers who have studied the distribution of lactase and maltase, suggests that Barfoed’s reagent may be used whenever it is wished to demonstrate the hydrolysis of disaccharides. 1. Cf. Plimmer, Joc. cit., p. 29. 185 A RAPID METHOD FOR SEPARATING HIPPURIC ACID FROM URINE By HERBERT E. ROAF, M.D., Lecturer on Physiology. From the Physiological Department, University of Liverpool (Received April 11th, 1908) When hippuric acid is precipitated by acid from a dilute solution — of one of its salts it usually separates slowly, and when obtained from urine it also carries down with it a certain amount of pigment.! The following method has been found a convenient way in which to prepare hippuric acid from (herbivorous) urine. It consists, in short, in adding ammonium sulphate to the urine before acidification. By this means the hippuric acid crystallises out rapidly, and usually contains only a comparatively small amount of adherent pigment.? This can be removed, and the crystals obtained free from pigment, by recrystallisation, after boiling the free acid (or its sodium salt) with animal charcoal. The following experiments will illustrate the results which can be obtained by altering the conditions of experimentation. “To samples of twenty-five cubic centimetres of cow’s urine varying amounts of salt and acid were added, and the time at which crystalli- sation commenced was noted. In some cases the crystals were filtered off after a definite period and the filtrate allowed to stand for twenty- four hours to see if a further crop resulted. 1. For the usual methods of preparing hippuric acid see Naubauer u. Vogel, Harn Analyse, roth ed. P+ 225, 1899. +2. Previous treatment by heating with milk of lime and then filtering did not appear to lessen the amount of pigment in the crystals. 186 BIO-CHEMICAL JOURNAL Urine Salt Acid Crystallisation Remarks — 1 c.c. 31 % HCl soe que ) Only one or two deeply : < oe -- 1 c.c. 31 % H,SO, — aaa re a at the : 266.0. 6 g. NH,Cl 1 ¢.c. 31 % H,SO, — No eine ae at 4°28 GC. 6g. (NHy), SO, 1 cc 31% H,SOq 15 mins. Good Tee 5 25 C.c. 6 g.(NH,4),S0, 2. ¢-¢. 31 Y HpSOq 20 mins. sage ad eis CH,-CH,NH, —> CH,CH,-OH —> CH,CHO -> CH,;COOH -» (CH,°CO),O By a similar series of changes we can then proceed to the formation of higher aldehydes, propionic, butyric, valerianic, etc., their acids and anhydrides. 1. Claus, Annalen d. Chemie, Bd. CXLV, s. 256. FORMATION OF LACTIC ACID AND CARBONIC ACID _ 197 By combining glycollic nitrile with NH, and subsequent hydration we obtain glycocoll! __-=-€H,(OH)‘CN + NH, = CH,(NH,)‘CN + H,O T9067 CH,(NH,)‘CN + 2H,O = CH,(NH,)-COOH + NH, glycocoll In the same way by combining it with methylamine we obtain sar- cosine | CH,(OH)-CN + NH,(CH,) = CH,(NH-CH,)‘CN + H,O CH.,(NH-CH,)‘CN + 2H,O = CH,(NH-CH,)-COOH + NH, sarcosine which combined with cyanamide forms kreatin. D.—The most interesting and important action, however, is the formation of amino-malonic nitrile from the condensation of three molecules of prussic acid 3HCN = CH(NH,)-(CN), amino-malonic nitrile Little attention has been paid to this substance, but I have reason to suppose that it plays an important part in the synthesis of the various proteids and of their derivatives. It is prepared in the following manner :— If a fragment of potassium cyanide be added to anhydrous hydrocyanic acid, condensation of three molecules gradually takes place,” forming amino-malonic nitrile, a crystalline body 3HCN = CH(NH,) (CN), amino-malonic nitrile a black insoluble substance being produced at the same time. The nitrile may be extracted with ether,; this, on evaporation, yields the crystals, which may then be dissolved in water, decolourised with animal charcoal, and recrystallised. ‘The yield by this method, however, is small. A much larger result, not less than §0 per cent. of the theoretical amount, is obtained by the following process.® 1. Eschweiler, Annalen d. Chemie, Bd. CCLXXVIII, s. 655. 2. Lescoeur, Regault, Bulletin de la Société Chimique de Paris, t. XLIV, p. 473. "3. Eug. Bamberger, Leo. Rudolph, Berichte, Bd. XXV, s. 1083. 198 BIO-CHEMICAL JOURNAL Add 2-6 grammes of pure dimethyl-aniline-oxide to 0-6 gramme of anhydrous prussic acid. Let the pale yellow mixture remain in the dark for twenty-four hours at the ordinary temperature. Extract’ with boiling water and separate the nitrile from the aqueous extract by shaking it several times with ether. It must then be recrystallised from boiling water and decolourised with animal charcoal. Warmed with barium hydrate, amino-malonic nitrile is resolved into glycocoll, ammonia, and carbonic acid CH(NH,)(CN), + 4H,O = CH,(NH,)‘COOH + CO, + 2NH, amino-malonic nitrile glycocoll the carbonic acid and ammonia being liberated in the same proportions as result from the decomposition of urea when heated with barium hydrate. The intermediate stages may be represented as follows :— CH(NH,)(CN), + 2H,0 = CH(NH,):¢ <=. : “CO NH, which with a further molecule of H,O NH, = CH,(NH,)‘COOH + COC 2 or, if we suppose that the NH; and CO, are discharged separately we may assume that they combine to form ammonium carbamate since the two gases if passed in a perfectly dry state into cold absolute alcohol are known to combine, the product separating as a copious crystalline precipitate. This, if filtered off and then heated with absolute alcohol in a sealed tube to 100°, on cooling appears as large crystalline laminae of ammonium carbamate. Under certain con- ditions this is converted into urea and ammonium carbonate CO, + 2NH; = NH,’COONH, : ammonium carbamate NH, 2NH,-COONH, = COC + (NH,),CO, . 2 ammonium urea carbonate 1. Kolbe and Basaroff, Chem. Soc. Fournal, [2] 6, 194. FORMATION OF LACTIC ACID AND CARBONIC ACID _ 199 If glycocoll be heated with acetic anhydride it is converted into acetyl-glycocoll.? —" NH NH-CO-CH, aH + (CH-CO),0 = 2CH + H,O RoR ( ee i een ‘ Glycocoll anhydride Acetyl-glycocoll If it be heated in a sealed tube to 160° C. with benzoic acid it is converted into hippuric acid? NH, NH-CO:C,H, CH.< + C,H,COOH = CH.¢ + H,O COOH benzoic acid COOH glycocoll hippuric acid which is a normal constituent of the urine of herbivorous animals, and which appears also in human urine after the administration of benzoic acid.? In the Lancet for December 16, 1905, and December 8, 1906, I described a new method for the synthesis of tyrosine from amino- malonic nitrile (that is from prussic acid) and oxy-benzaldehyde, the following being the reactions :— NH,-CH(CN), + 4H,0 = CH,(NH,)-COOH + CO, + 2NH, amino-malonic nitrile glycocoll CH,(NH,)COOH + C,H;,COOH = CH,(NH’CO-C,H;);COOH + H,O glycocoll benzoic acid hippuric acid OH’C,H,CHO + CH.(NH°CO-C,H;);COOH = p. oxy-benzaldehyde hippuric acid OH-C,H,-CH : C(NH-CO-C,H,)‘COOH + H,O oxy-benzoyl amino-cinnamic acid This heated in a sealed tube with 2KCN to 175°-182° is converted into p. OH-C,H,-CH,-CH(NH,)‘COOH, C,H,COO-NH,, and (COO),K, tyrosine ammonium benzoate _ potassium oxalate the 2KCN acting as the reducing agent ; when heated with 4H,O being converted into H, + 2NH, + (COO),K, 1. Curtius, Berichte, Bd. XVI, s. 757, and Bd. XVII, s. 1663. 2. Dessaignes, Fabresbericht der Chemie, s. 367, 1857. 3. Botis, Ure: Berz. Fabresbericht, Bd. XXII, s. 567: 200 BIO-CHEMICAL JOURNAL If, instead of using hippuric acid in the above synthesis, acetyl glycocoll, CH,(NH‘COCH,)COOH, is used, the same series of re- actions will ensue, ammonium acetate instead of the benzoate being among the final products. This experiment suggests that HCN ; CH(NHg)*(CN); (CH3"CO),0; together with OH*C;H,*CHO (or very probably NH,*CsH,*CHO) are among the earliest products formed in the synthesis of proteid. We have now to consider in what way we can synthesise methyl tartronic nitrile and its amino compound, namely, iso-aspartic or amino-iso-succinic acid from these products. By mixing together 16 c.c. of acetic anhydride and 74 c.c. of dry ether, placing the solution in an ice bath and after slowly adding 1o grammes of KCN, passing through the mixture a stream of HCl gas and leaving it at rest in a bath at o° for twenty-four hours, we obtain diacetyl-dicyanide (CH;*CO’CN),,' the polymeride of pyruvic nitrile CH;*CO°CN, or acetyl cyanide (CH,-CO),0 + 2KCN + 2HCl = (CH,:CO),O + 2HCN + 2KCl acetic anhydride = (CH:CO:CN), + H,O + 2KCl diacetyl-dicyanide © Diacetyl-dicyanide is readily soluble in alcohol, ether and benzol. If left at rest with fuming HCl for twenty-four hours and then boiled with dilute HCl it is transformed into acetic acid and iso-malic or methyl-tartronic acid. According to L. Bouveault? the following are the reactions : CH, | (CH,’CO-CN), = CH,;CO-C——C:CN diacetyl-dicyanide || | N——O CH, which with HO = CH,;- CO‘;C——C:CN | | N-OH OH 1. Brunner, Monatsheft. f. Chemie, Bd. XIII, s. 835. 2. Bulletin de la Soc. chimique [3] t. 1X, p. 557- . FORMATION OF LACTIC ACID AND CARBONIC ACID 201 CH, CH,;-;COOH + C ——C:CN ens acetic acid Il ————— N OH methyl-tartronic nitrile the latter being immediately saponified into methyl-tartronic acid and 2NH,. Judging from analogy, however, methyl-tartronic nitrile should also result from the combination of diacetyl-dicyanide with 2HCN ; (CH,'CO’CN), + 2HCN = 2CH,°C(OH):CN), diacetyl-dicyanide methyl-tartronic nitrile which by hydration is resolved into lactic acid, CO, and 2NH; CH,:C(OH)-(CN), + 4H,0 = CH,-C(OH)-(COOH), + 2NH, methyl-tartronic nitrile methyl-tartronic or iso-malic acid = CH,CH(OH)COOH + CO + 2NH a lactic acid or, the CO, and NH, in the latter case being liberated in the same proportions that result from the decomposition of urea—we may have CH,'C(OH):(CN), + 3H,O = CH,.CH(OH):COOH + co¢N a lactic acid NH, urea If, when the methyl-tartronic nitrile is decomposed, the intermediate products, methyl-tartronic acid and ammonia being formed, other substances are present such as the ketonic acids or the cyan-alcohols, R*CH(OH):CN, the liberated NH, will combine with them and on the decomposition of the methyl-tartronic acid, a lactic acid, and CO, will be alone set free. This I suggest is the action which takes place during muscular contraction, an explosive outburst of the two products being the result: the NH; combining with other con- stituents of the muscular tissue. The same products may be formed slowly when the muscle is at rest ; or if, when at rest, NH; is set free from some other constituent of the muscular tissue the NH, will combine with the methyl-tartronic 202 BIO-CHEMICAL JOURNAL nitrile forming a compound homologous with amino-malonic nitrile, namely, amino-iso-succinic nitrile, which by hydration can be con- verted into a alanine or amino-propionic acid and urea— CH,-C(OH):(CN), + NH, = CH,C(NH,)(CN), + H,O methyl-tartronic nitrile NH. and CH,'C(NH;):(CN), + 3H,O = CH,°CH,;CH(NH,)COOH + Cog ia amino-iso-succinic nitrile a alanine NH, urea or the intermediate products amino-iso-succinic acid and NH, may be formed. Before proceeding to prove that methyl-tartronic nitrile (or its components) are constituents of fibrin there are some extremely interesting points regarding amino-malonic nitrile, and its derivative tartronic nitrile, which are worthy of reference and consideration. By des-aminating amino-malonic nitrile in the usual way, that is by means of HNO,, it will be converted into tartronic nitrile CH - H,O CH a NH + H,O = ~ PEL eae oo” SO : CN CN CH CN with which the nitriles of malic acid | SoH and of oxyglutaric CH,-CN CH fh acid ‘oH are homologous. CH,°CH,°CN In a later communication I shall endeavour to prove that these three nitriles are constituents of living proteid, being the precursors of amino-malonic, aspartic and glutamic acids, and that in dead proteid they are transformed into the anhydrides NH NH NH CN'CH€ |, CN-CHyCH€ | and CN(CH,,CH< | co co co FORMATION OF LACTIC ACID AND CARBONIC ACID 203 ; a Agen Cw... I further suggest that amino-malonic nitrile CNCHQ is 2 constituent of living proteid, which in dead proteid is transformed a as follows— NC NH, N=C(NH,) N=C(NH,) | N\ dt ae \CH +> C | e°4 |) NH CN CN Cc . amino-malonic nitrile and that tartronic nitrile may be similarly transformed NC OH NH—CO NH—CO | anes N\da on: Nida a Ne Cc . NH CN CN \C tartronic nitrile and that if each of these is combined with 2HCN it is converted respectively into adenine and hypoxanthine N=C(NH,) N=C(NH,) rai | CG + 2HCN = CH bag te || DNH Sage | CH N—C—N adenine NH—CO NH—CO \ | | ; ae: + 2HCN = CH C—NH \ i NH lI HI en Cc N——C—N hypoxanthine the latter when oxidised in the tissues being converted into uric acid! NH—CO | | = CO C—NH bei > NH——C—_N 1. Burian, R., Zeitsch. f. physiol. Chemie, Bd. XLI. Pet 204 BIO-CHEMICAL JOURNAL ‘ NH . Another point of interest with regard to CN-CH€ | __ is-that CO by reduction and hydration it can be converted into diamino propionic acid H NH CN‘CHK | + H, = CH,(NH,)-CH< | Co CO» NH and CH,(NH,)CH< | + H,O = CH,:NH,°CH(NH,)COOH co diamino propionic acid a body ‘ related chemically to a whole series of physiologically very important substances.’ In the animal system this is transformed into glyceric acid? OH CHy (OH) CHC oo | and this by reduction is converted into glyceric aldehyde CH,:(OH):CH(OH)-CHO . I have thus endeavoured to shew how, in the earlier stages of the genesis of organic matter the formation or synthesis of amino- malonic nitrile, and methyl-tartronic nitrile, may take place. What proof have I that these compounds are constituents of the proteid molecule ? The proof rests upon the evidence furnished by the hydrolysis of albumin. / In 1875, P. Schutzenberger? communicated to the Academy of Sciences of Paris his first paper on the products obtained when albumin and its congeners are decomposed by simple hydration. This paper is a remarkable one, for had Schutzenberger been able rightly to ifiterpret the results of his experiments the synthesis of albumin would, I believe, have been numbered among his achievements. 1. P. Mayer, Zeitsch. 7. physiol. Chem., Bd. XLII, ’s. 59. 2. Comptes Rendus, t. LXXX, Pp. 232. FORMATION OF LACTIC ACID AND CARBONIC ACID 205 Unfortunately he adopted an erroneous molecular formula for albumin (Lieberkiihns), an error which he corrected a few years later. He also committed himself to the statement ‘that the albuminous molecule contains an urea group, and represents a complex ureide,’ which is not entirely borne out by the facts and has probably dis- couraged many from carefully considering the results which he obtained. ‘These experiments, as recorded in his first paper, consisted in heating 100 grammes of coagulated albumin with 200 grammes of crystallised hydrate of baryta and one litre of water (i) to a temperature of 100°C. under ordinary atmospheric pressure for 120 hours, (ii) digesting them in an autoclave at a temperature of 140-150°C. He determined in each experiment the amount of NH; liberated, and, from the amount of BaCO, formed, calculated the quantity of CO,. | In both cases the amounts of CO, and NH; liberated were in the same proportion as those obtained from the decomposition of urea. If Schutzenberger had been dealing only with such substances as amino-malonic nitrile, tartronic nitrile, methylamino, and methyl- ' tartronic nitriles his inferences would have been correct, but in his later experiments, in which the decomposition of albumin was more profound, this relationship between CO, and 2NH,; entirely disappears. On evaporating the solution of albumin, freed from the baryta, a series of crystalline compounds were obtained, to two of which I will now call attention. He says, ‘I have found at least two acids’ [in the solution of hydrolysed albumin] ‘ which by their presence prevent the complete precipitation of the baryta by means of carbonic acid, from the solution. They are either crystallisable with difficulty or not at all, are deliquescent and cannot be precipitated by nitrate of mercury. These characters make their examination and their separation extremely difficult. One of them has the composition of an isomeride of aspartic acid (C\H,;NO,), already found by Kessler in, the products of the decomposition of albumin by sulphuric acid, but differs from aspartic acid by being highly soluble in water. 206 BIO-CHEMICAL JOURNAL Another found in small quantities, gives numbers corresponding to the formula of a di-amido-citric acid.’ Unfortunately, in neither of these cases does Schutzenberger give his analyses, so that with regard to the latter substance we are left in doubt. ‘The first substance, the isomeride of aspartic acid, is, I suggest, iso-aspartic acid or amino-iso-succinic acid CH,-C(NH,):(COOH), = C,H,NO, derived from the proteid constituent diacetyl-dicyanide and NH, or from the combination of methyl-tartronic nitrile with NH, and. its subsequent saponification. The other compound ‘ corresponding to the formula of a di- amido citric acid’ is, I suggest, CsHigNoO,, differing from diamido- citric acid C,H,,N,O,; by one atom of hydrogen, and if so, it is a com- — pound of glycocoll with the above-mentioned iso-aspartic acid C,H,.N,O, = CH,(NH,)-COOH + CH,-C(NH,)-(COOH), glycocoll iso-aspartic acid resulting from the saponification of the proteid constituents amino- malonic nitrile and methyl-tartronic nitrile CH(NH,)(CN), + 3H,O + CH,-C(OH)-(CN), + 2H,0 amino-malonic methyl-tartronic nitrile nitrile which incompletely hydrolysed with baryta in a sealed tube, give CH,(NH,)‘COOH + CO, + NH, + CH,-C(NH,)-(CN), + 2H,0) = CH,(NH;)COOH + CH;'C(NH,)(COOH), + CO; + 3NH, eee glycocoll iso-aspartic acid - the CO, combining with the baryta and the NH, with other con- stituents of the proteid. 207 ON THE COMPLETE HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN AT 180°C. By P. W. LATHAM, M.D., Downing Professor of Medicine in the University of Cambridge (1874-1894). (Received April 26th, 1908) . The fact that a lactic acid and CO, should originate in the manner I have described in the previous paper? is highly suggestive. If these two substances are evolved from muscle during its contraction and have for their antecedent a compound of pyruvic acid or its nitrile with prussic acid, the question at once. presents itsel{—Can we, by combining pyruvic acid and its homologues, or their nitriles, with other substances produce a series of compounds of the same nature and constitution as are obtained by various processes from protein matter ; can we, in fact, in this way synthesise a number of, or even all, the protein derivatives ? The task I have set before me is to prove that an affirmative answer must be given to the question; and I propose to doso byshewing that the synthesis of a very large number of compounds which are obtained from protein, or albuminous bodies, including among them Schutzenberger’s leucéins (which I shall endeavour to prove are the imino-ketonic acids) and tyroleucine, as well as various pyridine and chinoline derivatives, can be effected by combining pyruvic acid and its homologues with prussic acid, with ammonia or with other substances, such as the fatty aldehydes, and then acting on these combinations in various ways. I shall then be able to determine the composition of certain compounds or combinations of the above mentioned bodies, which, 1. See pp. 194 and 2or. 208 BIO-CHEMICAL JOURNAL according to Schutzenberger!, result from the hydrolysis of albumin, viz. :— Cy,HygNgOQi, Or 3 CyHygN.O, acides amides fort Cy,HepNeOx2 oF 3 CoH, N20, leucines CyHeeN,O, or 2C,H,N,O, gluco-proteins and CrasHeogsNs20,, or 16 C,H,,N,O, gluco-proteins. The synthesis of tyrosine from oxy- or amido-benzaldehyde will then be discussed; and subsequently the synthesis of Schutzen- berger’s leucines, that is, of amino-caproic, -valeric, -butyric, -propionic and amino-acetic acids from the various aldehydes; and lastly the synthesis of the aspartic acid series from a similar source. With the knowledge of the composition of these bodies we shall be able to determine precisely and completely what substances are furnished by the hydrolysis of albumin, as well as their respective amounts. Moreover, knowing the constitution and synthesis of each product we shall then, as I shall endeavour to show, be within measurable distance of determining the constitution and arriving at the synthesis of albumin itself. On THE SynTHESIS oF Pyruvic AcID, AND ON SOME PROTEIN DERIVATIVES WHICH CAN BE SYNTHESISED FROM IT Synthesis of Pyruvic Acid (CH, *CO*COOH).—This acid may be obtained by carefully acting upon acetyl-cyanide with HCI and heating. CH, *CO-CN + HCl + 2H,O = CH,:CO-COOH + NH,Cl acetyl-cyanide _ pyruvic acid This method is applicable generally for the formation of the a ketonic acids from their nitriles. In the cold the amide is first formed. The acid may also be regarded as a condensation product of | 1. Annales de Chemie et de Physique, sme sér., t. XVI, p. 398. 2. Claisen, Shadwell, Ber. XI, 620 u. 1563. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 209 oxalic acid and acetic acid, and may be prepared in the following manner :— To one molecule of di-ethyl-oxalate dissolved in four parts of absolute ether add one molecule of sodium, and then in small quan- tities at a time a little more than one molecule of acetic ether, taking care that the temperature of the mixture does not rise above the boiling point of the ether. The sodium is gradually dissolved, hydrogen being evolved, and the greater part of the somewhat dark coloured fluid solidifies, after a few hours, into a yellowish crystalline semi-solid mass, which, when drained from the liquid portion, is washed with ether. In this way the almost pure sodium compound of oxalacetic ether is obtained, as a thick white mass.} C,0,(C,H;). + Na + CH, - CO, + C,H; diethyl oxalate acetic ether = CO, -C,H,CO- CHNa: CO,: C,H, + C,H;*OH + H sodium oxalacetic ester On adding dilute H,SO, (10 per cent.) to the ester mixed with H,O and ether, the sodium salt is resolved into CO, : C,H, * CO: CH, : CO, - C,H, oxalacetic ester which warmed with dilute H,SO, is converted into CH,:CO-COOH, CO, and 2C,H,: OH. pyruvic acid alcohol Some Protein DERIVATIVES THE SYNTHESIS OF WHICH CAN BE EFFECTED FROM Pyruvic -AcIpD The following substances have been obtained from protein :— Leucéine ea tee ye. Zi C,;H;NO, Gluco-protein ie x C,H,.N,0, Alanine or a amino-propionic ‘acid ees ... CH, + CH(NH,) : COOH Picoline or si as oa mT ... CH,;*C,H,N Pyridine... rap a iy? C;H,N Indol ore cee m dp et C,H,N C - COOH—CH Chinoline carboxylates “5 Ee ioe C,H, CH—CH and a R. Chinolines st es A one Ni==C:|R 1. Wislicenus, Annal. d. Chemie, Bd. 246, s. 315-327. 210 BIO-CHEMICAL JOURNAL I will now describe the methods by which, starting with pyruvic acid, these substances can be synthesised. On the Synthesis of Schutzenberger’s Leuceine (CsH;NO,).—This, I suggest, is imino-pyruvic acid, CH;* C(NH)* COOH; the ammonium salt of which is precipitated when to a solution of pyruvic acid in ether or alcohol, alcoholic ammonia is added to exact neutralisation.+ CH, : CO: COOH, + 2 NH, = CH, C(NH) - COONH, pyruvic acid ammonium imino-pyruvate The ammonium salt is very soluble in water, and reduces Fehling’s solution. On boiling it with water it is rapidly decomposed into CO,, NH, uvitaminic acid (CjH,,;NO,), and uvitoninic acid (C,H,NO,) or picoline dicarboxylic acid, (CH, * C;H,N(COOH),)? 6 CH, * C(NH) - COONH, + H,O ammonium imino-pyruvate C-COOH He On = CO, + 10 NH, + C,H,,NO, + isits. uvitaminic acid OS Oy C\A4e * COOH N o-picoline . op-dicarboxylic acid. Synthesis of a Alanine (CH,*CH(NH,) -COOH).—I have already described the method by which this substance can be synthe- sised from pyruvic nitrile (see page 203). - The synthesis can also be effected directly from pyruvic acid by the following process :— Take equal molecules of pyruvic acid and concentrated prussic acid and heat them in a sealed tube to 30°-40° C., then add two molecules of alcoholic ammonia and heat the whole to 70°C. Wash the resulting crystals of amino-iso-succinamide with alcohol and then dissolve them in water and recrystallise.? The following are the reactions :— CH,:CO-COOH + HCN -> CH,:C(OH-CN)COOH pyruvic acid CH, *C(OH -CN)COOH + 2NH, —> CH,* C(NH,) (CO NH,), + H,O- amino-iso-succinamide 1. Beilstein’s Handbuch d. org. Chemie, 3te Auf., Bd. I, 8. 587. 2. Bottinger, Liebig’s Annal., Bd. 188, s. 330; Bd. 208, s. 138. 3- Kérner, Menozzi, Gazetta chimica Italiana, Vol. XVIL,¥p. 426. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 211 The crystals appear as large rhombic tablets which are soluble in twenty parts of cold, or three parts of boiling water, but are only slightly soluble in-alcohol. On boiling the amide with hydrochloric acid it is decomposed into CO,, NH, and alanine. CH, ‘ C(NH,) - (CONH,), + 2H,O -—> CO, + 2 NH, + CH,~-CH(NH,) - COOH amino-iso-succinamide a alanine On the other hand by: boiling the amide with one molecule of caustic baryta or other alkali until ammonia is no longer evolved we obtain barium amino-iso-succinate or barium iso-aspartate.! CH, : C(NH,) : (CONH,), + BaH,O, —> 2NH, + CH,-C(NH,): (COO),Ba barium iso-aspartate On treating the barium salt with an exact equivalent of H,SO, to precipitate the baryta, and then evaporating the filtered solution im vacuo over H,SO, we obtain prismatic crystals of iso-aspartic or amino-iso-succinic acid, CH, * C(NH,) - (COORH),. : On boiling this acid in water it is resolved into CO, and a alanine CH, : C(NH,) (COOH), = CO, + CH, - CH(NH,)COOH iso-aspartic acid a alanine On the Synthesis of Schutzenberger’s Gluco-protein (CgH,,N,O,) :— Dissolve one molecular equivalent of a alanine in a small quantity of water, then add to it one molecular equivalent of alcoholic ammonia. To one molecular equivalent of pyruvic acid dissolved in alcohol add, very slowly, two molecular equivalents of alcoholic ammonia avoiding a rise of temperature, when the ammonium imino-pyruvate will be formed. Pour into this the solution of alanine, adding, if necessary, a little water to form a clear solution. Evaporate im vacuo, when crystalline nodules will be formed consisting of small prisms radiating from a centre the composition of which after crystallisation is a com- pound of equal molecules of CH,*CH(NH,) ‘COOH (alanine) and CH, * C(NH) - COOH (imino-pyruvic acid), or C,H,,N,O,, the basic NH, having been dissipated. 1. Ké6rner, Menozzi, loc. cit., p. 429. 212 BIO-CHEMICAL JOURNAL The analysis of the crystals by Mr. M. M. Pattison Muir, who kindly examined them for me, gave the following results :— Experiment Calculated for CoH,.N,0, C = 40-46 vee ae 1 gO-Ot H = 7-26 és C,H, ‘COOH + NH, > Cc; eis 6 CH + CO, + NH, NH NH ‘ indol carboxylic acid indol Synthesis of the Chinoline Carboxylates and the a R. Chinolines from Pyruvic Acid.—The chinoline carboxylates C,H», _is3NO,, con-. taining an alcohol radicle in the Py 2 . position, are formed by heating pyruvic acid with the aldehydes and aniline in alcohol for four to five hours in a water bath ;? the aniline being very slowly added during the process. 1. E. Fischer, Annalen d. Chem., Bd. 236, 8. 142. 2. Débner, Annalen d. Chemie, Bd. 242, s. 270. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 213 CH, - CO: COOH + R: CHO + C,H, : NH, pyruvic acid aldehyde aniline C- COOH=CH << = cH | + 2 H,O + H, N———_-C-_R a R. chinoline carboxylic acid If the experiment is made at the ordinary temperature neutral bodies are formed H:N-°C,H R-CHO + CH,-CO- COOH + 2C,H;-NH, = R:‘CH:C ay 3 2U,t15 2 CS nn ee By heating the carboxylates, either by themselves or with soda- lime, they are resolved into a R. chinolines and CO, C - COOH= CH CH=CH N=————-C:R ~ SX + CHK _b *R Synthesis of Picoline (CH, * C;H,N).—On heating picoline dicar- boxylic acid (obtained as above) to 274° C. it is resolved into CO, and mono-carboxylic acid? CoH, C - COOH CH, * C;H,N(COOH), = CO, + CH,*C;H,N - COOH or HC/ \cH picoline dicarboxylic picoline carboxylic | acid acid CH; ° 1 si 2-methyl pyridine -4-carboxylic acid On fusing the calcium mono-carboxylate with soda-lime, it is converted into CO, and picoline? CH, - C,H,N - CO,H + CaH,0, = CH, C,H,N + CaCO, + H,O picoline carboxylic 2-picoline or acid methyl pyridine Synthesis of Pyridine (C,H;N).—By oxidising 2-picoline with KMn0(, it is converted into pyridine carboxylic acid* :— CH,:C;H,N +O, —> C;H,N~COOH + H,O picoline pyridine carboxylic acid 1. Bottinger, Berichte, Bd. 14, s. 67; Bd. 17, s. 92. 2. Beilstein’s Handb. d. org. Chemie, Bd. 4, s. 166. 3. Weidsl, Berichte, Bd. 12, 8. 1992. 214 BIO-CHEMICAL JOURNAL On heating this with alcoholic potash to 240° C. it is completely transformed into pyridine? C,H,N- COOH + KHO -> C,H,N + KHCO, pyridine carboxylate pyridine Synthesis of Pyrrol (CsH,(NH)).—On combining uvitaminic acid C,H,,NO,, obtained as above from ammonium imino-pyruvate, with baryta and subjecting the dry salt to the action of heat, a large amount of pyrrol distils over, together with NH, and other bases.” On THE SynTuHEsIS oF AceTyt-Acetic Acip AND OF CERTAIN PROTEIN DERIVATIVES WHICH CAN BE FORMED FROM IT The next higher homologues in the pyruvic acid series are propionyl-formic acid, CH, *CH,*CO* COOH, and acetyl-acetic acid, CH, CO*CH,* COOH. It is to the products derived from the latter that I will, at present, direct attention. This acid exists in the animal economy, being found in the urine of individuals suffering from diabetes, and is then generally accompanied by 8 oxybutyric acid, CH,*CH(OH)+CH,*:COOH, and acetone CO: (CH,),. I suggest that these substances are derivatives of the albuminous tissues. Synthesis of Acetyl-Acetic Acid (CH, *CO+*CH,* COOH).—By acting upon acetic ether CH, * CO, * C,H, with sodium alcoholate, sodium acetyl-acetic ether is Prodaeeal 2 CH,: CO,°C,H, + NaO-C,H, -> CH,+CO+CH(Na)CO,C,H, + 2 C,H, (HO): acetic ether sodium acetyl-acetic ether On adding acetic acid to this and distilling at 130° C. acetyl- acetic acid CH, * CO CH, * COOH ° passes over. The free acid may also be obtained by mixing together 4-5 parts of the ethylic ester with 2-5 parts of KOH and 80 parts of H,O letting the mixture stand for twenty-four hours, then acidulating ~ with H,SQ,, and extracting the acid with ether.4 1. Beilstein’s Handb. d. Chemie, 3te Aufl., Bd. IV, s. 141. 2. Beilstein, loc. cit., Bd. I, s. 587. 3- Geuther, Zeit. 7. Chemie, 1868, s. 652. 4- Ceresole, Ber. XV, p. 139, 1872. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 215 Practically then, we may regard acetyl-acetic acid as the con- densed product of two molecules of acetic acid. 2 CH,‘ COOH = CH, CO- CH, - COOH + H,0. On the Synthesis of Schutzenberger’s Leucéine (C,H,NO,).—This compound is, I suggest, imino-acetyl-acetic acid CH, *C(NH) - CH, * COOH known also under the name of amino-crotonic acid CH, * C(NH,) : CH - COOH,! according to the view which is taken of the constitution of the compound. The ester of this acid may be prepared by mixing together, at the ordinary temperature, acetyl-acetic ether with strong aqueous | ammonia, and shaking the mixture, from time to time, for several — days.? CH, - CO: CH,: COOC,H, + NH, —> CH,:C(NH)-CH,-COOC,H, + H,O acetyl-acetic ester imino-acetyl-acetic ester or —> CH,- C(NH,) : CH - COOC,H, + H,O amino-crotonic ester It may also be prepared by passing dry gaseous ammonia rapidly into pure acetyl-acetic ester ;? mixed with double its volume of ether. The yield being larger if ammonium nitrate is first mixed with the dry ester. _ This imino- or amino-body crystallises in colourless thick mono- clinic prisms which are not very soluble in water but readily soluble in ether, benzol and chloroform. Its melting point is 34° C. (Collie), 37° (Conrad). It is resolved into its constituents by dilute HCl, and by NaHO. The composition of this body C,H,NO, merely differs from that of amino-butyric acid CH, ‘CH, * CH(NH,) ‘COOH or C,H,NO,, by two atoms of hydrogen. Synthesis of B Oxy-Butyric Acid (8 CH, * CHOH - CH, * COOH). If water is added to an alcoholic solution: of imino-acetyl-acetic ester until it is slightly opaque and the mixture then placed in an ice bath 1. Conrad, Epstein, Berichte, B. 20, s. 3056. 2. Duisberg, Liebig’s Ann., Bd. 213, 8. 166. 3. J. Norman Collie, Liebig’s Ann., Bd. 226, s. 294-301 ; and Conrad, Epstein, Berichte, Bd. 20, s. 3054. 216 BIO-CHEMICAL JOURNAL and treated for three days with sodium amalgam we obtain sodium 8 oxybutyrate’ CH, ‘ C(NH) * CH, ‘ COOC,H, + 2 Na + 3H,O imino-acetyl-acetic ester —> CH,*CH(OH) :CH,COONa + NH; + NaHO + C,H; ~* HO sodium oxybutyrate the solution of which, if exactly neutralised with HCl yields 8 oxybutyric acid, It may also be prepared from ethylic aldehyde by converting this by means of dilute HCl into aldol, and then oxidising the latter . with oxide of silver.” 2CH,:CHO -—> CH,:CH(OH)< CH,: CHO ethylic aldehyde aldol and CH,:CH(OH):CH,:CHO + 0 —> CH,-CH(OH): CH,COOH aldol B oxybutyric acid The Synthesis of Acetone (CO: (CH,),).—By heating imido-acetyl- acetic ester with dilute NaHO it is resolved into NH3, CO,, alcohol and acetone. CH, ‘ C(NH) - CH, * COOC,H, + 2 NaHO = NH, + Na,CO, + C,H, - HO + CO(CH,), imido-acetyl-acetic ester alcohol acetone Acetone may also be obtained from aceto-acetic acid by heating it to a temperature below 100° C., when violent action takes place and it is resolved into CO, and acetone.® CH, *CO*CH,*COOH -—> CO, + CO(CH,), aceto-acetic acid j acetone Synthesis of Collidine from Acetyl-Acetic Ester.—This is effected in the following stages :— (i) Synthesis of Hydrocollidine Dicarboxyllic Ester (CSH,N(CHs)s . (COOC,H;),.—This compound may be obtained by warming 52 parts of acetyl-acetic ester with 13-5 parts of aldehyde ammonia ;4 or by 1. Wislicenus, Liebig’s Ann., Bd. 149, s. 205. 2. Beilstein’s Handbuch d. Chemie, 3te Aufl., Bd. I, s. 1206. 3- Ibid., s. 591. 4. Hantsch, Liebig's Ann., Bd. 215, s. 8. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 217 combining 8 amino-crotonic ester, CH, *C(NH,) : CH - CO,C,H,, with paraldehyde and a little H,SO,.1__ In the latter case (CH, * CHO), we have _ — CH, CH, | CHO € CO, ‘C,H;—CH CH—CO, - C,H, CO,-C,H,—CH C—CO,-C,H,-+ I | > | H,O + NH, CH,—C-NH, C(NH,)- CH, CH,CH!: -Cate, —> C,H,N(CH;), (CO, - C,H;), + H,O + NH, hydro-collidine-dicarboxyllic ester (Gi) On treating this ester with nitrous acid H~ NO,, it loses H, and is converted into CH, Aw C,N(CH,), (CO,C,H,), or C,H,-CO,-C C—CO,C,H, I cd ee YY collidine dicarboxyllic-diethyl ester (ui) From this the mono-ethyl ester can be obtained by heating it with a molecular equivalent of alcoholic potash for some time in a flask, to which a reversible condenser is attached. ‘The solution is then freed from alcohol by distillation and the residue dissolved in water, any unchanged diethyl ester is then extracted with ether or benzol, and the solution neutralised by adding a molecular equivalent of HCl. The solution is then evaporated to dryness in a water bath and the ester recrystallised from absolute alcohol.? The result is -CO,H - C;N(CH,),CO, * C,H, collidine dicarboxyllic mono-ethyl ester. 1. Collie, Liebig’s Ann., Bd. 226, s. 314. 2. R. Michael, Lieb. Ann., Bd. 225, s. 124. 218 BIO-CHEMICAL JOURNAL If two molecules of alcoholic potash are employed CO, is disengaged and y collidine or trimethyl-pyridine is left behind. © 2.4.6. trimethyl pyridine or ¥ collidine. If the mono-ethyl ester prepared as above is heated to 255° C. it is resolved into CO, and C,NH(CH;), * CO, * C,H, collidine carboxylic ester which distils over. On saponifying this with alcoholic potash and then adding sufficient HCl to convert the whole of the potash into KCl, evaporating the solution to dryness and treating the residue with absolute alcohol, on evaporation we obtain short needles of C,NH(CH,), ‘CO,H + 2 H,O collidine carboxylic acid® Synthesis of Lutidine (C,H,N).—By the oxidation of one molecule of potassium collidine carboxylate with 2, 4, and 6 molecules of KMn0O, we obtain respectively? the potassium salts of — C,NH(CH,),(COOH), lutidine dicarboxylic acid C,NH(CH,)(COOH), picoline tri-carboxylic acid and C,NH - (COOH), pyridine tetra-carboxylic acid 1. Ibid., s. 131. 2. Ibdid., s. 134. 3. Ibid., s. 136. bo \ he “So HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 219 By fusing potassium lutidine dicarboxylate with lime we obtain! CsH;N(CHs), : lutidine “7a or dimethyl pyridine 8 Lutidine can also be obtained by mixing together 130 grammes aceto-acetic ester, 61 grammes aldehyde ammonia and 50 grammes of aldehyde? at 130° C., to form lutidine carboxylic ester. CH, - CO - CH, - CO, C,H, + CH, - CHO - NH, + CH, - CHO = (CH;),*C;H,N -CO,C,H; + 3 H,O + H,. From this lutidine carboxylate can be obtained, which fused with lime is resolved into CO, and 2°4 dimethyl pyridine or lutidine. CH, CH, CH, fis 5 Bigs 33 oe: RO . C- COOC,H,; II + 3H,O + H, Cc ox duo bo - CH, be C-CH, bf | CHO XY NH, —> 1 || + H, + 2 H,O + CO, + C,H; -OH CH ¢- CH, » 4 2'4 dimethyl pyridine or lutidine. On THE Syntuesis oF Tyroztructne (C,H,,NO,) Perhaps the most important of Schutzenberger’s experiments as elucidating the constitution of the leucéins is the one by which he succeeded in isolating tyroleucine C,H,,NO, and caproic leucéine C,H,,NO,. 1. Engelmann, Lieb. Ann., Bd. 231, 8. 54. 2. Michael, Ber., Bd. 18, s. 2022. a ot 7 1 ae ~1) Ry eee ME he 220 BIO-CHEMICAL JOURNAL The following are the details of his experiment? :— ‘Ten kilogrammes of albumen were decomposed by baryta in a large autoclave, and the liquid having been freed successively from ammonia and from the excess of baryta by a current of carbonic acid and by sulphuric acid, it was then concentrated till it crystallised. I was able to isolate about 2 kilogrammes of crystalline deposit corresponding to the deposit (4). ‘From this considerable mass I was only able to procure—by repeated crystallisations carefully carried as far as possible—first, the substances mentioned already, tyrosine, leucine, butalanine, amido-butyric acid, crystalline compounds of the type C,,H,,,N,O, (m = 12 and 10), and secondly, two new definite and crystallisable products, of which one, to which I give the name Tyroleucine, corre- sponds to the formula C,H,,NO,, and the other belonging to the type C,,H,,,-;NO, ; its composition is represented by the formula C,H,, NO,; it is a leucéine (» = 6), caproic leucéine. 'Tyroleucine belongs to the series C,H,, ,NO,; it plays only a very secondary part in the com- position of albumen, and appears in small quantities only in the products of its decomposition. With 100 grammes of substance, I completely overlooked its presence. By experimenting with to kilo- grammes I was able to obtain from 60 to 70 grammes in a pure state. The existence of a definite and crystallised substance belonging to the type C,H,, ,NO, tends to prove that the compounds of the type C,,H,,,N,0,, which are so abundant, should be regarded as molecular combinations of leucines and of leucéines. ‘The following experiment, however, seems to shew that the compounds C,,H,,,N,O, (m=10 or 12) cannot-always be separated, by successive crystallisations into leucines C,H,,,,NO, and leucéines C,Hy,-4NO,. A crystalline deposit in nodules or spherical masses radiating from a centre, gave the following result. (2) Substance ... ie 0°3495 Carbonic acid Si 0*707 Water vs rss 0:286 1. Annales de Chimie et de Physique, sme série, t. XVI, PP- 345, 352. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 221 «These crystals, re-dissolved in water and purified by a fresh crystallisation, gave ___ {by Substance... Sn 0-3105 an Carbonic acid oe OBE Waters: ).. ry 0-251 The percentage being Calculated for a b C,,H,,N.O, Carbon 55:1 55-42 55°38 Hydrogen 9:09 9:01 9:2 The composition remained unaltered, and we must therefore admit the existence of a body corresponding to the formula C,,H,,N,O, (m =12). This result has been frequently verified, not only as to the compound C,,H,,N,O,, but also for the lower homologues C,,H,,N,O,, CypHopN,O0,, CyH,.N,O, In the experiment with to kilos of albumen, tyroleucine (C;H,,NO,) and caproic leucéine (C,H,,NO,) were extracted from the aqueous solution of the crystalline deposit (4) by first separating the greater part of the tyrosine and leucine, by means of fractional crystallisations. The mother-liquor, freed from traces of baryta by sulphuric acid, was decolourised by animal charcoal, and then again concentrated. A fairly copious deposit of spherical masses took place. From this I was able to obtain a considerable amount of two new bodies. This deposit was again treated with hot water, which dissolved it all except a residue of tyrosine; the dark liquid was: decolourised by means of a little sub-acetate of lead, and filtered from the brown flocculi; the excess of lead was precipitated by sulphuretted hydrogen and the liquid filtered and decolourised by animal charcoal. This was twice concentrated and two crystalline products (B) and (C) in spherical masses or in granules were obtained. ‘'The crystals (B) purified and recrystallised were composed of tyroleucine C,H,,NO,. ‘At a temperature of from 250° to 280° in an inert gaseous atmosphere tyroleucine is decomposed, furnishing a white sublimate, water, and carbonic acid, together with an oily volatile base having BIO-CHEMICAL JOURNAL 222 the odour of horse-radish, which remains in the retort as an amorphous, transparent mass having a resinous fracture. ‘An analysis of the chloroplatinate of the oily base shewed its composition to be CgH,,N which represents collidine or an isomeric substance. ‘The vitreous residue on analysis furnished numbers corresponding with the formula C,,H,,N,O,. It appears from these results that tyroleucine heated above 250° is in part dehydrated and polymerised, while another portion is decomposed into carbonic acid, collidine and butalanine in accordance with the following equation :— . 2 C,H,,NO, = CO, + C,H,,N + C,H,,NO, Dehydration takes place as in the following equation :— 2C,H,,NO, = 2 H,O + C,,H,.N,0, Tyroleucine, therefore, must be regarded as a compound of butaldtine (amino-valerianic acid) with a body whose composition is CyH,,NO,, differing from tyrosine by one atom of oxygen. ie ‘An analysis of the crystals (C), obtained by concentrating the mother-liquor from which the tyroleucine had been obtained, shewed that it consisted of C,H,,NO, or leucéine caproic.’ From the above extract it is evident that tyroleucine is composed of amino-valerianic acid CH, *(CH,),* CH(NH,):COOH and collidine carboxylate CyH,,NO,,‘or HC C+COOH CH, Cc d - CH, Y which is obtained as shown on page 218 by heating collidine dicar- boxylate, a compound which results from oxidation of hydrocollidine dicarboxylic ester, the latter being obtained by combining ati sis with the ester of 8 amino-crotonic acid.! CH, ‘CHO + 2 CH, : C(NH,) : CH: COOC,H, = C;H,N(CH,),(CO, - C,H;), + H,O + NH, 1. Collie, Liebig’s Ann., Bd. 226, s. 314. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 223 Hicuer Homotocuts or THE Pyruvic Acip Series The next higher homologues of the pyruvic series are—Methyl- aceto-acetic acid CH,- CO:CH(CH,)*COOH, and ethyl-aceto- acetic acid CH,-CO~- CH(C,H,): COOH and two others isomeric with them, namely, acetyl-propionic acid or laevulinic acid CH, °CO:CH,:CH,*COOH, and_ jy acetyl-butyric acid fee) CH, *.CH, - CH, - COOH. On the Synthesis of Methyl-Aceto-Acetic Acid and Ethyl-Aceto- _ Acetic Acid.—These two bodies are obtained by the action of the iodides of methyl and ethyl respectively on sodium acetyl-acetic ester. The reaction takes place most readily by dissolving the a theoretical amount of sodium in ten to twelve times the weight of absolute alcohol and, after the mixture has cooled, adding the aceto- acetic ester and immediately afterwards iodide of methyl or ethyl (as the case may be) until the reaction is neutral. Distil off the greater portion of the alcohol, and then add sufficient water to the residue to dissolve the whole of the sodium salt.? The following are the reactions :— CH, : CO: CH, - CO, - C,H,+ NaO : C,H,—>CH, - CO: CH(Na)CO, - C,H, + C,H,(OH) aceto-acetic ester sodium aceto-acetic ester CH, - CO - CH(Na)CO, - C,H; + CH - I —> CH, - CO - CH(CH,)CO, - C,H; + Nal sodium aceto-acetic ester methyl aceto-acetic ester and— CH, - CO - CH(Na)CO, - C,H; + C,H; - 1 —> CH, -CO-CH(C,H;)CO,*C,H; + Nal sodium aceto-acetic ester ethyl aceto-acetic ester These two esters if acted upon by 2KHO are resolved into K,CO, +. HO’C,H; and CH,;*CO*CH,*CH, (methyl-ethyl ketone) and CH,*CO*CH,* C,H; (methyl-propyl ketone) respec- tively. The two other homologues of pyruvic acid, isomeric respectively with methyl-acetyl-acetic acid and ethyl-acetyl-acetic acid, viz., : 1. Conrad, Limpach, Lieb. Ann., Bd. 192, s. 153. 224 BIO-CHEMICAL JOURNAL laevulinic acid or Bacetyl-propionic acid (CH, CO* CH,*CH,* COOH) and yacetyl-butyric acid (CH,;*CO* CH, *CH,*CH,* COOH), ~ may also be obtained from sodium diacetic ester. Synthesis of Imino-Laevulinic Acid.—By acting upon sodium- diacetic-ethyl ester with chlor-acetic ester, acetyl-succinic diethyl ester is produced.! . CH, ‘CO: CHNa : CO, : C,H, + CH,Cl - CO,C,H, sodium diacetic ester chloracetic ester > NaCl + CH, : CO - CH(CH, : CO, : C,H,) CO, : C,H, _> NaCl + C,H, — CO, : CH(CO - CH,)CH, - CO, : C,H, acetyl-succinic diethyl ester Rash? prepared this by taking 52 grammes of acetyl-acetic ester, 9:2 grammes Na, 150 grammes C,H, * OH, and 125 grammes chlor- acetic ester. On boiling the acetyl-succinic ester with twice its volume of dilute HCl, or with baryta water, it is resolved into CO,, C,H, OH and CH,*CO*CH,*CH,*COOH (@acetyl-propionic acid) or laevulinic acid, together with a little acetic and succinic acids. This combines with NH, to form CH, ~-C(NH) : CH, : CH, ° COOH (imino-laevulinic acid). Synthesis of Imino-Acetyl Butyric Acid (CH; ‘ CO * CH, * CH, * CH, * COOH).—By acting upon sodium diacetic ester with B iodo- propionic acid, acetyl-glutaric ester is produced.° CH, : CO: CHNa O0,C lH. + (Cit Co — >. CH;- GH, ca, Gr | + Nal CH, - CO - CH - CO,C,H, acetyl-glutaric ester. This boiled for eight to ten hours with one part of concentrated HCl and two parts of water is resolved into CO,, alcohol, and » y acetyl-butyric acid.4 = CO, + 2C,H,Cl + CH, * CO - (CH,), * COOH Conrad, Annalen, Bd. 188, s. 218. a pay oe. aS Annalen, B. 234, s. 36. Wislicenus, Limpach, An. d. Chemie, p. 192, 128. Wolff, An. d. Ch., p. 216, 129. webs HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 225 Treated with a strong solution of alcoholic potash it is resolved into acetic acid and glutaric acid. CH, - CH * COOK = CH,;~CO,K + 2C,H,;: HO+ | er CH, * COOK pot. acetate alcohol pot. glutarate Combined with NH; acetyl-butyric acid forms CH, - C(NH) * (CH,)3 ° COOH (imino-acetyl butyric acid). SyNTHEsIs oF SCHUTZENBERGER’s Leucfines C;H,NO, and C,H,,NO, Each of these bodies may be represented by two isomeric com- pounds. The leucéine C,H,NO, may be represented either by— CH, - C(NH,) : C(CH,) ‘ COOH . 8 amino-a methyl-crotonic acid or by CH, * C(NH) » CH, - CH, * COOH imino-laevulinic acid The leucéine C,H,,NO, being represented by— CH, - C(NH,) : C(C,H,) - COOH 8 amino-a ethyl-crotonic acid or by CH, ~C(NH)- CH, - CH, - CH, : COOH yy imino-acetyl-butyric acid Each of these four leucéines being, I suggest, derivatives of protein. The synthesis of the ester of the two amino-crotonic acids may be effected by passing NH, gas into an ethereal solution of methyl- crotonic and ethyl-crotonic ester respectively,! as in the formation of @ amino-crotonic ester from acetyl-acetic ester. Or, the two esters may be formed from 8 amino-crotonic ester by acting upon it first by sodium ethylate and treating the resulting compound with CH,I and C,H,I respectively. CH, - C(NH,) : CH - CO,C,H, + NaOC,H, > CH, * C(NH,) : CNa - CO,C,H,; + HO- C,H; and CH,*C(NH,) : CNa+ CO,C,H, + CH,I —> CH, * C(NH,) : C(CH,)CO,C,H, + Nal 8 amino-@ methyl-crotonic acid 1. Conrad, Epstein, Berichte, Bd. 20, s. 3055 u. 3057. 2. See page 215. 226 BIO-CHEMICAL JOURNAL Acting on the esters of these two amino-acids with dilute HCl they are converted into the corresponding crotonic esters and NH,Cl; and these acted upon by KHO are converted into the corresponding ketones.? I therefore venture to submit that these imino compounds of pyruvic acid and its homologues are the compounds which, in combination with the amino-fatty acids, Schutzenberger obtained by the hydrolytic decomposition of albumin—and to which he gave the name of leucéins, the combinations with the amino-fatty acids being named by him gluco-proteins. ; Having made this advance we are now in a position to discuss the results which he obtained in a remarkable series of researches extending over several years. ‘These results are published in Comptes Rendus, tome 80, p. 233; tome 81, p. 1108; tome 84, p. 124; tome IOI, p. 1267; tome 102, p. 1289; tome 106, p. 1407; and tome 112, p. 198. "The most important paper is contained in the Annales de Chimie et de Physique, 5me série, tome 16, p. 289, and gives the results of innumerable analyses made with extreme accuracy. From this paper I shall take the details which are necessary for my present purpose. In his researches Schutzenberger employed white of egg coagulated by heat and with a slight excess of acetic acid. This was well washed’ with water, alcohol and ether—and dried at 140° C. Fifty or one hundred grammes of this mixed with water were treated with two to six times its weight of crystalline barium hydrate in a closed iron vessel, and heated to a temperature ranging from 100° C. to 250° C., for periods varying from eight to one hundred and twenty hours | (Joc. cit., p. 303). After the vessel had completely cooled down it was opened ; generally there was entire absence of increased pressure or of unabsorbed gas. Sometimes if the temperature had been raised to near 200° and if a large proportion of baryta had been used, a certain 1. See page 223. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 227 amount of hydrogen uncontaminated by carbon escaped from the digester. This Schutzenberger attributed to some secondary action of the baryta on the iron vessel.} 7 The contents of the digester were now :—(i) Subjected to distilla- tion and the amount of NH, determined. (ii) The insoluble deposit was separated fromthe liquid portion and the amount”of barium carbonate and oxalate in the deposit was determined. (iii) The baryta contained in the filtered liquid was precipitated first as far as possible by passing a current of CO, through the solution kept at the boiling point for a considerable time,” then filtering off the precipitate and washing it : concentrating the filtrate and the washings and adding the exact quantity of H,SO, necessary to precipitate the baryta still held in solution. (iv) The acid filtrate from these precipitates was then distilled and the amount of acetic acid contained in the distillate determined. In addition to acetic acid the distillate contained traces of formic acid, and an essential volatile oil consisting of pyrrol, etc. (v) The liquid was finally evaporated to dryness in a water bath. This dried residue was called by Schutzenberger the ‘ résidu fixe,’ meaning thereby a mixture of substances which do not sublime nor volatilize at a temperature below 100° C, Having advanced so far, Schutzenberger’s next step was to endeavour from the analysis of albumin to construct such a molecular formula as might serve as a working hypothesis. Taking tyrosine as a basis, the percentage of which can be readily determined, as it crystallises readily and is only slightly soluble in cold water, he found as the result of numerous most carefully conducted experiments that the amount obtained from too grammes of albumin ranged between 2-5 and 3:5 grammes, the mean result being 3-4. Now it is improbable that tyrosine results from the combination of several molecules of albumin ; consequently, if we assume that one molecule 1. Annales de Chimie et Physique, 5me série, t. XVI, p. 296. Later on see page 230. I shall endeavour to shew that this hydrogen resulted from the action of the baryta on formic acid converting it into oxalic acid and hydrogen. 2. Loe. cit., p. 298. 228 BIO-CHEMICAL JOURNAL of tyrosine (= 181) is produced by the decomposition of one molecule of albumin, and that this contains 3-3 per cent. of tyrosine, we baile as the molecular weight of albumin—: 181 X 100 = 5484 3°3 3 On this ground alone Lieberkiihn’s formula C,,H,,.NjgQ9, of which the molecular weight is 1612, cannot be accepted as correct. Moreover, as Schutzenberger points out, the proportion of nitrogen (15:8 per cent. in Lieberkiihn’s formula) is too small; the actual percentage being 16-6. Schutvenbenger s analysis of. albumin? gives the following results :— Carbon sak 2: be §2°57 Hydrogen ... tea ae 7:16 Nitrogen ir es ate 16:6 Oxygen ap As ott 21°8 Sulphur ‘ot ins ai 1°38 99°93 This agrees closely with those of Brittner®? and Fleitman‘ Coe 549 53°8 oe gcre 73 Ne 20066 16-2 S 1-6 1-4 Based on this analysis he adopts as the formula for albumin, CospHsg7No50,,53 having a molecular weight of 5473, and the per- centage composition of which is :— Carbon SORE STO 52-62 Hydrogen ke es 7°07 Nitrogen ni ae 16°62 Oxygen’... ise ood 21-94 | Sulphur ... = se 1-75 T00°00 Loc. cit., p. 383. Loc. cit., p. 384. Beilstein, Handb. d. Chemie, 3te Aufl., Bd. IV, s. 1590. Watt's Dict., Vol. IV, p. 738. > Ww N = ° - . . HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN — 229 The formula Cys 9H3,,N,;O,;S3, differing from the above by ten atoms of hydrogen, and having a molecular weight of 5483, corre- sponds more closely with Schutzenberger’s analysis, and is the one I shall adopt. Its percentage composition is :— Carbon ... a a ees Hydrogen ile “ij 7°24 Nitrogen es aie 16-6 Oxygen ... oe 48 21-88 Sulphur ... =e ar 1°75 100-00 and I will endeavour by its aid to interpret the analytical results which Schutzenberger obtained. Acting upon 100 grammes of albumin, in the manner previously described (see page 226), at a temperature of 180° C. with four or five parts of baryta he obtained Nitrogen (in the form of tiie -, ay 4°03 Barium oxalate... ke ome 17-6 Barium carbonate ... RY ots > II-O Acetic acid th ey sia a 4°6—49 which, for a molecular weight of 5483, correspond very closely to 16 molecules of Ammonia 4 e Oxalic acid 3 Z Carbonic acid and 4 a Acetic acid The ‘ résidu fixe’ obtained under the same conditions after separating the baryta with CO, and then with H,SO, and evaporating to dryness weighed 99-6 grammes, and on analysis yielded the following percentages very approximately? 48:63 797 12°58 30°82 OZTO Hou wd 100°00 1. Loc. cit., p. 385. 230 BIO-CHEMICAL JOURNAL giving for the ‘ résidu fixe’ the formula C,5;H43;N4gO49;, the calculated percentage of which is— 48-95 8-03 12-0! 31-01 O2ZzO ie || 100:00 Schutzenberger, moreover, determined that the decomposition of albumin by baryta under the conditions specified was one of hydrolysis, and that during the process one molecule of albumin at 180° C. combined with 60 molecules of water. With the results just given, the following equation represents the decomposition :— CopoHso7HesNz5S3 + 60 HZO = CooyHy3sNagOj05 albumin résidu fixe + (16 NH; + 4(COOH), + 3 CO, + 4 CH,‘ COOH + S,) + 5H, Schutzenberger’s polynomial the intermediate stage being :— CooHygsNyoO10s + 16 NH; + (COOH), + 6H (COOH) + 3 CO, + 4 CH, - CO,H + 2SH, + S* We have now to deal exclusively with the ‘ résidu fixe ’ C,5, Hag; NagO,o5- After trying by various methods to separate the constituents of this compound, Schutzenberger arrived at the conclusion that the only feasible plan was by fractional crystallisation from various neutral solvents such as water, alcohol, ether, etc. ‘The different crystalline products can be distinguished, to a certain extent, by their form and appearance, especially when examined under the microscope. But, as we are well aware, the bodies of which the “ résidu ” is com- posed belong to small groups, each members of a homologous series, and frequently there is very little difference in the characters of the neighbouring terms of any single group. Moreover, two terms of the same group, or of two neighbouring groups, have a general tendency to crystallise together in their molecular proportions. * 6H: COOH + 2SH, + S being under the action of the BaH,O, converted into 3 (COOH), + S$; + 5H, formic acid oxalic acid _ HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 231 Intermediate products, therefore, are frequently obtained, adding considerably to the difficulty of arriving at a proper estimate.”? The conclusions at which Schutzenberger arrived were based upon the results of upwards of five hundred analyses of the various products which he obtained from the ‘ résidu fixe.’ In order to separate these products he found that in the first instance the best plan was to pass through the primary solution freed from acetic acid and filtered from the barium carbonate and oxalate a stream of car- bonic acid for a considerable time, in order to precipitate the excess of baryta, and after filtering from the BaCO, to concentrate the solution, without precipitating the baryta which it still contains, with sulphuric acid. After a certain degree of concentration a crystalline pellicle forms on the surface and, on cooling, a copious crystalline mass (A) separated out, consisting of granules formed by the aggregation of crystals round a centre. Further concentration of the mother- liquor furnished additional crystals. Finally there remained a voluminous syrup (B) which required subsequent treatment to obtain its constituents in a crystallised form. From the crystalline mass (4) crystals of tyrosine CgH,,NO;, and leucine C,H,,NO,, together with amino-valerianic acid C;H,,NO,, were obtained. These two latter were combined in various ways with the leucéins C,H,,NO, and C;H,NO,, forming the crystalline compounds— C,.H,,N,O, composed of C,H,,NO, + C,H,,NO, leucine leucéine C,,H,.N,O, either C,H,,NO, + C;H,NO, or C;5H,,NO, + C,H,,NO, and C,,H,)N,O, composed of C;H,,NO, + C;H,NO, The last fractional crystallisation from the mass (4) on analysis, furnished numbers which agree with those of amino-butyric acid, C,H,NO,. Schutzenberger makes no mention here of the presence of amino-propionic acid C,H,NO,. From analyses and statements, however, which will be found further on it is evident that it was 1. Loc. cit., p. 332. 232 BIO-CHEMICAL JOURNAL present, probably combined with C;H,,;NO, and so forming the compound 2 C,H,NO,. Caproic leucéine, CgH,,NO,, crystallises in granules consisting of needles grouped round a centre, and is more soluble in water than leucine. It is soluble in boiling alcohol, and has a slightly sweet taste.* Amino-valerianic acid C,;H,,NO, is found in the primary crystallisations of tyrosine, tyroleucine, leucine and caproic leucéine, together with the intermediate products C,,H,,,N,O, (m = 12, IT, and 10). It is more soluble in water than leucine, and is most readily obtained from the mother-liquor after the separation of the above products. It is therein associated with a considerable proportion of amido-butyric acid C,H,NO, and traces of alanine or amino-pro- pionic acid, C,H,NO,. This mother-liquor also contains the lower members of the series C,,H,,,N,O, and C,H,,,,NO, (m = 10-7; n = 5-4).! For the extraction of amino-valerianic acid and the other substances referred to above, which are very soluble in water, the following process was adopted :— The mother-liquor or uncrystallisable syrup (B) was first completely deprived of baryta by means of sulphuric acid and then evaporated to dryness. The residue was treated several times with boiling alcohol, which almost entirely dissolved it, and on cooling furnished a crystalline deposit. ‘This deposit weighed about 3 or 4 per cent. of the albumin operated upon, and consisted of CyH,gN,O,, crystal- lising from the alcoholic solution in tufts (houppes) ; this is a mixture of amino-butyric acids. C,H )N,O, = C;H,,NO, + C,H,NO,. amino- amino- valerianic butyric acid acid These two acids have a marked tendency to crystallise together in their molecular proportions, especially from an alcoholic solution. A large number of analyses were made of the crystalline deposits from 1. Loc. cit, p. 352. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 233 the alcoholic solutions of the dried residue of the mother-liquid (B) obtained in the manner above described. These analyses always indicated the presence of a compound of two allied substances. The separation of these substances, however, could be accomplished by using water as a solvent, in the following manner :—After dissolving ‘the compound C,H,.N,O, [= C;H,,NO, + C,H,NO,] in water, the solution was decolourised with animal charcoal and then concen- trated im vacuo, at a gentle heat (40° to 50° C.). Butalanine crystal- lised out during ebullition in the form of white leaflets resembling those of caprioic alanine, which, if present, was also deposited. ‘The mother-liquid then, when highly concentrated, yielded crystals of amino-butyric acid. Amino-valerianic acid closely resembles amino-caproic acid, or leucine, in appearance. It is almost equally volatile and more soluble in water, and under the microscope it appears as crystalline nodules, formed by short needles radiating from a centre and not in flat plates. It has a somewhat sweetish taste. Amino-butyric acid is a comparatively large ingredient of the ‘ résidu fixe.’ It crystallises, from a hot alcoholic solution, on cooling in small delicate pearly leaves resembling amino-caproic acid. It is almost insoluble in cold, but is more soluble in hot alcohol. It is readily soluble in water from which, when sufficiently concentrated, it crystallises in the form of nodules composed of needles grouped round a centre. It has a sweeter taste than amino-valerianic, or amido- caproic acid. The extract obtained by boiling absolute alcohol furnished also the following products! :— (1) The leucines C;H,,NO, (amino-valerianic acid), C,H,NO, (amino-butyric acid), C,H,NO, (amino-propionic acid). (2) The leucéins C,;H,NO,, C,H,NO,, C,H;NO,, and (3) ‘Their intermediate products or combinations Cy9Hg9N,0,, C,H,,.N,O,, CsH,,N.0O,, C,HisN20,. 1. Loe. cit., pp. 359, 369. 234 BIO-CHEMICAL JOURNAL To these products, together with the two higher members of the — series C,,H,,N,O, and C,,H,,N,O,, Schutzenberger gave the name of gluco-proteins. After completely exhausting the ‘résidu fixe’ with boiling absolute alcohol there still remains, as stated on page 231, a certain quantity of matter, amounting to about 3 or 4 per cent. of the albumin, which is readily soluble in water, and has a somewhat sweet taste. The aqueous solution after concentration becomes after a time a mass of crystalline grains. Its analysis corresponds to the formula C,H,,N.O,. 3 These, then, are some of the substances (among many others) which, starting with pyruvic acid and its three higher homologues, can be synthesised in the laboratory. ‘Their compounds with NHg, viz. -— (i) CH, * C(NH) - COOH (imino-pyruvic acid) ; (ii) CH, * C(NH)* CH, * COOH (imino- acetyl-acetic acid); or CH;*C(NH,):CH*COOH (amino-crotonic acid) ; (ili) CH, * C(NH) * CH(CH;) - COOH (imino-methyl-acetyl-acetic acid); or, CH, » C(NH,) : C(CH,) COOH (amino-methyl-crotonic acid) ; and (iv) CH, * C (NH) - CH(C,H;) - COOH (imino-ethyl-acetyl-acetic acid) ; or, CH, * C(NH,) : C(C,H;) * COOH (amino-ethy]-crotonic acid), have the same ultimate composition as the four compounds which Schutzenberger obtained from albumin and to which he gave the name. of leucéins—a knowledge of the constitution of which was, in his opinion, the only thing wanting to solve the problem as to the general structure of proteid matter. He consequently endeavoured in various ways, but unsuccessfully, to effect their synthesis. Those which he was able to isolate presented the following characters! :—they crystallise with difficulty, or not at all, and are deliquescent ; they combine with baryta from which they are not completely disengaged by CO,; they are not precipitated by mercuric nitrate. Though uncrystallisable themselves, they form crystalline compounds with the amido-derivatives of the fatty acids Une. NO 1. Comptes Rendus, t. LXXX, p. 238. 2. Loc. cit., t. Cl, p. 1267. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 235 They differ, as he pointed out, from the leucines, amino-crotonic, amino-valerianic acid, etc., to which the general formula C,H,,,,,NO, may be given, by containing two atoms less of hydrogen than the corresponding leucine, and may be represented by the general formula C,H,,_,NO,. When combined we have— CAT NO, + C,Hop_,NO, = CUNO, where n + ? = mM. leucine leucéine These characters correspond with the salts of pyruvic acid and its homologues. ‘The salts of pyruvic acid if prepared at the ordinary temperature can be obtained in a crystalline form; but if these aqueous solutions are heated to the boiling point, their character is changed and, on evaporation, gummy uncrystallisable salts remain behind. If an aqueous solution of the acid itself is evaporated by heat, a syrupy non-volatile acid is left behind, which when heated with HCI to 100° C. is resolved into CO, and pyrotartaric acid.? 2 CH, *CO- COOH = CO, + CH; * CH(COOH) - CH, : COOH pyruvic acid pyrotartaric acid Acetyl-acetic acid is a viscid fluid mixable with water, with a strong acid reaction. Its barium salt is amorphous and very soluble in water. Methyl-acetyl-acetic acid or methyl-crotonic acid is a thick fluid mixable with water, which when heated is resolved into CO, and C,H; cog The barium salt is very soluble in water.” Ethyl-crotonic acid possesses similar properties. Having obtained from the ‘résidu fixe’ the various crystalline bodies to which I have referred, Schutzenberger then proceeds to resolve this latter into certain constituents. He arrives at the con- 1. Clermont, Ber., Bd. 6, s. 72. 2. Beilstein, Handb. d. Chemie, 3te Aufl., B. IV, s. 601. 236 BIO-CHEMICAL JOURNAL clusion, based on reasons which are fully set forth in his paper,! that the ‘ résidu fixe,” Cy5,H435NqgO405 is made up of the compounds :— Cott NOs 3%: a tyrosine + 3 C,H,,N.O, ... au mean of the strong amido-acids + 7 CANDO. = leucines + 2 C,H,,N,0, gluco-proteins +16 C,H,,N.O, gluco-proteins elites 5 (RS 2: ERE es waa résidu fixe or in other words, when albumin is digested with five or six times its weight of barium hydrate for forty-eight hours, at a temperature of 180° C., it combines with 60 molecules of water and Uneeteeae the following decomposition.” CoroH5g7Ne507553 + 60 H,O 16 NH, + 4 (COOH), + 3 CO, + 4 (CH, * COOH) + §, Cc .H,,NO, + 3 C,H,.N.O, + 3 ‘CH 20N20, 2 C,H,,N,0, + 16C "HNO, I shall now endeavour to resolve these terms into their respective constituents. The last term in the equation 16 C,H,,N,O, is the one which presents the greatest difficulty in this respect for its formula may be satisfied by an indefinite number of combinations. The following considerations, however, furnish us with material assistance + of in arriving at a satisfactory solution. In the first place we know that with regard to a large number of organic bodies, condensation of three molecules takes place forming compounds much more stable than those consisting of one molecule only : such compounds, for instance, as the cyanides, the cyanates, the cyanamides, the aldehydes, etc. It is consequently not an unwar- rantable assumption that in such a body as albumin this ternary con- densation or triple combination takes place. As a working hypothesis, at all events, it is useful, and we will assume that the constituents of 16 CsH,,N,O, exist in it either as single molecules or as compounds of three molecules or as multiples of three. Secondly, we will assume that in this compound the leucines 1. See Annales de Chimie, sme série, t. XVI, PP: 394-399. 2. Loe. cit., pp. 385 and 398. HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 237 C,H,,,,;NO, are combined with the corresponding leucéins C,H,,-,NO, forming the compounds C,,H,,N,O,. Thirdly, Schutzenberger! points out that the amount of the com- pound C,,H,,N,0, obtained from one molecule of albumin, and consisting of C,H,,NO, + C,H,,NO,, is about 15 to 16 per cent. ; 3 (C,H,,NO, + C,H,,NO,) corresponds to 14 per cent. If we sub- tract this from 16 (C,H,,N,O,), the remainder, he says, can only be resolved into 4 terms of C, and 22 of C,, that is, into 2 (C,H,,NO, + C;H,NO,) and 11 (C,H,NO, + C,H,NO).? In other words— 16 (C,H,,N,O,) = 3 (C,H,,NO, + C,H,,NO,) + 2(C,H,,NO, + C;H,NO,) + 11(C,H,NO, + C,H,NO,) In making this calculation, however, Schutzenberger leaves out of consideration the presence of alanine C,;H,NO, or CH, - CH(NH,) ° COOH which, as he shews,* is also contained in the ‘ résidu fixe ’"— * mais en petites quantités seulement.’ One molecule of this combined with the corresponding leucéine C,H;NO, or CH,(NH)CH,COOH, forms the compound C,H,NO, + C,;H;NO, = C,H,,N,O,. Taking this into consideration, and that 2 (C,H,NO, + C,H,NO,) may /represent (C,H,,NO, + C;H,NO,) + (C,H,NO, + C,H,;NO,), the compound 16 (CyH,,N,O,) or Cy44 HoggN5904, may be represented by 3 (CsH,;NO, + C,H,,NO,) = C,,H,,N,O), + 3(C,H,,NO, + C;H,NO,) = CapHeoNcO1. = A (C,H,NO, - C,H,NO,) t= CreAyasN 15036 + (C,H,NO, + C,H;NO, = CHjN,O; CyasHoggN a2 4 which satisfies the assumption made above as to the ternary com- binations of the molecules. It also satisfies the requiremént that amido-butyric acid and its complement C,H,NO, are the dominant factors of the ‘ résidu fixe,’ which is in conformity with Schutzen- berger’s analysis.* 1. Loc. cit., p. 397- 2. Loc. cit., p. 398. 3. Loc. cit., p. 358. It is found in the portion of the ‘ résidu fixe’ insoluble in alcohol. 4. Loc. cit., p. 398. 238 BIO-CHEMICAL JOURNAL The theory as to the ternary combination of the molecules can only be regarded as satisfactory if it holds good not only for this particular compound but for the general constitution of the albuminous molecule. The next term in the general equation 2 C,H,,N,O, would, therefore, at first sight seem to negative the theory, whereas the theory enables us to determine its composition. The gluco-proteins 2 C,H,,N,O, may be regarded as the com- bination of two molecules of alanine, or amino-propionic acid, CH, * CH(NH,) - COOH, with two molecules of the leucéin, imino- aceto-acetic acid CH,*(NH)CH,* COOH, which together form 2 C;H,,N,O, (gluco-proteins), and here the two molecules of amino- propionic acid with the one moluecle in the previous compound furnish the necessary triad. If we regard 2(C,H,,N,0O,) as the combination of 2 CH, * CH, * CH(NH,)COOH + z2CH,*C(NH)COOH the same amino-butyric acid imino-pyruvic acid argument might be employed with respect to imido-pyruvic acid, but as will subsequently appear two molecules of amino-butyric acid would not be in accordance with it. The next term 3 CyH,)N,O,, or the leucines, is satisfied by the combination of 3C,H,(NH,) ‘ COOH + 3C,H,(NH,) - COOH = 3C,H,,N,0, which, as it satisfies the ternary theory, is the one I adopt, though as 3C;H,,NO, = C,H,;NO, + C;H,,NO, + CjH,NO, and 3C,H,NO, = C,H,,NO, + C,H,NO, + C,H;NO, many other combinations of these four amino acids would satisfy the formula. i The term 3C,H,,N,O,, representing what Schutzenberger terms ‘les acides amidés forts,’ contains the acids belonging to the glutamic series. Combining glutamic, aspartic and amino-malonic acids a HYDROLYTIC DECOMPOSITION OF EGG-ALBUMIN 239 respectively, with imino-ethyl-acetyl-acetic, imino-methyl-acetyl- acetic and imino-acetyl-acetic acids we have CH(NH,) - COOH = + CH; C(NH) « CH(C,H;) - COOH = C,9H, .N,O, ===—— CH, ‘ CH, - COOH imino-ethyl-acetyl-acetic acid glutamic acid CH(NH,) : COOH + CH, . C(NH) - CH(CH;): COOH = C,H,,N,O, CH, - COOH imino-methyl-acetyl-acetic acid aspartic acid ; CH(NH,) - COOH + CH; * C(NH) - CH, COOH = C,H,,N,O; COOH imino-acetyl-acetic acid amino-malonic acid CypHysNeOis or 3C,H,,.N,O, Both glutamic and aspartic acids were obtained by Schutzenberger from the ‘ résidu fixe’ and consequently must appear in the equation. Amino-malonic acid is not mentioned by him. The explanation I suggest is, that in the combination of amino-malonic acid and glutamic acid the compound would have the same molecular composition as aspartic acid, and as, according to Schutzenberger, the amido-acids of the same series show a great tendency to crystallise together (see p. 230), we can have C-H,NO, + C,H;NO, = 2 (C,H,NO,) glutamic amino- acid malonic acid and in this way its presence might be overlooked. Taking, then, the formula C,,)H59,N,,0,;5, for the composition of albumin (differing from Schutzenberger’s by H,9) we find that by the action of BaH,O, at 180° C. it combines with 60 molecules of H,0O and is resolved first into (i) 16 NH, + 4 (COOH),"+ 3 CO, + 4 (CH, . COOH) + S, + Hy (Gi) + C,H,,NO, (iii) + 3 CsH,,N,O, (iv) + 3 CyH,sN,O, (v) + 2 C,H,,N,0, (vi) + 16 C,H,,N,O, and resolving (iii), (iv), (v) and (vi) into their components, the whole may be represented in the following tabular form :— _ urine o*H 09+ BSc 89 pj 6E 70FZy 10. "OES fe . S BS Ge © SIO MEE HM, oxy npisar kar tee ee LOT 6F yy POP Eye : ~ ; - ——- - - -s pappe °TH ats sutaqoid-oony3 suraj01d-oonys — sguronay $]10} sprue spor jeruroudjod ForN* H®O 91 40 Yotn”'H’d Z 10 "OmN®H®O £ 10 *OFN*TH®D £ 10 autsoi.9 $,19819qZU9INYPS bad @ \i TN ia © bdle®) Le) NeH" OD 819? NO HAD 8To? Neh ED £6 NT B9 8Q08 ov NHS pre oransAd-ourut Sara eas Sidae aise ae - ee Gm aid ee i ae = TES ieee = HOOD * (HN)O * “HO poe o19a08-]4q008-OUTWUT prov o1j00e-[4qaov-ourUt ptoe o1jaov-[Aja9v-ourwut HOOD **HO*(HN)O**HO® HOOD * SHO *(HN)O * “HO # HOOD * *HO * (HN)O ° “HO pre orjooe-,A300e-]AYJOW-OUTWUT proe o1jaoe-]Ajaov-[AY}OW-OUL WUT HOOD * (“HO)HO * (HIN)O* “HO £ HOOD * *HO)HO * (HN)O * “HO proe o19008-[Ajoou-[AYI9-OUTUT prov o1ja08-]4qaov-[Ay2-ourM HOOD * @H*9)HO'(HN)O * “HO t HOOO * CH*9)HO * (HIN)O **HO proe oruoyeur-oullule HOOD pre s1uordoid-ourme 9 prov oruordosd-ourue 2 | HOOD * GHN)HO * “HO HOOD * GHN)HO **HO @ HOOD * GHN)HO H+ “HS? +St prov onaedse pie. aneoF pioe o1143nq-ourue 2 plow o14gnq-ourme 9 HOOD **HO HOOD **HO t+ HOOD * GHN)HO **HO **HO © HOOD * CHN)HO **HO * “HO & SES HOOD * CHN)HO oo £+ pre oTuUvi19]/BA-OUTULe D P pre oruviio[eA-OUTUTe D HOOD * CHN)HO **CHO) * “HO £ HOOD * “HN)HO **@HO) * “HO € prow orereynys PP HOOD **HO**HO | “(HOOO) *+ ploe s1oidvo-outue p | aurso14} : * HOOD * GHN)HO **CHO)*HO € HOOD * GHN)HO 009 CHN)HO" *HO * (HO)"H?O ‘HN 91 (14) (4) (at) (mm) () ®) *) OSI LV aLVAGAP, woaltvg HLIM NINOATY Soy AO NOILISOdNOOACT DILATOUGAP ALATINO) aH], <3 = E. “S . 241 ON THE SYNTHESIS OF LIVING ALBUMIN By P. W. LATHAM, M.D., Downing Professor of Medicine, University of Cambridge (1874-1894). (Received April 26th, 1908) Having under the conditions given in the preceding papers determined the products which result from the complete hydrolysis of albumin, I will now proceed to consider what is the constitution of albumin itself, or in other words, what are the respective constituents of the albuminous molecule from which these products are derived. In dealing with the origin of lactic acid I have already indicated the way in which, in the earlier stages of the genesis of organic matter in plants, the following substances may be formed, hydrocyanic acid ; its condensed product amino-malonic nitrile, with its derivative tartronic nitrile; acetic anhydride; pyruvic nitrile; and methyl- tartronic nitrile. ‘The series of changes by which we can proceed to the formation of the various fatty aldehydes, commencing with the lowest, namely, formic aldehyde, have also been referred to.1 It remains now to consider what are the antecedents of tyrosine and in what form they exist in protein. Secondly, in what form do the antecedents of the amino-fatty acids exist therein, and lastly what are the antecedents of aspartic and glutamic acids. _ The answer to these questions will be obtained by considering the synthesis of the respective bodies. On THE ANTECEDENTS OF TYROSINE IN LiviING PROTEIN On page 199 I have already indicated the method by which the synthesis of tyrosine may be effected from p. oxybenzaldehyde, amino-malonic nitrile, and 2 HCN. Practically the result is— OH - C,H,: CHO + CH(NH,) : (CN), + 2 HCN + 7H,O p. oxybenzaldehyde amino-malonic nitrile =p. OH: C,H, - CH, - CH(NH,)COOH + CO, + 2 NH, + (COO), (NH,), tyrosine ammonium oxalate 1. See pages 195-8. 242 BIO-CHEMICAL JOURNAL By similar reactions we may from p. amino-benzaldehyde obtain p. amino-phenyl alanine p. NH, ‘C,H, * CH, ‘ CH(NH,) ‘COOH the hydrochloride of which treated with a single molecular equivalent of NaNO, at o° C. and then boiled yields tyrosine ; but if an excess of NaNO, is used the compound is decomposed into tyrosine and oxyphenyl lactic acid,t OH * C,H, * CH, *CH(OH) *COOH. Now when tyrosine is taken into the animal system it is converted into hydro-para-coumaric acid and . oxy-phenyl-acetic acid*. With this exception, however, all other para derivatives of benzene when taken into the animal system are immediately excreted, with very little change, in the urine’, so that it is improbable that either p. oxy- benzoic aldehyde or p. amino-benzaldehyde exist in living protein. On the other hand o. amino-benzaldehyde under certain con- ditions combines with aldehydes and ketones to form chinoline derivatives* CHO CH,—R CH=CeR can ree * can + BO NH, COR Nz ‘CoREO and this suggests that 0. amino benzaldehyde may be a constituent of living protein, since chinoline and its derivatives are obtained by the distillation of certain vegetable alkaloids which are derivatives of vegetable albumin. Some of the ortho-derivatives of benzene, moreover, under certain conditions, can be transformed into the para-compounds ; 0. oxybenzoic acid for instance when heated with potash to 220° is converted in some degree into p. oxybenzoic acid.® I shall therefore assume that in the living protein amino-benzalde- hyde exists in the ortho-form, and that in some way it is converted into the para-form before entering into the formation of tyrosine. In living protein then we have as the antecedents of tyrosine, amino-oxy- benzaldehyde, amino-malonic nitrile and hydrocyanic acid— NH, - C,H, - CHO + CH(NH,) : (CN), + 2 HCN which with 8 H,O form | p. OH - C,H, CH, - CH(NH,)COOH + CO, + 5 NH, + (COOH), tyrosine oxalic acid Friedlander, Annal., Bd. CCXIX, s. 223, u. Bd. CCXXIX, s. 227. Blendermann, Zeitsch. f. phys. Chemie, Bd. VI, s. 234. Schrotten, ibid., Bd. VII, s. 23. Friedlander, Berichte, Bd. XV, s. 2574, Bd. XVI, . 1833, Bd. XXV, s. 1752. Kolbe, Yourn. f. praktische Chemie (2), Bd. XI, s. 24. Cee ee Se ON THE SYNTHESIS OF LIVING ALBUMIN 243 On THE SYNTHESIS oF THE AmiINo-Fatry Acips Amino -Caproic Acid (CHy;°* (CH,)3 * CH(NH,)COOH).— If valerianic aldehyde is mixed with aqueous ammonia the aldehyde is converted into valeral ammonia and this digested with hydro- eyanic acid and hydrochloric acid, is converted into leucine. CH,(CH,), : CHO + NH, = C,H, - CH(NH,) - OH valerianic aldehyde valeral ammonia and C,H, - CH(NH,) -OH + HCN = CH, - (CH,),- CH(NH,)COOH + NH, normal amino-caprioic acid or leucine This is the usual way of obtaining leucine synthetically. ‘Tiemann, however, has shown! that the amido-acids, both of the fatty and aromatic series, may be obtained by converting the aldehydes and ketones into cyan-alcohols, then into amino-nitriles or cyan-amides and thence into the amido acids. We may consequently have the following changes :— OH CH, - (CH,),CHO + HCN = CH, - (CH,),CH valerianic aldehyde valerianic nitrile 1. Ber. XIV,s. 1985. ‘The amido-acids of the fatty series are easily obtained by the familiar reactions which take place on treating aldehyde ammonia with hydrochloric and hydrocyanic acids, and which led Strecker to the discovery of alanine. .. The reactions indicated by Strecker take place unquestionably according to the following general formulae :— (NH, (NH, R- CH: + HCN = R-CH- + H,O (OH (CN NH and cH * 42H,0 +'HCl = R- CH(NH,)COOH + NH,CL CN NH, The question arises, whether the cyanamide R- cH! could not be obtained more readily from (cn { OH the cyanhydrides of the aldehydes R- CH | by digesting them with ammonia, expecting the ultimate CN change to be as follows :— OH NH R- CH + NH, =R:- CH; + H,O (CN (CN The truth of this supposition has been confirmed by experiment.’—Ber. XIII, s. 382. BIO-CHEMICAL JOURNAL 244 (OH (NH, C,H, CH; + NH, = C,H, - CH- + H,O (CN (CN amino-caproic nitrile ( NH, (NH, C.H,-CH; + 2H,0 = C,H, - CH: + NH, (cn |COOH amino-caproic acid or leucine NH which by dehydration is converted into the anhydride C,Hy "CH¢ | CO This method is applicable generally to the production of the amino- acids and we have OH NH, NH, NH R-CHO>R- CHE RCH > R-cHE —>R-CHY | CN CN COOH oe) Practically, however, we may regard the cyan-alcohols as the constituents in living albumin, which are the antecedents of, and by hydrolysis are converted into the amino-fatty acids, since omitting the intermediate formation of the nitrile of the amino acid we have OH NH, R- CH + H,O = R-CH CN COOH cyanalcohol amino acid Tiemann has also shewn? that by treating the di-ethyl, ethyl- methyl, and di-methyl ketones in the same manner, other iso-amino acids are formed— (OH (NH, (C,H), CO + HCN = (C,H;),-C —> (C,H,)2 - C- fen (COOH (OH NH (CH,),- CO- + HCN (CH,),- C Ss.(CH). °C (CN COOH These amino-fatty acids can also be prepared? in the same way as tyrosine by combining the fatty aldehydes by means of Perkin’s 1. Berichte, Bd. XIV, s. 1975. 2. E. Erlenmeyer, Jnr., Annal. d. Chemie, Bd. CCCVII, s. 74. ON THE SYNTHESIS OF LIVING ALBUMIN 245 reaction with those derivatives of glycocoll which have one of the hydrogen atoms of the NH, replaced by an acid radicle such as NH - CO - CH, =~ acetyl-glycocoll CHC | | COOH | NH - CO: C,H; or hippuric acid CH, COOH NH - CO - C,H, NH: CO+ CH, R-CHO + CHL = R«CH:C COOH ‘. COOH = R-CH:C—N—CO: C,H; + 2 H,O VY CO + H,O forming a lactimide which heated with dilute aqueous solution of soda is transformed into an acid. This on reduction becomes NH - CO: C,H; me CH. - cH and can then be resolved into COOH NH, the a-amino acid R*‘ CH’: CHC and benzoic acid OOH, C,H, : COOH. On the Synthesis of Aspartic and Glutamic Acids.— CH(NH,) * COOH CH(NH,) COOH | ana; . | CH, -COOH ites Gite SOUL The lowest member of this series is amino-malonic acid CH(NH,) * COOH CH(NH,) - CN = the nitrile of which | is COOH CN formed as shewn on page 197, from the condensation of three molecules of HCN, and by desamination should be converted into tartronic nitrile. Theoretically it should be possible to convert these two bodies respectively into amino-malonic acid and tartronic acid. Hitherto this has not been accomplished. 246 BIO-CHEMICAL JOURNAL We have here, however, a demonstration of the existence of the nitrile of the lowest member of the aspartic series and this is a strong argument in favour of the existence of the nitriles of the two other members. ‘The facts also that the antecedents of the amino- fatty acids are the fatty aldehydes combined with HCN, and that the antecedent of tyrosine is an aromatic aldehyde render it most probable that aspartic and glutamic acids have a similar origin. I shall now endeavour to shew how, in this way, their synthesis may possibly be accomplished. Synthesis of Aspartic Acid.—Cyan-acet-aldehyde CH,(CN) ‘CHO can be obtained from chloracetal CH,Cl *CH(C,H,O), or, chlor- acet-aldehyde CH,Cl*CHO by converting the latter into iodo- aldehyde, and thence into the cyanogen compound. ‘Treating this compound with HCN and then with NH, we should obtain the corre- sponding cyanamide— OH CH,(CN) - CHO + HCN = CN- CH, - CH é CN cyanacetaldehyde oxy-succinic nitrile OH CN - CH, : cut + NH, = CN: CH, - CH(NH,)CN. + H,O CN aspartic nitrile which acted upon by acids or alkalies would be converted into asparagine or aspartic acid— CH(NH,) : CO: NH, CN - CH, - CH(NH,)CN + 3H,O= | + NH; CH, - COOH asparagine CH(NH,)COOH ~ or with +'4HO= | + NH; CH, - COOH . aspartic acid consequently if oxysuccinic nitrile, which, I suggest, is the antecedent’ of aspartic acid in living protein, is heated in a sealed tube with BaH,O, the following reaction would ensue :— CN + CH, CH(OH)CN + 3H,O = COOH : CH, : CH(NH,) COOH + NH, oxysuccinic nitrile aspartic acid ON THE SYNTHESIS OF LIVING ALBUMIN 247 If, however, the cyan-acet-aldehyde is first combined with NH, we should have— NH, CN - CH, - CHO + NH, = CN - CH, CHE Tapa OH igich reduced by nascent hydrogen (H,) becomes NH, — CH,(NH,) « CH, - cue OH - Combining this with HCN we obtain CH,(NH,) * CH, - CH(NH,)CN which on saponification becomes CH,(NH,) - CH. CH(NH,)COOH diamino-butyric acid Moreover, if by the Perkin’s reaction we combine cyan-acetic- aldehyde with acetyl or benzoyl glycocoll (derived from amino-malonic nitrile) as in the synthesis of tyrosine, the following reactions should ensue :— CN - CH, - CHO + CH,(NH - CO - C,H,)COOH — CN - CH.CH : C(NH - CO - C,H,)COOH which on reduction with H, becomes CH,(NH,) - CH, - CH, - CH(NH - CO - C,H,)COOH* benzoyl ornithin and is then resolved into CH,(NH,) - CH, - CH, - CH(NH,)COOH + C,H, - COOH 1-4 di-amino valerianic acid or benzoic ornithin acid By bacterial action Ellinger? succeeded in converting this into CH,(NH,)CH, - CH, - CH,(NH;) putrescine By combining cyanamide with ornithin, argenin is formed. The Synthesis of Glutamic Acid.—We may assume that with 8 cyanpropionic aldehyde CH,(CN) ‘CH, *CHO similar reactions will take place to those with cyan-acet-aldehyde. Treating it with * Which is excreted by birds after the ingestion of benzoic acid (Kossel). 1. Zeitsch. f. phys. Chemie, Bd. XXIX, s. 334. 248 BIO-CHEMICAL JOURNAL HCN, and then with NHg, the cyan-alcohol would first be formed and then the cyan-amide or CN - CH, : CH, - CH(NH,) - CN glutamic nitrile which by saponification would be converted into COOH : CH, : CH, - CH(NH,) - COOH + 2 NH, glutamic acid . If, however, the cyan-propionic aldehyde is first combined with NH, and then undergoes reduction the following reactions will take place :— OH CN: CH, CH,- CHO + NH, = CN CH CH, CH which reduced by H, = CH,(NH,) * CH, - CH, - CH, : CH 4 NH, combining this with HCN, and then saponifying, we again obtain ornithin or NH, OH CH,(NH,) : CH, - CH, : CH(NH, COOH 1-4 diamino-valerianic acid On the other hand if we combine the cyan-propionical dehyde with acetyl or benzoyl-glycocoll by the Perkin’s reaction and then reduce the resulting compound we should obtain CH,(NH,) - (CH,), - CH(NH,) - COOH I-5 diamino-normal caproic acid or lysin which combined with cyanamide forms Drechsel’s lysatinine. By bacterial action also lysin is converted into cadaverine NH,(CH,); - NH, It follows from this that aspartic acid and ornithin have a common . origin, which is also the case with glutamic acid and lysine. Further- more, that oxy-succinic or malic nitrile and oxy-glutaric nitrile are the antecedents in living albumin of aspartic acid and glutamic acid respectively, since by heating them in a sealed tube with BaH,O, these two acids would be produced. ON THE SYNTHESIS OF LIVING ALBUMIN 249 Finally, we have to determine the antecedents in the protein molecule, of Schutzenberger’s polynomial ; 16NH; ° +4CH;~-COOH + 3 CO, + 4 (COOH), + S; + [Hip] - acetic acid oxalic acid ‘The very Simplicity of this polynomial makes it difficult to determine its antecedents, for the possible ways in which they may arise may truly be said to be innumerable. It is, however, unnecessary for me to discuss the possibilities. I shall content myself with stating the result at which I have arrived, briefly indicating the grounds which have led me to it. In describing the synthesis of tyrosine I have shewn that its antecedents are— CH(NH,)(CN), + NH,+-C,H,-CHO- + 2(HCN) amino-malonic amido-benzoic nitrile aldehyde which with eight molecules of H,O give OH - C,H, - CH, - CH(NH,)COOH + (COOR), + CO, + 5 NH, tyrosine oxalic acid thus furnishing (COOH), + CO, + 5 NH, towards the polynomial. Again in describing the synthesis of glutamic, aspartic and amino- malonic acids I have represented these as resulting from oxyglutaric, malic and tartronic nitriles. ‘These under the action of baryta in a sealed tube become respectively— OH NH, cut 3 cut CN + 3H,0 = COOH + NH, CH, - CH, : CN CH, - CH, - COOH glutamic acid H NH, CH cut CN + 3H,O = COOH + NH; CH, : CN CH, * COOH aspartic acid OH NH, cut cut | CN + 3H,O = COOH + NH, CN COOH amino-malonic acid . “450 BIO-CHEMICAL JOURNAL furnishing three more molecules of NH. The remaining portion of the polynomial— 8 NH, + 4 CH, : COOH + 2 CO, + 6H-COOH + 2SH, + S is, therefore, all that remains to be considered. It has been shewn that all the compounds hitherto dealt with may be derived from the cyanides or nitriles. It is not improbable, therefore, that the sulphur in albumin is present also as a cyanide, namely, as sulpho-cyanide, and this assumption is rendered more probable as this compound is found, though in small quantities, in the saliva, and is also excreted in the urine.! Sulphocyanic acid CN~*SH is very unstable, and is quickly resolved into hydrocyanic acid and persulphocyanic acid” which under 3 CN- SH = HCN + C,N,H,S, certain conditions are resolved as follows :—* HCN + 2H,O = NH, + H- COOH and C,H,H,S, + 4 H,O = 2 CO, + 2 NH, + 2SH, + S This is the form, then, in which I suggest the sulphur exists in the albuminous molecule, and which furnishes these constituents of the polynomial. We have now only to determine the origin of the remaining terms 5 NH, +4 CH,- COOH + 5 H- COOH In a previous paper (see page 200) I endeavoured to show that diacetyl-dicyanide was a constituent of protein, being the antecedent of methyl-tartronic nitrile. Now diacetyl-dicyanide acted upon by alkalies in the cold is resolved into NHs, HCN and CH, * COOH, and when the temperature is raised we have (CH, -CO- CN), + 6H,O = 2 CH,- COOH + 2 NH, + 2H- COOH I have also shown that 3 HCN and (CH;*CO),O are among the 1. Gschleiden, Fabresb. uber die Fortsch. d. Chemie, s. 1001, 1877. 2. Watt's Dictionary of Chemistry, Vol. V, p. 505. 3- Ibid., Vol. IV, p. 379. ON THE SYNTHESIS OF LIVING ALBUMIN 251 earliest products in the genesis of protein. ‘These two substances acted upon by BaH,O, are hydrolysed as follows :— _ = 3 HCN + 6H,O = 3 H- COOH + 3 NH; - ra (CH, - CO),0 + H,O = 2 CH, - COOH The following compounds, therefore, may be regarded as the ante- _cedents of the polynomial :— 3 HCN + 6H,O = 3NH, + 3H-COOH 3 HCNS + 6H,O = 3NH,+ H-COOH + 2CO, + 2SH,+S ' (CH, - CO),O + HO= 2 CH, - COOH (CH;,;-CO-CN), + 6H,O = 2 NH, + 2H-COOH + 2CH,- COOH that is to say— 3 HCN + 3 CNHS + (CH,- CO),0 + (CH,;:CO-CN), + 19 H,O = 8 NH, + 6H- COOH + 4CH,- COOH + 2 CO, + 2SH, + § which with the terms CO,+5 NH,+(COOH), furnished by the hydrolysis of the antecedents of tyrosine, and with 3 NH, resulting frem the hydrolytic decomposition of tartronic, malic and oxyglutaric nitriles in a sealed tube make up— 16 NH + 4CH,-COOH + 3 CO, + (COOH), + 6H- COOH + 2SH, + § which, as 2 H* COOH heated in a sealed tube with BaH,O, is converted into H,+ (COOH), becomes 16 NH, + 4 CH,;- COOH + 3 CO, + 4 (COOH), + H, + 2 SH, + § acetic acid oxalic Ae acid From these data the composition of living albumin may be represented in the following tabular form :— 4 2 sPomennS = — > 8O'N™H"D "ONvao “olen *O'N"H"O ‘Kon HD ae ; . ] l | o'H°o9 | = OH9F OHT OH 9 o*H 6 Sas OHO: ee + + Oo + + -- cs + is BBL p08 G4 OPE = FFM NPE °0'N"H"D °o°N* HD So°n* HD : O'N*H™D §8o°N HD proe o1ans4d-ouruit HOOD - (HN)O - “HO plore otjaov-|43a0e-ourUl plow orjeor-[4q908-ourUut ploe o1jeov-[Ajo0v-ourtur HOOO -*HO-(HN)O-"HO® HOOD-“HO- (HN)O - *HO @ HOOO - *HO - (HIN)O - *HO pre sg i a aR, prow oaov-[Aqo0v-[AyowW-OUr LUT a HOOD - CHO)HO « (HNO - “HO £ HOO0 - (*HO)HO - (HN)O - “HO =< pre apase ae tap-Saa prov o1jade-[Ajaov-[4qq0-ouLwut S HOOD + @H*0)HO -(HN) - “HO € HOOO - (H°O)HO - (HN)O - “HO =) fe) suas) 4 HO -*HO HO - “HO Byieetae otuoeu-AXxo 10 ‘91U01}18} on HO: HO. ‘ NO apruedorp-[490281p en) | *(NO +00 - *HO) Y joyooye-uesd ouapryAdoad No : (HO)HO NO = Sw *H . "HO 6 joyooye-ueds suaprAdosd NO HO HO -*HO -*HO & aplaqru otursons-Axo 10 o1]eUr apipAquy 219008 NO - “HO o*(09 * *HO) joyooye-urdd suegng HO | NON No - (HO)HO Bey). Joyooye-uvdéo auayng Ka Ho) -*HO £ ies = HO’ ®H9) -*HO € NOH z + poe rund>-o1y3 joyooe-ue> auaquad HO Eyiselaes georen ourwe SHNO £ NO ayaqtu d1eynys-Axo *(NO)CGHN)HO ore . & * 6 Siw -*@HO) - “HOt nS: oy apAyaplezuaq-ourue mm HO? NO - (HO)HO OHO « "H°OCHN) NOH £ (4) (a) (at) (a) q) w) NINONG1IV DNIAIT AO NOILLISOdNOO ON THE SYNTHESIS OF DEAD ALBUMIN 25 o>) On THE ComposITION oF Dreap ALBUMIN In my Croonian Lectures in 1886 delivered at the Royal College of Physicians,! and again in 1897? I suggested that in dead proteid, the antecedents of the amino-fatty acids are their anhydrides, a triple union of each taking place :— NH CH,—NH—CO—CH, 5 CHC | Me lire NH eC) CO—NH—CH,—CO glycin anhydride or generally— NH R-CH—NH—CO—R - CH 3R-CHC | = | nu CO CO—NH—R - CH—CO compounds which, when the ring is broken, are now known as poly- peptides.® Further in accordance with Pfliiger’st view that ammonium cyanate is the type of living and urea of dead nitrogen, and that the conversion of the former into the latter is an image of the essential change which takes place when a living proteid dies, I suggested that NH when R:CH¢ | in the above triple molecule becomes part of CO ._OH living tissue it is transformed into the cyan-alcohol R° CH¢ CN and vice versa. On this assumption the cyan-alcohols in columns (vi), (v), and (iv) of the table giving the composition of living albumin, are in dead albumin transformed into their respective anhydrides. OH _OH NH, NH 3R-CH€ — —>3R-CHY > 3R- CH —>3R-CHE | CN ‘ CONH, OOH ee) 1. ‘On some points in the Pathology of Rheumatism, Gout and Diabetes.’ Deighton, Bell & Co., Cambridge, 1887. z. ‘On the Synthesis of Dead and Living Proteid.’ Deighton, Bell & Co., Cambridge, 1897. 3. For the mode of preparation of these polypeptides see E. Fischer and others, Ber., 1901, Bd. XXXIV, s. 2868 ; Bd. XXXV, 1095; Bd. XXXVI, sn. 2094, 2106, 2592, etc. 4. Pfliger’s Archiv., Bd. X, s. 337. 254 BIO-CHEMICAL JOURNAL and the three nitriles in column (iii) are also transformed in a similar way, into their respective anhydrides— NH < CH, : CH, . CN the nitrile of pyrollidin-carboxylic acid or its isomeride NH NH CHS | CH | | CO, and | CO CH , CN CN the last being the antecedent of di-amino propionic acid (see page 204). As the proteid passes from the living to the dead state the amido- benzaldehyde in column (ii) is converted into the para-form, and the amino-malonic nitrile + 2HCN is by molecular transformation converted into adenine (see page 203) a substance which according to Kossel! exists in all animal and vegetable cells. The com- position of adenine is isomeric with prussic acid, its formula being H,C;N, or 5 HCN, and since by the action of nitrous acid it can be transformed into hypoxanthine, it belongs to the uric acid series and may be represented by the formula— N = C(NH,) ae 5 HCN = CH C—NH ‘ee \cu Nac we. adenine its transformation into hypoxanthine bene represented by the oe equation— = C(NH), NH — CO | | ¥ . ey. + HNO,= CH C—NH + N, + H,O CH aes Sow Bice: NA: N i ee adenine hypoxanthine 1. Zeitsch. f. physiol. Chemie, Bd. X, s. 258. St a Ga line ts ae Me « x. ee i ree Wine ON THE SYNTHESIS OF DEAD ALBUMIN 255 Heated in a sealed tube with hydrochloric acid (sp. gr. 1-19) for from twelve to fourteen hours, adenine is resolved into glycocoll, formic acid, carbonic acid gas and. ammonia,! —~ -_—,C,N, + 8 H,O = CH,(NH,)COOH + 2 H- COOH + CO, + 4NH, adenine glycocoll formic acid precisely what takes place when amino-malonic nitrile and two mole- cules of hydrocyanic acid are treated in the same way :— CH(NH,)(CN), + 4 H,O = CH,(NH,)COOH + CO, + 2 NH, amino-malonic glycocoll nitrile and 2 HCN + 4H,O = 2H- COOH + 2 NH;- The relationship, therefore, between amino-malonic nitrile and adenine may be represented as follows :— N= =C(NH,) CH(NH,)(CN), —> | | )NH Cc and N== yt Hi) N=C(NH,) \D +2HCN = ee. > or 5 HCN l| )NH fl H Cc N—C——N Fé amino-malonic adenine nitrile The compounds 3 HCN, 3 CNHS, (CH, -CO~-CN), in column () are transformed as the protein passes from the living to the dead state into 2 HCN, (HCN),S, and 2 CH, - C (OH) - (CN),, the latter NH being further transformed into 2CH,*C¢ | the antecedent | CO CN of iso-malic acid (see page 201). The acetic anhydride remains unchanged. From the foregoing data the composition of dead albumin may therefore be represented in the following tabular form :-— 1. Martin Kriiger, Zeitschrift fir physiologische Chemie, Bd. XVI, s. 160. ng BWW ppEIPMED — MOM NCEE 8o'N“H"D Hon HD i Od ta = le) SON HTD hl SeBOON“ HYD P| | | on ~ OH +. ; OH? + OHO + O°H 6 + 7 Oe te o*H 61 + $8205 90 py 268 Fg OPE — HHH ERIM EIT °0’N“H"'D °° NP HO °o'N“H*O ON"HD 8s8o*N" HO proe o1anid-ourm! HOOD * (HN)O * “HO : ploe o1908-[Aja0e-ourut pio o1qeoe-[Ajaoe-ourut ~ ploe o1jaov-[Aja0e-ourult HOOO **HO*(HN)O*"HO® HOOD **HO * (HN) “HO @ 009 **HO * (HN)O * “HO plow o1jeoe-[A4j20v-]AYoUI-oUTUI prow o1j00e-[4qQa08-]Ay}9u-OUTUNT 2 HOOO *(H9)HO * (HN)O* “HO € ; HOOD * (*HO)HO * (HN)O * “HO | 2 poe Sneone Arado pine CulEG pioe omjacv-[4jaou-]Ay}9-ourtut 3 HOOO * @H°O)HO * (HN) * “HO € HOOD * @H*O)HO * (HN) * “HO | iS) inal setae = =, apiupAyue stuordoid-ourwe apupéyue oruordosd-ourme NO O 09 coe) OO’ | ; = | >Ho°*HO | Ho -*HO | | | Do: "Ho? fx} HN HN HN on r k NO auruope oprupAqur o1144nq-ourwe apupéyque o1144nq-ourwe ; O a ‘ 00 oo | os ae Se | Ho **HO**HO 6 | PHO" "HO ““HO € Bow wt | | po se HN HN a eee apupAyue oruvriajeA-ourure — STEEP A ORGS NO" + GHN)O==N / ae 3 5) sak OOo ree) ; c | >Ho° *fHO) -*Ho £ | >Ho%CHO)**HO & | Su fr HN HN HN HNC | NOHZz + RS) pre onre Aoonatanaa epupéyure stormdes-ourue NO °*HO**HO pa ®5*(NOH) 09 09 CHNJO=N + &(% + & | HO* *CHO)* “HO & >i apAyapyezueq-ourure © ae HN OHO *"H°OCHN) NOH? 2 S) (4) | (4) (at) (a) () 1) NINAGIV AvVad AO NOILISOdNOO ON THE SYNTHESIS OF DEAD ALBUMIN 257 Note Appep JUNE 5TH, 1908 The four imino-ketonic acids—imino-pyruvic, -acetyl-acetic, -methyl-acetyl-acetic, and -ethyl-acetyl-acetic—which in the preceding tables are indicated as being constituents of protein, represent, in my opinion, the simplest forms of those bodies. If, however, imino- methyl-acetyl-acetic, and imino-ethyl-acetyl-acetic acids in either columns (iii) or (vi), or in both, were replaced by imino-laevulinic acid CH, ~ C (NH) - CH, - CH, - COOH and imino-acetyl-butyric acid CH,°C (NH)-(CH,),;*° COOH (the formation of which is described on page 224) a different protein compound would result, having, however, the same molecular weight and the same ultimate composition as that given in the previous tables. Again, if for the amino-fatty acids in the tables, the iso-amino acids (the preparation of which from the ketones is in some degree indicated on page 244) were substituted, other protein substances would result ; having, likewise, the same molecular weight and ultimate composition. These facts appear to me to explain why so many protein substances have the same ultimate composition but vary considerably both as to their physical and chemical properties. 258 THE OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES TAKEN FROM SEA-WATER OF NATURALLY VARYING CONCENTRATION By W. J. DAKIN, MSc., ’51 Exhibition Scholar, University of Liverpool. (Received May 16th, 1908) It is now thirty-seven years since Bert (1), who was one of the first to consider the osmotic relations existing between the internal fluids of the animal body and the external fluids bathing their bodies, published a paper on the causes of death when freshwater fishes are plunged into sea-water. During the interval a great advance has taken place in physical chemistry, particularly with regard to the application of this branch of science to physiology and medicine, and numerous observers have turned their attention to the constitu- tion and physical properties of the ‘ internal’ and ‘ external media ’ both for invertebrates and vertebrates. Fredericq (4) published in 1885 an account of some investigations concerning the relation of the — salt contents of the blood of Crustacea to the salt contents of the sea or fresh water in which the animals were living, and from that date to the present time the osmotic conditions of the blood and other body fluids together with the nature of the bounding membranes have been studied either by chemically estimating the constitution. of the internal and external media or by determining directly the osmotic pressure with the aid of the Beckmann’s freezing point apparatus. It is not necessary in this introductory communication to go into the history of the discoveries made in this line of research, but it may be mentioned here that various problems which are linked together and stand in very close relation to the constitution of the internal and external media and to the bounding membranes have been con- sidered from several points of view. For example, the relation existing between the sea-water and the blood and coelomic fluids of marine invertebrates has been investigated from the purely OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 259 physico-chemical point of view, and again, the physiological action of various fluids, such as sea-water, on freshwater animals, and fresh water on marine animals with regard to duration of life in these media, has been considered. The results of the various experiments made by different observers, often differ considerably, as do naturally the theories deduced from these results. Fredericq (5), Rodier (12), Quinton (11), Garrey (6) and others have shewn that the blood and fluids of the body cavity, coelom, or haemocoele, of invertebrates have practically the same osmotic pressure as the water in which they live, and contain almost the same percentage of salts in solution. More- over, as the sea-water in the case of marine invertebrates varies in salt contents and osmotic pressure, so do the fluids of the body change accordingly. Turning now on the other hand to the vertebrates, we - find in the highest vertebrates, the mammals, a constant or practically constant osmotic pressure for the blood, amounting to about seven atmospheres, and this is held in defiance of alterations in the consti- tution of the food. For other higher vertebrates this also holds, and though the Amphibia have a somewhat lower osmotic pressure corresponding toa 0-7 per cent. salt solution, Overton (10) has demon- strated the action of the organism in keeping this pressure constant. One finds, however, on investigating the same question in the fishes that there is a surprising difference. The blood of Elasmobranchs possesses about the same osmotic pressure as the external medium, the sea-water, and further, this pressure is not constant but, as is the case for the invertebrates, varies with changes in the external medium. Teleosts, on the other hand, are quite different, and appear to resemble more the higher vertebrates in keeping a constant osmotic pressure. The freezing point depression of the teleost blood appears from Garrey’s work to average about 0-872°, though it is subject to slight variations. ‘The surprising feature here is that in the teleostei, though the blood is brought into close connection with the sea-water by the gills, the fish contrives to maintain an osmotic pressure which is only about one-third of that of the surrounding sea-water. It should be mentioned also that the Elasmobranchs, though resembling the invertebrates in having a varying osmotic pressure for the blood which 260 BIO-CHEMICAL JOURNAL is almost the same as that of the external medium, have a salt contents much less, resembling in fact the proportion of salts in the blood of the teleosts. The osmotic pressure, however, is brought up by the presence of considerable quantities of urea in the blood. We have therefore, roughly speaking, the following three groups of animals, members of which live in water but do not breathe air directly :— 1. Invertebrates—Osmotic pressure and salts contents of blood and internal media practically identical with external medium. 2. Elasmobranchs.—Osmotic pressure of blood practically identical with external medium, but salts contents much lower. | 3. Teleosts.—Osmotic pressure and salts contents of blood much lower than that of external medium. Thus it appears as if the independence of the constitution of blood is first established in the teleosts. What determines this differ- ence? Is the membrane, either gills or body wall, of the invertebrates so different in constitution from that of the teleosts that in the first case perfect osmotic conditions are set up, and the internal fluids are directly dependent on the external, whilst in the latter the membranes are absolutely impermeable to the external medium ? For invertebrates Fredericg and Quinton state that the bounding membranes are permeable to both water and salts, whilst Botazzi and Enriques (2) state that the membranes are semi-permeable, that is, they allow water to pass through and, therefore, bring about the osmotic equilibrium but are impermeable to salts. For teleosts, Dekhuyzen (3) states from a series of observations made at Bergen, that they have a definite osmotic pressure of the blood, and that any differences occurring are probably due to differ- ences in pathological conditions or variations in observation. Sumner (13) has, however, quite recently conducted an extensive series of experiments for the purpose of investigating the conditions of the external membranes in teleosts, the fish used being chiefly three species of Fundulus, small fishes which pass into brackish waters and one species of which occurs frequently in fresh water. By OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 261 weight determinations before and after placing a number of these fishes directly or gradually into fresh water or into sea-water diluted considerably with fresh water, he found that a considerable increase or decrease in the weight of the fishes resulted from changes in the salinity and hence osmotic pressure of the external medium, and came to the conclusion that though the osmotic condition of the blood _and the external fluids in the teleostei are so different, yet the membrane of the gills is both permeable to water and probably to a smaller extent to salts. This is opposed to the views of Fredericq (4) who states that the membranes of teleosts are effective barriers to the external medium, and to Garrey who also supposes an impermeability. Fredericq (4) makes, moreover, the statement that the blood of salt- water fishes does not taste or tastes scarcely more salt than that of freshwater fishes, and that the muscles and glands of a salt-water fish contain no more salt than that of a freshwater one. Griffiths (8) also states that the blood of a sole or haddock does not contain more soluble salts than that from freshwater fishes. With regard, however, to these estimations of chlorine and salts, there seems to be some con- fusion. Atwater gives figures which are 15 per cent. lower for chlorine in the constitution of freshwater fishes than in marine, his average being 0-235 per cent. Cl for marine fishes, Quinton (11) gives the chlorine percentage of the blood of eight species of marine teleosts as 0-651 and freshwater teleosts as o'411. ‘This, as Sumner (13) points out, is much greater than the chlorine contents of the body as a whole, and also shews that the salinity figures for marine fishes are almost 50 per cent. greater than for freshwater fishes. Sumner has, unfortunately, not given the osmotic pressures as determined directly by the freezing point method for the blood of the fishes in which the changes in weight occurred in his experiments. If the gills are semi-permeable, that is only permeable to water, or to a small extent for salts in addition to this, then the osmotic pressure and chlorine contents of the blood of freshwater fishes should be lower than that of marine fishes, and this change should be found to take place in those teleosts which pass from the one medium to the other, like the eel and salmon. Garrey states that transferring common eels 262 BIO-CHEMICAL JOURNAL from salt water to fresh water did not lower the osmotic pressure of the blood. Greene (7), on the Physiology of the Chinook Salmon, finds a lower osmotic pressure in those caught in fresh water, but thinks the small difference may be due to absence of food or to changes in the metabolism due to the changes in habits and the breeding period. In view of these conflicting results I was encouraged to take up this investigation through a suggestion of Prof. Brandt at Kiel, and to examine the osmotic pressures and chlorine contents of the blood of fishes living under natural conditions and not alone under the experimental aquarium conditions. ‘This is in many cases a matter of great difficulty, and most of the experiments previously made, including Sumner’s, have been performed under the somewhat artificial conditions of the aquarium. It seemed very important, therefore, to supplement the aquarium results, and I was fortunately able to do so by obtaining permission to take my apparatus on the German investigation steamer ‘ Poseidon’ on one of the expeditions from Kiel through the Kattegat and Skagerack to the North Sea. In this way I was able to examine fish from water of gradually varying density, and the number was only limited by the very bad weather this last February rendering it often both impossible to trawl for the fish and to perform any other experiments. The first determinations were made at Kiel University, where fish were easily obtained living, since they are brought into the harbour at Kiel in submerged boxes and kept in the water until the actual moment of selling. ‘The osmotic pressure has been determined by the freezing point method, and in order to make more certain of the correctness of the results, the freezing point of distilled water was determined before and after each series of observations. The Beckmann thermometer differed slightly from those in general use in having a shorter bulb so that only 10 to 15 c.c. of blood were necessary instead of the 18 to 20 c.c. usually required. This I found to be a most important acquisition, since it is often difficult to obtain larger quantities of blood from small specimens. The thermometer was made by Goetz, Leipzig. In almost all cases the blood was obtained by cutting the caudal artery and withdrawing the blood with a pipette. OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 263 It was always easy to obtain blood in this way, if the fish was actually living at the time and the heart beating. This occurred in every case, so that the blood was taken from the living fish and the osmotic pressure determined immediately, allowing no errors to creep into the results from decomposition. It has been stated by Hamburger (14) and Hedin (15) that it is the same whether one uses the blood, blood plasma, or serum, because the blood corpuscles in suspension have as little effect on the osmotic pressure as sand grains, but no decom- position should have taken place. Further investigations are being made with regard to this point but need not be considered here since for purposes of comparison the blood for the following experiments was always taken in the same way and the whole blood was used in every case. The depression of the freezing point is expressed here in the usual way as A and the comparisons are made in terms of this depression. The actual pressure in atmospheres can be found by multiplying the depression in degrees by eee After the determination of the A, the blood was removed from the Beckmann’s apparatus, and a quantity carefully weighed and transferred to a porcelain crucible. Powdered chlorine-free sodium carbonate was added and the whole evaporated to dryness and then slowly incinerated by moderate heat to prevent any considerable loss of chlorine. The residue was extracted with hot distilled water, filtered, and determined by Volhard’s method. To the filtrate nitric acid was added in slight excess. ‘To this solution N a definite quantity of To Ag NOs was added from a burette, taking care to use more than required to precipitate the chlorine so that in the solution there is an excess of silver nitrate. The precipitated silver chloride was filtered off, and the amount of silver nitrate in the filtrate determined by titrating with potassium sulphocyanide, using iron ammonium alum asindicator. This gives the quantity of excess, and by subtracting it from the quantity originally taken one has the amount of standard silver equivalent to the chlorine in the solution, from which the chlorine percentage can be easily reckoned out. 264 BIO-CHEMICAL JOURNAL The experiments made have been grouped into series according to time and place, beginning at Kiel and ending at Helgoland. In every case where the A is given, this is the average of three deter- minations, the degree of ‘ under cooling’ being kept small and, as far as possible, the same throughout. Series I—Feb. 5th, 1908, Kiel Harbour :— Sea water from Kiel Harbour A — 1-093° Chlorine contents of harbour water I-125 per cent. Salt contents 2-033 per cent. Blood from cod (Gadus morrhua) A — 0-720° Chlorine contents of blood o-50 per cent. Series II, Feb. 6th, K1el Harbour :-— Blood from Gadus morrhua A — 0-750° Blood from Gadus morrhua A — 0751 Chlorine contents of blood 0-50 per cent. and 0-503 per cent. respectively, Series III, Feb. toth, Kiel Harbour :—Blood taken from three specimens of Pleuro- nectes platessa (plaice), a very small quantity of blood is obtainable from a single fish. Depression of the freezing point A = — 066° Chlorine contents of the same blood 0-500 per cent. The difference in the osmotic pressure between the blood of the cod and the plaice is here notable, and illustrates the differences which are found to occur amongst different species of teleosts from the same water. Series IV, Feb. 12th, Kiel Harbour :—Blood from three large Pleuronectes platessa taken as in the previous experiment— Depression of the freezing point A = — 0:650° Chlorine contents of the same blood 0-531 per cent. The blood for both determinations was a mixture of that from the three fishes used. Depression of the freezing point for ovarial fluid from the same fishes A = — 0-630. Series V, Feb. 16th, 1908—On S.S. ‘ Poseidon.’?—Baltic Sea, just outside the Kieler Bucht. Depth 27 metres. Temperature at bottom 1-88°C. Salts contents of bottom water 2:6 per cent., and of surface water 1-46 per cent. Depression of the freezing point for bottom water A — 1-3° The following determinations were made :— Gadus morrhua A — o-758° Gadus morrhua A — 0-710 Gadus morrhua A — 0-730 For each of the following determinations, three fish were used :— Pleuronectes platessa A = — 0-718° Pleuronectes platessa Aan 0*720 OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 265 Sertes VI, Feb. 17th, 1908, S.S. ‘ Posetdon.’? — Station, 21-24 metres. Between Jutland and Seeland. Temperature at bottom 2-99° C. Sp. gr. at bottom’ 24-6... Salt contents: surface 2-93 per cent., and bottom 2-97 per cent. A sample of bottom water gave Pleuronectes flesus (2 specimens used) Gadus morrhua Pleuronectes platessa (4 specimens used) ... Raia radiata Kattegat II]. Depth A = — 1°665° A = — 0-96 A= —0715 Ave SR O79 A= —I°51 This last named was the first elaamobranch examined, the great difference from the teleosts and the resemblance to the A for sea-water is noticeable. \\ Lie J ~~ 4 59 s-- i ‘ / \ ; 2 ! reg ies ~ NY) : d Me he na 5: sues } “ \s \ C rp a >. <2 57 Nolin TH ‘s, \ Ww \ SEA Sal. N \ Zz m XN rk \ Pt \ 8 \ ae j ss << 7 ‘I \ v4: om pig Ce / ~., vo S i f N Helgcland ~4 ~ \ oor 4 7 2 % é > 10 WR Cruise or ‘ Poserpon,’ Feb.-March, 1908.—The stretches in black connected by the dotted line indicate the places where the trawl was used, and the numbers the series of determinations referred to in the text. I. The figure given above (and in the succeeding determination for the Specific Gravity of the sea water) + 1000 equals the true Specific Gravity, that of distilled water being considered as 1000. 266 BIO-CHEMICAL JOURNAL Series VII, Feb. 17th, 8.S. * Posetdon.—Kattegat, near coast of Sweden and about thirty miles North of Seeland. 37-57 metres deep. Temperature at bottom 3:7° C. Sp. gr. at bottom 26-0, Salt contents: surface 1-93 per cent., bottom 3-15 per cent. A sample of bottom water gave api = + 171° 1. Gadus morrhua ths ze voce A= —08 2. Gadus morrhua 32 “ ae) Alen O77 Gadus aeglefinus re ae roe A= —075 Raia batis ... F wip 56 A = — 1-820 1. Acanthias vulgaris... iv en A = — 1°820 2. Acanthias vulgaris... kat Ys A= — 1795 For the determination of the blood from Gadus aeglefinus, five or six fish were used. All the remaining fish were large, the cod being 1-3 metres in length. The increase in the depression of the freezing point for elasmobranch blood is to be noticed here, and the likeness between the two species. Series VIII, Feb. 18th, 8.8. ‘ Poseidon.’—Kattegat, between Denmark and Sweden, and direct East of Frederikshavn. 24-40 metres deep. ‘Temperature at bottom 4-9° C. Salt contents: surface 3-447 per cent., bottom 3-445 per cent. Sp. gr.: surface, 28-2, bottom 28-2. A sample of bottom water gave A= — 1°86° 1. Gadus morrhua oa i w A= —0O75 2. Gadus morrhua A = —0-76 Rhombus laevis oe oe . A= —oo71 Series IX, Feb. 19th, S.S. ‘ Poseidon. —In Kattegat, direct East of and not far from Frederikshavn, Denmark. Conditions practically the same as above, both catches were trawled in the same stretch of water and no further observations taken. Rhombus maximus... es .. As —0-79° Series X, Feb. 20th.—In Skagerack, West of and not far from Skager Point. Depth g2 metres. ‘Temperature at bottom 5°02°C. Sp. gr., surface 28-4, bottom, 28-4. Salt contents, surface and bottom, 3-472 per cent. A sample of bottom water gave a A = — 1°893° Gadus morrhua A= —0-74 1. Anarrhichas lupus A= — 0:84 Anarrhichas lupus = a2 A= —-073 Cyclopterus lumpus ... ee Re A= — 0:66 The Anarrbicas were large specimens, length respectively 78 and 71 centimetres. The rather remarkable difference between these two fishes with regard to the osmotic pressure is another case of the variation sometimes noticed for the same species, in the same water. The other interesting feature here was the low osmotic pressure of the Cyclopterus blood. 2 an rey: ere = 3 OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 267 Series XI, S.S. * Poseidon,’ Feb. 21.—Skagerack, middle of channel, between Norway -and Denmark. Depth 130-192 metres. Bottom temperature 6-76°C. Sp. gr: surface 28°1, bottom 28-6. Salt contents: surface 3-434 per cent., bottom 3-519 per cent. Freezing point of the bottom water... A = — 192° Lophius piscatorius ... Fs wi at o63 Gadus aeglefinus Bie A cs A= —0-74 The Lophius weighed 14 kilos and was, therefore, of considerable size ; the blood was easily obtained, and the extremely low osmotic pressure is rather remarkable for a marine fish. Series XII, S.S.‘ Poseidon,” Feb. 26th—Middle of channel on the boundary of Skagerack and North Sea. Depth 54 metres. Bottom temperature 5-31°C. Sp. gr. : surface 28-55, and bottom 28-65. Salt contents: surface 3-499 per cent., bottom 3°511 per cent. A sample of bottom water gave A = — 1-96° Anarrhichas lupus A= —o0-74 Lota molva ... , A= — 0-66 Hypoglossus vulgaris ... a aay A= —078 Lophius piscatorius ... eh oy A= — 068 Gadus pollachius ee ahs ree A= —073 Raia valonia vas ie 3 vas A= —2°0 The fish used for the above determinations were all of very large size, the Lota molva being 131 centimetres long and weighed 17-5 kilos. Series XIII, S.S.‘ Poseidon,’ March §th.—North Sea about 100 miles West of Stavanger, Norway. Depth 105 metres. Bottom temperature 6-78°. Sp. gr.: surface 28-2, and bottom 28-6. Salt contents: surface 3-467 per cent., and bottom 3-512 per cent. Bottom water ide a wee OFS A! aay ge? Gadus morrhua bi s¥d aK A = —0o70 Gadus virens oak Pry 3s A= —o7I Gadus aeglefinus uo ix "pes A = —o-78 Lota molva a ons me A = — 068 The Gadus morrhua has a remarkably low osmotic pressure here, which is probably pathological ; extremély few specimens were present in the trawl. 268 BIO-CHEMICAL JOURNAL Series XIV, S.S. ‘ Poseidon,’ March 7th.—North Sea, about 70 miles North-West of Helgoland. 40 metres deep. Bottom temperature 4°53° C. Sp. gr.: surface 28-5, bottom, 28-5. Salt contents: surface 3-479 per cent., and bottom 3-485 per cent. Bottom water A = — 190° 1. Gadus morrhua t A= —0-73 2. Gadus morrhua A = —0-79 3. Gadus morrhua A= —0-75 4. Gadus morrhua A= —077 Rata clavata A = — 1-99 With the exception of the Ray and the specimens of Gadus morrhua, the contents of the trawl were small plaice. ‘The four specimens of Gadus morrhua were used to determine to what extent variations might occur in the same species caught in the same water. Series XV, Helgoland, April, 1908.— Blood from three specimens Pleuronectes platessa A= =—0-78° ” ” three » ” ” A=-— 0-848 ” »” five ” »” ” . A=-— 0750 ” ” three ” ” ” A=-— 0°773 Chlorine estimation for blood used in the second and third of the above determina- tions 0-60 per cent. Chlorine estimation for blood used in the last determination 0-537 per cent. Series XVI, Helgoland, May, 1908.— Gadus morrhua—blood as me . h'= Blood from two specimens Gadus morrhua ora A = —0-778 Blood from five specimens Gadus morrhua A= Chlorine estimation for blood from the first specimen 0-507 per cent. Chlorine estimation for blood from the cod used in the last two determinations 0°530 per cent. Freezing point for bottom water A = — I-go. Salt contents of bottom water 3-485 per cent. The first point to be noticed in this series of estimations, is the great variation that occurs both amongst different specimens of the same species as well as between the different species themselves. It shews the great necessity of making a number of determinations before deducing any theories. For the teleosts examined the A varied from — 0-63 to —o-96 and this was not due to any great difference in the external medium since the fishes with the average — 0-96 came from water with a lower A than the fish with A 0-63. It was therefore “obvious that the different species could not be compared directly together unless specimens of the same species had been caught at all — OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 269 or at many stations. Now the most important determinations are those made at Kiel and Helgoland where the convenience of alaboratory was at hand, and unfortunately only two species could be obtained living at Kiel, namely Gadus morrhua and Pleuronectes platessa. Fortunately, however, the codfish were caught at almost every station, and so form an interesting series; and plaice were also caught in Helgoland to allow of a comparison with the Baltic Sea specimens. The following table gives the determinations made on the plaice :— Salt contents of Place where fish were caught A for blood A forsea-water sea-water Chlorine in blood per cent. per cent. Kiel 1 apa peor Smee — 0°66 \ ; {O*500 — * “oO 2 < — 0°65 093 3 (0-531 Series V.—In Baltic Sea ... — 0-718) ns v ae —o72 ) Serres VI.—Kattegat ne — 073 — 1°66 2°97 — Helgoland ... as Ae —o-78 | — 0848 Koyere) pg 3°48 — | (0°537 — 0°773 It will be seen that a direct increase in the osmotic pressure ‘of the blood takes place as the density and osmotic pressure of the sea-water increases. The average for Kiel is — 0-655, and for Helgoland — 0-787; that is, whilst the sea-water bathing the fish has increased in osmotic pressure so that the A has changed from — 1-093 to — 1-90, an increase of 74 per cent. or almost 10 atmospheres, the osmotic pressure of the blood has increased by 20-1 per cent. or about 1-5 atmospheres. This is a very interesting result and shows that the plaice at least have an osmotic pressure which is dependent to a certain extent on the sea-water, though nothing like the Elasmobranchs, since an increase of 74 per cent. in that of the sea-water produces an increase of but 20-1 per cent. in the fish. The chlorine contents also shew an increase corresponding to the increase in the osmotic pressure; the average for Kiel being 0-515 per cent., and for Helgoland 0-557 per cent. The relation of these changes will be discussed after the results for the cod are given in tabulated form. 270 BIO-CHEMICAL JOURNAL Results of Chlorine and Osmotic Pressure Determinations for the Cod (Gadus morrhua) Salt contents of Place where fish were caught A for blood A for sea-water sea-water Chlorine in blood per cent. per cent. Kiel ves res ‘ink — 0°720' fe 790 Rees F935 0°503 Aber: sire bags Series V.—In Baltic Sea ... —o758 — 0-710 — 1°30 2:6 _ — 0°739 Series VI.—In Kattegat ... —O°715 — 1:66 2°97 — Series VII.—In Kattegat ... — 0-80 — 1-71 3°156 -= ~tO77 Series VIII.—In Kattegat _ ee 2 hie ee Series IX .—In Skagerack ... — 0°74 — 1:893 3°472 — Series XIII.—North Sea ... — 0°70 — 1°95 3°S12 — Series XIV —North Sea ... — 073 riO79 0-75 1-90 3°485 rig hf Helgoland ... He + — 0-748) — 1:90 3°485 0:507 — 0778 — 0°748 thee" The figures in the above table form a rather startling contrast to those for the plaice. More specimens have been used for the esti- mations of codfish blood than that of any other species, perhaps forty or more in all, and never are two alike. ‘There seems to be a very considerable variation in specimens of the cod taken from the same place, considerable when compared with other teleosts that I have examined since, and this variation is greater than the actual change of the blood between the two places Kiel and Helgoland. If the average be taken for the first two series Kiel and Baltic Sea specimens the A is — 0-73; the fish are from water of A — 1-093 and A — 1-30, In the same way the average for the fishes from Series VI, VII and VIII is —.0-759, the sea-water being A— 1-66— A — 1-86, and lastly Series IX—Helgoland gives an average A —0°757 (leaving out the fish with A 0-70 caught in Series XIII and which a OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 271 is probably in a pathological condition), with sea~water A —1-89— BiEQO) 200 = ~ Thus if the averages of these three sets are taken, there is a slight increase between the fishes of the Kiel and Baltic Sea examined, and those of the Kattegat, Skagerack and North Sea, but instead of an increase of 20 per cent. as occurs with the plaice, there is only an increase of about 3-9 per cent. in the blood for an increase of 74 per cent. in the sea-water. I mention these averages in order to shew that if any steady variation can be said to take place it is in the direction of an increase in the denser water of the North Sea. The difference in the averages of the Kiel and Helgoland fishes taken separately from the others is as follows :—Kiel average A = — 0-740, Helgoland A — 0-758—an increase of A — 0-018, whilst the variation amongst specimens caught at the same place is in one case A — 0-73—A — 0-79. The conclusion arrived at therefore here, is that a slight increase of osmotic pressure occurs in the blood of cod caught in water of higher salt contents, as the North Sea, but that this increase is only small compared with that taking place in Pleuronectes platessa under the same conditions, and is furthermore overshadowed by individual variation. Another point to be noticed in the series of experiments made on the ‘ Poseidon,’ and which has been verified by further observations made at Helgoland, is the differences occurring between. the species. The numbers given by previous observers, often from single observa- tions, have not sufficiently emphasised these differences, since they may be put down in the same category as those occurring between ‘individuals of the same species. If, however, averages are taken for different teleosts, it will be found that in spite of variation there is a ‘mean’ for eath species which is not the same. ‘Thus, for example, Lota molva and Lophius piscatorius have a low osmotic pressure average A = — 0°65, whilst that for Pleuronectes flesus is much higher averaging about A = — 0°85, for fish caught in sea-water of A — 1-go. The figures for the Elasmobranchs agree very satisfactorily with what: was expected from the results of Botazzi; there is little variation between different species caught in the same water, the 272 BIO-CHEMICAL JOURNAL osmotic pressure is almost the same as that of the surrounding sea- water, and as this increases in density so does the osmotic pressure of the blood: change. Thus the first Elasmobranch caught on the voyage, a specimen of Raza radiata, gave A — 1-51, the A for the sea-water being — 1°66, and later when the osmotic pressure of the sea-water had increased and the A was — 1-95— — 1-98, two specimens, R. valonia and clavata, gave for the blood A — 2:0 and —.1-99 respectively. | In view of the small amount of change taking ces in the blood of the cod and on account of the above experiments. generally, it was thought advisable to determine the osmotic pressure and chlorine contents of some freshwater teleosts that were obtainable in Kiel, and particularly for the eel, in order to see what difference existed in the blood of typical freshwater fishes. The specimens had all been kept in fresh water in large well-aerated tanks for a few days from the time that they had been removed from the breeding ponds and freshwater lakes in Schleswig Holstein in the neighbourhood of Kiel. : Tank water in which fishes were living w. A = 0°020° Care vids as 0. 14° blood A — 0-487 Abramis brama ... ny ean » A—osIo Eel (Anguilla vulgaris) A = »» , & —0:570 The chlorine estimations for the blood of the above fishes gave. the following results pes / Carp blood contained... Maas) 12 per cents Cll prin Abramis brama do. ead ez rity eye 0°253. 55 ” Eel do. — tee oO 277 »” re. Thus the osmotic pressure and chlorine contents of the blood of freshwater fishes is much below that of marine teleosts, and the state- ments of Griffiths and Fredericq with regard to the salt contents of freshwater fishes cannot be regarded as correct, for the marine teleosts have about 50 per cent. greater chlorine and, therefore, salts contents. These figures agree with those of Quinton’s for other freshwater teleosts, and it must be observed that the chlorine contents of the blood is much higher than the chlorine contents of the body, or the whole fish. These determinations were completed by investigating the OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 273 changes taking place in the blood of the freshwater eel when placed in sea-water, and I am at present engaged on the complementary experiments with the same species of eel caught in the sea. A large eel-taken from the freshwater tank was placed abruptly into slightly diluted North Sea water, the A for which was — 1-59. This abrupt change was followed by a copious secretion of mucous, but after a few hours the specimen appeared quite normal. After an interval of six hours in this water the blood was taken and examined. . By Blod ... ... A—o66° prenn “bor. ...-- Chlorine ... a 0°367 per cent. _ There is evidence here of considerable change, for, compared with: the A for the cel in fresh water, the freezing point is 16 pet cent. lower and the chlorine contents have also increased considerably. | _ Another still larger specimen was therefore taken, as before, directly from fresh water and placed this time in sea-water rather more diluted with fresh (sp. gr. 20:3). This specimen endured the change more calmly and without such a mucous secretion. The following was the procedure :— | ; 10.30 a.m.—Eel placed abruptly from fresh water into diluted sea-water, sp. gr. 20°3. | 3.45 p.m.—Sp. gr. of water increased to 25-5 by addition of North Sea water. 6.45 p-m.—Sp. gr. of water, further increased to 29-3 by addition of some concentrated sea-water. The eel remained overnight in this water and at 10.30 a.m. the next morning, after-a sojourn of twenty-four. hours in sea-water, the eel being quite healthy in appearance, the osmotic pressure of the blood was determined. - Blood from eel after 24 hours in sea-water .... . A= —0-745° Hence the osmotic pressure had increased until it attained a pressure which is about an average for marine teleosts. The osmotic pressure of the Teleostei.in fresh water averages about A —o-527, and for those in North Sea water the A is about — 0-750, hence for those fishes like the eel and salmon which pass from the sea ‘to the rivers there must be a place where the external water is isotonic with the blood. ‘These fishes are, in short, capable 274 BIO-CHEMICAL JOURNAL of passing from a hypertonic fluid to a hypotonic fluid without injurious effects following. Of the marine teleosts which do not pass far into the rivers there are many which are found in the Baltic Sea, far East of Kiel, for example, Cottus scorpius, Pleuronectes flesus, Gadus morrhua, Lota vulgaris, Nerophis ophidion, occurring frequently together with others only occurring as visitors. Whether these species that occur frequently have passed their whole lives in this area is a question. Inall probability they do pass their whole lives and lay their eggs in this region. Now it will be seen from hydrographical tables that in the North-East Baltic where the fishes named above occur, the water has a salt contents below that of the fishes’ blood and is hypotonic. ‘Thus marine teleosts are often found in waters isotonic and hypotonic to the blood. ‘The Elasmobranchs on the other hand are only rare and as visitors in the West Baltic, and alto- gether absent from the South-East and North-East Baltic. This appears to be correlated with the fact that the constitution and osmotic pressure of the blood is much more dependent on the osmotic pressure of the external media than is the case in the Teleostei, but further experiments on the resistance of teleosts and elasmo- branchs to changes in the constitution of the sea-water are being made and the results will be discussed later. CONCLUSION AND SUMMARY - It has been shewn by previous observers that the marine teleosts have an osmotic pressure and salt contents differing to a great extent from that of the sea-water bathing their bodies, and in this respect shew a remarkable contrast to both the marine elasmobranchs and invertebrates. Certain workers have assumed from this that the body membranes separating the blood and internal fluids from the sea-water were impermeable barriers to water and salts. Sumner, by weight and chlorine analyses came to the conclusion that this was not the case, and that to a certain extent both water and salts could pass through these bounding membranes. Now we may assume here four possibilities for the bounding membranes :— OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 275 The membranes are impermeable to water and to salts. 2. ‘The membranes are semi-permeable, allowing water to pass _- through but impermeable to the dissolved salts. 3. The membranes are permeable to water and in a slight degree to salts. 4. The membranes are permeable to both water and salts. -+ If the first of these possibilities held good, it would be easy to understand how the teleosts retained their low osmotic pressure and salt contents, against the influence of the sea-water, but no change either in osmotic pressure or weight should then take place if the animals are placed in different media. This change does take place, for the experiments shew that in natural conditions the freshwater teleosts have a lower osmotic pressure than the marine forms, that this changes with the alterations in the water (as for example in experiments with the eel), and that even alterations in the salt con- tents prevailing in the sea, influences the osmotic pressure of the blood. Thus we may consider the first possibility, of the teleosts having impermeable membranes, as quite disproved. If the fourth possibility held good and the membranes were directly permeable to both water and salts, then the conditions for an osmotic change would not prevail and no change in weight should occur if the fishes were taken from salt-water and placed in fresh water. Sumner has shewn, however, that a change does occur and that water passes osmotically into the fish, increasing the weight. Moreover, if this was the case it would be difficult to conceive of the alterations in the osmotic pressure of the external media—for example the difference between that of the Baltic Sea water and the North Sea water, or the fresh and salt water in the case of the eel experiments—producing such a little change in the constitution of the blood. Furthermore, it would mean that some organ or organs were continually at work to such an extent that the osmotic pressure and salt contents of the marine teleosts were kept regularly at about one-third of that of the water bathing the outside of the membranes under consideration. This fourth possibility, is therefore, also impracticable, and we are reduced to the second and third. 276 BIO-CHEMICAL JOURNAL If the limiting membranes of the body are semi-permeable, that is, permeable to water but not to salts, then perfect osmotic conditions are set up, and any such increase of the osmotic pressure on the outside of the membrane as takes place in passing from the Baltic Sea to the North Sea, would cause a corresponding increase in the osmotic pressure and chlorine contents of the blood, since more water would pass out from the blood and body fluids through the membranes into the sea and leave the blood and fluids more con- centrated. Similarly, when placing an eel from fresh water into salt water, the great increase in osmotic pressure in the external medium should cause water to leave the blood and body fluids, making the concentration and osmotic pressure of these latter higher than before. This is exactly what takes place in the experiments. Sumner assumes also a slight permeability to salts, but only slight, so that the osmotic conditions are preserved and exactly the same state of things would occur. Hence we must conclude that the bounding membranes of the teleostei are either semi-permeable, permeable for water and not for salts, or to a small extent for salts. I am of opinion at present that the permeability is only for water, and that Sumner’s experiments do not indicate conclusively a permeability for salts; this, however, will be discussed in a later paper. ‘The increase in chlorine contents in these experiments does not prove that chlorine has passed into the blood, since the same result would be obtained by water passing out, and therefore increasing the concentration. If now we conclude that either of these two possibilities holds good, and the experiments prove this to be the case, then how do the marine teleosts contrive to maintain an osmotic pressure and salt contents much lower than that of the external medium, whilst freshwater teleosts have a higher osmotic pressure and salt contents than the external water? Allowing that the membranes are permeable to water, then. there must be a continual stream of this fluid from the bodies of marine teleosts outwards into the sea-water and vice versa in the freshwater teleosts, but in spite of this the constitution of the blood remains practically constant. I believe this to be accounted for by the three following assumptions :— : + ‘oes OSMOTIC CONCENTRATION OF THE BLOOD OF FISHES 277 1. The permeability for water is not very great. 2. ‘The permeable membranes are of small extent. ~ 3. The actual loss or gain in water by the blood is counteracted by resorption and secretion. The permeable membranes of the teleosts appear to be confined to the gills, the body walls of the fish being impermeable, because fresh water has practically no harmful effect on a marine teleost if the body part only is immersed in it, and sea-water flows over the gills. The permeability of the gills for water must not be great or one would have the secreting organs working continually at ‘ high pressure’ to keep the blood under constant conditions against the action of the external media. Since, however, an increase of 74 per cent. in the osmotic pressure of the external fluid produces a much less increase in that of the blood, only 3-9 per cent. in the case of the cod, the amount of water passing from or into the fish must not be large. It is, however, large enough to show that the blood of the teleosts is not altogether independent of the external medium and a considerable alteration in the constitution of this latter is accompanied by a corre- sponding alteration in the constitution of the blood in defiance of any organs working to maintain this constitution constant. SUMMARY 1. The blood of marine teleosts has a considerably higher osmotic pressure than that of freshwater teleosts. 2. The change of density in the sea-water from Baltic to the North Sea is accompanied by a change, though small, in the osmotic pressure of the blood. 3. This same change occurs when a common eel is taken from fresh water and placed in sea-water, the osmotic pressure changes from that typical of freshwater teleosts to a much higher pressure, about the average for typical marine teleosts. 4. ‘These changes indicate a permeability of the bounding mem- branes, probably only the gills, to water. 5. The teleosts contrive to maintain, partly by physiological 278 BIO-CHEMICAL JOURNAL means, an osmotic pressure for the blood which is almost independent of the external water, and only great changes in the constitution of the surrounding medium affect this constancy. 6. Though considerable. variations occur in any one species of teleost, in water of the same density, yet there is a ‘mean’ for the osmotic pressure of the blood which is distinct and peculiar to the respective species.” ! 7. Different species react differently to the same changes in the outer medium. REFERENCES (1) Bert, P. ‘ Sur les Phénoménes et les causes de la mort des animaux d’eau douce que I’on plonge dans Peau de mer.’ Comptes Rendus de l’ Acad. des Sciences, t. LXXIII, 1871. (2) Bottazzi, F., and Enriques, P. ‘ Uber die Bedingungen des osmotischen Gleichgewichts und des Gleichgewichtsmangels zwischen den organischen Fliissigkeiten und dem Ausseren 1 Medium bei den Wasserthieren.’ Archiv. fur Anat. u. Phys., Supp. Band, 1got. (3) Dekhuyzen. Ergebnisse von osmotischen Studien, Bergens Museums, Aarbog, 1904. (4) Fredericq, L. ‘Influence du milieu ambiant sur la composition du sang des animaux aquatiques.’ Archiv. de Zool. Exper., 2 série, t. IIT, 1885. (5) Fredericq, L. ‘Sur la concentration moléculaire du sang et des tissus chez les animaux aquatiques.’ Archiv. de Biol., t. XX, 1904. (6) Garrey, W. E. ‘Osmotic Pressure of Sea Water and of Blood of Marine Animals.’ Biol. Bull.,: ' Vol. VIII, 1905. (7) Greene, C. W. ‘ Physiological Studies of the Chinook Salmon.’ Bull. U.S. Bur. of Fish., 1904. (8) Griffiths. Physiology of the Invertebrata. London, 1892. (9) Hamburger. Osmotische Druck u. lonenlebre in den medicinischen Wissenschaften. (10) Overton. ‘ Neununddreissig Theses iiber die Wasserdkcnomie der Amphibien und die osmotischen Eigenschaften der Amphibienhaut.’ Verband. der Phystkalisch-medicin. Gesell. xu Wirzburg, N.F. Band XXXVI, 1904. (tr) Quinton. ‘Communication osmotique chez l’Invertébré marin normal, entre le milieu intérieur de l’animal et le milieu extérieur.’ C. R. de. ? Acad Sc., t. CXXXI, 1900. *“Perméabilité de la paroi extérieure de l’invertebre marin, non seulement a l'eau mais encore aux sels.’ C.R. de P Acad. Sc., t. CK XXI, 1900. (12) Rodier, E . ‘Observations et expériences comparatives sur l’eau de mer, le sang et des liquides internes de animaux marins.’ Travaux des lab. de la Station Zoologique d’ Arcachon. (13) Sumner, F. B. ‘ Physiological effects upon fishes of changes in the density and salinity of water.’ Bull. U. 8. Bureau of Fishertes, 1905. (14) Zentralblatt. }. Physiologie 11 (1897). (15) Zentralblatt. f. Physiologie 7 (1894) and rr (1897). 279 THE EFFECTS OF VARIATIONS IN THE INORGANIC SALTS AND THE REACTIVITY OF THE EXTERNAL “MEDIUM UPON THE NUTRITION, GROWTH, AND CELL-DIVISION IN PLANTS AND ANIMALS By BENJAMIN MOORE, M.A., D.Sc., Fobnston Professor of Bio-Chemistry ; HERBERT E. ROAF, M.D., Demonstrator and Assistant Lecturer im Physiology ; anv ROBERT E. KNOWLES, M.D., University of Liverpool. From the Bio-Chemical Department, University of Liverpool ( Received May 20th, 1908) The experiments of which the results are here recorded may be regarded as a continuation of the work of Moore, Roaf, and Whitley’ on growth and cell division as modified by the presence of alkali or acid sufficient to cause only slight changes in the chemical reactivity of the cell. . The earlier experiments were carried out on the fertilized eggs of Echinus esculentus, and it was found that while small traces of acid repressed growth and stopped cell-division, small traces of alkali stimu- lated the division of the cell, and slightly increased amounts rendered the division of the cell and growth of the organism highly irregular and pathological. In the present experiments attempts were made to carry on these artificial stimulations of the cell, in higher plants and animals, including mammalia, and although the results are not in all respects like those in the simpler and unprotected organism of the echinus, they are in our opinion sufficiently interesting to merit description. In the earlier stages of the developing echinus, one has practically a naked mass of protoplasm, the cell membranes being very thin and delicate, and under such conditions the cells have little or no protection against the attack of the chemicals placed in the water in which it is suspended, so that the result obtained is the immediate reply of the t. Proc. Roy. Soc., B. Vol. LXXVII,p. 102, 1905. 280 BIO-CHEMICAL JOURNAL living cell to the added reagents. In the higher animal, and probably in the highly developed plant, however, there is the secretory and — excretory activity of the cells themselves to be reckoned with, and the mutual protection and assistance that they give to one another, whereby alkali is neutralised by synthesised acid and vice versa, and where excess of either alkali or acid is rapidly drained out of the circu- lation and out of the tissue fluids by the action of the excretory organs. Although, as will be shown later, by giving large doses of alkaline or acid salts, the reactivity of the blood may for the time be con- siderably changed, yet this occurs in a very mediate fashion, and not with the crude directness found when the alkali or acid are added to the fluid in which the cell is bathed. One circumstance which may be specially mentioned is that the fluid in which the cells of the higher organisms live is full of organic matter and especially of proteins, these possess the property of acting according to the demands of the situation either as acid or as alkali, and so effecting the corresponding neutralization of either alkali or acid. ‘This powerful factor against the disturbance of the natural equilibrium has to be constantly borne in mind in such experiments as we have to deal with here. Asa result of its operation, the concentration of either hydrogen or hydroxyl ions can never, fortunately for the living cell, be raised very high, and it is difficult to judge from changes in reactivity after incineration what the values were before incineration and in the presence of the proteins.’ Unfortunately, there exists no method by which the ionic con- centrations, or even the reactivity to indicators, can be satisfactorily determined in the natural conditions of the medium bathing the cells. We may infer from the experiments on echinus that the range of variation to either the acid or alkaline side compatible with life at all, is very short, and hence in the experiments recorded below the varia- 1. The question as to the maintenance of a constant reaction has been studied by. L. J. Henderson, Amer. Fourn. of Physiology, Vol. XXI, p. 427, 1908. This author has investigated the H and Ho concen- trations in mixtures containing HgCOs3, NaHCOs, NagHPO,, and NaH2PO,, and he shows that these are very efficient for maintaining a low concentration of H and Ho. Other substances, such as the proteins, play a minor part in the reactivity regulation. INORGANIC SALTS AND CELL STIMULATION 281 tions must have been well within this short range in spite of the large doses sometimes given. -—There is at work a compensatory mechanism much like that which regulates the body temperature, and the effect of even our large doses of alkali or acid, is but to set the level a little higher or a little lower, as exposure to extreme variations of temperature will set the body temperature a degree or so up or down. Even this slight change in the level of the gauge of the reactivity of the blood, if it may so be expressed, has, as will be shown, a profound effect upon the life and functions of the cells, so sensitive is the cell to the reaction of the medium in which it lives. Just as in a warm-blooded animal a few degrees above the normal temperature causes the most violent disturbance in the metabolism, and disarranges the whole mechanism, so in the animal where the reactivity or ionic concentrations in the medium are nicely regulated even the small disturbance in the balance which can be brought about in spite of the regulating mechanism, leads to profound effects. ‘The experiments have been made on such widely different types of living cells that it will be convenient to describe them in separate sections. A.—Own THE Errects or Actps AND ALKALIES AND OF ACID AND ALKALINE SALTs oN GROWTH AND CELL-Diviston IN VEGETABLE CELLS The plants used were a common variety of hyacinth (Hyacinthus orientalis) with pink florets, and the common onion (Allium cepa). Healthy bulbs of as nearly a uniform size as possible were chosen for the experiments. These bulbs were at first placed upon clean damp sand until theroots had commenced to grow, when they were transferred to hyacinth glasses which had been blackened on the outside with black lacquer to prevent action of light upon the rootlets. The hyacinth glasses held about 500 c.c., and the various solutions mentioned below in the desired concentrations were filled in until the solutions came just short of wetting the bulbs. The solutions were made up of fractions of gramme-molecular strength in Liverpool tap water’ and 1. This is a very pure surface water, and practically free of inorganic salts. 282 | BIO-CHEMICAL JOURNAL were changed from time to time. A few of the ends of the growing roots were cut off at certain intervals for the purpose of studying the effects of the solutions on cell-division, and nuclear changes, and all these were immediately fixed with Flemming’s strong solution; cut in paraffin; and stained for nuclear figures by Heidenhain’s ‘ Iron- alum, Haematoxylin Method.’ (See Plates.) In addition to preparing the rootlets in this manner measurements and observations were made at intervals of the gross growth and condition of the plants, the points noted being the length of the green leaves, the length of the flower spike, the condition of the roots, the stage of development of the flower, and condition of the florets, whether all green, pink, open or closed, form of whole flower head, etc. These observations and measurements as well as the concentrations of the solutions used both in gramme-molecular strength and in per- centages are given for the hyacinths in the accompanying able (Table I). The results obtained in the case of the onions, where the work was chiefly confined to the preparing and examining of the root sections, are precisely similar to those with the hyacinth, and hence need no separate description. The figures in the table printed in thick type show the stinnaiel of growth above the normal, which occurs chiefly with the alkaline solutions, potassium hydrate (No. 8), and alkaline sodium phosphate (Nos. 18 and 19); on the other hand the marked deleterious effects of even slight traces of acid is shown by the low figures marked sal brackets in the case of Nos. 5 and 6. Attention may be especially directed to several interesting points regarding rate of growth brought out by the table. First, the very small amount of free alkali or acid necessary to produce most marked effects is seen, and it is also shown that the range of variation in hydrogen and hydroxyl ions compatible with growth is a very narrow one. Secondly, much more of the carbonates and phosphates can be borne, but here the swing in hydrogen and hydroxyl ionic concentration is much less sharp, which i is the probable explanation of this result. There is nowhere seen a stimulating action with even the weakest ' : As °, os J of o-oor M = 0-012 %... INORGANIC SALTS AND CELL STIMULATION 283 —— ayy 12 a i or Acip AND ALKALINE SUBSTANCES. on GROWTH oF HyAcINTHS * (a) After seventy-one days’ period of growth (November 14th, 1905—January 24th, 1906). Substance and Concentration 1. Control = a 2. Sodium hydrate (N20H) o O-0o!r M - = 0004 54% née ee iY i; oy o-oo15 M = 0°006% ... 4. Hydrochloric Acid (HCI) o-00075 M = 0:00274% 5. ” . ” ” o-oo M = 000365 % 6. ” ” ” o-0015 M = 0-00547% 7- Potassium hydrate (KOH) o-oo1 M = 0-0056% 8. re = o-oo15 M = 00084 % -g. Sodium bi-carbonate (NaHCO,) o-oo15 M = 0:0126% Io. - J i 0-003 M = 00252 % II. a = z e005 M = 0042 % ... 12. » ” ” oor M = 0-084.% 13. Sodium carbonate (Na,CO,), o-0015 M = o-0159 % 14. ” ” 0-003 M = 00318 % 15. Mono-sodium shieohake (NaH,PO,) (acid phosphate) 070005 M = 0:006% 17. = i Pe coors M = 0-018 % 18. Di-sodium phosphate (Na,HPO,), (alkaline phosphate) o-0025 M = 0:035 % 9. ” ” ” 0-005 M = 0:07% 20. ie ”» e oor M =0:14% LENGTH IN CENTI- METRES Green Flower leaves spike 8-5 4°4 8-2 4°6 79 44 76 50 (28) (x0) (2-0) _ 7? 3°7 10°3 37 77 41 5°6 2°6 CE: 31 a3 32 £? 44 6°6 4°1 8-9 50 79 38 48 gt 7°6 61 11 5°6 8-3 48 Remarks on condition of plant All green Slight traces of pink Very slight traces of pink Very slight trace of pink ; some roots macerated Very shortened green leaves and abortive spike; roots all macerated Practically no growth; - no spike formed; root rotted off All green All green - All green All green All green All green All green All green All green All green All green; roots slightly macerated Great deal of pink colour in closed florets Many pink fiorets, and some commencing to open A few florets, very faintly pink 284 | BIO-CHEMICAL JOURNAL* — (b) The following observations were made on the 76th, 79th, and 86th days of growth, the growth and development of the flowers having occurred rapidly ; the solutions and their concentrations being given above are not here repeated, and can be identified from the numbers. Seventy-six Days’ GrowTH SEVENTY-NINE Days’ GrowTH E1euty-six Days’ Growrn Length in cm. of Remarks Length in cm. of Remarks_ Length in cm. of Remarks ae es ee — - — WW at - Leaves Stalks Leaves Stalks Leaves Stalks et os | 5:3 Floretsturning pink 12-3 6:1 Lowerfloretsopen; 12:9 7:2 As before intermediate ones green to pale yel- low and fading 2. I0°0 6:1 One or two florets 11-4 8-5 Almost all open 13-3 11°3 All open nearly out 3. 12-7 5:9 Five florets open 12-99 68 More florets open 13:8 7-2 One side still green, than control, but rest open; closely green on one side packed and irregu- Jar flower 4. 93 6:6 Five florets open 105 =: 84s Nearly all open 11-4 10°4 All open 5. (3:1) (2°5) Small, green, abor- (3:1) (2°6) No development (3°1) (2:8) No development tive 6. (2°5) — Small, green, abor- (25) — No development (2-5) — No development tive 7. Elk 46 All green 125 (5:3 Slightly pink 136 5:8 Afewopen 8. 14°2 4:9 All green 15°9 5:8 Many pink, butnone 17°7 8-0 A fair number open open 9. -9°5 499 Afew floretstinged 105 5:3 A few pink, none 11-7 6-2 Lower open; top with pink open green 10. 8-8 4:1 All green 94 42 All green tog = 45,_—s Al: green, but well developed and healthy. Soa a 46 All green 8-2 5:5 A few pink; three 8-7 7:5 Nearly all open almost open , 12. 7°0 3:9 All green 8-3 44 All green gt 5:0 All green, but : healthy 13. 10°4 50 Afew showing pink 11-5 60 Three open, anda 131 72 A fair number open few slightly pink 14. 9'7 5:3 All green 104 67 Two open, some 108 8-5 Nearly all out — others pink 15. 114 7°9 All pink, ten open 128 11°0 Allopen - 14°9 12°0 As before 16. 10°7 54 All green 114 6 Several pink; none 13-9 7:5 Lower open; top open green by fat ai 28 All green 8:0 «= 4:2—s All green 8-1 49 All green 18. 103 12°38 All florets open 11°3 17°2 As before 14°6 21°3 All out and well expanded ; best of series by far 19. 15°0 9°0 Nearly all open 15°6 11°9 All open 17°2 146 As before 20. 108 5°6 Lower florets open, 13:0 6:3 Lower florets out, 14:0 6:6 As before top of flower but flowers irregu- yellowish and larly developed ; withered florets packed to- gether from non- development _ of the stalk, and un- healthy looking INORGANIC SALTS AND CELL STIMULATION 285 acid concentration used, and the histological examination (see next section) showed that even this minimal concentration in hydrogen ions damaged the root-tips and caused degeneration. On the other hand with the lower concentrations of alkali and alkaline phosphates (Nos. 18 and 19), there is distinct stimulation to increased rate of growth, which is confirmed by the histological investigation of the rootlets. ; It is very noteworthy that the metallic ion seems to have a specific effect upon the rapidity of growth of the green leaves as distinct from the flower stalk. ‘This is quite visible in the plants grown in the potassium hydrate solutions as No. 7, and more obviously still in No. 8. The phosphatic ion appears to have a more special effect upon the flower stalk, causing a great increase in length at the optimum strengths, -and a peculiar crowding together and irregular inflorescence due to packing together of the florets on a dwarfed stalk. This arrested development of the flower stalk, and very irregular and unequal development of florets was particularly well marked in No. 20, the strongest concentration of the alkaline phosphate used.. B.—Histrotocicat INVESTIGATION OF THE GRowING CELLS AND Divipinc Nuciet UNpErR THE INFLUENCE OF THE ABOVE REAGENTS The accompanying micro-photographs show clearly the profound changes in the growing cells and in the rate and character of the nuclear divisions in the rootlets, accompanying the changes in the external medium. In general terms it may be stated that the smallest amount of acid used arrests the growth and nuclear divisions at the root tips; causes the nuclei to become very obvious, but at the same time destroys their finer structure ; the cyptoplasm becomes hyaline, shrinks, and is destroyed, and the cell walls are clear and well marked. In addition to these effects the individual cells become enormously swollen, the linear dimensions being nearly doubled so that the cell volume is about eight times as great. The result is that the whole root tip becomes thicker. ‘Thus all the micro-photographs in each set are of the same magnification, but the tip in B and J, the acid preparations, is enormously enlarged, and the higher magnifications 286 BIO-CHEMICAL JOURNAL in the Sections B, and J, show that this is due to the enormous increase in the size of each cell. The dilute alkali, on the other hand, stimulates to excessive nuclear division, the lines between the cells become almost or quite invisible, and the whole root tip comes to look like a syncytium with rapidly dividing nuclei. But examination further up the root shows that the cell outlines are visible and hence that the syncytium appearance is probably fallacious. The amount of chromatin under the influence of the increased hydroxyl ion concentration, becomes largely increased, the chromosomes becoming much thicker and more massive. ‘There is also a great tendency to shortening of the chromo- somes into rounded masses or dots, and the size and amount of chromatin in the different nuclei become exceedingly variable. It has been impossible for us to make out with any certainty the number of chromosomes in the dividing nuclei either in the normal rootlets or in those treated with the alkaline salts, but both the number and the shape of the chromosomes appear to vary much more widely in the rootlets treated with alkali than in the normal rootlets. There appear to us to be two distinct forms of nuclei present in two different types of cell in both normal rootlets and chemically treated rootlets, one the normal parenchyma cell occurring in long rows parallel to the length of the root, and the other a cell with a longer and more prominent nucleus occurring intercalated between these rows, and appearing to possess a larger development of chromatin and a much greater number of chromosomes. The following are brief notes of the histological examination of slides stained by Heidenhain’s method of each of the Nos. I to 20, grown as above described. ‘The roots were cut off and fixed, all at the same time, after seventy-two days’ growth in the solution. Control (No. 1).—The cells are arranged in regular rows with very clearly seen walls marking off their outlines. The number of actively-dividing cells is small. Most of the nuclei are in the resting condition, and in nearly all these resting nuclei there are seen two darkly staining dots resembling nucleoli each surrounded by a small clear space, | INORGANIC SALTS AND CELL STIMULATION 287 In addition to the ordinary parenchymatous cells, with round or oval nuclei which make up the greater part of the section, there are, especially in the median portion of the rootlet, cells visible with very elongated nuclei, but little cytoplasm, and no very obvious cell outline or cell wall. These cells are much more frequently in cell division compared to their numbers, and the chromosomes and division figures appear to be different, the amount of chromatin being greater than in the ordinary cells, and the chromosomes longer, more bent, and twisted upon themselves, and less regularly arranged. In the subsequent sections treated with alkali the number of these cells is often enormously increased. In position these cells appear to be intercalated between the continuous rows of ordinary cells, to form discontinuous rows. “Sodium Hydrate (Nos. 2 and 3).—In No. 2 there is no very marked increase in the number of actively-dividing cells; but where division is in progress the chromosomes appear to be thicker and shorter. ‘These dividing nuclei are found chiefly in the second type of cell above mentioned, which has increased in number relatively to the other type, and many of the nuclei are exceedingly elongated. The nuclei of the resting cells are very prominent and longer than the normal, and the chromatin appears somewhat granular.* In the mid region and at the tip the cell outlines are entirely invisible, and the nuclei appear to be closely studded together in a kind of syncytium. The above differences are exaggerated in the stronger alkali of No. 3 (see micro-photographs B and B,), the number of dividing cells being greater ; the chromosomes in some cases are shorter and thicker, in others they are long and thin, and curved upon themselves into loops and very irregularly disposed in the division. In the resting nuclei the black dots are very conspicuous, and the size of the surrounding clear space is increased; occasionally the dot persists amongst the chromosomes in the dividing nucleus, but in the majority of cases it is missing. Hydrochloric Acid (Nos. 4, 5, 6).—There is not a single dividing cell to be seen in any of the three strengths of acid, and most of the rootlets are degenerated. Thus even the most dilute acid, less than 288 BIO-CHEMICAL JOURNAL 0-003 per cent. (000075 M.), is an intense deterrent to cell vitality. In the few cases where degeneration is not absolute the cell walls are most clearly visible and thickened, the cytoplasm is granular, and the nuclei shrunken and rounded. ‘The volume of the cell is also much increased (see micro-photographs C and C,, which are exactly the same magnification as the others in each series). ‘ Potassium Hydrate (Nos. 7 and 8).—In these sections the effects of alkalinity are more pronounced than in those from sodium hydrate 4s if the potassium ion had an additive effect (see micro-photographs D and FE, and D, and E,). The number of dividing cells is greater even in the more dilute strength than in the stronger solution of sodium hydrate. In the stronger potassium hydrate solution, there is seen a blurred mass of dividing nuclei in all stages of division on a common back- ground of finely granular cytoplasm, showing no indication of discrete cells. ‘The nests of chromosomes seem scattered about without any definite order; there are all grades of thickness and length of chromosome and spireme threads, and the contortions and shapes taken by the twisted chromosomes are very manifold. The black chromatin dots in the nuclei are of very variable sizes, and the surrounding clear space very conspicuous. Sodium Carbonate and B1-carbonate (Nos. 9 to 14). (See micro- photographs F, G, H and F,, G,, H,).—The same effects are seen as in the case of the free alkalies, but occurring more gradually and only with much more concentrated solutions. ‘The number of dividing nuclei is in all cases greater than in the normal, and in the bi-carbonates (Nos. 9 to 12 inclusive) a progressive increase in the number of dividing nuclei is seen pari passu with the increasing concentration of the sodium bi-carbonate. In the bi-carbonate series there is no appreciable effect upon the cytoplasm or cell boundaries, the walls are clearly marked, the rows of cells are beautifully regular, and there is no obvious increase in the number of the elongated nuclei of the median cells above referred to. ‘The number of dividing nuclei of the ordinary parenchymatous cells is, however, very obviously increased above the number in the control, the increase throughout the series is also striking. In some members of the series the nucleolus-like dots are very INORGANIC SALTS AND CELL STIMULATION 289 conspicuous, two or three in each cell surrounded by very clear Spramesz 1 9 fae “In the case of the normal carbonate, as might be perhaps expected from its more marked alkalinity, the effects are much more pronounced. The cytoplasm is much more granular, the regularity of arrangement of the cells in columns is considerably interfered with, the number of cells at some stage in the process of division is remarkably large. Even in the resting condition, the chromatin is very coarsely granular, and the chromatin threads in the fine skeins are very distinctly beaded in appearance. ‘The chromosomes wherever present vary enormously in length and thickness, and in the shapes into which they are twisted and contorted. There is also no regularity in arrangement of the chromosomes into any dividing figure. Mono-sodium-Phosphate or Acid-Phosphate (Nos. 1§ to 17). (See micro-photographs I and-J, and I, and J,.)—In all three of these pre- parations the regularity of arrangement of the cells is beautiful, and although the two more dilute solutions show more nuclei dividing than the control, there is none of that disordered and incoordinated division of the alkaline preparations, the arrangement of the planes of division is perfect and regular, and there is no such variation in the chromosomes. ‘The cell walls are more marked and clear than in the normal, and this culminates in the strongest solution in very much thickened walls which are brownish in colour in distinction to the blue- black staining of the chromatin of the nuclei. There is not a single dividing nucleus visible anywhere in the highest concentration, and the chromatin is very distinctly granular. There is a clear space, resembling the retraction space of the acid preparations, around each nucleus, and the cytoplasm is also granular, and has a precipitated appearance. A good many of the rootlets in this concentration are completely degenerated. On the whole there is a close resemblance between the strongest acid phosphate and the acid preparations, while the weaker acid phosphates probably demonstrate the effects of the more minute increases in hydrogen ion concentration. Di-sodium-Phosphate or Alkaline Phosphate (Nos. 18, 19 and 20). 290 BIO-CHEMICAL JOURNAL (See micro-photographs K and L, and K, and L,.)—The alkaline effects are not so characteristic as in the case of the free alkalies or the normal carbonate, as might perhaps be expected from the low degree of alkalinity (or hydroxyl ion concentration) of the solutions of this salt. Still, there is a noticeable increase in the frequency of dividing nuclei, but the divisions are well coordinated and the cells arranged in quite regular rows. ‘There is no dimming or disappearance of the cell outlines which are as well marked as in the control. There are, however, more variations in length, thickness, and arrangement of the chromatin in the chromosomes and skeins than in the control, and an increase in the number of the median elongated nuclear cells, which are nearly all at some phase in division. C.—Tue Errects or PHosPHATIC SOLUTIONS ON THE RATE oF GROWTH in AmMpuHIBIA (JAPANESE Newts, Triton pyrrhogaster) The marked effects of phosphates upon growth in both vegetable and animal organisms which we had observed in the sea- urchin eggs’, in the tadpole®, and in the experiments recorded in Section A in the hyacinths made it desirable to test the action more thoroughly in more highly organised animals. There are two different effects to be thought of chiefly in such experiments with acid and alkaline phosphates, the first being the variation in the reactivity of the medium, and the second the excess of the phosphatic ions, which is present both when acid and when alkaline phosphate are being used. ‘The change in reactivity due to variations in hydrogen and hydroxyl ion concentrations, is largely met in the more highly organised animals by the altered metabolism of the animal, and hence whether acid or alkaline phosphate be. employed, apart from the necessary effect upon metabolism, the chief factor of change in the inorganic constituents of the medium of the cell is the excess of phosphatic ions. | It is from this cause that the effects observed bear such a close 1. Moore, Roaf and Whitley, loc. cit. 2. Roaf and Whitley, Bio-Chem. Four., Vol. I, p. 88, 1906; and Roaf, ibid., Vol. I, p. 383, 1906. INORGANIC SALTS AND CELL STIMULATION 291 resemblance for the two series of phosphatic salt solutions, both in the experiments in this section, and in those in Section C on mammals, unlike the experiments on the echinus eggs and in lesser degree on the vegetable tissues where the most prominent change is the change in reaction, and the less prominent one the phosphatic excess. For this series of experiments a gross of Japanese newts were purchased, and a dozen of each placed in each of a dozen of large flat circular specimen jars, such as are used for mounting anatomical or museum specimens. The jars gave ample room for air and water, being ten centimetres in depth and thirty centimetres in diameter. The glass lid of each dish was supported on small chips of wood so as to allow free access to air without allowing the newts to escape, and in each jar a piece of clean old brick was placed so that the newts could at will leave the solution and rest upon this brick, the top of which stood out of the solution. At first the animals were kept in 400 c.c. of solution in each dish, which was just enough to cover the bottom, and the bodies of the newts, their heads being well out of the solutions. But later 1600 c.c. of solution was used. The animals were fed on minced raw beef twice a week or occa- sionally on worms instead, and the solutions were always changed the same day after they were fed, to prevent putrefaction of unused food in the solutions. The animals lived in good health under these conditions except in one vessel, where there was an accident, probably due to poisoning by a small amount of meat decomposing in the vessel; as a result the population of this vessel was reduced to two newts. In all the others the mortality was low and there were ten or eleven newts left alive in each at the end of the experiment which went on for over ten months time. The animals were weighed about once every fortnight, but in order to save space in the table only monthly weighings are here recorded. The weighing was done under exactly similar conditions in each case in the following manner. The superfluous moisture was drained off all the newts in any given solution and these were placed in a tared beaker and weighed. The total weight was divided by the 292 BIO-CHEMICAL JOURNAL number of newts, so giving the average weight of the newt in that particular solution. Table II gives for each date the percentage gain in weight of the average newt calculated as a percentage on the original average weight of the newt at the commencement of the experiment. This table gives the best idea of the rate of growth in the various concentrations of solution, but for purposes of comparison the actual average newt weight in each solution is shown in Table III. Regarding the conditions obtaining in this series of experiments it may be remarked that it is quite impossible to feed by hand such small animals with the phosphates. It is improbable that the skin served to any appreciable extent as a medium for uptake of the phosphates, but the food of the animals was placed in the solutions : and there is no doubt that a good deal was taken up in this way, and that the amount of phosphate taken up would be proportional to the concentrations of the dissolved salts. The strengths of solution taken were designedly low so as not to rapidly injure the newts, and to produce a slow chronic effect. Two controls were used in the series of twelve dishes in which tap water only was used ; these were numbers 1 and 12 in the Tables ; No. 12 did not grow rapidly at first but caught up later, and the per- centage increases at the end of 60-4 per cent. in No. 1, and of 62-0 per cent. in No. 12, are quite close together. Acid phosphate of sodium (NaH,PO,) was added to the set lots of newts in Nos. 2, 3, 4 and 5, in concentrations of 0-0005, 00015, 0:0030 and 0-0050 gram-molecular concentration respectively ; these figures correspond to the percentages of 0°006, 0°018, 0°036, and 0'060 of the dried NaH,PQ,. Six lots of newts were grown in the alkaline phosphate of sodium (Na,HPO,) in concentrations of 0°00025, 0°00075, 0°00150, 0700250, 0700500, and 0-00750 gram-molecular concentration respectively, corresponding to percentages of the dry salt Na,HPO, of o° 0035, 0'0106, 0°0213, 0°0355, 0'0710, and 0°1065 respectively. These concentrations were chosen from the experience of our previous work as those compatible with cell life, and below the limits’ likely to cause rapid damage. When regard is paid to the small per-. INORGANIC SALTS AND CELL STIMULATION 293 centages added to the solutions, the effect on growth recorded in the Tables is remarkable. Both the acid and the alkaline phosphates at a certain optimum concentration cause a well-marked increase in the rate of growth. The reason why both act alike in this respect has already been touched upon. ‘The optimum for the acid phosphate lies at 0-0015 to 0-0030 M, and that for the alkaline phosphate at about 0:00075 to o-oo150 M. Attention may further be drawn to the fact that the weights in all cases rose to a certain maximum and then declined; the maximum being reached for all early in August after which there is a general decline lasting to the end of the experiment on October 26th. It is also highly interesting to note that the newts in the controls, and in solutions of concentrations both above and below the optimum, never did attain to the amount of increase in weight seen in the optimum, but merely increased at a certain slower rate until the August maximum and then went down with the others. Effects of Higher Concentrations of the Phosphatic Solutions.—At the end of the experiments with the more dilute solutions which had been well borne throughout so many months, the same animals were used to test the more lethal effects of increased concentrations. Two sets of newts were taken, one in the acid phosphate and the other in the alkaline phosphate, and in each case the concentration was increased by definite amounts at regular intervals until lethal effects were produced. | Alkaline Phosphate.—The newts in 0-022 M. concentration were placed in the following successive concentrations at the intervals named :—o-030 M. for twenty-five days ; 0-037 M. for fourteen days ; 0-044 M. for three days; 0-050 M. for four days; 0-056 M. for five days. At the concentration of 0-056 M., the newts became strongly affected, and showed marked hyper-excitability passing into tonic con- tractions of a spasmodic character accompanied by excessive ophis- thotonus when handled, and rapidly died off in this condition if left in the strong solution. If removed to water, they recovered. More exact experiments were instituted with more dilute solutions, BIO-CHEMICAL JOURNAL 294 *(€ asn3ny) 3y81aM 4s03v018 Jo porsad ayy ye GyFram UI sasvoIDUT aSezUIOIEd JY} SUT] [LI1GIVA dy UT samn3y yoryy oy3 pue ‘yes Youe Jo UONeIQUIDUOD WNUTdo oY} YIeUF saUT [eJUOZIIOY ay} UI saIN3y yory oY], zo-z7g 0g-0L 86 gh.oL §=6—gg- gy «= d-z@L Ss SE-zqg Ss oS-1$ S Sz-0S = SEbz Ee IN TAY: ae Be "** Tomuos ‘ZI E145 ghzl Shogo gag, 98-62 zgSS thLb +$89.S€ og1f +192 Loto osdoo.o = s “II 46.$£L S.zg +1-£€g JoHRQ gz-bg ~ of-1Z $0.85 oz-oh +2.4€ 9f.0z zlo-o J 00S00.0 “ ‘“ ‘OL zo-1g gz-bg «=o. 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SOR ae 403s as oist sug> 408 ee kos 386 * Dosage in grammes per kilogramme of body weight in each case. Date 2and_day~ 343 grams. 23rd 24th 25th 26th 27th 28th 29th bb] ” > »? ”? ” ” ” » INORGANIC SALTS AND CELL STIMULATION 299 Taste IV (Continued) I II iit IV Vv 0°38 gram.*. 0°35 gram.* 0°35 gram.* 0°24 gram.* 0°30 gram.* 410 grams. — 578 grams. 386 grams. 343 os 400, = 607, 377s 343° <*> 416 ,, = Gis Lys: ae 39s 8 430s, — ioe os ee ss 354» 42555 — ys ae 476 6<2 345 ss 428, 0% 597» 336, 353» 421s, — 638 ~—_—,, Death. 339s 419 5, — 582 ,, Decrease in body 353 ” 407 ” es 587 tn weight 36°5 per 379» 4355s “= 604 ,, cent. 461, A265 a 596 ,, ey No phosphate given Lee 426-4; — 622 ay i 428, a 626° = co 348 ”? 414 2? $i) 613 2? Teak: 334 ”? 374 2 int, 513 ” hate 354 ” 390 ” are 558 ” Te 356 ”? 348 2” CIR 545 cB ete No phosphate given 355 ”» 358 ”? pe 579 ” iT 5 ee 360 =, — 540 yg abe 350 2? 372 ” = 565 3 — 344 2 386 ”? a Ss 587 ”? = 346, 382s, = 573» - 361 ” 385 ”? ae 571 ”? a, se oem No phosphate given Death. cs Se — 55875; ee Decrease in body Death. — 569) >: al weight 28 per Decreasein body — ey aoe 2, cent. "weight 29°5 per 9 — 550° 5, nYS = cent. — 568, pen BOK a al JAP 93 ae ih — — pl: rages =e Death. - Loss of body weight 29-8 per cent. The decrease in body weight in each case is very considerable, amounting on an average toa loss of 29-16 per cent. The other symptoms and results of post-mortem examinations and histological changes are given later. * Dosage in grammes per kilogramme of body weight in each case. 300 BIO-CHEMICAL JOURNAL Experiment IJ.—The next table illustrates the changes in weight of four guinea-pigs which were fed with the crystalline salt of the acid phosphate (NaH,PO,, 8 H,O). The amount of this salt given to each animal was 0-4 gramme twice daily. This dose corresponds to 0-11 gramme of P.O; or to 0:22 gramme for the daily amount administered, which for the approximate average weight of guinea-pig of 850 grammes used in the experiment corresponds to 0:26 gramme per kilogramme of body weight. Taste V.—Errects oN Bopy WeEIGHT or ADMINISTRATION oF AcID Puospuate (NaH,PQ,, 8 aq.) VI VII Vill a Date 0°24 gram.* 0:26 gram.* 0:26 gram.* 0:29 gram.* Ist day 927 grams. 846 grams. 846 grams. 757 grams. and ,, 851. < 55, 841, B30 3; 771» ad. | No phosphate given sth 35 878, 833, S18... Vi FOO. ae sth ,, 852 776 55 7785s 708 yy 6th ,, 860. xf 7a0- 55 34 gee 708s, ath oi 820° =f eee fee ata 676 ,, Sth. 800 __—,, (a eS 6615) gth ,, ey 708: | 55 iio, eee 6Eg Ioth ,, No phosphate given 11th .,, 835 5s 672 5, 725°» 664s, Fath, ce Cele Cas da a 656° ‘ings 59th... 866. 3 629. ;, a 634 if as 14th _,, 796) 53 O33 55 28S 620-ne sth ,, 793. 5 615, 667, 605, 16th ,, 784 5, 573 9s 679s 598 . «,, No phosphate Death. No phosphate oy eee given Decrease in body given 18th ,, Foes as : weight 32-3 per Goes. 59}\ am 19th ,, 806, cent. 709 ~,, 510i ee 20th ,, 70. © — iS ae Death. Sist 9 Sere — 4 Mase Decrease in 22nd ,, TOU xs _- O70 5 body weight 23rd_,, 7s eee — O08) <4, 32-7 per cent. 24th ,, No phosphate given 7 ta 2 eee — eee — 2008, cs ere — 690. — arth S:, paces eo 5 pale == * Daily dosage in grammes per kilogramme of body weight. RCD rie =~: Date 28th day 29th ,, 30th ,, ast 5, 32nd _ ,, 33rd, 34th ,, 35th ,, 36th ,, 37th ,, 38th ,, 39th ,, goth ,, INORGANIC SALTS AND CELL STIMULATION 301 Taste V (Continued) VL vi VIII Ix =F 14 gram.* 0°26 gram.* 0°26 gram.* 0°29 gram.* 750 grams. — 600 grams. — 761 ” aes 596 ” Ta 734 ” Rem 561 ” a No phosphate given 753 ” as 597 ”? eat 745 ” iw 622 ” a 747 ” +. 645 ” a 715 , 2? a 608 ” 73 738 ” TERK 625 ” car No phosphate given 744 ” erat 626 ” Bt 728 99 = 615 ” nee 745 ” aa 600 $3 — Death. Death. Decrease in body weight, 19-6 per cent. Decrease in body weight, 29-1 per cent. Nore.—The table gives the decrease in weight under acid phosphate administration, the average loss in weight being 28-4 per cent. ; other details are given later in the text. Experiment IIJ.—During the time that the animals in the above experiment were being treated with the acid phosphate, two more guinea-pigs were put on the alkaline phosphate ; in this instance the animals were given double the quantity, 7.c., o-8 gramme of Na,HPO,, 12 aq., equivalent to 0-16 grammes P,O; twice daily. food. ‘The results are shown in Table VI. Taste VI.—Errects on Date Ist day 2nd ,, 3rd, 4th ,, 5th ., ALKALINE Puospuate (Na,HPQ,, 12 aq.) x XI 0°37 gram.* 0°39 gram.* 855 grams. 821 grams. 818 > 855 9? No phosphate given 838, 8o2 | -’,, 752) in 745» * Daily dosage in grammes per kilogramme of body weight. One guinea-pig was kept as a control and not given anything except its ordinary Bopy Wei1cHtT oF ADMINISTRATION OF XII Control 802 grams. 827», 842 5, 835 (is 302 BIO-CHEMICAL JOURNAL Taste VI (Continued) x XI : XII Date 0°37 gram.” 0°39 gram.” Control 6th day Death. 722 grams. 755 grams. 7th ,, Decrease in body i, eer 804. "5 8th ,, weight 12-1 per O50 2a Biz ae gth ,, cent. 649», $28 ow roth _,, No phosphate given 13th: ,, a JOO oo 794 12th ” cans 737 oF) 794 ” 13th ” ae 754 ” 812 ” 14th ” ed 77° ” 805 ” rth: — 7 ae 803° 3 16th ,, — FOO cys : 808 si, Fgth = 5, No phosphate given 16th; — oy mae? 800 y 19th ” aa 722 ”? 813 ” arn. *s a FO 700 = eter: x; —- 62; 1.) Maer 2and ” rai 685 ” 787 ” Or eae _- O60. 5S At iy as a ee No phosphate given 25th ,, ee 673» 7783 26th 5 we 731 ” 809 ” 27th ” Bay 728 » 789 ” 28th 35 =e 685 ” 792 ” 2gth_,, — 795 os 8075, 30th ” pos 634 ” 79° ” i) ee No phosphate given 32nd ”? roy 652 ” 760 ” 33rd ” aoe 725 ” 765 ” 34th ,, Bar 669 783s 35th a 661, TTT 36th ” Sine 685 ” 79° ” 37th 4, No phosphate given 38th _,, —- mo, 800g, 39th 2» pees . 645 ” 800 ” 4oth ”? ey 645 ” 804 ” Death. Killed, Decrease in body and tissues fixed weight 21-5 per cent. and sectioned for control Symptoms OpserveD DurING THE PROGRESS OF THE ABOVE EXPERIMENTS As is shown by the tables given above the animals in all cases decreased in weight, the decrease being most rapid in the beginning and then more slowly with occasional variations in the upward direction, but on the whole showing a downward tendency until *Daily dosage in grammes per kilogramme of body weight. INORGANIC SALTS AND CELL STIMULATION 303 death ensued. The emaciation involved all the adipose tissue and also very extensively the skeletal muscles, the limb-muscles becoming excessively shrunken. Accompanying the decrease in weight a gradual enfeeblement of the animals occurred, so that they became torpid and remained almost motionless. It is interesting to note that although rapid decrease in weight occurred in the beginning of the experiment the animals were consuming more than the usual quantity of food, the appetite being so much increased that more than double the usual amount of food was taken. At a later stage the excretion of water by the bowel became increased so that the faeces lost their usual hard consistency and became very soft. It was noticed soon after the appearance of this soft condition of the faeces the animals lost their increased appetite and from this onward the amount of food taken decreased till it fell below normal, but the animals never reached a stage at which food was entirely refused. Reactivity of the Blood.—In order to determine whether the drugs administered had altered the reactivity of the blood, a series of experiments was carried out by the method described by Moore and Wilson. Determinations were made in the normal animal, ‘in animals under alkaline phosphate, and animals under acid phosphate. Taste VII Guinea-pig Drug administered Alkalinity of blood N Number g sis Control 30 H,SO, = Normal alkalinity N Number 2 ree Na,HPO, 25 H,SO, = Increased 5 N Number 10 744 Na,HPO, 25 H,SO, = Increased is N Number 7 ‘bbe NaH,PO, 33 H,SO, = Decreased 2 N Number 11 oes NaH,PO, 33 H,SO, = Decreased “ x Number 12 es NaH,PO, : 33°5 H,SO, = Decreased _,, 1. Bio-Chemical Fournal, Vol. 1, p. 297, 1906. 304 — BIO-CHEMICAL JOURNAL From the above results it will be observed that there was a marked increase in the alkaline reactivity of the blood in the animals receiving the alkaline phosphate, whilst on the other hand there was a decrease in the animals receiving the acid phosphate. The animals receiving the alkaline phosphate were not manufacturing sufficient acid to neutralise the alkali, and vice versa those receiving the acid phosphate were not manufacturing sufficient alkali to overcome the excess of acid. It is hence obvious that the amounts of drug administered had been sufficient to alter the reactivity of the plasma in the direction intended in each series of experiments. Post Mortem Examinations.—Post mortem examinations were made in twelve cases, in each case practically no adipose tissue was found subcutaneously or in the mesentery, the emaciation of the skeletal muscles was extreme, and the muscular substance pale in colour. On opening the abdomen, fluid was found in quantity in excess of normal in the peritoneal cavity. In some cases several cubic centimetres of clear amber coloured fluid was obtained. ‘The fluid quickly coagulated spontaneously on removal from the body. It showed an alkaline reaction to litmus and ‘ di-methyl,’ and acid to phenol-phthalein. In three cases small ulcers were found in the stomach, three in number in two cases and two in the third case; the ulcers were usually situated near the lesser curvature, were oval in shape and one to one and a half centimetres in diameter. Histological examination showed a close resemblance to acute gastric ulcer, the base was smooth, the edges clean cut, and the penetration extended to the submucosa. The liver was soft and friable, in some cases mottled and showing small yellowish patches on the surface. On cutting into the organ, small pale patches were noticeable in places. Portions of the organ were fixed (a) in Muller’s fluid and formol in equal parts, and (0) in Flemming’s fluid for histological investigation: The sections when cut and stained showed extensive and interesting changes which will be described in a separate section. Extracts of the liver were made and tested for glycogen and INORGANIC SALTS AND CELL STIMULATION 305 sugar, which were found to be abnormally low in quantity, the usual qualitative tests giving negative or doubtful results. The gall bladder was much distended in every case. The kidneys were usually pale in colour and in two cases showed small cysts on being opened. Aiistological Examination of the Tissues The Liver (Alkaline Phosphate).—The sections of the guinea-pig livers show in all cases degeneration of the liver cells, but the amount of degeneration varies in different animals and in sections from different parts of the same liver. In general the cells are vacuolated, and masses of round cells and polymorphonuclear leucocytes are observable throughout the tissue but especially well developed in masses lying around the vessels and about the portal canals, the vacuolation in certain places has progressed to the formation of larger cavities occupying nearly the whole of the cytoplasm. Complete destruction of cells is observable in places, and in certain situations rounded cavities larger than the liver cells are seen. In some sections well marked patches of tissue in process of necrosis are plainly marked off from more nearly normal tissue ; these patches vary much in size and in the extent to which necrosis has advanced ; they are usually marked off at their periphery by a thick layer of young connective tissue thickly studded with round cells and polymorphonuclear leucocytes. ‘The tissue within the patches at times contains liver cells staining normally but showing vacuoles and cavities of different sizes; in other cases the cells stain badly, taking on only a diffuse pink staining with eosin and showing no nuclei. In certain cases, on the outer margin are seen clusters of small round masses much larger than a liver cell which stain a deep blue with the haematoxylin. These are probably derived from nuclear chromatolysis of the liver cells, for similar dots are to be observed deeper in the necrosing islet - spread out more uniformly in liver cells which are not so far advanced in degeneration. In some cases the growth of young connective tissue is enormously increased so that it occupies nearly the whole of the tissue. Between the strands of the growing connective tissue, large clear spaces are observable, and interspersed with large cells 306 BIO-CHEMICAL JOURNAL occurring singly or in small groups which have the staining and appearance of liver cells; many of these cells are, however, larger than the normal liver cells, and in many cases they are multi-nucleated. The Liver (Acid Phosphates).—The liver is enormously congested, and the cells are vacuolated. The vacuolation of the cells is, however, different from that seen in the case of the alkaline phosphates, the nucleus often being surrounded by a clear space or the entire cyto- plasm of the cell takes on a very light staining. A considerable development of young connective tissue is seen around the portal canals. | The Kidney.—In sections of the kidney with both alkaline and acid phosphates there is acute nephritis particularly well marked in and around the glomeruli. Gastric Ulcers.—The ulcers resemble an acute gastric ulcer with erosion of epithelium and formation of granulation tissue. ExperRIMENTs ON Carnivora (Docs) Administration of the phosphates accompanied by determination of the metabolism of various products was undertaken in two. pro- longed experiments on dogs. One animal was given alkaline phosphate, and the other acid phosphate (See Tables IX and X). The animals were kept in boxes of the usual form for experiments in metabolism. ‘The animals were offered a diet of five hundred grammes of horse flesh daily, and were given as much water as desired ; the total amount of meat was not always eaten; the daily amount of phosphate given is recorded in the tables. ‘The urine was carefully collected, measured and analysed daily for chlorides, phosphates, total acidity, and total nitrogen. The chlorides were determined by titration with standard solution of silver nitrate; the phosphates by the uranium acetate method ; the total acidity by the phenol-phthaléin, and the total ~ nitrogen by Kjeldahl’s. The method of feeding was the same as that employed in the case of the guinea-pigs. . The animals were weighed twice weekly. 397 INORGANIC SALTS AND CELL STIMULATION ‘suv ut u23017IN ££g.bz 000.61 gt6.91 bSo.Ez og$ Lz of 1.0% ZS1.92 zgi-f1 019.7% gtg.g1 gzS-b1 oL6.41 tot ti obZ.Z1 009-81 gS g-Sz ZQO.SE 966.92 £96.17 940.17 tgz.Sz z1Z.61 06z.0£ Hre.$1 tHg.Ez 26.61 139-91 698.1 6zS.21 €fS.11 ghl.2i 4gs-£1 01.6 eo 09Z.$1 169-£1 S£t.g1 6tg-S1 gzit1 — bt 122.4 gfSg.0 og$ os S.o Soy u oz ozo 8 8 ogt £260. gzig.o 6 oft ~ §.0 Stee 7 eee Oe — — _ —_ — 7 Lf.0 ene, 3 eR oLog L6£ gzll.e oLLL.o ors he $z.0 bien

bb] bb) ” o> It is noticed that in the 1 in 1,000 solution there was enough silver nitrate left after all the chlorides had been precipitated! to still make the solution strongly germicidal. 1. The Liverpool Water Supply is almost free from chlorides. ACTION OF METALS UPON BACILLUS TYPHOSUS 331 In the case of the I in 10,000 solution the result cither indicated (i) that there was still enough silver nitrate left after the precipitation of the chlorides to make the solution sufficiently toxic to kill the typhoid bacilli in less than six hours, or (ii) that if all the silver nitrate was precipitated as chloride, there must have been a sufficient quantity of this so-called insoluble salt dissolved in the water to produce the toxic effect. In the 1 in 100,000 solution, the toxicity was very greatly diminished by making it with tap water instead of with distilled water. Series VIJ.—ExperimMeNts witH LeEAp SALTs This series was done with solutions of lead nitrate made with (a) distilled water and (4) tap water, collected after being allowed to run for five minutes. Four flasks were used in each case, and were incubated at about . 40°C. One cubic centimetre of the emulsion of typhoid bacilli was added to each of the flasks. (a) Solutions made with distilled water : Leap NITRATE SOLUTIONS M M M ~ Sub-cultures taken Control "1,000 "10,000 100,000 At once aad Infinite Infinite Infinite —dInfinite 2 hours after... ace Hs Negative Positive a 4 hours after ... ie a A: Negative Positive 6 »” ” Si ad ” >? ” > 24 bP bP] weeds = bP] > >? ”? (879 colonics) 48 ” 2 fee one ” » ” (53 colonies) (6) Solutions made with tap water, collected as previously :— Leap NItrrATE SOLUTIONS M M M Sub-cultures taken Control 1,000 _ 10,000 190,002 At once Eyn aids Infinite Infinite Infinite Infinite 2 hours after ... bys rf Negative e 44 7 ee ARES = a a Positive a > 9? pinky a ath » 2? > 9? 24-5, sar kat 43 y, p x Positive. - 48 ” 2 ee tee + ” 3° »” 332 BIO-CHEMICAL JOURNAL It is seen that the lead nitrate solutions both with distilled and tap water were decidedly toxic to the bacilli, but that the solutions made with distilled water were much stronger and quicker in their action than those made with tap water. ; Here it is interesting to note the result of experiments by Bassett- Smith, done with metallic lead, to see whether solutions containing B. typhosus could be rendered sterile by being placed in (a) a lead tube, and (d) a lead box. In both cases the solutions still contained many organisms at the end of forty-eight hours, and it could not be said that the lead had any germicidal action. On the other hand, Nageli did experiments with lead, and was satisfied that it possessed ‘ oligodynamic ’ properties similar to copper. Conc.usions To Part I The following conclusions were arrived at as the result of the foregoing experiments :— (1) All the salts tested manifest a decided toxic action to typhoid bacilli, but there is a great difference in degree, shown in the case of the stronger solutions by the time taken by the corresponding solu- tions of the various salts in clearing the water from bacilli; and in the failure of the more dilute solutions of the less toxic salts to clear within the limit of forty-eight hours. | (2) The toxic action is most marked when the solutions are prepared with distilled water, and are incubated at a temperature of 35° to 40°C. (3) The toxic action of the solutions made with tap water and sea water is much less than in the case of those made with distilled water, and this diminution is due to the fact that the tap and sea water both contain other substances which modify the action of the salts employed—-probably by lessening the ionization. (4) ‘The copper sulphate and silver nitrate solutions possess the greatest toxicity; the lead nitrate, ferrous sulphate, and ferric chloride solutions being next in order of toxicity ; whilst the zinc sulphate, nickel and cobalt chlorides and manganese chloride solutions take a ACTION OF METALS UPON BACILLUS TYPHOSUS 333 much longer time before the corresponding solutions are free from bacilli. —{5) The toxicity of water to which either copper foil or certain other metals (¢.g., silver, iron, tin, lead, etc.) have been added is probably due to a solution of some salt of that metal, and the so-called oligodynamic action of the solution is due to the presence of the ions of this salt, and not to the metal itself. Parr II EXPERIMENTS ON PuRIFICATION OF SHELL-FISH Having determined by means of the experiments detailed in Part I the salts most likely to prove successful in purifying infected shell-fish, an endeavour was made to purify Anodons (freshwater mussels) which had previously been strongly infected with typhoid bacilli. It was first necessary to discover the actions, if any, of the solu- tions it was proposed to use on the Anodons themselves, and a series of experiments was first carried out in each case with this end in view. In these experiments a number of glass dishes was used, in which the Anodons were placed in known quantities and strengths of various solutions. The Anodons used were of medium size and good vitality, and all the glass dishes were very thoroughly cleaned after each series of experiments. When it was required to infect the Anodons with typhoid bacilli the process was carried out as follows :— An emulsion of typhoid bacilli was first made in 100 c.c. of sterilized tap water, exactly as in the experiments in Part I, with the exception that tap water was used instead of distilled water. This emulsion was made up to two litres with tap water (collected after being allowed to run for five minutes) in one of the glass dishes, and into this solution twelve Anodons were placed and left for at least twenty-four hours. At the end of this time they were taken out as required, and put, without any preliminary cleansing, into the various solutions. 334, - BIO-CHEMICAL JOURNAL The solutions containing the Anodons were kept at laboratory temperature, and in the case of the silver nitrate experiments they — were kept in the dark, as it was found that, unless this precaution was taken, the silver became reduced and the solutions turned dark brown. | iy The determination of the number of organisms in different parts of the Anodons before and after treatment with the solutions was made by passing a sterile capillary tube (a) into the stomach, (8) into the rectum, and (c) over and among the gills. In each case the water in the capillary tube was added to a sterile test-tube, containing litmus red taurocholate agar which had previously been melted and kept at 40°C. by means of hot water in a beaker, and the contents were then placed at once in a sterile Petri dish and incubated at about 40° e. The average capacity of the capillary tubes was 0-05 c.c., but they varied slightly in size. ‘Before these cultures could be made it was necessary to open the shell of the Anodon, and to do this the anterior and posterior adductor muscles had first to be cut through with a sharp knife. When it was possible to slightly open the shell this could be done by passing the knife along inside, keeping close to the shell, and first cutting the anterior and then the posterior adductor or vice versa. When, however, it was impossible to open the cat even a little, it was first necessary to cut the edge with a sharp pair of scissors, when the knife could be used as before. The knife and scissors used were carefully sterilized after each experiment, as were also the hands of the operator. MetTattic AND CoLLoIDAL CoppPpER I. Experiments with (1) Anodons and (2) Anodons infected with typhoid bacilli, in (a) tap water containing a piece of. bright copper foil 15 cms. by 12 cms., (6) a solution of colloidal copper sulphate. As a preliminary to this series, a solution of colloidal copper was prepared by passing an electric current through distilled water, using ACTION OF METALS UPON BACILLUS TYPHOSUS 335 copper electrodes, which were so arranged as to form an electric arc under the water. The current was cut off at frequent intervals to prevent the solution from getting hot, as when this occurs the copper becomes coagulated and precipitated, and then appears as flaky masses in the solution. Altogether nearly three litres of this solution, which is of a slightly greenish colour, were prepared. The next step was to estimate the amount of copper in the colloidal solution, and this was done as follows :— Five hundred cubic centimetres of the solution were evaporated to dryness in a clean glass evaporating dish, the residue being dissolved in about twelve drops of strong nitric acid, diluted with a little water, and this solution was again evaporated to dryness. This residue was dissolved in distilled water containing a drop of hydrochloric acid. The solution was transferred to a clean glass beaker (the evaporat- ing dish being twice washed out with a little distilled water to dissolve any residue), heated to boiling, precipitated as hydrate, and filtered and weighed in a Gooch crucible. Weight of Gooch crucible alone... ... ... ... +s. 60960 grammes fA ie containing residue ... ey ... 6-1098 grammes =e ie weight of CuO ESR ar Ore. ,. z nd ” Cu og = av OOTIO”” This represented the amount of copper in 500 c.c. of the colloidal copper solution, and .*. the amount in one litre was 0-0220 grammes. This represents 1:1 grammes of copper in 50,000 c.c. A series of experiments was next done to discover the degree of toxicity of this solution. - Four Erlenmeyer flasks were taken, as under, and to each of them was added 1 c.c. of typhoid emulsion, prepared as in the experiments in Part I. | The flasks were left at laboratory temperature, and sub-cultures made: at regular intervals. 336 BIO-CHEMICAL JOURNAL (1) Containing 100 c.c. of distilled water. (2) - 100 c.c. of the colloidal copper solution. (3) - 10 c.c. =z ie made up to 100 c.c. with distilled water. (4) ‘3 » A ear a me ee? vm CoLtompaL Copper SOLUTIONS Sub-cultures taken Control Original I in Io I in 100 At once Be Infinite Infinite Infinite Infinite 2 hours after ne ‘s Negative Negative Negative rape = Ae a a ¥ Si : aa 4 5s Re fee Cat i i “* ‘. It will be seen from the above table of results that the colloidal copper solution had a marked toxic action on typhoid bacilli, and even when diluted 100 times it killed them in less than two hours at laboratory temperature. The experiments with dilutions exceeding I in 1,000,000 of (a) copper in the ionic form, as present in dilute solution of copper sulphate, and (b) of colloidal copper, as in the above experiment, — agree in giving complete removal of the bacilli within the two hours time. It is almost impossible to follow with accuracy the toxic properties beyond these limits, and hence we may for the present regard the two forms of copper as being approximately equally toxic, and both as lying at the upper limit of high toxicity. ) It is not at all easy to determine the exact state of the copper in this colloidal solution, for although, since the classical experiments of Graham, it has been customary to apply the name colloidal to those substances which will not pass through animal membranes, more recent researches have shown that there are two sub-classes of colloidal mixtures—the one having the characteristic properties of true solutions, 7.¢., possessing osmotic pressure, diffusibility, and usually a limited solubility at some temperature: the other being without these properties, and being in the nature of macroscopic or microscopic suspensions. The fact that this colloidal copper solution possesses the property ACTION OF METALS UPON BACILLUS TYPHOSUS 337 of permeating colloids, as the cell wall and the organized contents of the cell, thereby producing marked disturbances in the cell and exerting its toxic action, tends to show that it in some ways resembles the crystalloids. Whatever may be the true nature of the solution, it is certain that its toxicity is not merely due to this ‘ colloidal’ state, for Roaf and Whitley (7) have shown that whereas colloidal silver prepared in the above manner was exceedingly toxic to tadpoles, a solution of colloidal platinum had absolutely no effect upon them. It must also be borne in mind that the bacilli present in the solution are producing carbonic acid and weak organic acids which are capable of acting upon the colloidal copper and causing it to pass from the colloidal form to true solution in the ionic condition. When regard is paid to the excessively minute quantity of copper present in the solution, it is obvious that the bacterial products would be quite sufficient to cause the copper to form a salt, become ionized, and act in the same way as the excessively dilute solutions of salts of copper used in the former experiment in Part I with copper sulphate. Further, when once the colloidal copper entered the cells it would meet there with substances capable of converting it into a copper salt; so that whether the copper be present in the solution as a colloid or as an ion it would, in the end, produce its effect as copper ion; and the results obtained with copper sulphate, when present in so high a dilution as one part of copper in 10,000,000, show that copper, qua copper ion, is capable of producing all the effects obtained with colloidal solutions, so that no peculiar ‘ oligo- dynamic properties’ or ‘ colloidal condition’ need be assumed in order to explain the results obtained. Having determined the amount of toxicity of the colloidal copper solution, the experiments with Anodons were now proceeded with in solutions containing different forms of copper. Two Anodons were placed in each of the following dishes to 338 BIO-CHEMICAL JOURNAL determine the action of the different solutions on them. ‘The dishes | were kept at laboratory temperature. (1) Containing 2 litres of tap water, collected after being allowed to run for 5 minutes. (2) ma 2 & 0 with a piece of bright copper foil. (3) & 1 litre colloidal copper solution. (4) “ 2 litres — copper sulphate solution, made with tap water. M (5) ” 2 » 5,000 ” ” ” M (6) ” 2 5 10,000 mf ” ” It was noticed that there was soon a good deal of mucus in the solutions. 24 hours 48 hours 72 hours 1 week 2 weeks 4 weeks Ist dish 2 alive 2 alive 2 alive 2 alive 2 alive 2 alive 2nd ” 2 ” 2 99 2 bP) 2 oP) 2 bb] 2 ”° 3rd ” 2 » 2 » 2 55 2» 2 » 2 4th ,, 1 dead 1 dead 2 dead — — - SEE 55 2 alive 2 alive 2 alive I alive 2 dead — 6th", eh apa ap 2 hss 2 dead a It will be seen from this table that the copper foil and colloidal copper solutions did not visibly affect the Anodons in four weeks. This is probably due to the fact that the Anodons secreted a good deal of mucus, which in some way precipitated the copper in the solutions and thus stopped or greatly modified its action. It was noticed that the copper foil rapidly lost its lustre and became dull; and in the colloidal copper solution the mucus at the bottom of the dish was tinged green, whilst the water itself was quite clear. In the case of the copper sulphate solutions, although the Anodons secreted mucus on account probably of the irritation from the solu- tions, and thus, no doubt, modified their toxicity somewhat, the solutions still remained too powerful for them, and even in the I in 10,000 dilution both the Anodons were dead in less than two weeks. The dead Anodons from the two strongest copper sulphate solutions were distinctly blue in colour, due to the staining with the salt. ACTION OF METALS UPON BACILLUS TYPHOSUS 339 It is interesting to remember in connection with the above experiments that Herdman and Boyce (8) found in their experiments with oysters that salts of copper always had a deleterious effect on : the oysters themselves. | A series of experiments was next done with Anodons which had been strongly infected by being placed in a solution containing typhoid bacilli (prepared as previously described) for twenty-four hours. Three of these infected Anodons were placed in each of the following dishes :— (1) Containing 2 litres of tap water, collected as usual. (2) os a piece of bright copper foil, 15 cm. X 12 cm., in tap water collected as usual (3) ts 1 litre of colloidal copper solution. ® - 2 litres of yee SORES sulphate solution made with tap water. M (5) ” 2 Li 9 100,000 39 bb ”? ” Cultures were made at intervals from the stomach, gills, and rectum (indicated in the following tables, $., G., and R. respectively), and the results are shown below :— Cultures taken Control Water with copper foil Colloidal copper solution S. G. R. S. G. R. 3 G. R. mpronce *! S57 Saye 563 233 — — a —- — — 24 hours after — _ — 211 294 178 53 107.2 48, » 243. 4710187 «147, 207 eae 9 aie Ly Meet Cultures taken Control ois Copper Sulphate scans Copper Sulphate S. G. R. S. G. R. S. G. R. At once Gea Ec 868 233 — — — _: — — 24 hours after — _- _ 43 89 52 163: 48g 1937 48 4,» 243 471 187 47. «i 23. «143, 352-103 _ It will be seen from the above table of results that only the colloidal copper solution and the 1 in 10,000 copper sulphate showed any marked power in dealing with the infected Anodons, that the maximum effect of these solutions was manifested in twenty-four hours, and that the next twenty-four hours’ treatment did not seem to have any further action in diminishing the number of the bacilli. This seems to show that the different solutions practically lost their toxicity in twenty-four hours, as far as the bacilli were concerned, 340 BIO-CHEMICAL JOURNAL and this was probably due to the secretion of mucus by the Anodons and the consequent precipitation of the copper salts. As regards the Anodons themselves, in the 1 in 10,000 solutions, this explanation is hardly sufficient, for if this solution lost all its toxicity in twenty-four hours, the reason for the death of the Anodons in less than fourteen days is not apparent. It is possible that the Anodons were so injured by the solution in the first twenty-four hours as to be unable to recover from the effects, and it is certain that the toxicity of this solution to typhoid bacilli was much diminished when the Anodons were present in it. Series I[].—ExperIMENTs wiTH IRON SALTs Experiments to discover the action of ferrous sulphate and ferric chloride solutions on (1) Anodons, and (2) Anodons infected with typhoid bacilli. (a) A series of dishes was again used containing different solu- tions, and in each were placed two Anodons. | (1) Containing 2 litres of tap water collected as usual. ; M : ’ Seer ye Ferrous sulphate solution made with tap water. 1,000 M (3) 9? 2 9 10,000 9% 9”? ” ? M : ; (4) $5 R53y Ferric chloride * Fs 1,000 (5) = 2 — 39 ” 10,000 ” ” ” ” The result of the above series was that at the end of five weeks all the Anodons were still alive, and apparently quite healthy; and whereas at the time of starting the series one or two seemed a little sickly, these revived in the solutions and seemed to benefit by the presence of iron salts in the water. These results also coincide with the observations of Herdman and Boyce, who found that salts of iron seemed to have a favourable action on oysters. (2) A series was now done with infected Anodons, three of ACTION OF METALS UPON BACILLUS TYPHOSUS 341 which were placed in each of five dishes containing solutions similar to the above, and-the results are tabulated below :— Sub-cultures Control Ferric Chloride Ferric Chloride 1,000 10, Se G. R. Ss. G. R. S. G. R. At once 24 hours after _ — _ fe) 54 fe) 70 93 49 48 oo 463. 153 S 53 1 EE ae ee Sub-cultures Control Ferrous Sulphate io Sulphate I, 10,000 S. G. R. Ss; G. R. S. G. R. At once 24 hours after _- _ — fe) 7 fo) 61 104 53 Ya os 251. "263 153 fe) ) fe) Ce) 57 II It is seen from the above table that ferrous sulphate and ferric chloride solutions both had a decided action in clearing the Anodons from the bacilli, and this was especially noticeable in the case of the ferrous sulphate solutions which were freshly prepared. It is remarkable that these salts should have such a decided action on the Anodons infected with typhoid bacilli, but the explanation is probably to be found in the fact that the iron solutions were not only not irritating to the Anodons but were even beneficial, and so they did not attempt by secreting mucus or in other ways to nullify the toxic action of the solutions. There was a certain amount of mucus secreted by the Anodons, however, but not nearly so much as in the copper solutions, and it was of a ‘rusty’ colour, due to the iron. These results were considered satisfactory, as these salts could easily be used on a large scale for purifying oysters; for it has already been shown by Herdman and Boyce that they are likewise benefited by the presence of iron salts in the water. Again, it was noteworthy that the chief action took place in the first twenty-four hours, and this fact was the basis of another series of experiments. (c) Another batch of infected oysters was taken and three of them placed in each of a series of glass dishes :— (1) Containing tap water collected as usual. (2) e Ferric chloride solution made with tap water. 3000 M (3) 99 eee Ferrous sulphate __s,, ie : > 342 BIO-CHEMICAL JOURNAL An Anodon from (1) was opened immediately, and the cultures showed stomach 314, gills 517, and rectum 271 colonies respectively. At the end of fifteen hours the Anodons ftom each dish were taken and rapidly rinsed in. clean water, and placed respectively in three other dishes containing fresh solutions of the same strength and were left for ten hours longer. Cultures were then taken from one Anodon out of each dish, and the result was as follows — M Ferric Chloride M __ Ferrous Sulphate ares , — 1,000 ~=——— Solution _ 1,000 Solution ; S. G. R. S. G. RO Se G. Re, At once oie ee 517 271 a — — — —_ a 25 hours after 207° 384 .—s-159 fo) fe) fe) Oo fo) fe) This change into fresh solutions made the action much more marked, and both the Anodons tested from the iron solutions at the end of twenty-five hours were free from typhoid bacilli. This action of ferrous sulphate and ferric chloride may possibly prove of great practical value in the treatment of suspected oysters, and a series of experiments will be carried out, shortly, using sea- water solutions of iron salts and oysters —— with phon bacilli with this end in view. 7 | Serres [I].—ExprerimENtT witu SILver SALTs III. This series was started to find out whether it would be possible to use silver nitrate solutions to purify infected Anodons. A series of three dishes was used, and they were kept at laboratory temperature in the dark. | : 3 It was noticed that in dishes 2 and 3 the solutions were milky, due to the precipitation of the chlorides in the water as silver chloride. Two Anodons were placed in each of the following :— (1) Containing tap water, collected as usual, (3) ”° 9 bb) ” . > In four days it was noticed that the Anodons were sickly in ACTION OF METALS UPON BACILLUS TYPHOSUS 343 dishes 2 and 3, and they were all dead in these two dishes six days after being placed-in the solutions. | "The Anodons in dish 1 remained healthy. No attempt was.made to purify infected Anodons with these solutions, which had proved so harmful to the Anodons themselves. Series [V.—ExperRIMENTS WITH ZINC AND Leap SALtTs IV. ‘This series was conducted to discover the action of zinc sulphate and lead nitrate on Anodons, and, if feasible, to try and purify infected Anodons with solutions of these salts. A series of five glass dishes was used, and into each of them two Anodons were put. (1) contained 2 litres of tap water collected as before. (ao ?™,, ties: sah zinc sulphate solution made with tap water. > M (3) ” 2 > 10,000 bb ” ” ” pre, tg oe lead nitrate solution made with tap water. 1,000 ) 2 5 2” bP 10, ” ”? 3 The Anodons in dishes 2 and 4 were all dead in less than one week, and so it was concluded that these solutions could not have any practical application. ~The Anodons in dishes 1, 3, and 5 were still living, and apparently healthy after three weeks. _ There was a fair amount of mucus in all the dishes, but less in the control. Another batch of Anodons was now infected with typhoid bacilli, and three of them were placed in each of the following dishes :— (1) Containing 2 litres of tap water, collected as before. dries 2155 zinc sulphate solution, made with tap water. 10,000 : M : a oe lead nitrate # i ps 10,000 344 BIO-CHEMICAL JOURNAL Sub-cultures were taken from the stomach, gills, and rectum of Anodons from each of the above dishes at certain intervals, as shown in the following “table :— Control _M __ Zinc sulphate _M _ Lead nitrate Sub-cultures 10,000 solution 10,000 __ solution S. G. | ales * G. R. eects R. At once (ot ee 719 327 24 hours after. 179 435 192 743) 100. ae Both the zinc sulphate a lead nitrate showed a decided amount of toxicity, but the Anodons still contained many bacilli at the end of forty-eight hours. The lead nitrate proved itself to be more toxic than the zinc \ sulphate solution. To briefly sum up :— Of all the different salts experimented with, it would seem that only those of iron are likely to be of use in the purification of suspected ° ‘infected shell-fish. Salts of all the other metals used, either acted detrimentally on : the Anodons themselves or were not able to free them from the. bacilli in a reasonable time. 3 The conclusions to be drawn from Part II of this paper may be tabulated as follows :— . (1) A solution of colloidal copper made by passing an electric current through distilled water, using copper electrodes so placed as to give an electric arc in the water, is exceedingly toxic to typhoid bacilli, and even when diluted 100 times will kill Them in legs than © two hours at ordinary temperature. : (2) Salts of copper have a harmful effect on Anodons, and the stronger solutions rapidly kill them, whilst the weaker solutions are not able to purify Anodons infected with typhoid bacilli. It is, therefore, impracticable to attempt to use salts of copper in the purification of shell-fish. 3 (3) Ferrous sulphate and ferric chloride solutions exert a bene- ficial action on Anodons placed in them, and the 1 in 1 000 solutions’ are able to practically free infected Anodons from typhoid bacilli in twenty-five hours. ACTION OF METALS UPON BACILLUS TYPHOSUS 345 (4) It is probable that a practicable method of purifying oysters contaminated with typhoid may be discovered by employing some salt-of iron for the purpose. (5) Salts of silver are very harmful to Anodons, and even I in 10,000 of M silver nitrate will kill them in less than a week. (6) Sulphate of zinc and lead nitrate in the stronger solutions are harmful to Anodons, and in weaker solutions, although they exert a decidedly toxic action on typhoid bacilli, are unable to entirely free infected Anodons. BIBLIOGRAPHY (1) Nageli, ‘ Ueber oligodynamische Erscheinungen in lebenden Zellen.’ Neue Denkschriften der schweixerischen naturforschenden Gesellschaft (Vols. XXXIII, XXXIV), pp. 1-51, 1903-1905. (2) Israel and Klingmann, ‘ Oligodynamische Erscheinungen an pflanzlichen und thierischen Zellen.’ Virchow’s Archiv, Vol. CXLVII, pp. 293-340, 1897. (3) Locke, Fourn. of Physiology, Vol. XVIII, p. 319, 1895. (4) Moore and Kellerman, U.S. Department of Agriculture, Bureau of Plant Industry Bulletins, Vol. LX1V, 1904; and Vol. LXXVI, 1905. (5) Kraemer, Amer. Four. Pharm., Vol. LXXVI, 1904, pp. 574-581 ; Amer. Medicine, Vol. IX, pp. 275- 277, 1905 ; Proceedings of Amer. Philos. Soc., Vol. CLXXIX, pp. 51-65, 1905. (6) Basset-Smith, ‘ Experiments to demonstrate the Germicidal Power of Copper and Copper Salts on Pathogenic and Non-pathogenic Organisms.’ our. éf Prev. Med., Vol. XIII, page 7, 1905. (7) Roaf and Whitley, Bio-Chemical Fournal, Vol. 1, No. 2, pp. 88-115. (8) * Herdman and Boyce, Lancashire Sea Fisheries Memoirs, No. 1, ‘ Oysters and Disease,’ Pp- 34- 346 NOTE ON THE CHEMICAL COMPOSITION AND PHYSICAL PROPERTIES OF RENAL CALCULI By J. SYDNEY ROWLANDS, M.D. (Liverpool), M.R.C.S.; L.R.C.P., Thelwall Thomas Fellow in Surgical Pathology, University of Liverpool. From the Bio-Chemical Department, University of Liverpool (Received Fune 20th, 1908) A series of twenty-two calculi in all was investigated, derived from a fairly wide distribution in South-west Lancashire and North Wales, the object of the study being chiefly to determine whether there was anything in the appearance of a calculus which would give a fairly reliable clue to its chemical composition. The results are concisely recorded in the subjoined table, which shows that analysis alone can determine the nature of a calculus, the pigmentation, surface, and hardness which are usually relied upon for the classification of calculi being exceedingly untrustworthy. An important result obtained as far as this series at least is con- cerned is that uric acid either free or as urates is an exceedingly rare constituent, for uric acid or urates were completely absent in nineteen out of the twenty-two stones, and only traces were found in the three cases where a faint positive result was obtained with the murexide test. Of equal importance is the unexpected fact that oxalic acid, combined usually with calcium, was found in every one of the stones examined, although the previous literature places this as a very much rarer constituent than uric acid. Phosphates were also commonly present, although this is usually described as only an occasional constituent. In two of the stones only were carbonates found, but in these two they were present in considerable quantity. ‘These two stones also contained oxalates and phosphates. In regard to the physical property of hardness, on which great NOTE ON COMPOSITION OF RENAL CALCULI 347 stress has been laid by earlier observers, in relationship to chemical composition, I have found as far as the simple test of crushing goes that the hardness is very little, if any, related to the chemical com- position, and would suggest that the rate and process of formation of the stone is of much more importance, the compactly laid down glossy stone, probably of slower growth, being most difficult to crush, whatever its chemical composition. In the previous literature—in addition to rarer forms of calculi which need not here be discussed, since none of them were found in the series—there were three chief forms of calculi recognised : (a) the pure uric acid calculus, usually described as of a yellowish or brownish colour, rough and crystalline on the surface, and brittle ; (4) the urate calculus, containing urates chiefly of magnesium and ammonium along with excess of uric acid, and oxalate of calcium and ammonium magnesium phosphate (these stones were supposed to grow larger than the pure uric acid stones, and were described as having a smooth surface and pale colour) ; (c) the oxalate calculus, which was described as having a brown colour and a spinous surface full of irregular projec- tions of a rounded or club-like form (these are said to be much less frequent in occurrence). The present series of analyses furnishes no experimental evidence to support this classification. As above stated, oxalates are the constituent par excellence in this series, being present in every one of the twenty-two calculi examined, and in the majority of the cases in preponderating amount above all the other constituents present. Further, the smoothness and pigmentation give no information as to chemical constitution in the series here examined, and these appear to me to depend much more on the rate of deposit and the natural amount of pigment present in the urine during the period that the stone is being deposited. It would have been quite impossible for anyone not knowing the three stones in the series which contained uric acid to pick these out from the remainder from their physical appearance, and their pigmentation was very like that of the rest of the series, in which, as above stated, uric acid was entirely absent. 348 BIO-CHEMICAL JOURNAL In conducting the analyses, the excellent table of Haller was followed as a routine. Since the amount of oxalates in nearly all the calculi examined was preponderating, it was thought that this might mask the presence of small amounts of uric acid, and in order to test this point the following experiment was carried out with an artificial mixture. A solution of calcium chloride was added to one of oxalic acid, and the insoluble calcium oxalate was separated off. To this precipitated calcium oxalate the smallest trace of uric acid was then added, the proportion of uric acid in the mixture being not more than I in 1,000. ‘This mixture was placed in a porcelain dish, a drop of nitric acid added as in the murexide test, and the whole evaporated to dryness. ; The residue was quite white, and did not show the usual reddish brown residue of appreciable amounts of uric acid, so that the small trace of uric acid obviously was here concealed by the excess of oxalates. However, on the addition of ammonia even this trace showed up positively. For a pink coloration could now be detected; this turned purple on the addition of caustic soda. ‘The murexide test was hence here quite positive, and shown to be a very delicate test. The delicacy of this control assured me that no appreciable amount of uric acid was present save in the three cases in which positive results were obtained. Tasie or ANALYsEs with Suort Description oF Eacu CAtcu.us Percentage Percentage of Incom- of Com- Car- Oxal- Phos- Uric Moisture Dustible —_bustible bonates _ates phates Acid Material Material _ Percentage Initials of I.—Mrs. G. 6 43°6 56-4 Absent Present Present ‘Trace Brownish colour, spinous projections II.—M. L. 2°89 =. 267 73°3 Absent Present Present Present Shape-cast of kidney pelvis, chocolate colour, with whitish crystals on surface Ili.—E. B. 1:92 43°86 5614 Present Present Absent Absent Brownish, glossy, smooth IV.—W. H. H. 9°7 62:2 37°8 Absent Present Present Absent Greyish, spinous projections NOTE ON COMPOSITION OF RENAL CALCULI 349 Taste or ANALYsES WITH SHorT Description oF Eacn Catcutus—Continued ——_- Percentage —-fnitials of Moisture V.—A. E. 33 Oval, greyish rind, cut surface brownish and glossy VI.—F. H. 4°13 Greyish, brittle VII.—E. K. 3°8 Shaped to renal pelvis, brittle, greyish-brown VIUL—W. G. B. 2-09 Greyish and spinous IX.—W. E. P. 12:2 Light brown and spinous X.—Mrs. T. 2°32 Brownish, irregular, brittle XI.—P J. H. 2°15 Greyish and spinous XIIL—E W. 5-92 Whitish, irregular surface XII.—H. C. 2°3 Chocolate colour, projections correspond to calyces, glossy XIV.—G. W. 7:6 Large, whitish, brittle XV.—J. D. 5°4 Oval, greyish, spinous, brittle XVI.—H. H. S. 4°7 Oval, whitish, and smooth XVII.—M. C. 55 Greyish, irregular, brittle Percentage Percentage of Incom- bustible Material 34°5 7tor 72 55°94 28°1 59°97 59°14 75°6 44°5 65°5 62°8 76:2 66:9 of Com- bustible Material 65°5 28-09 28 41-16 709 42°93 40°86 24°4 54°45 34°5 37°2 23°8 33°1 Car- bonates Absent Absent Absent Absent Absent Absent Absent Trace Absent Absent Absent Absent Absent Oxal- ates Present Present Present Present Present Present Present Trace Present Present Present Present Present Phos- phates Present Present Present Present Present Present Present Large quantity Present Present Present Present Present "Uric Acid Absent Absent Absent Absent Absent Absent Absent Absent Present Absent Absent Absent Absent 350 BIO-CHEMICAL JOURNAL Taste oF ANALYsEs Witt! Suor1 Description or EAcn’ CaLtcuLus—Continued Percentage Percentage Percentage ane ? ye of Incom- of Com- Cie Oxal- Phos-,;... Uric initiale M - , _ bustible bustible bonates __ ates phates Acid eee Material Material XVIII.—Dr. H. 5°67 47°73 52:27 Present Present Present Absent Brownish and spinous XIX —A. L. 2°02 40°6 59°4 Absent Present Present Absent - Surface chocolate and ° smooth XX —Nurse D. 3:07 385 61°5 Absent Present Present Absent Rind light brown and spinous, cut surface chocolate XXI.—E. L. 2:27. 433 56:7 Absent Present Present Absent Oval, chocolate colour, glossy : XXII.—Mrs. W. 1-6 32°8 67 Absent Present Present Absent Rind chocolate, cut surface greyish Notr.—Regarding other constituents present calcium and sodium were always found in every calculus of the series. Xanthin cystin and other rare constituents were usually tested for, but were invariably found absent. The twenty-two samples examined were all from cases occurring in the practice of Mr. W. Thelwall Thomas, F.R.C.S., to whom my thanks are due for the suggestion of this work, as also for his kindness in placing the material at my disposal. My thanks are also due to Professor B. Moore for allowing me to work in his department, and for his help at all times. CoNncLusions _1. The commonest constituent by far in this series of calculi is oxalate, present chiefly as the calcium salt. 2. Uric acid in any form was found to be extremely rare, and absent in nineteen out of twenty-two cases. 3. Neither pigmentation, hardness nor surface of the stone are any criterion of its chemical composition, and depend in all probability more upon the physical and physiological conditions while the stone is being deposited. 351 ON THE PRESENCE OF AN OXIDISING-ENZYME IN THE LATEX OF HEVEA BRASILIENSIS By D. SPENCE, Pu.D., A.L.C. From the Bio-Chemical Laboratory, University of Liverpool (Received Fuly ist, 1908) The following note is intended rather as an addition to a recent paper published in this Journal on the presence of oxydases in india- rubber than as an independent communication on this subject. The question of oxidising-enzymes in indiarubber and in the latex secretion from which this is derived has been fully dealt with in my original paper,’ and will not, therefore, be discussed again here. As this subject appears to me, however, to be an important one, both biologically in regard to the function of the caoutchouc in the latex of the plant, and commercially in view of possible improvements in the preparation of raw rubber, and furthermore as only indirect evidence was available at the time for the presence of an oxydase in the raw Para rubber examined, it may not be out of place to record here that since the previous communication was written I have had the opportunity of examining four separate samples of latex from Hevea brasiliensis (Para rubber), and have found in each marked evidence of the presence of an oxidising-enzyme. The latices investigated were collected on the Jugra Estate, Ceylon,? and were preserved according to my directions by methods which will be described in their proper place. The samples arrived here in good condition, and showed no signs of coagulation macro- scopically or microscopically. 2 For the examination of the latices for oxidising-enzymes the same methods were employed as were described in connection with Funtumia elastica.® ‘The latex was dialysed for twenty-four hours 1. Bio-Chemical Fournal, Vol. II, No. 4, p. 165, 1908. 2. I should like to take this opportunity of expressing my thanks to Messrs. Edward Lawrence & Co., of Liverpool, for the care which they have taken in collecting, and for their kindness in providing me with this material. 3. Loc. cit., p. 175. 352 BIO-CHEMICAL JOURNAL into running water in order to remove products which might interfere with the reactions. ‘The dialysed latex was then tested with the reagent for detecting the presence of oxidising-enzymes, both with and without hydrogen peroxide. Controls in this case consisted of latex which had been made very faintly alkaline and carefully raised to 80° C., and kept at this temperature for five minutes, then cooled and neutralised immediately before use. In this connection it may be well to point out that only the alkaline or neutral reagents are suitable for the direct determination of oxidising-enzymes in the latex, for those reagents which react best in a faintly acid medium or in alcoholic solution (tincture of guaiacum) produce rapid coagula- tion of the latex, and give uncertain results. Each of the four samples of Hevea latex examined was found to give a marked positive reaction without hydrogen peroxide, and a still more intense reaction in presence of this chemical when the indophenol mixture of Réhmann and Spitzer, the hydrochinon and the p-phenylene-diamine reagents, were employed as indicators. Furthermore, by diluting the latices with water and then coagulating with 40 per cent. alcohol, it was found possible by addition of absolute alcohol to the watery mother-liquor to separate the oxidase in an impure state in the form of a gummy mass, which, on drying in vacuo, left a small quantity of a brown vitreous solid. This solid when dissolved in water gave all the reactions of a powerful oxydase. The chemical properties of this oxidising-enzyme in the latex of Hevea brasiliensis have not yet been studied, but the present note serves at least to show that in spite of the negative results of Schid- rowitz and Kaye! the latex of Hevea brasiliensis does actually contain an oxydase enzyme, so that the chain of experimental evidence for the existence of such an enzyme in the so-called insoluble constituent of Para rubber is now complete. 1. India Rubber Fournal, Vol. XXXIV, No. 1, p. 24 (1907). 353 EXPERIMENTAL EVIDENCE OF THE LOCAL PRODUCTION OF ‘OPSONINS’ By H. LEITH MURRAY, M.D., Pathologist, David Lewis Northern Hospital, Liverpool, R. STENHOUSE WILLIAMS, M.B., D.P.H., Assistant Lecturer on Public Health Bacteriology, University of Liverpool, anp J. ORR, L.R.C.P. & S.E., D.P.H. From the Department of Hygiene, University of Liverpool (Received Fuly 2nd, 1908) These experiments were originally undertaken in the hope that we might, by the firm application of an elastic bandage and the subsequent inoculation of a vaccine, be able to demonstrate the local production of specific ‘ opsonins.’ Before proceeding to inocu- late we felt that it was necessary to test the local and general effects upon the opsonic index produced by the bandage alone. The altera- tions in the index were so marked that our original purpose was frustrated, as it would have been impossible to say to what extent variations obtained were due to the vaccine. We are, therefore, left with a series of experiments demonstrating the local and general effects upon the opsonic index resulting from the application to a normal individual of a Bier’s bandage. The experiments may be divided into three groups. A. Evidence that the index of the subject was normal to the Diplococcus Intracellularis Meningitidis (Weichselbaum).} B. Local and general effects upon the index when an elastic bandage was applied with extreme severity. C. Local and general effects upon the index when an elastic bandage was applied with less severity. In all the experiments the emulsion was made from a twenty-four hours’ old ‘ nasgar * culture. Leucocytes (2 vols.), emulsion (1 vol.), 1. We selected the Meningococcus because we had already worked with it continuously for nine months in the treatment of patients, and were also so engaged at the time of commencing the experiments. 2 A. Ascitic fluid, 15 c.c.; distilled water, 35 c.c. ; nutrose, 1 gramme. B. Ordinary peptone agar. Mix one part of A with two parts of B, steam thirty minutes, filter, place in tubes. Sterilise. (Gordon) 354 BIO-CHEMICAL JOURNAL and serum (1 vol.) were always freshly prepared (not more than four hours old), and were incubated for ten minutes at 37°C. One hundred leucocytes were counted. In the majority of the experiments the counter did not know until he had finished to which persons the various films referred, as the slides were mixed with those of patients - under treatment. As a rule, he did not know his figures at all, as they were taken down for him by another person. The slides were stained by the following method :—Twenty drops of Leishman’s stain for thirty-five seconds; ten drops of distilled water added, carefully mixed and left for six minutes; washed in three changes of distilled water for twenty seconds altogether, and dried high up over a bunsen flame. A.—Proor THAT THE INDEX OF THE SuBJECT was NORMAL TO THE MENINGOCOCcCUS Six preliminary observations were made, four on successive days. (See No. 1 on chart.) The culture employed on the first four occa- sions had been derived from a case seven days previously ; that on the last two had been isolated 138 days before the first of these experiments. A different control blood was used on each occasion. Twice there was only a difference of one coccus between the subject and the control, viz., 135-136 and 229-228. ‘The subject had, then, an index normal (0-9-1-1) to two strains of meningococcus of markedly different ages. ‘The subsequent experiments confirmed this, as the index was always taken before the application of the bandage, and invariably fell within these limits. B.—Locat anp GeneraL Errects upon THE INDEX WHEN AN ELasTic BanpacE was AppLieD wiTH ExTREME SEVERITY (See Nos. 2, 3 and 4 on chart) Five yards of Esmarch’s plain rubber bandage were applied to the left forearm just below the elbow for an hour. Severe pain was induced in the hand during the experiment, and tingling and numb- ness for several hours afterwards. The subject was covered with cold perspiration, and was somewhat collapsed. The limb below ~LOCAL PRODUCTION OF ‘OPSONINS’ 355 the bandage became intensely cyanosed at first, but towards the end of the hour was leaden-coloured.- The pulse was absent at the wrist, and sufficient blood for the index was only obtained by pricking deeply the pulps of all the fingers. Results.—In each case there was a very distinct rise in the index of the left hand at the end of the hour (1-7, 1:5, 1-9). In one case (Experiment No. 4) this rise had already begun at the end of half an hour. In this experiment indices were taken from both hands, and showed that the rise was confined to the left. Experiments Nos. 2 and 4 demonstrate that twenty-four hours later the index had returned to normal. C.—Locat anp GENERAL EFFECTS UPON THE INDEX WHEN THE BanNpDAGE was APPLIED wITH Less SEVERITY (See Nos. 5, 6, 7, 8, and g on chart) In these experiments the bandage was applied with sufficient firmness to produce apparently the same amount of stasis and dis- coloration, but relatively little pain. That the constriction was sufficient to isolate the limb from the general circulation is seen in the chart, which shows that variations in the index, where they occurred at all, were confined to the left hand. Experiments Nos. 5 and g show no result. In No. § the index readings are worth reporting in detail, as showing the consistency of the results. Control: 77 cocci in 100 leucocytes. Four readings from the subject (both hands) at the various stages of the experiment : 79, 76, 74, and 78 cocci in 100 leucocytes. In No. 9 a rise to I-2 on the second day can hardly have been due to the application of the bandage. If we consider Experiments Nos. 5, 6, 7, 8, and g together, we find that under the conditions of the observations the rise in the index is never more than moderate, and may be absent. In none of the experiments does the effect last more than twenty-four hours. 356 BIO-CHEMICAL JOURNAL CoNCLUSIONS It would appear that to produce a marked rise in the index a Bier’s bandage when applied for one hour must be sufficiently firm to cause very considerable pain. ‘This leaves untouched the question of a more moderate application extending over a longer period of time; but, from a consideration of the above experiments and the variations in the rises obtained, it may be doubted whether an applica- tion however long which does not isolate the limb will have any appreciable effect on the index. It is certain that the results will be irregular in character. ‘The irregularity of the indices seems to us to be of interest, if only from the fact that it confirms the clinical experience of those who use Bier’s bandage as a therapeutic measure, and find that the beneficial effects vary greatly. A further point is brought out, namely, that the alteration in amount of the ‘ opsonins’ (or such part of them as can be gauged by the index) was purely local, and that, therefore, no other tissues of the body were required for its manifestation than those present in the bandaged forearm. 357 LOCAL PRODUCTION OF ‘OPSONINS’ *sfup Sutpaaoons uo pur adepurg jo |vaoutar JgyyBAnoy suo puvyddyjiajoxepuy @ ‘adepurq Jo [vaowtad Jo aut] ye puvy’s9qI9 Jo xapuy oO ‘asepurg jo uonvordde s9qye anoy uv jeg pury Yat Jo xopuy xX LUYVHO XHANI SOINOSdO “‘pueg iqsLt jo daim9 xopuy *puvy Yel jo aaind xapuy ‘Juauttiadxa a10joq xapuy -— = ft astannases aSSHREESSHEERESAREGEE: asEARESG EaEEGEESASGRzi eRREGN i ta TT} ] HH 4 Bests aa gos seesess seus : H wai i i Ht yu" J HEH HEH | ss HT iti ait Pitesti HEH Sasaeteseddatttretstore: HATH TT Ei it iti] H | a lef HH eratite Aetrestee tee ; E t +H MD at TaBEOE ‘ah, oer HTH } f by ‘ i ii i spedeesadaeeceseuead ess Bsessassacccsussceessassasseceees HLTH SOOpEAG shanuaeauee’¢ ; Ht ; Hea: lif] &1 sive HTH HH H ages SSSessepensess E HH ; 1 aay ag “TTT aeeaa 66 ani saan E S55; REGERRRGRI Sanu eases Os Bat SoRe scaas sabehect ss Bat 8 H / SUUHTESE) FOSRy BEE es HHH as soeseesee! eeeree sess H BS5s Because BEGst seune SSSRSERS RRessbS Best seeuse Bpeeee . { { | Hatet ta sat H H HHH H HH Sessscs: sons HH W seeeeeseet coscones Ha : t aE rs Hit aigusas (eeeadeedi faussssens eatin HHH HH aeGGHE SHEEEESH HERE BREEH Stade SSEdHEGHE (HUaddeads tgsaaseas feaiteatit HH} RE 4 eae is rt HTH . z i eee si as age tt Tht +} +++] | aes Basse: +4 res « HH HH HH ssi 455 Hitt H HHH is} sesssduess sesseceees tee He so feses isessecees tee : it, { i Ht tH H sases tescsseese see Hiatt +H +H HH Soebesest Sones 1 SoSeseeeeSeccs HH | +H s | sesee: ees HH : HTH HH Seseess watt 1 hd 3 i seers | iH -O : si gs poses ss sss. ss eseseee ss tees HH THAT st es iegsegeses: H ses teeass seseese: bo Ope iessitets snnEEH seas Sidi SERIEGGH] GiG0Ni; G21 oet ieee eee et ette Sih cages ute Se neiiae ARETE tine eect Ht HH HH HE Sess Sessesees’ Seessese : Sh iee HH : Sf / Bs tbeeaeeeee tessecsses (seeps HH Seeeceees Sessseeest casesuesct tecstta. ee tess ] tH sg t iBe - I t one rT { + : sobaes sce 2 f t r] eaaen . eee te seen . 4 Ly e by hy is seeecs ts asses Be sigs te behing S88 (EEEP. 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Stee te ; ; oH sees iG iagei seeas (sees iseza seses teats TT + t + t t + pebae rf r t tt T t ttt t t t { ot seco ss pose ideas sess sages sean: tt t I tit t + + + 359 EXPERIMENTAL INOCULATION OF MENINGO-COCCIC _ VACCINE By R. STENHOUSE WILLIAMS, M.B., D.P.H., Assistant Lecturer on Public Health Bacteriology, University of Liverpool, H. LEITH MURRAY, M.D., Pathologist, David Lewis Northern Hospital, Liverpool, anv. J. ORR, ia. &.$.E., D.P.H. From the Department of Hygiene, University of Liverpool (Received Fuly 2nd, 1908) While treating cases of epidemic cerebro-spinal meningitis by the vaccine method we were struck by the difficulties experienced in estimating the dosage. The following investigation, therefore, was undertaken to determine the limit of dosage for a normal adult, and in the hope that at the same time light would be shed on the degree of immunity produced. Three points seem to be established by the experiments :— (1) That a dose of 222 million organisms was as much as the subject could bear with safety. (2) That the inoculation of a vaccine is not likely to be of much value as a prophylactic. (3) That a vaccine three weeks old has not lost its potency. Technique. A. Method of Preparing and Staining Slides for the Index.—Two volumes of corpuscles were mixed with one volume of emulsion and one volume of serum, incubated at 37°C. for ten minutes, and films stained in the following way :—Leishman’s stain twenty drops for thirty-five seconds ; distilled water ten drops added, carefully mixed and left for six minutes ; film washed in three changes of distilled water for twenty seconds altogether, and dried high over a bunsen flame. The emulsion was on each occasion prepared from a twenty-four hours’ sub-culture on ‘ nasgar 4 of a strain 201 days old on the date of the first inoculation (December gth, 1907). B. Method of Counting—The preparation of the films was always done by one person, and the counting also by one person ; 1. For composition, see previous paper. 360 BIO-CHEMICAL JOURNAL but before the counting was begun the slides were re-labelled by someone not concerned, and mixed with the slides of patients under treatment at the time. Every effort, therefore, was made to reduce any possible mental bias. One hundred leucocytes were counted on every occasion. C. Preparation of the Vaccine-—The vaccine was prepared from twenty-four hours old sub-cultures on ‘ nasgar’ of the same strain as that employed for the index. Normal saline was the diluting fluid, and after standardisation sterilisation was effected by heating in a water bath to 60° C. for thirty minutes. Sterility was confirmed by incubating for twenty-four hours on ‘nasgar.’ Until used, phials of vaccine were preserved in a cool dark place. D. Method of Standardising V accine.—All the vaccines prepared in this laboratory are now standardised by this method.t A ‘Thoma- Zeiss cell is employed, covered in by a thin slip chosen in the following way :—A series of ordinary thin (No. 1) 7/8 inch slips are carefully cleaned, dried, and applied lightly to the plate glass slip provided with the apparatus, after gently breathing upon the surfaces to be opposed. ‘The least pressure that will cause them to adhere is used, and the slip chosen which shows when held up to the light the best distribution of Newton’s rings. Only a few slips from each ounce are satisfactory in this respect. The slips so selected may, of course, be used many times over. ‘The diluting fluid is sodium chloride O-I per cent., Giemsa’s stain 5 per cent., in distilled water; filtered or preferably allowed to stand for a few days. Before use 5 per cent. of formalin is added. The organisms gravitate to the bottom, the formalin inhibiting movement, and the trace of salt assisting the staining. We are indebted to Major Harrison for the suggestion that formalin should be added. Either the red or the white corpuscle pipette may be used, according to the dilution required. A small plug of cotton wool is placed in the end, and the pipette is sterilised in the hot-air steriliser. The usual precautions as to the size and placing of the drop are observed, and the 1/12 oil immersion 1. The method was described by one of us at a Pathological Meeting of the Liverpool Medical Institution, on March 5th, 1908, and a short description appeared in the Lancet of March 14th, 1908, in its report of that meeting. , INOCULATION OF MENINGO-COCCIC VACCINE 361 lens is applied after dropping excess of oil on the slip. It is advisable to first of all bring the scale into good position with the 1/6 lens. Counting, with the condenser somewhat lowered, may be commenced within an hour; and as a rule it is quite sufficient to examine three sets of sixteen small squares. It is well to focus through the depth of the cell, as an occasional organism may be found floating, but the vast majority lie placidly at the bottom and may be rapidly counted. The method has been controlled by using for the same vaccine different dilutions in two similar pipettes, and by using simultaneously both the red and the white corpuscle pipette. That the error from irregularity of the coverslip or possible cupping from surface tension is negligible can be judged from the even distribution of the organisms in the small squares. Using a red corpuscle pipette and taking vaccine up to the second cross-marking from the point (z.e., a dilution of 1 : 500), the average number of organisms per small square is five iN a vaccine containing ten millions per cubic millimetre. By using the fullest dilution with the red corpuscle pipette it would be possible to standardise a vaccine containing 100 millions per cubic millimetre. Control Experiments.—The index of the subject of the experi- ments was tested eight times against five different sera before com- mencing the inoculations, and five times against four different sera afterwards. On every occasion it was found to be normal—that is to say, between 0-9 and 1-1. Although we accept this range as a basis, in actual fact our results so closely approximate to unity that we feel it is advisable to give them for these thirteen observations—especially so in view of the criticisms that have been persistently levelled at the accuracy of the index :—193-214, 135-136, 229-228, 202-219, 62-59, 30-32, 70-72, 76-78, 80-81, 76-74, 98-97, 71-77, 77-80. Experiment No. 1.—9th December, 1907. Four cubic millimetres (containing 32 millions) of a vaccine prepared four days previously were injected into the left forearm. At the end of one hour an index from each hand showed a very marked drop, which was recovered from one hour later. Till 13th December there was a well-marked positive phase, with a maximum of 1-5 (each hand) on 11th December. A second sub-normal phase was present at the next examination (17th December). One day later 362 BIO-CHEMICAL JOURNAL the index was normal (normal also to staphylococcus aureus), and remained so on the following day, when the next injection was given. Reaction.—There was no general reaction. Local swelling appeared one hour after inoculation, increased steadily during the next few hours to the size of a pigeon’s egg. There was not much redness. "Twenty-four hours later the swelling was still marked, but flatter and more diffuse ; the skin for about four square inches was hard and tender to pressure, but otherwise painless ;. the edges were distinctly raised, and the colour bright red. On 11th December the redness around the puncture was less, but its area had increased by one-half inch all round ; less hardness was present, except immediately around the puncture, where there was still a little swelling. On 12th December there was merely a little thickening and bronzing round the site of inoculation. Experiment No, 2.—19th December, 1907. Eight cubic millimetres (containing 68 millions) of a vaccine prepared fourteen days previously were injected subcutaneously into the left forearm. One hour later the index of each hand had risen (1°3 L., 1-2 R.). Readings till 26th December were within the normal range, but at its upper limit; on the 2nd January, 1908, 1-2; and on the 7th and 8th January, normal. The index on the 23rd December was I-I to two strains of the meningococcus (215 and 112 days old respectively). - Reaction.—There was no general reaction. Local swelling about one inch in diameter appeared one hour after the inoculation; twenty-four hours later there was swelling and uniform redness for six inches around the puncture; degree and course — very like the first inoculation. Experiment No. 3.—8th January, 1908. Twenty cubic millimetres (containing 100 millions) prepared two days previously were injected subcutaneously into the left forearm. ‘Twenty-four hours later the index had risen to 1-4, and it remained above normal on the toth, 13th, and 14th (maximum 1-5 on the roth). It was normal on the 2oth, and then rapidly fell to 0-5, in which region it remained for four days. It then rose to normal, within a range of 0-2 of which it oscillated until the next inoculation on February 4th. During the second sub-normal phase indices taken against the staphylo- coccus albus and citreus were found to be normal. Reaction.—There was some headache and malaise for twenty-four hours. On the day after the injection there had developed round the puncture a tender but otherwise painless area of brawny redness four by four and a half inches in extent. The axillary glands were unaffected. On the roth the area of redness was larger, but the colour less intense ; on the 11th the area of redness was only two inches in extent around the puncture. By the 21st January the area around the puncture had gone through stages very similar to calf lymph vaccination ; on this day the scabs were removed. Local tenderness had ceased. Experiment No. 4.—4th February, 1908. Thirty cubic millimetres (containing 222 millions) of a vaccine prepared eighteen days previously were injected subcutaneously into the left forearm. A negative phase appeared on the 5th, and was pronounced on INOCULATION OF MENINGO-COCCIC VACCINE 363 ~ the 6th. On the 8th (ninety-six hours after the injection) the index was once more within the normal range. The indices on the roth, 11th, 12th, and 14th showed a well- marked rise(highest 1-6 on the roth and 11th). - 'To this again succeeded an oscillating but mainly sub-normal phase lasting at least a week. As confirmation that this oscillation was actually present, it is of interest to note that the blood of a patient under treatment at the same time gave, with a control common to both, a very even curve. Reaction.—The injection was given at 5.30 p.m.; the subject had felt quite well all afternoon. At 6.15 p.m. there was a feeling of nausea accompanied by severe vertical headache and rigor. At 7.30 p.m. vomiting occurred, and some diarrhoea ; the tempera- ture was 103 F., and the pulse rapid ; headache was very severe, and a restless night was spent. In the morning the temperature had fallen to normal, and the subject felt better except for the headache, which persisted for two days. "There was considerable pain behind the eyes for thirty-six hours, and pain, but no stiffness, at the back of the neck for two days; this persisted to a slight extent for six days. ‘There was no pain in the back. ‘The skin over the head and neck was tender. On the second day’after the inocula- tion severe herpes appeared on the right side of the lower lip and left ear, which were greatly swollen and covered with vesicles. Locally there was a tender area five inches in extent ; redness appeared around the puncture very similar in appearance and course to the previous experiments. No local vesiculation occurred ; the axillary glands were unaffected. SUMMARY These experiments form a series, with a longest interval during which no indices were taken of seven days. Succeeding inoculations were only given when the index change from the preceding inocula- tion had returned to normal. 7 Primary Negative Phase—Apart from a drop in Experiment No. 1, lasting less than two hours but confirmed in each hand, there was no initial negative phase after the first three inoculations, but a very marked one (four days) after the fourth. Positive Phase.—All four inoculations produced a positive phase, lasting in No. 1 not longer than a week—certainly four days. No. 2 (? fourteen days), since a series of four consecutive indices at the upper limit of the normal range may be considered a rise, especially when preceded and followed by an undoubted rise, the former confirmed in each hand. 7 364 BIO-CHEMICAL JOURNAL No. 3, not longer than twelve days—certainly six days. No. 4, six days. In every case the highest point was reached within two days of the development of the positive phase. Second Negative Phase.—This followed the positive phase in three cases (Nos. 1, 3, and 4), lasting one to two days, four to five days, and a week or more respectively. With the larger doses this sub- normal phase showed a very distinct and somewhat prolonged oscilla- tion, with, however, a sub-normal average—that is to say, the original positive and negative phases were merely the first in a series of which the amplitude steadily diminished. ‘The number of days after each injection before the index had settled at normal worked out in— No. 1—Ten days. ,», 2——Nineteen days. 5 3—About twenty-four days. 5 4-—About twenty-six days. Reaction.—The reaction, local and general, increased part passu with the dosage of vaccine, and irrespective of its age, up to three weeks, A gglutination.—This was tested on the 6th and 9th of December, and the 15th February, by the loop method, at laboratory tempera- ture, 1/6 lens, time four hours. In dilutions of I-10, 1-20, 1-40, the results were negative. Apparently the extent of saturation required to produce agglutination was much greater than our experiments reached. CoNCLUSIONS Limit of Dose.—There can be little doubt that in this particular subject a dose of 222 millions even after three preceding inoculations was markedly excessive, leading to prolonged sub-normal phases, with a positive phase no longer than in the preceding experiment. Even the dose of too millions produced a second sub-normal phase to be avoided. It would seem, therefore, that the maximum useful — dose for an adult is well under 100 millions; as by the smaller doses INOCULATION OF MENINGO-COCCIC VACCINE 365 the rise obtained was as good, and the sub-normal phases were less marked. - Immunity Produced.—It appears that the immunising power of a meningococcus vaccine so made and administered is not of much practical service as a prophylactic, and might, indeed, do harm from the production of supersensitisation. ‘That the immunity conferred by an attack of the disease in a human subject is not of very long duration is shown by a case which we had under vaccine treatment in July, 1907, in collaboration with Dr. Rundle and Dr. Williams, of the Liverpool City Hospital, Fazakerley. During the illness a dose of 21 millions produced no local reaction. In November, 1907, the patient came to see us; the index then was 1-4 (tested once). During December, January, and February it fell to normal, and as the patient was complaining of variable but apparently increasing deafness a dose of 30 millions was injected subcutaneously. The result obtained- was in all respects comparable to a similar dose in the subject of these experiments. ‘That is to say, six months after her illness the patient had returned to the normal state as tested by the local reaction after the inoculation of a moderate dose of vaccine. The patient’s serum agglutinated her own organism once during the illness in a dilution of I : 25 at laboratory temperature, but on no occasion after recovery. 366 CHANGES IN THE CHEMICAL COMPOSITION OF THE HERRING DURING THE REPRODUCTIVE PERIOD By 'T. H. MILROY, Professor of Physiology, Queen’s College, Belfast. (Received Fuly 7th, 1908) The chemical changes which occur in the salmon during the various periods of its life history have been worked out with great care and thoroughness by Miescher,! Noél Paton? and others, and the results which have been obtained have greatly increased our knowledge of the relationship between the reproductive organs and the muscles of this fish. It is natural to suppose that a somewhat similar cycle of changes might occur in the herring, and as this fish constitutes such an important article of diet, it was deemed advisable to study the changes in its composition at various seasons of the year. At the request of the Scottish Fishery Board I examined fish obtained at various seasons and in different stages of reproductive activity. In the first place I examined fish caught on the West Coast of Scotland (Loch Fyne District) for a period of one year, starting with fish with immature ovaries or testes, and ending with spent fish. | The fish caught in May showed very immature genitalia, al as summer and autumn proceeded, the ovaries showed a continuous increase until December, when full maturation was attained. In January and February the fish were either spawning or spent. _ During March and April the fish were spent, but began to show commencing growth of ova in the old ovarian capsules. A similar investigation was subsequently carried out in the case of East Coast fish, and the results of this examination will be given in a subsequent paper. The fish in all cases were sent in a double-walled carrier, an ice- salt mixture filling the space between the walls. 1. Die Histochemischen u. Physiologischen Arbeiten, by Miescher, 1897. 2, ‘The Life History of the Salmon,’ Scottish Fishery Board Reports, 1898. ~CHEMICAL COMPOSITION OF HERRING 367 The fish were measured and weighed, and the genitalia then removed and also weighed. The ova were examined microscopically, and their diameter measured with the ocular micrometer. The length was measured from end of snout to end of tail fin, while the girth was taken in front of the dorsal fin around the thickest part of the fish. The fish were then skinned, and the muscles passed through a mincing machine. It was found impossible to do more than analyse the muscles and the ovaries. Some analyses were made of different muscles, but the variations in composition were so slight that the plan was abandoned and specimens taken of the general musculature instead. Metuops or ANALYSES It was impossible to adopt many precautions which might have been desirable, owing to the necessity for carrying out a large number of analyses rapidly. The water content of the muscles and ovaries was arrived at by drying the minced substance in vacuo over sulphuric acid. The total nitrogen was estimated by Kjeldahl’s method, and the P,O; by incineration with pure NaOH and KNOs, and subse- quent treatment by the ammonium molybdate method. The protein percentage in most cases was simply arrived at by converting the total nitrogen into terms of protein, but in the case of the East Coast fish previously mentioned the coagulable protein was estimated by the anhydrous sodium sulphate method. The fat was estimated in the powdered dry material after thorough admixture with pure silver sand, the powder being in the first place extracted with hot alcohol, and then with ether by Soxhlet’s method. | It-was thought advisable not only to give the percentage amounts of protein, fat, and P,O,;, but also the absolute amounts in- the collective muscles and genitalia respectively of the same fish. 368 BIO-CHEMICAL JOURNAL Tables are also given showing the ratios between the weight of the fish and the reproductive organs at different periods, and also those existing between the more important constituents of the muscles and genitalia. Analyses of herrings! have been made by different investigators, but so far as my knowledge goes no systematic examination of the fish has been made at the different periods of its reproductive life. Atwater gives the maximal, minimal, and average amounts in the herrings examined by him, but makes no statement as to the condition of the genitalia. His numbers are the following :— Water Water-free Protein Fats Ash substance t.e., N X 6:25 Maxima 7611 30°97 19:12 IT-o1 19 Minima 69°03 23°89 15°31 4°89 z Average 72:19 27°90 17°75 8-02 1-69 Resutts or ANALysses or West Coast Herrincs Those fish which were sent for examination during May were smaller than those received at later periods. The following table gives the necessary information with regard to their condition :— Taste 1.—Locu Fyne Herrinc, May torn, 1906 Length Girth Weight Condition cm- cm, cm. 25 11} 113 Very immature, previously 234 11 101 spent herrings. 221 12 104 There were large numbers 23% Il 102 of very small ova present, 23 10} 93 varying in size from 0:07 to 23 10 go o-15 mm. in diameter 23 10} 88 214 10} 80 223 10} 35 22 10 80 21} 10 74 22 10 76 Average 22-9 cm. 107 cm. 90-4. gm. Weight of genitals (fresh) of 12 herrings, 2-5 grammes. ‘The water percentage of these genitals was 71-52. 1. Payen, Substances Alimentaires p. 488; Konig, Nabrungsmittel, Bd. 1 . 201-7; and Atwater. U.S. Commssioner’s Report on Fish and P isheries, 1888, pt. XVI, 4 is - CHEMICAL COMPOSITION OF HERRING 369 Analyses of the muscles of these herring :-— Female— (2) Amounts stated in percentages of fresh material. Water Protein Fat P.O; 72°69 18-98 725 0-68 (6) Grammes in the total muscles of the average fish of this series. Protein Fat PO, 11-38 4°35 0-40 These herrings had probably spawned some time between February and April on the Ayrshire coast (Ballantrae spawning beds), and had then passed up Loch Fyne. ‘They were caught near the opening of that Loch. Judging from the amount of fat present, they had, however, probably resumed feeding for more than a month, but the collapsed condition of the large ovarian capsules showed that within a comparatively recent time the fish had discharged their ova. The high water percentage of the muscles is also characteristic of fish that have been recently spawning. On the following day some herrings were caught in the same neighbourhood, and these showed even more marked signs of recent spawning. (Table II.) They were on the average smaller fish, with one exception. This one (No. 10) was examined with the rest because it was evidently in the same condition as the others, which were on the average thinner and lighter than those of the previous set. As will be seen from the table, the water percentage is higher and the fat lower than in the previous set, while the phosphorus percentage is higher than in any other muscles which were examined. 370 BIO-CHEMICAL JOURNAL Taste II].—May Length Girth Weight Condition cm. cm. gm. I 20 8°5 58 The ova were very 2 20°5 9 60 immature. The 3 20°5 9°5 67 weight of the Io 4 20°5 9°5 67 pairs of genitals 5 20°5 9 64. was 2°4 gm. in the 6 21 9 72 fresh condition. 7 215 10 75 8 22'5 9 75 9 at 9°5 79 10 25 II 116 Average 21°3cm. + 9:4.cm. 72°4 gm. Muscle. (2) Amounts stated in percentages of fresh material. Water Protein Fat PO; 73°o! 17°55 5°85 0°82 (6) Grammes in the total muscles of the average fish in the series. Protein Fat P.O; 8-47 2-82 0°39 Fish caught during June and July were practically in the same condition, there being only slight growth of the genitalia; the weight of the fish gradually increased, but the percentage composition of the muscles altered very slightly. It is scarcely necessary to give the numerous analyses cf fuee fish which were made, because they were practically identical with those mentioned under the first set of the May fish. An example is here given of fish caught at the end of July. The ovaries were, although still immature, sufficiently large to +e of their analysis. July.—The water percentage of the muscles is still high, slain slightly below that occurring in the May fish. ° The protein percentage is practically the same, and the fat slightly higher than in the average May fish. The ovaries were larger, with the ova in a slightly more mature CHEMICAL COMPOSITION OF HERRING _ 371 condition, averaging about o:2 mm. in diameter. Analyses were therefore made of the genitalia. Taste II].—Jury Length Girth Weight Weight of Genitals cm. cm. gm. gm. 24 12 116 0°35 22 I1°5 96 0-46 23°5 12. 99 0-20 23 13 120 0:67 4%" 115 gI 0-20 22°5 12 105 0-74 23 12 107 0°33 24 12°5 124 1°18 21°5 II 82 0°49 22°5 12 105 0-43 22°5 vig BERG 102 0°39 22 II 92 0:22 23 12 97 oS 24 13 127 0:50 225 12 100 O-44 25 13 137 0°53 24 12°5 11g o-81 22 II 88 "0:20 22 II g2 0:20 23 13 120 0-65 Average 22°9 11-9 100°9 0-45 ° Analyses of the Muscle (fresh) :— (a) Percentages. Water Protein Fat PO; 71-60 18-18 7°32 0-45 (6) Grammes in the total muscles of the average fish. Protein Fat P.O; 12-18 4:90 0-30 Analyses of the genitals (fresh) :— (a) Percentages. Water "Protein Fat P.O; 72°5 13°52 8-92 0°77 (2) Grammes in the amount of genitals present in the average fish of series. Protein Fat PO; 0:05 0:03 0-003 September.—By the middle of September the ovaries were almost I gramme in weight, and the ova measured about 0-2 to o-45 mm. in diameter. | 372 BIO-CHEMICAL JOURNAL It will be sufficient simply to state the percentage composition of the muscle flesh at this period. Water Protein Fat P.O; 63°68 19:28 11-81 0-64 In all the fish examined about this time the water percentage was at its lowest, and there was also a distinct increase in the amount of fat and protein. The ovaries at this period had the following percentage com- position :— Water Protein Fat P.O; 66-02 18-91 7°34 1'23 The total amounts present in the ovaries of the average fish of this series were, of course, very small, namely :— Protein Fat P,O; O17 0:07 O-Orl The relatively high fat percentage of the ovaries indicates that the degenerated ovarian tissue of the spent fish has not yet been entirely used up. October.—During this month fish were obtained in different stages of maturity, some with ovaries weighing from 2 to 3 grammes, others weighing about 9 grammes; while the ova varied from 0-3 to o-6 mm. in diameter. | By far the larger number of the fish caught at this period were heavier than those obtained at an earlier season, but the changes in composition were of the same kind in all. As will be seen from ‘Table IV, the most marked alteration in the muscles is the great increase in the fat, the highest percentage being observed in fish caught at this season. It will be observed from a study of the later series that as the ovaries begin to increase rapidly in size the fish begins to use up the store of fat which has been accumulating in the muscles during the earlier months. ‘T'wo series of fish are given in the following table, one (Series A) including those caught during the earlier part of the month, the other (Series B) towards the close of October. (Table IV.) CHEMICAL COMPOSITION OF HERRING 373 Under Series B the results of the examination of male fish caught at this season are also given. As will be seen, they correspond very closely to the females. Taste [V.—Series A (Octoser 7TH) Length Girth Weight Condition cm. cm. gm. 28 15 206 The ovaries from 27 13°5 150 these seven herrings 25 13 147 weighed collectively 24°5 12°5 130 17 gm., and the 23°5 12 115 average size of ova 23°5 12°5 122 was 0-28 mm. 26 13 148 Average 25 cm. 13 cm. 144°5 gm. Sertes B (Ocroser 20TH) Length Girth Weight Condition cm. cm. gm. 29°5 16 257 The ovaries from 28-5 15°5 220 these eight herrings 27°5 13:2 167 weighed collectively 24°5 12°5 129 74 gm., and the 24 - 12-2 119 average size of the 25 14°5 220 ova was 0°59 mm. 29°5 15°5 229 28 14 197 Average 27 cm. 14 cm. 192 gm. Series A. Muscles. (a) In percentages. Water Protein Fat P.O; 69°97 12-78 14°25 0°53 (2) In total muscles of average fish. Protein Fat P,O; 12°39 13°82 o-51 Ovaries. (a) In percentages. Water Protein Fat P.O, 78-21 17:26 2°53 o-71 (6) In ovaries of average fish. Protein Fat PO; O-41 0°33 o-olI 374 BIO-CHEMICAL JOURNAL Series B. Muscles. (a) In percentages. Water Protein Fat P.O; 70°46 14°84 12°70 0°57 (b) Per average fish. ) Protein Fat P.O; 18-99 16°25 0-73 Ovaries. (a) In percentages. Water Protein Fat P.O; 68-02 22°45 4:80 0:93 (b) Per average fish. Protein Fat PO; 2:08 0°44 0°05 Males— Muscle. (a) In percentages. Water Protein Fat PO, 68-91 16:18 12°63 0-50 (b) Per average fish. . Protein Fat PO; 17-91 13-98 0°55 Testes. (a) In percentages. Water Protein Fat PO, 72°13 22°62 Le ee 0-86 (6) Per average fish. Protein Fat P,O, 5°42 O54 0:20 November.—During November there was a rapid increase in the size of the ovaries, the ova measuring about o-8 mm. in diameter on the average. The water percentage of the muscles was lower than in the preceding month, and the fat showed the first indication of being used up by the fish from the distinct decrease in its amount, while the protein showed a rise. It is probably about this period that the winter spawning fish begin to take a smaller quantity of food, and, therefore, they fall back upon their fat store as a source of the necessary energy. The muscles of the male fish are practically of the same com- position as those of the female. (Table V.) . CHEMICAL COMPOSITION OF HERRING 375 Taste V.—NovemBer Fisu I. Females ___.--Eength Girth Weight Weight of ovaries ee cm. cm. gm. gm. 30 16°5 275 35°7 31 16 270 38-2 33 17°5 326 51-6 31 16 255 31-8 30 - 1 261 25°6 31 16 260 18-9 32 16:5 297 31-6 32 EZ 290 35°2 32 16°5 275 31-4 30 16°5 276 28-8 32 16-5 304 2 29 15 210 20°1 30 15 204 15*4 29 14 192 15°8 Average 30-8 cm. 16 cm. 264 gm. 29°5 gm. The ova were from 9:8 to'r mm. in diameter. II. Males Length Girth Weight Weight of Testes tm. cm. gm. gm. 3f 17 279 44°5 31 17 316 52 31 16 277 40°6 29 14 215 34 30 16 257 40°9 33 16 296 37°3 32 16 287 40°I 30 17 290 4II 31 17 303 46-6 31 16 252 35°6 31 15-5 230 26:8 29 14°5 ¥95 a4°4 30 16 255 44°6 Average 30°7 cm. 16 cm. 265°5 gm. 38-9 gm. Females— Muscle. (a2) In percentages. Water Protein Fat P.O; 66-34 19°87 10°85 0°59 (2) In total muscles of average fish. : Protein Fat PO, 34°97 19°09 eNOS 376 _ BIO-CHEMICAL JOURNAL Ovaries. (2) In percentages of fresh material. Water Protein Fat PO; 68-04. 25°04 2°85 0-91 (5) Per average fish. Protein Fat P.O; 7:38 0°84 0-26 Males— Muscle. (a) In percentages. Water Protein Fat P.O; 68-22 17°94 10°84. 0-46 (b) Per average fish. Protein Fat PO; 31°77 19°18 o-81 December.—The fish caught during this month were either ready for spawning or had commenced to spawn. An example will first be given of fish which although probably about to spawn still show a moderately high percentage of fat, and a low percentage of water in the muscles. ‘They were, in fact, in very good condition. ‘The genitalia were, as will be seen from the table, of the maximum weight, and the ova were mature. Taste VI.—Earty December Fisu Females Length Girth Weight Weight of ovaries. cm. cm. gm. gm. 31 17°5 326 63 30°5 15 268 43 30°5 16°5 272 30 31 16°5 330 48 30°5 16 285 47 31 17°5 318 51 Average 30-7 cm. 16°5 cm. 2998 gm. 47 gm. Males Length Girth Weight Weight of testes cm, cm. gm. gm. 31 17 298 61 31 17 a ae 55 Average 31cm. 17 cm, 309 gm. 58 gm. CHEMICAL COMPOSITION OF HERRING 377 Analyses :— Females— Muscle (a) In percentages. Water Protein Fat POs 67°36 20°56 8-18 0-68 (>) Per average fish. Protein Fat P.O; 41-12 16:36 1°37 Ovaries. (a) In percentages. Water Protein Fat P.O; 67°33 25°72 2:89 1-03 (>) Per average fish. Protein Fat P.O; 12-08 1°35 0-48 Males— Muscle. (a) In percentages. Water Protein Fat P.O; 68-31 21°45 9°24 0°73 (b) Per average fish. Protein Fat PO; 44°18 19°03 I-51 Testes. (a) In percentages. Water Protein Fat PO; 72:10 22°05 3°73 2:10 (>) Per average fish. Protein Fat P,O; 12-78 2°16 I-21 The following table (VII) gives the results of the examination of herrings obtained farther North. They had commenced spawning ; in fact, had discharged probably about the half of their store of ova. The ova present were, of course, fully ripe. The fish were smaller and in much poorer condition than those of the previous series. As they are not from the same neighbourhood they are not strictly comparable with the preceding series, but it was impossible to obtain herrings from Loch Fyne during late December and January. These, however, belong to the same class of herrings as those which are given in the subsequent January and February series. 378 BIO-CHEMICAL JOURNAL Two herrings (females) were taken for analysis from a batch containing fish of approximately the same size. ‘Their measure- ments were :— Taste VIT.—Late DecemsBer Fisu Length Girth Weight Weight of ovaries cm. cm. gm. gm. 29 ey Saar 195 26 29 145 194 35 Average 29cm. 14°5 cm. 194°5 gm. 30°5 gm. Size of ova, 08 to I:'2 mm. Analyses :— Muscle. (a) In percentages. Water Protein Fat P.O, 72°50 23°01 2°75 0°77 (b) Per average fish (total in muscles). Protein Fat P,O; 28-76 3°4 0:96 Ovaries. (a) In percentages. Water Protein Fat P.O, 65°73 27°76 3°34 I-19 (b) Per average fish. Protein Fat P.O, 8-46 1-02 0°34 The most striking changes in the muscles of these herrings are the marked fall in the fat percentage and the rise in the water per- centage. This marked decrease in the fat was always found to take place either just at the onset of spawning or after spawning had been in progress for some time. In the case of the fish from the East Coast of Scotland spawning in early autumn, the fat percentage did not sink so low, and had often reached its minimum in the so-called ‘ full’ fish—that is, the herring with mature ovaries. . This will be seen from a study of the analyses of these fish which will be given in a later paper. Secu ils { a - CHEMICAL COMPOSITION OF HERRING 379 The fat percentage of the ovaries has risen slightly above that of the mature fish prior to spawning, this being characteristic of the spawning process. _Fanuary (Table VIII).—Some of the late December fish showed the same characters as those taken in January. They could not be obtained at this time in Loch Fyne, but some were analysed which came from farther North. ‘They were in _nearly all cases slightly smaller fish. ‘Two series are given, one with spawning proceeding rapidly, the other in the spent condition. ‘The male fish were not examined. Tasre VIII.—Janvary. Series A (spawning) Length Girth Weight Weight of ovaries cm. cm. gm. gm. 27°5 13°5 165 18 26°5 14 157 14°8 26 13°5 141 25 26 ¥2..; {2 127 115 26°5 13 168 20°3 26 14 160 18-3 26 13 150 19°4 25 12 115 10°5 26 13°5 150 18-8 Average 26cm. ct ete 148 gm. 17-4 gm. Series A. Muscles. (a) In percentages. Water : Protein Fat | PO; 74°12 18-91 2:02 0-66 (6) Per average fish. Protein Fat P.O, 18-15 1-93 0-61 Ovaries, (a) In percentages. Water Protein Fat P.O; 79°00 24°75 By fs 93 (b) Per average fish. Protein Fat PO; 4°23 0°63 o-16 380 BIO-CHEMICAL JOURNAL These fish were in much the same condition as those of the preceding series, but they had a higher water and a lower protein and fat percentage. Among the fish obtained at this time there were two which were completely spent, the ovaries being collapsed, and only a few remaining ova seen in the collapsed tissue. Taste 1X.—Series B (spent) Length Girth Weight cm. cm. gm. 23 12 153 28 12 140 Average 25°5 cm. 12 cm. 146°5 gm. Series B. Muscles. (a2) In percentages. Water Protein Fat P.O; 75°30 19°69 1°55 "77 (6) Per average fish. Protein Fat PO; 18:53 Ig! 0°75 ‘The water percentage has risen, and the fat fallen to a very low level. February (Table X).—Practically all the fish sent during this month were found to be spent, and in even poorer condition than those obtained in January. As they were obtained from the same place as the January fish, the two series are readily comparable. TABLE X.—FEBRUARY Length Girth Weight cm. cm. gm. 26 II 115 28 12 143 28 12 155 29 13 156 29 12 150 28 12 136 Average 28 cm. I2 cm. 142°5 gm. CHEMICAL COMPOSITION OF HERRING 381 I.—Muscles. (a) In percentages. a _ Water Protein Fat PO; 78-97 18-05 0-68 0-73 (6) Per average fish. Protein Fat P.O; 16-60 0-62 0-66 Il.—Ovaries (spent). The average weight of the ovaries per fish was I gm. (a) In percentages. Water Protein Fat P,O; 82-07 3°78 11-83 o-gI (6) Per average fish. Protein Fat P.O; 0-29 0-09 0-016 From these analyses it is seen that the fish had used up practically all their store of body fat. The muscles contained a very high per- centage of water, and were in the poorest condition. The spent ovaries showed the characteristic high percentage of fat seen in that condition, in this case an extremely large amount being present. It was impossible to obtain West Coast herrings in March, but I examined fish caught in April, and they were practically in the same condition as those taken in May, and of which analyses have been given. Before studying the changes in the chemical composition of the muscles and ovaries, as shown in the preceding tables of analyses, it is important to note certain points of general interest with regard to the growth of the ovaries. If one arranges the weights of the fish and ovaries in such a table as the following, one gets a clearer idea of the various periods in the life history of the herring. (Table XI). 382 BIO-CHEMICAL JOURNAL Taste XI.—Ratios or Weicut oF Fish to We1cut oF Ovarizs aT DirFERENT SEASONS Period Weight of fish Weight of ovaries Condition May a 434°6 : I Immature May yn. Sere I s July ae 224-2 I os September... 84:2 I ” (Ova 0-15 to 0-45 mm.) October A... 60:2 I es (Ova 0:28 mm.) October B_... 20°8 I ‘“ (Ova 0:59 mm.) November... 8-9 I Almost mature (ova 08 to I mm.) December __... 6:3 I Mature (ova 0-9 to I:2 mm.) December... 6°3 I Spawning PB a January 8-5 I ti : ie 2 January = — — Spent February s — — om It is interesting to note that the main growth of the ovaries takes place after the most active feeding period is over. These fish, which spawn in January and February, have their principal feeding time between April and September. From April to June they feed mainly on copepods, from June to September on schizopods.' Food is, however, taken also during October, November, and December, but in smaller amount. The spawning, according to Brook and Calderwood, takes place. six to eight months after the period of richest feeding. There is no doubt that with the increase in the development of the genitals the desire for food diminishes until spawning time arrives, when no food is taken. The most interesting period to study carefully is that included under the October, November, and December series. If one select from each of these tables the fish that are evidently comparable as regards length and girth, namely, in the October Series B the two fish 29:5 cm. in length, in the November series the three fish 30 cm. in length and 16 to 16:5 cm. in girth, and the three 30°5 cm. fish in the December series, one notices that there is certainly no loss in the weight of the fish, but rather a gain as the season advances. ‘That is to say, during the ‘period when the greatest increase in the development of the ovaries takes place, there is no evidence of this growth of the reproductive organs occurring entirely 1. Brook and Calderwood, ‘ Report on the Food of the Herring,’ Fourth Annual Report, Scottish Fishery Board, 1886, Appendix F, No. VIL. pp. 102-128. CHEMICAL COMPOSITION OF HERRING 383 at the expense of the other tissues, seeing that there must be in addition some combustion of food material stored in the tissues to cover the energy requirements. ‘There can be no doubt that during this period the fish is beginning to use up its store of fat instead of increasing it as was the case only a short time before. That there is a distinct loss in the total fat of the muscles of the December fish compared with the October ones is shown by again comparing the amounts of this constituent present in fish of similar size caught during these months. ‘Taking again the same fish from the October, November, and December series as was done for the comparison of weights, one finds that the total fat content of the muscles in the October fish is 20-56 grammes, in similar November fish 19°57 grammes, and in December fish 14:99 grammes. ‘There ean be no doubt that this loss of fat cannot be accounted for by a transference to the growing genitalia. The probability is that it is being used to furnish the necessary energy required for the work which the fish is performing. Tue Nature oF THE CHEMICAL CHANGES IN THE MUSCLES OF THE HERRING The Water Content of the Muscles——The percentage of water in the muscles rises when spawning commences, and reaches its maximum in the spent fish. There are, however, exceptions to this rule, as, for example, in the case of the late December fish. The lowest water percentage is, as a rule, to be found when rapid growth of the ovaries is taking place. Protein.—The changes in the amount of protein are difficult to follow. ‘This may be due in part to the fact that it really was the total nitrogen which was estimated in the case of the analyses given in the preceding tables, or it may be due to the variations in the water content. In the first place, the lowest protein percentage is found at the time when the fat is at its highest level. There seems to be a gradual transformation of protein, or possibly glycogen, into fat when the stage of active feeding passes into that 384 BIO-CHEMICAL JOURNAL of reproductive activity. The average protein percentage between May and September is approximately nineteen, while in October it is fourteen. During the period of rapid growth of the ovaries, and before full maturity, the protein percentage again rises, being due probably to the loss of water from the muscles during this time. Fat.—Reference has already been made to the variations in the amount of this constituent, but it is necessary to refer to them in more detail. It is the one constituent which shows regular alterations in its amount during the various stages of reproductive activity, although it is quite possible that glycogen may show similar changes. It has, however, not been possible up to the present to carry out a satisfactory series of glycogen determinations at the various periods of reproductive activity. ‘This may be said, however, that the amount of glycogen in the spent fish was- extremely small in the analyses which I have made. | During the months of May, June, July, August, September, and the early part of October there is a gradual increase in the percentage amount of fat in the muscles. This increase is most marked from August up to the beginning of October. It is most likely that during the summer months, when the young ova are just beginning to appear, the food material is mainly stored in protein form in the muscles, while in late autumn the main storage form is fat. In November and early December, when the ovaries are increasing most rapidly in size, the fish falls back upon its store of muscle fat to supply the necessary amount of energising material. By the time that active spawning has commenced the fat shows a great decrease, and in the spent fish of February it reaches its minimum. The reason why there is not a more rapid fall in fat during October and November is that the fish during this time is still feeding, although, probably, not to the same extent as at the earlier periods. During the process of spawning the: feeding ceases altogether. There can be no doubt that the fish feeds voraciously during CHEMICAL COMPOSITION OF HERRING 385 the summer months, hence the frequent occurrence of distended stomach and intestines in fish with immature ovaries. ‘This may lead to the abnormal condition called by fishermen on the West Coast of Scotland ‘gut-poke.? Such fish rapidly undergo decomposition owing to the deficient absorption of the intestinal contents. This condition is never found in fish with mature ovaries. It is interesting to notice the ratios of protein to fat in the muscles at the various seasons. The following table gives these ratios. (Table XII.) Taste XIJ.—Proretn: Fat 1n THE Muscies Period Protein Fat Condition May Ae 2-61 I Immature May ao 3°00 I - July BE: 2-48 I 3 September... 1-63 I fe (Ova 0-15 to 0-45 mm.) October A... 0-89 I =! (Ova 0-28 mm.) October B_.... 1-16 : I pe (Ova 0-59 mm.) November... 1°83 ; I Almost mature (ova 0-8 to I mm.) December... 2°51 I Mature (ova 0-9 to I mm.) December... 8-36 I Spawning January ‘ 9°36 I Pi January ie 12-70 I de February oie) Tizheee I Spent Phosphorus——The variations in the amount of phosphorus are somewhat irregular and difficult to account for. In October and November fish with rapidly increasing ovaries the average percentage is approximately 0-56 P,O;, while during the preceding months from May to September it may be taken as 0-68. The peculiarly low percentage present in the July fish is difficult to account for. When the ovaries have reached maturity, and also after spawning, the P,O; percentage in the muscles again rises.. CHEMICAL CHANGES IN THE OVARIES The ovaries of the fish examined showed an increase in size from July to December, but as there were great variations in the size of the fish caught at the various seasons, it is more important to study the amount of the various ovarian constituents per 100 grammes of muscle rather than per average fish. | 386 BIO-CHEMICAL JOURNAT. The following table gives this information :— Taste XIIJ.—Ovarian ConsTITUENTs PER 100 GRAMMES MuscLe Period Protein Fat PO, July on 0-074 0-044 0-0044 September oe 0°318 O-131 0:0206 October A ae 0°425 C342. 0:0103 October B ve 1-625 0°343 0:0625 November — 4°193 0°477 0°1477 December =e 6-040 0-675 0°2400 December or 6:523 0-799 0:2621 January a 4°286 0°638 0-1621 There is thus a gradual increase of these constituents until spawning occurs. When we compare these figures with the per- centage composition of the muscles at the same seasons, one notices that during the time when the protein of the ovaries is showing a distinct increase—for example, from November to December—there is no corresponding decrease in the muscle protein. It is true that between September and October there is a fall in the muscle protein percentage, but it is more readily accounted for by the local increase in fat than by a withdrawal of protein to the genitals. The herring is thus in a different position from the salmon, at least if one compares the salmon caught in the estuary of a river with those caught in the upper reaches. The salmon during its sojourn in the river abstains entirely from food. If one, however, studies the composition of the early and late estuary salmon examined by Néel Paton, one notices that there are but slight variations in the protein percentage and in the total amount per standard fish, whether the ovaries be immature or mature. The herring undoubtedly does feed practically until spawning occurs, although probably much less food is taken in the later months. : 1. Scott, ‘ The Food of the Herring,’ Scottish Fishery Board Reports, Part IIL, p. 260, 1907. CHEMICAL COMPOSITION OF HERRING 387 There can, therefore, be no doubt that the herring is not entirely dependent upon its muscle proteins for the growth of the ovaries. ai ‘The appearance of fat in large amount in the ovaries of spent fish is similar to that noted by Miescher in spent salmon. This formation cf fat was regarded by Miescher as of great nutritive value to these fish. Male Herrings.-Somewhat similar changes take place in the muscles of the male fish, the highest fat percentage being found just before rapid growth of the testes takes place. During the later period the fat percentage falls just as in the female. Composite tables are subjoined, which enable readily a com- parison being made between fish caught at various seasons. Before giving these tables one may shortly summarise the results of the investigation into the life-history of the herring by dividing up the year into the following three periods :— 1. The Restitution or the Feeding Period.—This continues for three to four months after spawning, and constitutes the principal feeding time. The spent, thin fish recovers, and accumulates during this time a large store of fat. 2. The Ripening or Maturation Period.—This continues for six to seven months. ‘The herring still takes food, but gradually with less desire, and the sexual organs increase as the store of muscle fat diminishes. 3. The Spawning Period.—This continues probably for about two months, and during this time feeding stops. Hence there results a very great fall in the fat, accompanied by an increase in the water content of the muscles. Heincke (Naturgeschichte des Herings, p. 48) has shown that the herring after spawning seeks a place where it can get ample food to recuperate. For example, the herrings of Schley, after leaving the spawning beds in June, take three to four months to feed up in Kiel Bay. In September and October they are fattest, and then begins 388 BIO-CHEMICAL JOURNAL anew the development of the reproductive organs, which up to this time was checked. ‘This takes up the whole autumn and winter, - the fish still taking food, but not using it for the building up of fat, but for the development of the genitalia. With the increase in the development of the genitals the desire for food diminishes until spawning time arrives, when no food is taken. Hence Heincke, although basing his statements on the life-history of the herring simply on general observations, comes to conclusions which are practically identical with those arrived at by me from a study of the chemical composition of the herring at various seasons. 389 910.0 | 60.0 | 6z.0 | 16.0 | Sg.11 | gZ.€ fo.zg 99:9 | 29.0 | 09-91 | £4.0 | gg.0 | So.gr | 6.92] quadg | gz.1 | S.zb1 ZI 4 ae a ve Areniqaq — — a= — —- — — [Slo | 18.1 | €S.g1 | 22.0 | $$.1 | 69.61 | of.$2 quodg — | S.otr 21-1 6.82) ge “ Ch 91-0 | £9.0 | feb | £6.0 | 2Z.€ | SLbz] co.0L}19.0 | 6.1 | S1-g1 | 99.0 | zo.z | 16.g1 | zi-bZ | t.1- go] +21} gi 1 gt | Vy Sottag ‘Arenuef S bE. | coor | og | Ore | EE | gL. rg) percent. hours oki dezastes per cent. Cancer pagurus — 1845° — 1915” —0'575 0575 II — 1402 105 (blood) Cancer pagurus —1°845° —1:915° — 3070 3°075 iS oe — 2785 273 (blood) Echinus esculentus — 1:860 — 076 — 34 —1'77 Note.—Cl (sea urchin) ~ BL ia oe : os ot a the norma (coelomic fluid) — 1:860 — 2°98 34 2°065 Sisod ieee per cent. Asterias rubens — 1*860 —0°965 _ 34 — 1°36 (starfish) (coelomic fluid) — 3:00 -— 34 So much water was withdrawn by the hyper- tonic solution, that the amount of coelomic fluid was insuffi- cient for deter- mination In all cases, in the above experiments the normal equilibrium of the blood or other internal fluid with the sea water was disturbed, and an attempt was made to set up a new one. Eleven hours were insufficient for the blood of the crab to attain its normal relations to the external medium, and any longer immersion than this would have been fatal; just as longer immersion than three and half to four hours would have caused death of the Echinus or Asterias, and also without an equilibrium having been reached. The osmotic pressure of the blood of invertebrates has been regarded as wholly dependent upon the external medium, that is, identical with it, the relations of the external and internal media being determined simply by osmosis through the body membranes. It is more likely that here also, there is a definite equilibrium between the blood and internal media on the one hand and the sea water on the other, even though normally the osmotic pressure of both is almost identical. Small differences very often occur, and I do not consider these due to errors in observation. Furthermore, it has been often forgotten that the blood of the freshwater crayfish has a 4 of —0-8° though living in water whose depression of the freezing point may be almost ‘nil. This is a perfect case of an equilibrium between CHANGES IN OSMOTIC CONCENTRATION OF BLOOD —§ 477 the blood of an aquatic invertebrate and the external medium, _yet-both fluids differ considerably in concentration. The freshwater crustacea are, therefore, very similar to the freshwater fishes (Teleostei). Osmotic Pressure oF BLtoop oF TELEOsTs AND ELASMOBRANCHS AND Cuances Propucep sy ALTERATIONS IN THE CONCENTRATION OF THE EXTERNAL MeEpDIA It has already been shown by me? that the concentration of the blood of teleosts is not altogether independent of the external medium. Thus the teleosts in the fresher water of the Baltic have a slightly lower osmotic pressure for the blood, than the same species in the North Sea. Freshwater teleosts also have a slightly lower osmotic pressure and salt contents than marine species, and the eel (Anguilla vulgaris) when placed in sea water was shown to change so that after several hours the osmotic pressure of the blood had risen to about the average for marine teleosts. The following are the results of experiments made to determine the effects of dilution of the sea water upon the blood. Pleuronectes flesus (flounder).—This fish though a typical marine flat fish, often penetrates a considerable distance into river estuaries, so that it may even be found in fresh water. It returns, however, to the salt water of the sea at spawning times, and hence resembles the eel to a certain extent, since both move back into the sea—a _ reversal of the rule of the salmon, which ascends the rivers for spawning. : The freezing point and Cl contents of the blood have been determined for fish caught round Helgoland in water whose A = —1-g1°, and also for some fish caught in the fresh water of the Elbe and forwarded by steamer. Hetcotanp.—P. flesus— A for blood from caudal artery — 0°883°, — 0-903°, — 0-903”. River Exse, Hamsurc. P. flesus— A for blood from caudal artery — 068°. 1. Bto-Chem. Fourn., Vol, III, p. 258, 1908. 478 BIO-CHEMICAL JOURNAL There is, therefore, a very considerable difference in the osmotic concentration of the blood of the flounder, according as it is living in fresh water or sea water respectively. The following experiments were -all carried out in two large concrete tanks into which sea water or rain water from a reservoir, could be pumped as desired. The rain water was examined and found to be much purer than the fresh water from springs on Helgoland. When fresh water or a mixture of this and sea water was used, the water was well aerated by compressed air driven through pieces of cane. With sea water, the water was also kept running. Cyclopterus lumpus (the Lump-sucker)— | Normal fish from aquarium tank. Blood A — 0-648° Normal fish from aquarium tank. Blood A — 0-658° Experiment I—One specimen placed in a mixture two-thirds fresh and one-third sea water, six hours, then in three-quarters fresh and one quarter sea water for two hours. Killed after eight hours in diluted sea water. Blood A — 0-620°. Experiment II.—One specimen placed in diluted sea water (A — 070°) for two hours and finally in fresh water for 24 hours. Blood A — 0:597°. Experiment III—One specimen in diluted sea water (A — 0-37°) for 48 hours. Blood A — 0-610°. In each case a reduction of the osmotic pressure took place, or in other words, a new equilibrium was either set up or in process of being setup. ‘Taking the mean for the normal blood as A — 0-653°, the amount of change in the three experiments was — 0-033°, — 0:056°, and —0-043° respectively. The coelomic fluid and fluid from the stomach of the last specimen of Cyclopterus was also examined in order to determine their relations to one another and to the external medium. ‘The results were :— From a normal fish in sea water— Fluid from coelom A — 0:617° Fluid from coelom A — o0-6go0° Fluid from stomach A — 0:36° Fluid from stomach A — 1-593° Thus, the coelomic fluid has its own equilibrium, the freezing point being a little lower than that of the blood but very similar. The contents of the stomach on the other hand have a A which is directly dependent upon the external medium swallowed, the CHANGES IN OSMOTIC CONCENTRATION OF BLOOD 479 food and secretion. The very different A’s of the coelomic fluid and-eontents of the alimentary canal in the last experiment are striking, since both are only separated by the thin walls of the intestine.! The Eel. Anguilla vulgaris—In my previous paper, the results of some experiments made on the eel, taken from fresh water, were given, and it was shown that the concentration of the blood increased when the fishes were placed in sea water. The blood of three freshwater species then examined gave the following figures :— Carp oe ee a SS ogg Abramis brama ... ‘ne A —o0-510° Bel 2%. ee Ss ye A — 0°570° After twenty-four hours in concentrated sea water, sp. gr. 29°3, the A of the blood of the eel had increased to —0-745°. The following supplementary experiments have been carried out with the same species of eel, but caught in the sea off Helgoland :— Blood from normal specimens taken direct from sea water ... A — 0-649° A — 0-620° These figures are very interesting, for in the first place they confirm the aquarium experiment at Kiel in showing that the concentration and osmotic pressure of the blood of the eel is greater in the sea than in fresh water, and further show that the osmotic pressure reached by the blood (A — 0-745°) in the Kiel experiment was too high. Perhaps the rapid change from fresh water to sea water at Kiel caused the blood to pass beyond the normal equilibrium 1. It had been noticed that when marine fish were kept in fresh water, there was a gradual, but small, lowering of the osmotic pressure until death. Just before death, however, the diminution was considerable. It was in fact as if the teleost resisted the action of the external medium as far as possible. This was partially confirmed by a determination of the freezing point of a Cyclopterus in aquarium water, but in a pathological condition. Cyclopterus appears in the shallow water round Helgoland, often adhering to the rocks and left dry by the tide, during the spawning season in spring only. In May and June it begins to disappear, and in summer seems to have left for deep water, since very few are caught. The fish kept in the aquarium, though living well in spring, almost all die as the time approaches when they should normally leave for deep water. Death comes gradually, and the jaws and bony scutes appear to decay, so that the fish presents for some time a very unpleasant appearance. The freezing point of the blood of all the normal specimens of Cyclopterus was about — 0658". One of these pathological specimens which was examined had the remarkable value A — 0-765, or — o-107° more than the normal. It may be said, therefore, that the concentration of the blood of pathological specimens tends to approach that of the fluid in which they are living. 480 BIO-CHEMICAL JOURNAL point for sea water, and after a longer immersion in that water it would probably have attained the more normal A — 0-634°, the mean for those specimens taken direct from the sea at Helgoland. As converse experiments to those at Kiel, some specimens were placed in aerated fresh water, the change being gradual. Eels placed direct in mixture half sea and half fresh water, and fresh water pumped in at intervals until the whole was perfectly fresh. ‘The eels finally remaining in fresh water for three days. Results :—(a) Blood A — 0-580° (b) Blood A — 0:583° (c) Blood A — 0:582° Thus the osmotic pressure had decreased, and the freezing point ascended until it was practically. the same as that recorded at Kiel for the fish from fresh water.’ All the experiments, therefore, made on the teleosts show that the blood is in equilibrium with the sea water or the fresh water in which the fish are living, and a change in the external medium is followed by processes setting up a new equilibrium. ‘This change may or may not prove fatal to the animal. The relation of the osmotic pressure of the blood of teleosts to the external medium is very like the relation of the. temperature of mammals to their surroundings. In the mammals the temperature is normally nearly constant, yet not altogether independent of the temperature of their environment, for a great rise or fall in the temperature of the latter produces a corresponding but smaller change in that of the body. In short, the temperature of the body is in equilibrium with the temperature of the surroundings though both may or may not be identical. 1. It is interesting to note that the freezing point for the eels from the sea is the highest of any marine teleostean examined by me, and further that the freezing point of the blood taken from freshwater specimens was lower than that from the other species experimented with. Hence, under normal conditions, the concentration of the blood fluctuates but little, compared with the change it would have to pass through if the blood of eels from sea water had an osmotic pressure equal to the average for marine teleostei. This feature can be correlated perhaps with the power the eel has of resisting sudden changes in the constitution of the external medium. CHANGES IN OSMOTIC CONCENTRATION OF BLOOD 481 ELASMOBRANCHS ~The blood of these fishes has usually the same osmotic pressure as the surrounding sea water. Experiments were made at Helgoland to determine how long elasmobranchs could live when transferred directly to fresh water, and the condition of the blood just before death. Mosso,! states that sharks die after some hours, and further, that after half an hour in fresh water, no more blood flows out from the caudal artery if the tail be cut, even though the heart pulsates. He states further that if a solution of NaCl is injected it will not pass through the gills, showing they are no longer permeable and that the blood of the fishes dead in fresh water is almost normal, death being due to stoppage of the gill capillaries. The first experiments - were performed with very large Acanthias vulgaris (piked dog fish). Average A for blood of normal fish from sea water — 1-g0° and Cl 0-88 per cent. ZI.—Specimen placed directly into fresh water and after 4 hours Ito minutes blood taken. The fish was nearly dead, but even after this long period blood was easily obtained from the tail and gave A — 1-455° and Cl 0-48 per cent. I].—Acanthias vulgaris. Placed directly in fresh water— Blood abstracted after 3 hours 45 mins. A — 1-448°. II1_—Acanthias vulgaris. Placed directly in fresh water— Blood abstracted after 44 hours A — 1-400°, Cl 0-455 per cent. In all these cases, after more than three hours in fresh water, blood was easily obtained from the tail, though not in such large amounts as from the normal fish. Moreover, it can hardly be said from the above results, that at the time of death almost no change had taken place in the blood. ‘There is no doubt, however, that the result varies greatly with the species, and perhaps that experimented with by Mosso was more like the Rays, the results of which follow. With regard to the power of resisting fresh water, Acanthias almost if not quite equals the cod in this respect, and if the cod only was used as typical of the teleosts, and Acanthias as an elasmo- branch, the great difference existing between these groups would not be so conspicuous. 1. Mosso, Biol. Centralblatt, 1890. 482 BIO-CHEMICAL JOURNAL Another interesting point in the above results is that the reduction in the salt contents of the blood, as indicated by the chlorine contents, is much greater than the lowering of the osmotic pressure would lead one to expect. Experiments with other Elasmobranchs.— Raia clavata—Average A for blood from normal specimens, = — 1-go°. One large specimen was placed in well aerated fresh water. "This fish secreted large quantities of mucus, and at the end of two hours was almost dead. The heart was still beating, and blood was obtainable from the tail, but the quantity was small. A of blood — 1-645° An elevation of the freezing point had thus taken place, though at the time of death it was much lower than that of the Acanthias blood. The same speedy death was exhibited by other specimens of the same species. ‘I'wo specimens (small) of Raza batis died within two hours after direct immersion in fresh water, but not enough blood was obtained from the caudal artery to allow of a determination of the freezing point. At the suggestion of Dr. Franz, some of the fluid was taken from the eyes of Acanthias. ‘These were removed carefully from the fish, the sea water removed, and the fluid abstracted. The freezing point was remarkably low, lower even than that of the blood, viz. A — 2-028°, and the amount of chlorine, 0-946 per cent., was greater. The flesh of elasmobranchs, like that of teleosts, contains much less salt than the blood, the figure for the muscles being only o-15 per cent. Some very interesting experiments made by Baglioni! show that to sustain the normal beat of the heart from an elasmo- branch (Scylliwm catulus), an artificial solution containing 2 grammes urea and 2 grammes NaCl for every 100 c.c. of tap water (in which calcium is present) is necessary. The urea aided contraction and the 1. Baglioni, Zeit. fiir allgem. Physiol., 1907. CHANGES IN OSMOTIC CONCENTRATION OF BLOOD 483 NaCl regulated the relaxation. Hence the urea is present not only to bring the osmotic pressure up to that of sea water, but as a necessary constituent of the blood, without which the regular beating of the heart is impossible. It follows, therefore, that the alterations in the constitution of the blood in the preceding experiments would interfere with physiological processes in addition to any blocking of the gill capillaries that occur as stated by Mosso. The average results of the resistance of elasmobranchs and teleosts to fresh water, show that the latter can resist much greater changes than the former, and this agrees with the distribution of the elasmo- branchs for they are typically marine fish and do not extend far into the Baltic. Bridge’ states that some species are permanent inhabitants of fresh water; it would be most interesting to determine the freezing point and salt contents of the blood of these represen- tatives of a group so characteristic. VaRIATIONS IN THE Btioop oF Pleuronectes platessa (Puatce) WHEN ‘TRANSFERRED FROM THE SEA TO THE AQUARIUM Some experiments were made to observe the change in the osmotic pressure of the blood of this marine teleost when immersed in diluted sea water the osmotic pressure of which was not less than that of the blood of the normal fish. ‘These experiments led to a remarkable change being discovered, which took place under apparently normal conditions in the aquarium. ‘The first experiments were as follows :— A number of large plaice, caught off Helgoland, were brought back living, and eight set in a mixture of sea and fresh water, the proportions being about two-thirds sea and one-third fresh. After two hours this was changed by addition of more fresh water until the A was — 1°125°, and the fish remained in this for fourteen hours. Blood from three plaice, out of this mixture A — 0-640° Blood from eight plaice, out of this mixture A — 0-630° Blood from plaice out of same catch but kept in normal sea water in aquarium :— Blood from three specimens A — 0°665° Blood from four specimens A — 0:695° Two things are noteworthy here: (1) The osmotic pressure 1. Bridge, Cambridge Natural History, Vol. VII, London, p. 432, 1904. 484 ~ BIO-CHEMICAL JOURNAL of the blood has decreased in this diluted sea water ; (2) the osmotic pressure of the blood of those fishes kept in the aquarium, but in- sea water, is much lower than the figures obtained previously’ for the same species direct from the sea. A second experiment was consequently performed ; a considerable number of fishes were brought back, and divided into two lots. First Lot—Examined immediately. Blood gave— For three specimens A — 0-715° For three specimens A — 0:710° For four specimens A — 0-705° Second Lot.—Kept in sea water in aquarium for 34 hours. Average result blood A — 0:630° That is, for those specimens in sea water, under apparently normal conditions in the aquarium, the freezing point was higher by —0:08° than the specimens examined immediately after capture. A third experiment was made to confirm the two previous ones by taking some of the blood from the fishes just after capture, the salinity of the bottom water being determined at the same time. The catch was divided into three lots. ‘Two of these were brought back alive to land, the blood was taken from the others and brought back in tubes kept in ice. Of the two lots brought to land, one was examined immediately, and the other lot placed in an aquarium tank. The salinity of the bottom water and the aquarium tank water was about the same, viz.: Bottom water 36:55 per thousand, Aquarium water 36:00. | Results :— Blood taken from fishes immediately trawl was brought on deck— First Series— A —0735° Second Series— A — 0°753° Third Series— A — 0-700° Blood taken from fishes of same catch brought back to the Biological Station in running sea water. ‘Time taken on way, 24 hours— First Series— A —o-741° Second Series— A —0-742° Blood taken from fishes of same catch brought back to the Biological Station in running sea water, and kept for three days in large aquarium tanks— First Series— A —0-645° Second Series— A — 0-655° 1. Dakin, Bio-Chem. Fourn., Vol. 11, p- 264, 1908. CHANGES IN OSMOTIC CONCENTRATION OF BLOOD 485 It will be seen from the above series of experiments that on every occasion there was a very considerable alteration in the freezing point after the fishes had been kept in the aquarium. Moreover, there was but little variation in these results; the fishes taken from the sea water direct had almost all the same A for the blood,. those kept in the aquarium also agreed between themselves, but differed, as the results show, very much from those examined directly they were brought to land. | This remarkable change could not have been due to differences in salinity of the water, since analyses showed the salt contents was practically the same. If the fish had been pathological or weak through the change, the freezing point for the blood would be expected to have been lower, that is, more like the sea water. Instead of this, it was much higher in the aquarium fish, and they were quite healthy. It is possible that this change was due to the difference in hydro- static pressure, since in the sea the fish must have been living under about four atmospheres pressure. It is obvious that this would have no effect directly, but it may be responsible indirectly for changes resulting in the blood. It was not possible at Helgoland to determine whether this change took place generally with fishes living on the sea bottom, when brought into the aquarium, but I hope to continue this series of experiments shortly. I have, in order to compare the action of fresh water on marine teleosts, in this paper compared the results with the freezing points of blood from fish in the sea water tanks of the aquarium, though it does not seem from the figures for Cyclopterus that they differ from those caught in the sea. In any case, it will be seen that great care must be taken when examining the blood for purposes of com- parison, to make certain the fishes are under the same conditions in each case. 486 BIO-CHEMICAL JOURNAL INFLUENCE OF THE OsmoTic CONCENTRATION OF THE SEA WATER or THE Bitoop upon Petacic Eccs The eggs of many marine teleosts are pelagic, and their specific gravity is so adjusted that but slight changes in the density of the water, if the salt contents is reduced, are sufficient to cause the eggs to. sink. Thus the eggs of the plaice or other pelagic eggs found floating twenty miles west of Helgoland, sink in the slightly less dense water round that island. In the Baltic Sea, where the difference between the surface and deep water in salt contents is very considerable, the eggs are very rarely found floating at the surface, but remain suspended at some point nearer the bottom. It has been found from measurements made chiefly by Ehrenbaum and Strodtmann,* that the diameter of these pelagic eggs is greater, the less dense be the sea water (with one exception—the plaice). For example, the eggs of the flounder from the Baltic Sea had a diameter of 1-054 and 1:216 mm., and they occurred in water with salt contents 11:24 to 17:54 per thousand. ‘The eggs of the same species from the North Sea had a diameter of 0-915 and 0-970 mm. It is still more interesting to find that the eggs while still in the ovary are larger in Baltic Sea specimens than in those taken from the salter North Sea water. For example, the ovarial eggs of Clupea sprattus have, according to Schneider, a diameter of 1-2 mm. in Baltic Sea specimens. ‘The diameter of the eggs of the same species in the North Sea is, however, much less than this, and since these extruded eggs are larger than the ovarial, the ovarial eggs must be still smaller than those from fishes in the Baltic waters. Ehrenbaum gives 0-94 mm. as the diameter of the ovarial eggs of Pleuronectes limanda in the Baltic Sea, whilst the floating eggs of this species in the North Sea only reached a diameter of 0-840. The probability of this difference in size being correlated with the difference in salt contents of the blood was first suggested by Ehrenbaum and Strodtmann, who said: ‘ Vielleicht beeinflusst das 1. Wiss. Meeresuntersuch, Abt. Helgoland, Bd. VI, H. I, 1904. CHANGES IN OSMOTIC CONCENTRATION OF BLOOD 487 umgebende Wasser die Fische in der Weise, dass in der Blutfliissigkeit und-vor allen Dingen in dem Liquor sich der Salzgehalt andert. In der éstlichen Ostsee befinden sich die Fische in der Regel in einem Wasser von geringem Salzgehalt, und hierdurch erhalt auch die die Eier umgebende Fliissigkeit des Ovars eine geringe Konzentration. Ist nun die zur Bildung eines Eies erforderliche Salzmenge eine konstante, so wird in diesem Falle die enstehende osmotische Spannungsdifferenz eine Vergrésserung des sich bildenden Eies bewirken. Die Volumenzunahme hat dann den Vorteil, das spez. Gewicht zu verringern, so dass die Eier auch noch in weniger salz- haltigem Wasser zu schwimmen vermégen.’ This suggestion has been justified, since I found that the salt contents of the blood does differ, though slightly, in the fishes from sea water of varying density. The ovarial fluid, like the blood, has a salt contents much lower than that of the surrounding water. It is usually slightly lower even than the blood, and also dependent to a small extent, like that fluid, on the concentration of the sea water. These differences might account for differences in the size of the eggs in the ovary, but they would not account—they are so small—for differences in the specific gravity of the eggs from two places so near, as previously mentioned. Ehrenbaum and Strodtmann state, moreover, that by taking fertilized eggs of Ctenolabrus and Sprat, from North Sea water, gradually down to almost fresh water, no noticeable increase in size was found to take place. Hence, I think, the specific gravity of the eggs is determined to a large extent by the density of the water in which the fish are living at the moment of extrusion of the eggs, when an increase in size takes place; and the larger size of the eggs in the ovary in fishes living in much less dense water is due to the action of the outer medium on the blood and ovarial fluid. CoNCLUSION The osmotic pressure of the blood of teleosts is, as we have seen, subject, though to a small extent only, to changes in the density 488 BIO-CHEMICAL JOURNAL of the outer medium.! The river eel has a lower osmotic pressure for the blood in fresh water than in sea water, though the A for this fish from sea water is higher than that of any other marine teleost. Moreover, there is a greater percentage of salt inthe blood of marine teleosts, and Sumner considers that both water and salts may pass osmotically through the bounding membranes. He states? that control experiments excluded the possibility that the water or salts passed from the body through the alimentary canal, leaving as the only possible alternative an osmotic exchange through one or more of the limiting membranes. Further, he remarks (p. 97) ‘The lining of the alimentary canal is, of course, readily permeable to fluids and to various substances in solution, but this, it is needless to say, is not freely exposed to the surrounding medium.’ Now I do not consider the alimentary canal excluded, since water must pass in with the food; and my freezing point determinations on the fluid of the alimentary canal of Cyclopterus show that in sea water this fluid has a salt contents almost the same as sea water. Again, when Cyclopterus was immersed in fresh water, the fluid in the alimentary canal had a A much less than the blood and rather approaching the surrounding water in composition. This being the case, there is present the possibility of water or salts passing though the membranes of the alimentary canal by simple osmosis or by the more complicated chemical processes of - physiological absorption. On the other hand Sumner showed that keeping freshwater 1. The low osmotic pressure of the teleost blood, and its small range of variation compared with that of invertebrates, has led Dekhuyzen to suggest (Ergebnisse von osmotischen Studien, Bergens Museums Aarbog, 1904, No. 8, p. 7), the probability of the teleostean fishes having descended from Ganoids which lived in brackish water. ‘They are assumed to have taken the osmotic pressure of the water there, and then having evolved the power of keeping this constant, wandered on the one hand to the sea and on the other to fresh water. One would then have to assume also that the elasmobranchs had inherited the salt contents of their blood from ancestors living in brackish water, because though the osmotic pressure is the same as the surrounding medium, the salt contents is only about half that of the North Sea water. It is much more likely that the teleostei have acquired the low osmotic pressure in the sea. Perhaps, as suggested by some authors, it represents the concentration of the primeval seas, which had a much lower density; or what is more probable, since all the vertebrates from teleosts to mammals have evolved the power of maintaining a definite salt contents for their blood, in defiance of solutions possessing higher or lower concentrations bathing their bodies, and passing through their alimentary canals, it is possible that at the same time the teleostean fishes acquired this low salinity, which is characteristic of vertebrates even though living in water of much higher concentration. 2. Bull. of the Bureau of Fisheries, p. 104. CHANGES IN OSMOTIC CONCENTRATION OF BLOOD —§ 489 fish with the body immersed in fresh water, but passing salt water “over the gills, caused death, and there was a considerable loss of weight. No experiments, however, that have been made prove the permeability of the membranes, e.g. gills, for both water and salts by simple osmosis, though it seems certain that they are permeable for water, which would suffice to a certain extent for the toxic action of the salt water on the gills of freshwater fish. Botazzi and Enriques,! experiments indicate an impermeability of the alimentary canal walls of inverte- brates to salts in solution except by nutritive processes, and Overton? after a number of experiments on the frog, as an example of the Amphibia, concludes that the skin is only with difficulty penetrated by salts, though very permeable for water. Schiicking,? on the contrary, finds that salts leave the body of Aplysia, when it is immersed — in fresh water, though the mouth and anus were ligatured, and that the amount of water or salts lost or gained is affected by injection of small quantities of nicotine, strychnine, etc. He concludes that the membranes are permeable to both water and salts. It is impossible to say whether the increase in salt contents of the blood of teleosts, in water of greater concentration, is due to the passage of salts through the gills by diffusion or through the walls of the alimentary canal by diffusion or absorption. The sensitiveness of the teleosts to changes in the chemical constitution of the external medium, is indeed apparent, and it appears that the toxicity of fresh water for marine teleosts, and vice versa, is due more to the alteration in the chemical constitution than to differences of osmotic pressure, though in all probability both are concerned. | This certainly points to a permeability for salts, but does not indicate whether through the gills by diffusion, or the alimentary canal by absorption. Under normal conditions the A of the urine of the teleosts is Jess than that of the blood, yet at the same time the kidneys appear to regulate to a certain extent the concentration of the blood, for it was generally found that marine fishes immersed in fresh water excreted large quantities of urine. 1. Archiv. f. Anat. u. Physiologie, Abt. Physiol., Supplement-Band., gor. 2. Overton, Verhandlung. der Physik-Medic. Ges. xu Wiirzburg. N.F., Bd. XXXVI, No. 5, 1904. 3. Schiicking, Archiv. f. Anat. u. Physiol., 1902. 490 BIO-CHEMICAL JOURNAL Whatever be the path taken by the water and salts, it is evident that a passage of both does take place both in vertebrates and inverte- brates, and further that under normal conditions in fresh or sea water the blood and other fluids of the body are in equilibrium with the external medium. ‘This condition is very like that suggested by Moore and Roaf' for the qualitative differences between the electrolytes of the cell and those of its environment, an equilibrium consisting not necessarily in an equality or isotonism of the total osmotic pressure. It has already been pointed out that the relation of the osmotic pressure of the blood of teleostean fishes to the water in which they live, is very like the relation of the temperature of ‘ warm blooded ’ vertebrates to that of their environment. This comparison might be carried still further and the aquatic invertebrates likened to the so-called ‘cold blooded’ animals whose temperature is practically identical with that of their environment. Finally, it has been shown that any change in the chemical con- stitution of the surrounding water brings about for both invertebrates and vertebrates, sooner or later, an alteration in the electrolytes and non-electrolytes of the internal fluids. This is followed by an alteration of the chemical constitution of the cell, which draws inseparably after it an alteration or disturbance of its function. I should like here to express my thanks to Professor Heincke, Director of the Biological Station on Helgoland, for his kindness in allotting me a work place and for providing me with the large quantity of material necessary, and also to the Staff of the Station, and in particular Professor Ehrenbaum for his interest and helpful suggestions. I have also to thank Professor Benjamin Moore for his many suggestions. 1. Moore and Roaf, Bio-Chem. Fourn., Vol. III, p. 55, 1908. yi (te 4 i. Se oat ee aera : i) Sa be QP The Biochemical journal 501 B47 v.3 cop.4 Biological & Medical Serials PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY 4 » na ¥ i at iy Hie Wik ‘ KN ne a ea aes eae fi penal " Hien CRY NOLSCn ra : Se

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(2) 35 _ I M_ Copper sulphate solution made with distilled 1,000 water.’ (3) ” >? I M 2”? >? ”? ” 10,000 (4) 23 3? I M ”? ”? 3 > 100,000 (5) ”» > I M ” 2? 2? ” 1,000,000 ; The flasks were incubated at 35° C., and sub-cultures taken at regular intervals. DisTILLED WATER Copper SuLPHATE SOLUTIONS . M M M M Sub-cultures taken Control 1,000 10,000 100,000 ¥,000,000 At once nus: . Infinite Infinite Infinite Infinite Infinite I5 minutes after... a Negative Positive Positive vs 1 hour after ra ia Negative Negative Positive : (5 colonies) 2 hours after a a = = Negative 6 hours after _24 hours after ? >? >? 9 ” ”? 9 ” 3? >”? 1. The extreme dilution of these solutions is seen when it is remembered that M strength means 63 parts of copper per 1,000, and therefore, means 63 parts in 1 million, and ao means 63 parts in . .* . ? . 3 2 1,000 millions. It may be pointed out that the solutions used throughout-are much more dilute than the I - ~ qipoo! etc., of previous authors, since these most probably mean parts by weight of the crystalline salts and I ‘ not » etc., gram-molecular solutions. 1,000 320 BIO-CHEMICAL JOURNAL It will be seen by the above table that in the copper sulphate solutions all the bacilli were destroyed in one hour with the exception of the 1,000,oooth dilution, in which five colonies per c.c. persisted, but even this solution was quite sterile at the end of two hours and remained so. ‘The number of bacilli on the control plate at the end of twenty- four hours was still ‘ infinite.’ I (6).—This was in all respects similar to I (a), with the difference that the solutions were made with sterilised tap water instead of distilled water. It was noticed that the contents of flasks 2 and 3, 7.2., the 1,000 and Fo.c00 dilutions, had a distinctly blue colour and there was a >) thick sediment at the bottom of these two flasks. Tap WATER Copper SuLPHATE SOLUTIONS M M M M Sub-cultures taken Control 1,000 10,000 100,000 1,000,000 At once cas ... Infinite Infinite Infinite Infinite Infinite 15 minutes after... re Negative Positive - a 1 hour after es s 2 = Positive is (14 colonies) 2 hours after a 7s os Negative rl Positive 4 hours after sae en »” > > (173 colonies) 8 hours after res Negative = 24 hours after PA ” ” ” ”? (193 colonies) ” ‘ Pa a Positive (147 colonies) 48 hours after I (c).—This was similar to I (a) and I (8), excepting that the solutions were made with sterilised sea water, instead of distilled or tap water. A thick precipitate was thrown down in all the flasks, but especially in No. 2; The first two dilutions were distinctly blue. ACTION OF METALS UPON BACILLUS TYPHOSUS 321 Sea WATER Copper SULPHATE SOLUTIONS M M M M Sub-cultures taken Control 1,000 10,000 100,000 1,000,000 At once i: ... Infinite Infinite Infinite © Infinite Infinite 2 hours after is r Positive Positive 53 ES 6 hours after 5 de we Negative ~ Positive ms 24 hours after ors . a Negative Negative Positive 48 hours after hy is 9% » % » 72 hours after vay ” ” ” ” > The most striking fact in the above series of experiments is the exceedingly small quantity of copper sulphate which was sufficient to kill all the typhoid bacilli in a few hours. _ This is especially noticeable in the results obtained with solutions made with distilled water, when a dilution of one in a million of gram- molecular was sufficient to kill all the bacilli in less than two hours. The actual amount of copper in this dilution was 63 parts in 1,000 million parts of water, or roughly one of copper in sixteen million parts of water. In the solutions made with tap water and sea water the action was still very marked, but less so than in the case of the distilled water. This is probably due to the fact that the tap and sea water both contained other substances, which in some way modified the action of the copper sulphate, thus weakening the solutions. In this connection it is of interest to remember that Moore and Kellerman (4) have shown, in their paper, the relative decrease of toxicity of copper sulphate solutions, depending on the amount of organic matter present, the amount of carbon dioxide in solution, or the temporary hardness of the water. NAgeli showed that the oligodynamic action of copper solutions was lessened by the introduction of many insoluble substances, and True and Oglevee have studied the influence of insoluble substances on the toxic action of poisons and have confirmed many of Nageli’s observations. The manner in which the presence of such adventitious substances, inorganic or organic, in the water to which the copper salt is added, produce their effects in diminishing the toxic action, is not discussed 322 BIO-CHEMICAL JOURNAL by previous authors, with the exception of Kraemer, who concluded that the toxicity is due to some salt of copper, which ‘is probably in the form of a crystalloid rather than that of a colloid.’ It is, however, fairly obvious that the toxic action depends upon the concentration in the solution of the copper ion, because all salts of copper possess the toxicity, which must, therefore, depend upon the presence of the free kation in the solution and not upon the non-ionized molecule of the copper salts, which is different in each case. When once it is considered that the toxicity is dependent in all cases upon the concentration of the copper ion in the solution, the varying toxicity, when the copper is added to (a) distilled water, (b) tap water, (¢) sea water or (d) water containing other added salts or organic matter, becomes easy of explanation. ‘The salts present in the water cause the degree of ionization of the copper to vary. For instance, when distilled water only is present and then copper sulphate is added, at the degree of dilution here concerned, the copper sulphate practically completely ionizes into copper ions and sulphion ions, and therefore the concentration in copper ions is proportional to the total amount of copper sulphate added. But when salts such as the phosphates are present, as in tap water or sea water, the ionization in the solution becomes that of the much more feebly ionized copper phosphate and consequently the concentration in copper ions is no longer indicated by the amount of copper sulphate added, but is reduced correspondingly to the low degree of ionization. Similarly the addition of apparently inert organic substances, such as cellulose, silk, wool, glue, etc., will produce a like effect by forming feeble combinations or adsorptions with the copper ion and thus reduce its concentration. Since the toxicity, in the case of salts of the heavy metals, depends on the metallic kation entirely and not on the anion of the particular acid in combination in the salt employed, it was considered sufficient in most cases to test only one salt of each metal. Also, since the amount of added salt is so small, any low degree of toxic action of the anion can be disregarded, and the most convenient and easily accessible salt of the heavy metal can be employed. ACTION OF METALS UPON BACILLUS TYPHOSUS 323 Secondly, the results of this series tend to strengthen the theory of those observers who contend that in the case of the copper foil in water, the metal itself is not actually in a state of solution as a colloid in the water, but that it forms salts and that it is the action of these salts which determines the toxicity of the solution; for these con- centrations of copper as salt are less than the amount of copper in colloidal solutions. These experiments with such excessively dilute solutions of copper sulphate appear to show very clearly that copper in the ordinary condition of solution as a salt (that is to say in the ionized condition and not in colloidal solution) is capable of exhibiting all the so-called ‘ oligodynamic’ properties, produced when metallic copper is immersed in water in which living organisms are present. Accordingly, no foundation is left for the term oligodynamic as applied to copper brought into solution by the latter method, and the results obtained are merely an index of the high toxicity of copper ions upon some forms of living cells. Attention may here be drawn to the fact that the solutions, con- taining the typhoid bacilli were incubated at a temperature of 35° to 40° C., and this applies to all other experiments with the exception of the ferrous sulphate and ferric chloride series, in which the flasks were kept at room temperature, i.¢., about 15° C. Attention is drawn to this matter, because it was proved by Israel and Klingmann that when the solutions of copper containing the organisms were placed in an incubator at 35° to 40° C. the toxic effects were manifested in one hour, but if the solutions were kept at ordinary temperature the toxic effects were not produced until two hours had elapsed ; so that, according to this statement, these experiments: were con- ducted under the most favourable conditions. Series 1].—ExpreriMENTs wITH Iron Satts Experiments with dilutions of gram-molecular solutions of ferrous sulphate and ferric chloride made with (a) distilled water, (b) tap - water, after being allowed to run for five minutes, and (c) sea water. 324 BIO-CHEMICAL JOURNAL In this series seven flasks were used, and 1 c.c. of the typhoid emulsion was added to each of them :— (1) Containing 100 c.c. of distilled water. (2) % 9 Iw Ferrous sulphate solution made with distilled. 1,000 water. I (3) ” ” a0 pes 9 as ia se : > I . (4) > 29 100 a ” > 33 ” > I : : (5) 99 9 ; og Ferric chloride an - > I (6) : 39 ” ite) Soo ” 99 9 ” > I (7) ” 9 100 soo ? > 3° ” > The flasks were kept at room temperature, about 15°C. Nos. 2, 3, 5, and 6 had a distinct colour, 2 and 3 being at first a pale green, and 5 and 6a rusty brown, but in twenty-four hours all the solutions were brownish in colour, due to the oxidation of the ferrous sulphate. DistTILLED WATER Ferrous SULPHATE SOLUTIONS Ferric CHLORIDE SOLUTIONS . M M M M M M Sub-cultures taken Control “1,000 10,000 100,000 1,000 10,000 100,000 At once ... Infinite Infinite Infinite Infinite Infinite Infinite Infinite 2 hours after ... re Positive Positive aA = Positive = (314 colonies) 6 hours after ... Hs Negative Negative Negative Negative Negative is 24 hours after ... a5 * 5 Positive Negative »» Positive 48 hours after ... ‘ x a Negative Negative », Negative The above table shows that both with the ferrous sulphate and ferric chloride solutions, the 1 in 1,000 and I in 10,000 gram-molecular dilutions completely destroyed the bacilli in less than six hours, with the flasks at laboratory temperature. | In both cases the 1 in 100,000 solutions still contained over 1,000 bacilli per cubic centimetre at the end of twenty-four hours, but were quite free in less than forty-eight hours. Il (6).—This was similar to II (a), with the difference that the solutions were prepared with tap water instead of distilled water. Flasks Nos. 2, 3, 5, and 6 have a more distinct colour than in the ACTION OF METALS UPON BACILLUS TYPHOSUS 325 previous series, and the ferrous sulphate solutions have turned yellow. There is a precipitate thrown down to a slight degree in all flasks, _-but more marked in 2 and 3. Tar WarTER Ferrous SULPHATE SOLUTIONS Ferric CHLORIDE SOLUTIONS M M M M M M Sub-cultures taken Control T,00c0 ~~~: 10,000 100,000 1,000 10,000 100,000 At once ... Infinite Infinite Infinite Infinite Infinite Infinite Infinite 2 hours after ... ms Positive Positive a Positive Positive va + hours after ... 2? 29 = d 29. a 29 29. 8 hours after ... » Negative Negative Positive Negative Negative Positive 24 hours after... ” ” ” ” ” ” ” 48 hours after... ” 29 ” ”? ” 2 ”? (227 (335 colonies) colonies) It will be noticed on comparing the above table with II (a) that the action of the solutions made with tap water was slower than that made with distilled water, and the I in 100,000 solutions were still positive at the end of forty-eight hours. II (c).—Similar in details to II (a) and II (4) except that the solutions were made with sea water instead of distilled or tap water. There was a thick brownish deposit in all the flasks, after they had been incubated, with the exception of the control, in which the deposit was less marked and not coloured to any extent. Sea WartTER Ferrous SULPHATE SOLUTIONS Ferric CHLORIDE SOLUTIONS M M M M M M _ Subcultures taken Control 1,000 10,000 100,000 1,000 10,000 100,000 At once ... ... Infinite Infinite Infinite Infinite Infinite Infinite Infinite 2 hours after ... ia Positive Positive Positive Positive Positive Positive 53 hours after a Negative oi ‘ iol Z a (17 colonies) 24 hours after... $f ¥ Negative 4 Negative Negative 3 48 hours after... 29 ”? 2”? "9 2 ”? ”? It is seen from this table that the toxicity of the solutions made with sea water is distinctly less than was the case with the distilled water, and slightly less than that of the solutions made with tap water. © This is probably explained in the same way as in the case of the copper sulphate solutions, and is no doubt due to salts, etc., in the 326 BIO-CHEMICAL JOURNAL water which modify the action of the solutions—in the latter two cases by lowering the ionization as previously described, In this series the marked toxicity of the solutions of salts of iron is strikingly demonstrated. ‘This is all the more remarkable, firstly, because this series was conducted at laboratory temperature, 7.¢., 15° C. instead of about 40° C. as was the case with Series I (it has been previously shown that the incubation of the solutions greatly increases their killing power), and secondly, because salts of iron are not poisonous in the ordinary sense of the word. It will further be seen that the action of the two salts was very similar in each series, the ferrous sulphate solutions being slightly more toxic than the ferric chloride. This is rather remarkable, as the ferrous sulphate always became converted into a ferric salt after the lapse of a few hours, but the result may be due to increased ionization of the ferric sulphate formed. It is interesting to compare these results with those obtained by Bassett-Smith in experiments with water infected with typhoid bacilli and contained in (a) an iron tube, and (4) an iron box of much larger capacity. In the iron tube all the bacilli were killed in less than twenty-four hours, and in the iron box in less than 18 hours; but in the latter case the solution was not so heavily infected with bacilli as in the former. In both cases the water, at the end of twenty-four hours, was quite a rusty colour, due to the formation of oxide of iron. The results of this series are important for several reasons. In the first place, although the toxicity of the salts of iron is less than that of the copper sulphate solutions, it is still sufficient to be of great practical importance, and whereas the safe dose for human beings has still to be determined in the case of copper salts, it is well known that not only are salts of iron not deleterious to man, but that many of them are even beneficial and are used medicinally. ‘This fact is also of importance in regard to the purification of drinking water by addition of salts of iron or by storage in iron tanks, and also in the purification of suspected shell-fish. 3 | It is, moreover, possible to employ iron in the manufacture of ACTION OF METALS UPON BACILLUS TYPHOSUS 327 tanks for water purification when the use of copper could not be entertained owing to its great cost. ~The results of the experiments made with these salts of iron in attempting to purify Anodons infected with B. typhosus are tabulated in Part II of this paper. SERIES ITJ].— Experiments with NIckEL AND Cospatt SALTS Seven flasks were used to contain the different solutions, which were made with distilled water. ~~ The 1 in 1,000 solutions were coloured green and red respectively, but the others were colourless. One c.c. of the typhoid emulsion was added to each of the flasks and they were then incubated at 40° C. Cozsatt CHLORIDE SOLUTIONS Nicket CHLoRIDE SOLUTIONS : M M M M M M Sub-cultures taken Control 1,000 10,000 100,000 1,000 "10,000 100,000 At once a Infinite Infinite Infinite Infinite Infinite Infinite Infinite I hour after ban Ss ” ”° ”° bb] bP] >? b>] 3 hours after ... # Positive so zs Positive = os 8 hours after ... & = Positive “a Pe Positive = 24 hours after... os Negative =a Positive Negative oe Positive (107 colonies) (203 colonies) 48 hours after... os A Negative rm a Negative ,, It will be seen from this table that although cobalt and nickel chlorides have a toxic action on typhoid bacilli, it is much less marked than in the case of the copper or iron salts and takes a much longer time ‘to manifest itself. _ This is interesting because these metals belong to the group of heavy metals, and yet possess a comparatively low toxicity. Taking the ‘ distilled water’ series in each case and comparing the results it is seen that whereas the copper sulphate solutions, including the I in 100,000, were clear at the end ofan hour, and the first two dilutions of the ferrous sulphate and ferric chloride experi- ments were clear in less than six hours, at room temperature the corre- sponding cobalt and nickel solutions still contained over 1,000 organisms per c.c. in each case at the end of eight hours, and the 1 in 10,000 solutions were not clear at the end of twenty-four hours. As the toxic effects would have taken still longer to manifest 328 BIO-CHEMICAL JOURNAL themselves in solutions made with tap or sea water, this series was not continued, as it was considered that the results would be of no practical value, even under the most favourable conditions. Serres [V.—ExpEerRIMENT WITH MANGANESE SALTS The solutions were made with distilled water and were incubated at about 40° C. To each of four flasks was added 1 c.c. of the typhoid emulsion, prepared as in the previous experiments. MANGANESE CHLORIDE SOLUTIONS M M M Sub-cultures taken Control 1,000 10,000 100,000 At once as ae Infinite Infinite Infinite Infinite 1 hour after iy a ms PY: ee 4 hours after oe = os i re 8 hours after feeds _ (403 colonies) ACTION OF METALS UPON BACILLUS TYPHOSUS 329 These results were not exactly what were expected, having regard to those obtained by Bassett-Smith with water infected with typhoid bacilli and placed in (a) a zinc tube, and (d) a zinc box. In the former case the water was cleared of bacilli in eighteen hours, and in the latter in less than twenty-four hours, the water in this case being more heavily infected than was the case with the zinc tube. As against this, however, the same observer conducted a special experiment with strips of zinc foil, to see if they would be able to clear water infected with typhoid bacilli, as the copper foil had done. Strips of zinc foil, having a superficial area of 50 square centimetres were added to a flask containing 100 c.c. of sterilized tap water, and the latter was infected with a broth culture of typhoid bacilli and kept at room temperature in the dark. ‘Up to forty-eight hours this did not seem to be effective against any of the tested organisms.’ Furthermore, it must not be forgotten that the emulsion of typhoid bacilli used in all these experiments contained very many more bacilli per cubic centimetre than did the broth cultures used in the experiments cited above. : (>) The above series was repeated with solutions made with tap water instead of distilled water, and the flasks were kept at room temperature. Zinc SULPHATE SOLUTIONS M M M Sub-cultures taken Control 1,000 10,000 100,000 At once Ss =e Infinite Trtinite _ Infinite Infinite 2 hour: after ... a > ‘3 st s — Pr ce is Positive “ = 8 2? > - be att £3 be > >» a a eis oxi = Negative Positive - - ie aks oad > ee ee Positive It will be seen from the above table that with the solutions made with tap water and kept at room temperature (about 15° C.) the toxic action was even less marked. 330 - - BIO-CHEMICAL JOURNAL Series: VI. —ExperRIMENTS WITH SILVER SALTS Experiments were carried out with different strengths of silver nitrate made with (a) distilled water and (b) tap water, after being allowed to run for five minutes, © (a) Four flasks were used, and the solutions were made with dis- tilled water and incubated at 4o° C. One cubic centimetre of the typhoid emulsion was added to each of the flasks. Strver Nitrate SOLUTIONS M M M - Sub-cultures taken Control “7,000 10,000 100,000 } At once oil tek Infinite Infinite Infinite Infinite 2 hours after ... ae S Negative Negative Negative 4 hours after ... = ‘ % 3 = aa 6 >? 9 oe. pe 9? bP) ”? oF? 24 99 9 Kg Mtge 9 bP) 2? 9 48 ,, edo: nex The results of this series were very definite and proved the silver nitrate to have a very decided germicidal action on the B. typhosus. Comparing the above table with that showing the results of the copper sulphate solutions made with distilled water, it is seen that the action of the silver nitrate is practically as great as that of the copper sulphate. (b) ‘This series was the same as (a), except that the solutions were made with sterilised tap water collected as described before. It was immediately noticed that the solutions were inclined to be milky, the 1 in 1,000 and the I in 10,000 decidedly so, and the I in 100,000 faintly milky. . This was of course due to the precipitation of the silver salt by the chlorides present in the water; the result being the formation of 99 9 ” ” silver chloride which is practically insoluble in water. Sttver Nitrate SoLutions M M M Sub-cultures taken Control 1,000 10,000 100,000 At once set oe Infinite Infinite Infinite Infinite 2 hours after ... ae i Negative Positive ee. 4 2 do tebe adia S 7. Se 9 ”