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Physiological chemistry


作者: W. D. Halliburton,  


期刊: Annual Reports on the Progress of Chemistry  (RSC Available online 1908)
卷期: Volume 5, issue 1  

页码: 210-241




年代: 1908




出版商: RSC


数据来源: RSC



PHYSIOLOGICAL CHEMISTRY.THE output of biochemical papers shows no sign of abatement,and one has also to chronicle the appearance of several new text-books on the subject during the past year. Carl Oppsnheimer, ofBerlin, is editing a very complete Handbuch der Biochemie, whichis to consist of twenty parts, and of these six have already appeared.The various articles in it are written by those who have paidparticular attention to the portions of the subject on which theywrite, and the chapters deal with the questions involved from boththe chemical and biological point of view. Another new text-bookis from the pen of Professor Rohmann. Abderhalden’s book hasbeen translated into English, and a second edition of the Germanversion is just to hand. Messrs. Longmans have commenced theissue of a series of important biochemical monographs under theeditorship of Drs.F. G. Hopkins and R. I€. Aders Plimmer; thefirst of these is written by Dr. Bayliss, and treats of the enzymes,particularly emphasising the catalytic nature of their action. Thenext two monographs are by Dr. Plimmer, who deals with theproteins, first as a whole, then of their cleavage products, andfinally of the attempts which have been to synthesise them. Dr.H. M. Vernon, of Oxford, has published the lectures he gave atthe University of London in the form of a book, entitled “ Intra-cellular Enzymes ”; here the part played by fermer,ks in the innerlife and metabolism of the cell is described, so far as we can a tpresent recognise such action. Dr.Vernon himself, by his dis-covery, among other things, of tissue-erepsin, has done much toelucidate this obscure and comparatively new aspect of fermentactivity, and of the importmame of such action there can be noquestion. There is, however, a tendency just now to attribute allthe chemical transformations which occur during life to enzymaticaction, and there is a danger that this idea may be overdone.Ferments may be the exclusive agents which protoplasm employs,but the proof that such agents are really ferments is, in many cases,very insufficient, and it is quite possible that, as knowledgeadvances, other mechanisms may be brought to light. In thPHYSIOLOGICAL CHEMISTRY. 211eighteenth century ‘ I vital force ” was supposed to be a t the bottomof all that could not be otherwise explained, and the conceptionof a force which no one hoped ever to understand delayed theprogress of science. We must take care in the twentieth centurythat the adoption of a new phrase, “ferment action,” is not con-sidered in itself to be a final solution of vital problems.To labelany particular chemical change as due to enzymatic activity shouldbe rather a signal for the commencement of renewed research inattempts to understand it still further.The journal familiarly known as Hofmeister’s Beitriige has dis-appeared in the struggle for existence, having been absorbed in thecomparatively youthful Biochemische Zeitsciirif t. During the fewyears this last-named publication has existed, fourteen volumes havealready appeared, and if we judge fruitfulness by quantity alone,certainly the Biochemische Zeitschrif t has been successful in aphenomenal way.I f we examine the articles published there, thequality is also of a high standard.I n selecting from the papers of the past year subjects for moredetailed and critical review, the difficulty, as usual, arises from anenzbai*ras des ?ichesses, for hardly any aspect of biochemical know-ledge has been omitted from the mass of material published. Mostof this relates to subjects which have been previously noticed inthese Reports, and works out further details which support orcorrect views already expressed. This patient exploration ofportions of the field which had escaped, or almost escaped, noticebefore, is most necessary and most praiseworthy, but it must beconfessed that, as a rule, it is also extremely dull, and hardly lendsitself for interesting treatment in an article of this nature.Theactivity of many workers among the ferments has already beenalluded to. I n the forthcoming index Abderhalden’s name will loomlargely, and his work on the polypeptides and cleavage productsof the proteins progresses in a steady stream of published papers;each one of these is a brick in the edifice of knowledge which isslowly being reared, but it may be many years before we are ableto obtain a clear view of the final construction. I n America,Osborne and his colleagues are pursuing similar work, especiallyon the vegetable proteins, and data there are being accumulated,the final evaluation of which is also for the future.Nucleic acid,that important appendage of many proteins, has received its dueshare of attention, and here it does seem that we are in a positiona t last to reduce the chaos of former years to something like order;nucleic acid, therefore, will form the subject of a fuller paragraphlater in this Report. The work of London and his collaborators intheir important study of digestion, which formed the subject of aP 212 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.lengthy section in last year’s Report, progresses rapidly, and thislist of papers promiscs to rival in number those of Abderhalden;here again, however, the work has been the pigeon-holing of freshdata, and presents nothing of really new importance; this passingreference will, therefore, be all that I shall attempt this year inrelation to this branch of research.Although so little is known of creatine and creatinine as test-tube products of protein fission, their importance as products ofthe body’s katabolism is rising into prominence. The work directedfrom so many quarters to the study of these two substances datesa few years back to Folin’s investigations, and to the simplemethods he then introduced for detecting and estimating them.Although I devoted some pages last year to this subject, it will benecessary to return to it again, because an entirely new light hasbeen shed upon it by the remarkable work which Mellanby isresponsible for.Perhaps, however, the most noteworthy feature of biochemicalresearch to-day is the recognition of the importance of the lipoidsin cell life, and, therefore, a section of my Report will be occupiedwith these materials.Fresh information on the functions of the pituitary body,hitherto a subject on which we have been almost completelyignorant, has shown us the importance of this gland, both in healthand disease. Although the chemical side of the subject is as yetfar from clear, I propose to conclude my Report with a briefsummary of our present knowledge on this outgrowth of the brain.Without, however, enumerating all the sub-headings of my Report,let us a t once proceed to break it up into its various sections, andI will commence with a brief consideration of one I have not yetmentioned, namely :Protein Nomenclature.Fellows of the Chemical Society will be well acquainted with thedifficulties which attend any efforts at uniformity of terminology,and, although in our own Journal the rules about the use of suchterminations as in and h e , or of 02 and ole, are enforced, therecommendations of the Nomenclature Committee are more oftenhonoured in the breach than in the observance in other publications.This difficulty is enhanced when people speaking and writing otherlanguages are concerned.Although the nomenclature of theproteins is, and probably for long will remain, very unsettled untiltheir constitution is better known, a joint committee of theChemical and Physiological Societies sat, a year or two ago, toattempt a settlement of the more salient points, and the reporPHYSIOLOGICAL CHEMISTRY.213which this committee presented was sent over to America in orderto try and obtain uniformity in the use of names between the twogreat sections of English-speaking men of science. The report wasalso considered at the International Congress of Physiology, whichwas held last year a t Meidelberg, and although it was not receivedwith enthusiasm, we may, a t any rate, claim that the adoption ofthe word Protein as the title for the great group of albuminoussubstances by German writers is, a t any rate in part, due to ourefforts.But to return to America, for there a t least we should hope tobe successful, the report was considered by a joint committee ofthe American Physiological Society and the American Society ofBiological Chemists, and during the last year they have publishedtheir views.1 One notes with satisfaction that the American reportis in substantial agreement with our own.The points of differenceare small ones, and the chief improvement in the American classi-fication is the inclusion of the chief classes of the vegetable proteins;here the hand of T. B. Osborne, who was a member of the Americancommittee, is traceable.The main points of difference and agreement may be best statedby the following quotations from a report prepared by the ProteinNomenclature Committee of the Physiological Society, which wasadopted a t a meeting of that Society in May last 2 :“The term AZbum‘noid is retained for the sub-class namedSclero-proteins in the English report.We think the adoption ofthe new word is preferable to the retention of the old one, becausethe name albuminoid is still largely used by English and Frenchchemists as synonymous with Protein:‘ I The only noteworthy difference in the arrangement of the sub-classes is the transference of the phospho-proteins (vitellin-caseinogen group) from the simple to the conjugated proteins. Weadhere to the opinion that our own arrangement is better, becausethe phosphorus-containing group of the phospho-proteins is notsplit off from them as a true prosthetic group is, and the cleavageproducts of this class of protein still contain phosphorus.3“ A minor difference is the substitution of the term hzemoglobinsAmw.J. Physiol., 1908, 21, sxvii-xxx ; J. Biol. Chem., 1908, 4, xlviii-Ii;Proc. physiol. Soc., 1908, xxxii-xxxv ; J. Physiol., 37.This view, that phosphorus is part of the protein molecule, and not part of agroup such as nucleic acid linked to the protein, is emphasised in Dr. Plimmer’sbook already mentioned. See also paper by Plimmer (Trans., 1908, 93, 1500),which treats of vitellin, and also of a second protein in egg-yolk (livetin) which isalmost free from phosphorus. See also Plimmer and Scott on the distinctionsbetween phospho-proteins and nucleo-proteins (ibid., p. 1699).A., i, 301214 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.for the class called chromo-proteins in the English report.prefer the latter name as having the wider significance.the American report. These are:Wc“ Five additional substances are introduced a t various places in“ 1.The glutelins, the alkali-soluble proteins of vegetable origin.(‘ 2. The alcohol-solzcble proteins found in the vegetable world.“We think the inclusion of these two new classes of simpleproteins (and especially the latter) is advantageous, as it makesthe system more complete. We are prepared to accept, provision-ally, the term glutelin for the proteins soluble in alkali, althoughthey are doubtless closely allied to the globulins. The termalcohol-soluble proteins strikes us as rather cumbrous ; these sub-stances might, as Rosenheim * has suggested, be termed gliadins ;that is, the name of the principal member of the group might beextended to serve as a class name.(‘ 3.The Zecitho-proteins are added to the classes of conjugatedproteins. As it has not yet been decided whether these aremechanical mixtures, adsorption compounds, or true chemical com-binations, we see no reason for the inclusion of this group.4. The proteam : insoluble products which ‘ apparently resultfrom the incipient action of water, very dilute acids or enzymes.’‘( 5. Coagulated proteins: which result from the action of heator of alcohol.“Sub-classes 4 and 5 are placed among the primary proteinderivatives; they are of an ill-defined nature, and we see no objectin singling out for special mention a few of the infinite varietiesof insoluble modifications which proteins exhibit.”The final result of this consultation with our American confrtresis the classification of proteins into the following groups:1. Protamines.2.Histones.3. Albumins.4. Globulins.5. Glutelins.6 . Gliadins.7. Phospho-proteins.8. Sclero-proteins.9. Conjugated proteins :a. Nucleo-proteins.6 . Chromo-proteins.c. Gluco-proteins.Proc. ph?ysio7. Soc,, 1908 ; J. Physiol., 36, Iv ; Scimcc Progress, 1908. 2,696PHYSIOLOGICAL CHEMISTRY. 21510. Protein derivatives :CL. Meta-proteins (acid-albumin, etc.).b. Proteoses.c. Yeptones.d. Polypeptides.It is, of course, impossible, a t this stage in the history of protein-chemistry, to obtain absolute unanimity on minor points of classi-fication and nomenclature; but the system is now complete forpractical purposes, and its utility can only be proved by giving ita fair trial.I n the new text-books on the subject, it is alreadybeing adopted.Caseinogen and Rennin.It is an undoubted fact that the milk provided by Nature forthe growing offspring is different in the various classes of theanimal kingdom. The quantitative variations are often enormous,and it has been shown that the milk best adapted for the nutritionof the young animal is that which comes from its mother, or, a tleast, from an animal of the same species. The practical applica-tion of this rule comes home to most of us when dealing with thefeeding of children, and it is universally acknowledged that, afterall, cows’ milk is but a poor substitute for human milk.Cows’milk is, of course, diluted, and sugar and cream added, so as tomake it quantitatively like mothers’ milk, but even then thequestion arises whether the essential difference between the twokinds of milk is not deeper than one of mere quantity; and, inparticular, the pendulum of scientific opinion has swung backwardsand forwards in relation to the question whether the principalprotein, called caseinogeq, in both is really identical in the twocases. The caseinogen of human milk curdles in small flocculi inthe stomach, so contrasting with the heavy curd which cows’milk forms; and even although the curdling of cows’ milk bemade to occur in smaller fragments by mixing the milk withbarley-water or lime-water, its digestion proceeds with comparativeslowness in the child’s alimentary canal.These are practicalpoints well known to every clinical observer, and in the past theyhave been attributed, not so much to fundamental differences inthe caseinogen itself, as to accidental concomitant factors; theexcess of citric acid in human milk, for instance, or its paucityin calcium salts, having been held responsible for the differencesobserved in the physical condition of the curd and in its digesti-bility.This question is far from settled even to-day, but there ar216 ANNUAL REPORTS ON THE PROGRESS OF CHEMJSTRY.some data now available that point to a qualitative differencehetween caseinogens. Some of these depend on the application ofthe “ biological test ” carried out on the line of immunity experi-ments, which method has been so signally successful in the distinc-tion between the blood-proteins of different species of animals.Thedifferences, however, which lead to the formation of specific pre-cipitins are so slight, that ordinary chemical methods of analysisare, at present, unable to reveal them. But, in the case of milk,there are differences which the chemist can detect. One cannotlay much stress on mere percentage composition, although differ-ences have been noted in that, because we have no guarantee thatthe proteins investigated were separated from all impurities.Differences are also noticeable in the yield of monoamino-acids, butthe methods a t present employed in the estimation of these cleavageproducts are far from perfect. A deeper chemical distinctionnoted is, however, mentioned in the recent work of Bienenfeld,5who find that human caseinogen contains a carbohydrate complex,which, as is well known, is absent from that of the cow.A few years ago it was stated that human caseinogen will notcurdle with rennet, and Bienenfeld upholds this view; but itappears to be a mistake.The conditions of rennet curdling aresomewhat different in the two kinds of milk we are considering,and the factors concerned in this phenomenon in human milk havebeen worked out by Jacoby? who has paid special attention tothe action of anti-rennin, by Fuld and Wohlgemuth,’ who criticiseBienenfeld’s observations, and by Engel,* who deals mainly withthe influence of reaction.Another problem closely related to the preceding is the viewwe are to take of the ferment rennet or rennin itself.Rammar-sten’s authority is the one usually relied on for the statementthat gastric juice contains two distinct enzymes, pepsin,the proteoclastic ferment, and rennin, the milk-curdling one. Itwas Pawloff who first suggested that the rennetic action was thework also of the peptic enzyme; and Ehrlich’s convenient side-chain theory was considered to explain the double action, thecurdling being the result of the activity of one or more moleculargroups in the pepsin molecule. It is a little early to argue inthis manner, for, as Emil Fischer so wisely said in his Faradaylecture, nobody has yet ever been successful in separating out anyenzyme in a state of purity.For the same reason, one must feelchary in accepting as proved the recent statements made byRioclrem. %oitsdt., 1907, 7, 262 ; A . , ii, 121.Ibid., 376 ; A., ii, 311.ti Ibid., 1908, 8, 40 ; A . , i, 236.F Ibid., 1908, 13, 89 ; A., ii, 873PHYSIOLOGICAL CHEMISTRY. 21 7SScala.9 that rennin is a weak base consisting of a proteose nucleusand amino side-chains. However this may be, Gewin10 has cham-pioned Pawloff’s view, and maintains that pepsin and rennin arcone and the same ferment. He regards the rennet action as thefirst stage in the digestion of caseinogen. I f hydrogen ions areabsent and calcium ions are present, digestion stops and a curdseparates, but if a sufficient number of hydrogen ions are present,ordinary peptic digestion proceeds.Unfortunately, there appear to be as many and as eminentauthorities ranged on the opposite side, and during the last yearI.Bang 11 and Hammarsten 12 himself have published papers whichconclude that the two ferments are not identical.The question is an interesting one, and, perhaps, in some futureyear, we shall be abIe to relate how it has been finally settled.PZasteins.-It has long been known that the addition of rennetto a solution of “peptone” causes the formation of a precipitate,and the name plastein was given to the precipitated substance.The same occurs when gastric juice, or pepsin-hydrochloric acid, isadded to ‘( peptone ’’ solution, so the formation of plastein is notdistinctive of rennet, even if that enzyme is not identical withpepsin. When the discovery was first made, it was supposed thatone of the actions of the rennetic enzyme was to produce theregeneration of albumin ” ; this was a t a date when the exclusiveseat of the synthesis of protein from its cleavage products wassupposed to be the wall of the alimentary canal, the seat of absorp-tion.Recent research has done something to confirm this view ofthe composition of plastein, but in modern terminology we speak ofit now as the reversible action of the enzyme concerned. This viewis taken by Sawjaloff,l3 and the analytical figures he gives indicatethat the reaction is either bimolecular or termolecular. Plastein is,therefore, the result of the union of either two or three molecules ofthe proteoses from which it is formed, and its molecular weight is, onthe average, twice that of the proteoses. The plasteins used wereprepared from a large range of proteins, and an attempt is madeto classify them.Levene and van Slyke14 have made similarexperiments, and obtained the cleava,ge products of plastein ; theseinclude both mono- and di-amino acids; the total yield, however,only amounted to 39 per cent. Their conclusion as to the com-position of plastein is different from that of Sawjaloff; they regardit as a complex protein not far removed in composition from fibrin,Stnz. .~periin. agrar. itmb., 1907, 40, 129 ; A., i, 236.lo Zeitsch. physiol. C7~e?n., 1907, 54, 32 ; A., i, $1l1 Ihid., 359 ; A ., i. 236.l4 Bioclwnz. Zeit,oc77., 19OP, 13, 458 ; A.: i, 932.I? J l d . , 56, 18 ; A . , i, 5%Ibid., 54, 119; A., i, 231218 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.but they were unable to decide whether it is a synthetic product,or a coagulated form of one of the fibrin proteoses.Lawroff 15 appears to have been more successful in formulatinggeneral views of the composition of these substances, although itis to be regretted that he has introduced confusion by the coiningof a new name for them. He obtained these precipitates (whichhe calls coaguloses) by the peptic digestion of proteins, as well asby their digestion in dilute mineral acids. He recognises twotypes of coagulose-yielding substances; the first are of the typeof proteoses, and the coaguloses which arise from them yield, onhydrolysis, both monoamino-acids and basic cleavage products.The second type of coagulose-yielding substances are polypeptides,and the coaguloses which arise from them yield, on hydrolysis,only monoamino-acids.The proteins which Lawroff first workedwith were caseinogen and haemoglobin, but subsequent work 16 withcrystallised egg-albumin showed that the same two types ofcoagulose-yielding substances could also be obtained from thisprotein after relatively short peptic digestion.These facts and theories are worthy of record, but I do notfancy the last word on plasteins or coaguloses has yet been written.Nucleic Acid.Nucleic acid has, in the past, been prepared from many differentsources, and differences have been noted in its percentage com-position and in its decomposition products.It has, therefore,been assumed that there are many varieties of nucleic acid, allresembling each other in containing phosphorus, in yielding purineand pyrimidine bases, and in containing a carbohydrate radicle,usuaIIy described as a pentose. The differences observed havebeen considered to be mainly due to the proportion in which thesevarious groups are combined together, and more especially to thenature of the purine and other bases contained within the mole-cule. It need hardly be said that if this view is correct, the com-plexity of the subject is enormous. But, as the years go by,and better methods for the separation and purification of nucleicacid are introduced, these difficulties are beginning to becleared up, and nucleic acids previously supposed to be differentare now to be regarded as identical.Schmiedeberg17 has beenable to give an empirical formula for the acid; and Levene andMandell8 have even advanced views as to its constitution, whichl5 Zeiitseh. physio?. Chem., 1907, 53, 1 ; A . , 1907, i, 995.l7 Arch. exp. Path. Pharm., 1907, 57, 309 : A . , i, 70.Ihid., 1908, 56, 343 ; A . , i, 844.Ber., 1908, 41, 1905 ; A . , i, 587PHYSIOLOGICAL CHEMISTRY. 219probably should be regarded more safely as provisional than final.Schmiedeberg, also, in his work has drawn attention to what heregards as a distinction between the hydrated and anhydrousforms of the acid, and to the power which the latter possesses ofgelatinising.This recognition of a gelatinous condition sometimespresented by nucleic acid and its salts is of importance, whetherthe explanation that the absence or presence of water in themolecule is the correct one or no€.A greater advance than this, however, was made a few years backby I. Bang. He prepared, from the nucleo-protein of the pancreas,a nucleic acid which is much simpler in composition than themajority of those previously investigated. It yields on decom-position three substances, namely, phosphoric acid, pentose, andonly one purine base, guanine. For this reason, he bestowed uponit the name of guanyPic acid. He also mentioned a fourthcleavage product, glycerol, but this has not been obtained by subse-quent workers, for instance, by Steude1,lg who otherwise confirmsBang’s results.The question was also taken up by v. Furth andJerusalem,20 who a t first denied the existence of guanylic acidaltogether. Bang21 pointed out how they had erred, and onre-investigating the matter they acknowledged their mistake,22and confirmed Bang’s statement, with the exception, again, thatglycerol was not found among the decomposition products ofguanylic acid.Since then, guanylic acid has been found in other organs, forinstance, in the liver, by Levene and Mande1.23Both liver and pancreas, however, contain, in addition toguanylic acid, what we may term ordinary nucleic acid, whichyields on cleavage other bases.These observers have, therefore, cleared up one previous sourceof error; there is no doubt that the older workers investigatedmixtures of nucIeic acid proper and guanylic acid, and thusobtained divergent analytical results. The discovery of guanylicacid a t first seemed to complicate the subject, but, really, it helpedto elucidate it.The next step was taken by Walter Jones,24 who, following upthe work of Schmiedeberg already referred to, has established theID Zeitsch.vhysiol. Chem., 1907, 53, 539 ; A . , i, 70.2o Beitr. chem. Physiol. Path., 1907, 10, 174; A!., 1907, i, 993.Ibid., 1907, 11, 76 ; A . , i, 70.22 Ibbid., 1908, 11, 146 ; A . , ii, 119.y5 Biochenz. Zeitsch., 1908, 10, 221 ; A., i, 587. They also describe a new methodGnanylic acid has also been for the separation of gnanine (ibid., 215 ; A., i, 586).discovered in the spleen, mammary gland, etc.24 J.Biol. Ckem., 1908, 5, 1 ; A., i, 744220 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.identity of at least three ordinary nucleic acids, namely, thosefrom the thymus, splqen, and pancreas. W-e need not follow himinto the interesting historical review of the curious “ comedy oferrors” which led to previous confusion. Most of these were dueto the different methods employed for isolating these substances,to the difficulties in estimating their cleavage products, to the non-recognition that certain bases are not primary cleavage productsbut arise by secondary reactions, and last, but not least, to admix-ture with guanylic acid.The special points worked out in deter-mining the identity of these acids were their specific rotationunder varying conditions, and the degree of viscosity of theirsodium salts. The so-called gelatinous sodium salt and the non-gelatinous salt are readily convertible one into another, andthis reversible action is believed to be a simple explanation, ifit occurs in zlizlo, of the physiological localisation and migration ofnucleic acid.He also states the following propositions, derived partly fromSchmiedeberg’s work and partly from his own, as being, a t anyrate, not far from correct:1. All “ ordinary nucleic acids ” (that is, nucleic as opposed toguanylic acid) yield the same two purine bases (guanine andadenine) and in the same proportion. Xanthine and hypoxanthine,when present, are due to the secondary action of de-amidisingferments.2.All yield the same pyrimidine base, cytosine.3. All yield lzevulic acid. This was first demonstrated byKossel, and points to the existence of a “ hexose ” carbohydrate.The previous statements about the presence of a pentose are, nodoubt, due to admixture with guanylic acid.4. There is, therefore, no insurmountable difficulty in acceptingthe hypothesis that the nucleic acids of different mammalian organsare identical substances. One must, however, be cautious a tpresent in applying this generalisation to all nucleic acids, for ithas been shown that those derived from plants and from fish eggsyield uracil, and that from the spermatozoa of fishes yieldsthymine, another pyrimidine base.Uracil obtained from mam-malian nucleic acid is derived secondarily from cytosine.5. The furfuraldehyde reaction stated to be given by nucleicacids is probably owing to admixture with guanylic acid, and dueto the guanine and pentose present.Tissue Metabolism.Metabolism was at one time only studied by what is called theThe total ingesta and egesta were measured balance-sheet methodPHYSIOLOGICAL CHEMIS'I'XY. 221and analysed, and by this means it was ascertained whether thebody was in equilibrium, or whether a deficit or the reverse wasoccurring in connesion with the main groups of body-constituents.Valuable as this method was, more important information still isobtainable by the investigation either of individual tissues ororgans, or in relation to the utilisation of one or other constituentof the food.It is this modern method of working at the subjectwhich has been successful in determining, not only the specialfunctions of each organ under varying conditions, but also ther8le played by the individual food-stuffs in their internal chemicalchanges.Many gaps exist in our knowledge still, but these are, year byyear, becoming less numerous, and from the large mass of workwhich has accumulated during the last twelve months, I am onlyable to select a, few papers which appear to be of exceptionalinterest.Respirution.-The work of Haldane and Priestley25 on theimportance of the chemical factor in breathing has been a greatstimulus to renewed work on the subject of respiration generally.Those who attend the meetings of the Physiological Societywill know the frequent discussions that occur there on the pointswhich still remain in dispute.I think, however, that Haldane'scontention, that variations in the tension of carbon dioxide in thepulmonary alveoli form the essential factor, has been very generallyconceded. The differences in the alveolar tension of this gas affectthe respiratory centre vici the blood-stream, and carbon dioxide isthe chemical stimulus par excellence which regulates the work ofthe respiratory centre in the brain. The points on which differenceof opinion still prevails are: (I) the part played by a diminutionof oxygen; (2) the relative importance of the nervous factor; and(3) whether fatigue products, such as lactic acid, may assist thecarbon dioxide in stimulating the respiratory centre.One notes an interesting piece of work relating in the main tothe second of these disputed problems by F.H. Scott.26 Fromthis work it appears that the principal respiratory nerves (thepneumo-gastrics) regulate the rate or rhythm of the xespiratorymovements, whilst the chemical factor specially regulates theamount of pulmonary ventilation, that is, the depth of the indi-vidual respiratory efforts; for when these nerves are divided, a risein the alveolar tension of carbon dioxide (or great diminution of25 See Ann. Xeport, 1905, 230.pci J. Phpsiol., 1908, 37, 301 ; A., ii, 865. A series of papers by Haldane a i dothers on the question of pulmonary ventilation has also just been published(J.Phpiol., 1908, 37, 355 ; A., 1909, ii, 66) too late for fuller iiotice here222 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.the oxygen in the respired air) increases the depth, but not the rateof breathing. The vagi in reference to respiration are regardedin the same light as the sensory nerves of muscle; without thesenerves, muscular movements are excessive and even ataxic.Such researches have also cleared up the mechanism of Cheyne-Stokes breathing, that curious waxing and waning of the respira-tion which is seen slightly during normal sleep, and markedly inthe hibernating state of winter-sleeping animals, as well as inmany pathological states in man (Pembrey 27).The condition common to all these states is a decreased excita-bility of the nervous system, and especially of the respiratorycentre.The man or the animal does not breathe for a certainperiod, a.nd during this apnceic stage, carbon dioxide accumulatesuntil its amount is sufficient t o stimulate the depressed cells of therespiratory centre to execute shallow and inefficient efforts atbreathing; but as the accumulation of the gas goes on, the respira-tions become more and more forcible, and culminate in forced ordypneic breathing; this sweeps out the carbon dioxide, and theconvulsive movements become quieter, the breathing gets shallower,and, finally, it once more ceases for a period, until the same seriesof events is again repeated.The Part Played b y Leucocytes in Protein Absorption.-Theentrance 0.f oxygen by the lungs is, of course, only the start ofthe true respiratory process which occurs in the tissues.Theentrance of protein by the alimentary tract is, also, only theinitial stage of protein metabolism. In last year’s Report I dealtwith this question, and so it will not be necessary to repeat myself.Dr. Pavy, in some recent lectures published in the Lancet,2* how-ever, dissents from the view now gaining credence among physio-logists, that the proteins are absorbed as amino-acids. He holdsthe older view, that the intestinal wall is the seat of proteinsynthesis, and, further, that the lymphocytes play an importantpart as protein-carriers. This last hypothesis has received supportfrom the work of Cramer and Pringle,29 who emphasise, as Pavydoes, the increase in these cells which occurs whether the proteinbe introduced in the usual way or parenterally.The blood ofdigesting animals shows a small but distinct increase of (‘ residualnitrogen ” over that of fasting animals, and the major part of thisis in the corpuscular elements. It is impossible to deny that theincrease in lymphocytes during absorption has some significance,but their total bulk is so small that it is difficult to believe that37 J. Patla. Bact., 1908, 12, 258 ; A . , ii, 204.29 November am1 December, 1908.zy J. P h y s d . , 1908, 37, 146 ; A., ii, 709PHYSIOLOGICAL CHEMISTRY. 223they carry the whole burden, and, therefore, Cramer’s conclusionthat these cells “ partly, a t any rate,” may perform a share of thework, is probably all that it is safe to affirm at present. It will,however, be noticed that such increase in nitrogen as can be deter-mined is in ‘‘ residual,” not in “ coagulable nitrogen.” Cramer,therefore, does not go as far as Pavy, for protein synthesis, accord-ing to him, does not occur at the seat of absorption, or even afteringestion by the colourless corpuscles.Respiratory Metabolism of the Sphal Cord.-The gaseous meta-bolism of nervous tissues is, perhaps, the most difficult of allsubjects to investigate, but by the use of Thunberg’s micro-respiro-meter,30 it has been shown that even peripheral nerves participate,to some extent, in respiratory interchanges.We should anti-cipate, OK a priori grounds, that the more vascular central nervousmaterial would be more active in this direction; we, a t any rate,know that if the brain is deprived even momentarily of its duesupply of fresh oxygen, unconsciousness or fainting is the result.MOSSO, also, some years ago stated that the temperature of thebrain is a high one, and during the last year Winterstein31 hassubjected the isolated fresh spinal cord of the frog to quantitativeexperiment; he found that, on stimulation, it has a high respiratoryexchange; per unit of weight this is two or three times greaterthan that of the body as a whole. Curiously enough, stimulationby the administration of strychnine was not found to have anyeffect.Rate of Conduction in serve.-An indirect means of approach-ing the question whether any given phenomenon is chemical orphysical is the method of determining the temperature-coefficientof its velocity.Arrhenius showed that the rapidity of a chemicalreaction is a t least doubled by a rise of loo in temperature,whereas a physical reaction is not accelerated in nearly so greata proportion. Synder 32 has used this method in relation to anumber of physiological phenomena, and although his data are, inmany cases, insufficient to draw conclusions from them, he never-theless showed that, in cases where it is known that metabolismdoes occur, the coefficients observed are those of chemical reactions.The method, therefore, appears to be one which possibly maysettle the vexed question whether the propagation of the nervousimpulse is chemical or physical.Maxwell 33 made experiments onthe pedal nerve of a giant slug; he selected this, first, because it:30 A m . &:port, 1905, 231.:I1 Z c n t ~ . Pl~ysLoZ., 19OS, 21, 869 ; A, ii, 509.Ante?.. J. Physiol., 19OS, 22, 309 ; A., ii, 768.‘j3 J. Bid. C h w . , 1907, 3, 359 ; A., 1907, ii, 977..>,2% ANNUAL REPORTS ON THE PROGRESS OF CNENISTRY.is a sufficiently long nerve, and secondly, because the normal rateof conduction is sufficiently slow for purposes of measurement.From his figures he concludes that the nerve impulse is a chemicalphenomenon, although he doubts whether it is an oxidationprocess. These experiments have been repeated by Woolley34 onamphibian nerve, and although he obtains the same figure asMaxwell (1.78 to 1-79), he is, unfortunately, not so clear as toits interpretation, for he doubts whether the high figure is anecessary proof of it chemical as opposed to a physical process.The conduction rate in amphibian muscle has about the samecoefficient (1.79 to 2-01>.It is pretty certain that muscular con-duction has an underlying chemical basis, and probably theconduction process is similar in both tissues. The coefficient forthe latent period of muscle is distinctly higher (3.26 to 3*3), andthis result strengthens a supposition previously advanced on othergrounds that conduction in muscle is a propagation, not of thecontractile change, but of an independent disturbance which elicitsthe contractile change a t each point on its passage.Glycogen.-This subject is one of perennial interest in relationti0 metabolism, and some observations on rabbits made by Lochheadand Cramer35 furnish a useful contribution to the chemistry ofgrowth.In the fetal condition, the greater part of the placentalglycogen was found in the maternal portion of the placenta; thisdiminished from the eighteenth day of fatal life onwards, whereasthat in the fatal liver increased. A distinct parallelism wasfound between the growth of the fetus and the amount of glycogenwhich it contains. In the earlier stages of intra-uterine life,the fatal liver does not possess the power of storing glycogen;this power is not acquired until the last week of gestation.Duringthe earlier period, the placenta fulfils the hepatic function so faras glycogen is concerned. Investigations on the effect of diet andphloridzin appear to show that the glycogen metabolism of theplacenta and fetus is independent of that of the mother.Pfluger3G seems to have published only one paper on glycogenthis year; he finds that the administration of kvulose leads tothe formation of glycogen in the liver, but the glycogen formed isnot laxorotatory; the liver cells have, therefore, the power of trans-forming the sugar given into dextrose, and it is this from whichthe glycogen is formed.His colleague a t Bonn, I(. Grube,37 has continued his interesting3‘ cJ. Physiol., 1908, 37, 112, 122 ; A., ii, 711.22 Proc.Eoy. ,SIN., 1908, 80, K, 263 ; A . , ii? 710.PJiiger’s Archiu, 1908, 121, 559 ; A., ii, 307.37 LM., 636 ; A., ii, 307PHYSIOLOGICAL CHEMISTRY. 225experiments on the perfusion of the tortoise’s liver, and, in answerto the question what is the smallest molecule from which the livercan make glycogen, finds that by perfusing that organ with aweak formaldehyde (O.Ol-O*OZ per cent.) solution, the liver is ableto make glycogen from it.The R61e of Sagar in Muscular Activity.-In confirmation ofthe view, now so generally held, on the importance of sugar as thesource of muscular energy, Locke and Rosenheim’s 38 results on theisolated mammalian heart must be noted. They describe aningenious new perfusion method 39 by which a solution of dextrosein oxygenated Ringer’s solution can be repeatedly circulatedthrough an excised rabbit’s heart.Five to ten centigrams of thesugar disappear in from eight to nine hours. This is not due tominor metabolic or fermentative by-processes, but is associatedwith the main chemical change that underlies cardiac activity. Ifthe activity of the heart is lessened by the omission of the calcium,or still more by the omission of both calcium and potassium fromthe circulating fluid, the amount of sugar used up is lessened.The amount of carbon dioxide formed runs parallel with the dis-appearance of sugar. No evidence was found of the formationof disaccharide or of lactic acid, or of the storage of glycogen inthe heart. Nitrogenous waste was not fully investigated, but thetotal is extremely small.The last aspect of the subject has been taken up incidentally ina piece of work just published by Howell and Duke.*O Theseauthors had previously stated that an output of potassium fromthe heart’s substance occurs during vagus inhibition, and sug-gested that the increase in cardiac activity which follows stimula-tion of its accelerator nerves may be due to an output of calcium.Using the Locke-Rosenheim method of perfusion just referred to,their findings, however, were negative; neither the calcium nor thepotassium in the circulating fluid showed any variation in amountafter a perfusion lasting for hours, nor after long-continued excita-tion of the heart through its accelerator nerves.I n relation to nitrogenous metabolism, they found no output ofhypoxanthine, such as Burian has described in the case of skeletalmuscle.This may be a fundamental distinction between the twoforms of muscular tissue, or it may be due to the presence ofdextrose in the circulating fluid used by Howell and Duke,which was absent in Burian’s experiments. The heart, however,3* J. Physiol., 1907, 36, 205 ; A . , ii, 120.39 Brodie and Miss Cullis have also described a new apparatus for the heart-perfusion (ibid., 1908, 37, 337 ; A., ii, 865).1 O Amer. J. Physiol., 1908, 23, 174; A,, 1909, ii, 72.REP.-VOL. V. 226 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.gives off creatinine (or creatine) to the circulating fluid, and itwill be interesting to determine how far this elimination is asso-ciated with functional activity.Creatine and Creatinine.Creatine is absent from normal urine and creatinine is alwayspresent.I t is, in fact, next to urea, the most abundant nitrogenoussubstance found there. Amid all the inconstancies of urinary com-position, it appears to be the substance which is most constant inamount, diet and exercise having no effect on it. The old idea that;the bulk of the urinary creatinine is derived from the creatine offlesh food has been entirely abandoned, and Folin’s view that it is ameasure of endogenous nitrogenous katabolism has steadily gainedground, The close chemical relationships of the two substances inquestion led physiologists to the erroneous conclusion that creatine isthe source of the excreted creatinine.The discovery of the mistakehas led some to the equally hasty conclusion that the two materialshave no physiological connexion a t all. A relationship does existbetween them in. vivo, but it is a different one from that previouslysupposed to exist. During the past year, the papers relating tothis subject have been numerous, and although differences of opinionon points of detail are noticeable, the general trend of most ofthe results is the same.Taking Mellanby 41 first, he took as his starting-point an investi-gation of the contradictory data relating to the proportion ofcreatine and creatinine in muscle. Monari’s authority is usuallyquoted for the text-book statement that the latter increases atthe expense of the former when muscle becomes active.Mellanbyhas shown that this is not the case. Monari’s technique affordsan opportunity for the change to occur, and his precipitates wereimpure. Creatinine is never present in muscle a t all, even afterprolonged muscular work ; the original amount of creatine remainsunaltered by work, and even (in frog’s muscle) after survival forthree days.42I n addition to this, aseptic or antiseptic autolysis causes nochange in either creatine or creatinine. When, however, the musclebecomes septic, all the creatine disappears. The statements ofGottlieb and Stangassinger 43 regarding the tissue enzymes theyterm creabase and creatinase were in no respect confirmed.4441 J. Physiol., 1908, 36, 447 ; A., ii, 308.42 Cathcart and Graham Brown, however, discovered a slight increase of creatininein frog’s muscles after stimulation ; hut if the circulation is intact there is a decrease(Proc.physiol. SOC., 1908, xiv--xv ; J. Physiol., 37 ; A . , ii, 516).43 Ann. Report, 1907, 239.44 Gottlieb and Stangassinger have published a seooncl paper on these ferments PHYSIOLOGICAL CHEMISTRY. 227Invertebrate muscle contains no creatine, and Mellanby selectedthe developing bird in which to study the biochemical history ofcreatine. It is absent from the musculature of the chick up to thetwelfth day of incubation; after this date, the liver and themuscular creatine develop pari passu. After hatching, the liverstill continues to grow rapidly, the creatine percentage in themuscles increases also, although the development of the size ofthe muscles occurs very slowly.This led Mellanby to the con-clusion that the muscular creatine has its origin in the liver. Theliver is thus continuously forming creatinine from substancescarried to it by the blood from other organs; in the developingmuscles this is changed to creatine, and then, when the muscle issaturated with creatine, excess of creatinine is excreted by thekidneys. His view is, therefore, an absolute reversal of thosepreviously held; the muscles do not change creatine into creatinine,but creatinine into creatine. I f creatine (an innocuous neutralsubstance) was converted by the muscles into creatinine (a stronglybasic substance), it would really be contrary to all that is knownof the chemical changes which occur in the body.This view is upheld by experiments in which creatine andcreatinine were added to the food; feeding with the latter substanceleaves the muscles still free from that material. Feeding withcreatine has also no effect after the muscles have reached a certainpoint of saturation.The small amount of creatinine excreted in diseases of the liveralso supports the view that that organ is responsible for creatinineformation.The excretion of creatine in cancer of the liver isexplained by supposing that the muscle cells break down, and thatcreatine is liberated without conversion into creatinine beforeexcretion takes place.Verploegh and van Hoogenhuyze 45 have published an extensiveseries of observations on creatinine excretion ; they confirm Folin’sviews in the main, and also agree with Mellanby concerning theimportance of the‘liver in its metabolic cycle.Their reference toMellanby’s views, however, appears to contain a misunderstanding,for they allude to the conversion of creatinine into creatine by theliver, which was not Mellanby’s point a t all. This change theythey attribute the destruction of creatiniue and the consequent appearance of creatinewhich liver extracts accomplish t o the action of creatinase, and the subsequent dc-struction of creatine to creatasc (Zeitsc’c. physiol. Chcm., 1908, 55, 295, 322;A., ii, 515). Their statements regarding the non-participation of putrefaction inthis ferment action are confirmed by Rothmnnn (ibid., 1908, 57, 131 ; A ., ii, 967).That the transformation of creatine into creatinine in the liver is the work ofsoluble ferments is also confirmed by van Hoogenhuyze and Verploegh (ibid., 161 ;A., ii, 971). 46 Loe. cit.Q 228 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRT.attribute to the creatinase of Gottlieb and Stangassinger,46 and inconditions where hepatic activity is lowered (cancer of the liver,certain fevers, and hunger), the appearance of creatine in the urineis explained by its non-conversion into creatinine.Mellanby’s views will, of course, have to be subjected to the usualtests of criticism and renewed research, and, until they have stoodthis, cannot be accepted as proved. They nevertheless provide astimulus to fresh work, and their novelty, and the fact that theydo explain some of our previous difficulties, render them attractive.He entirelydissents from Mellanby’s theory that creatinine is formed in theliver at all; neither does he agree with Folin that its amount isan index of endogenous protein metabolism.He, however, confirmsprevious statements regarding the constancy of its amount, and heplaces the figure at from 7 to 11 milligrams of creatinine nitrogenexcreted per diem per kilo. of body-weight. The excretion isconstant, not only from day to day, but from hour to hour; it isnot influenced by the volume of the urine, nor by the total nitrogenexcreted. The creatinine coefficient is parallel to the muscularefficiency of the individual, and its source is some special processof muscular metabolism.But in acute fevers, he states that itsexcretion is increased, and admits that here it is not parallel tomuscular efficiency. Creatine is absent from normal urine, but itmay be excreted in acute fevers, in women during involution ofthe uterus, and in certain other conditions in which there is arapid loss of muscle-protein.Shaffer is not the first who has pointed out a parallelism betweencreatinine excretion and muscular development.48 The smallamount in infants’ urine corresponds with the smaller amount ofmuscular development in the child. The amount of creatinine ininfants’ urine is so small that some previous observers, using theold zinc chloride method, missed it altogether.Funaro4g hasalways found it present, and its amount constant, in spite ofvariations in the food.Another lengthy paper on creatinine metabolism has recentlyappeared by G. Lefmann.50 The following are its principal con-clusions: The excretion of creatine and creatinine is prettyconstant in well-nourished animals. I f either substance is addedto the food, it is excreted unchanged. I f creatine is given by themouth, or parenterally, it is never changed into creatinine, andShaffer,47 for instance, has independent opinions.46 See footnote 44.47 Ainer. J. Physiol., 1908, 23, 1 ; A., ii, 971.49 Biochcm. Zeitsch., 1908, 10, 467 ; s4., ii, 716.5o Zeitsch. physiol. Chew., 1908, 57, 476 ; A , , ii, 1050.See Spriggs, also Amberg and Morrill, Ann.Report, 1907, 238PHYSIOLOGICAL CHEMISTRY. 229in inanition it is almost completely excreted as such. Disease ofthe liver or increased protein katabolism produces, first an increase,then a decrease in creatinine excretion, and when it is lessened, theamount of creatine excreted rises. The liver is the probable seatof creatinine formation. I f nephritis is induced by chromates,nearly all the creatinine is changed into creatine, probably by thealteration in the reaction of the urine.I have been content merely to enumerate Lefmann’s conclusionswithout comment; some agree with, some differ from, those enun-ciated by others, and the last one opens up a new possibility.None of the papers written (except that of Mellanby) haspresented a clear and consecutive view of the history ofcreatinine, and although some of his views may have to be modifiedin the future, there is a general consensus of opinion now that theliver plays an important share on the constructive side of its meta-bolism.The Lipoids.61The term lipoid is one of recent origin; it appears to have beenfirst employed by Overton in 1901 for a group of substances con-tained in the protoplasm of all cells, especially in their outer layeror cell-membrane.Overton pointed out, in his work on narcotics,that materials which act as anzesthetics (such as ether and chloro-form) are capable of obtaining an entry into the cell, because theyare soluble in the lipoids of the cell-membrane. Whether this isa correct explanation for the narcotic properties of all drugs ornot, the fact which is undoubted is the solubility of many anaes-thetics in lipoid substances, and the solubility of the lipoids inthese anzesthetic reagents.I n their solubility in such reagents asether, chkroform, alcohol, etc., the lipoids resemble the fats andfatty acids; hence their name.The interest recently bestowed on the lipoids is due, not onlyto their chemical properties, but also to their biological importance.Although present in smaller amount than proteins, they appear tobe essential constituents of protoplasm, and the labile character oftheir molecules, in many cases, is a property they share in commonwith the proteins. There seems to be a good deal of truth in theopinion expressed by Bang that the importance of proteins as“ carriers of life ” (Triiger des Lebens).has been over-estimated,whilst that of the lipoids has been neglected. The lipoids are con-tained in special abundance in that tissue, which, above all others,51 In the preparation of this section, I have been much helped by two courses oflectures delivcred a t King’s College by 0. Rosenheim, who has devoted much of histime to a study of the lipoids. The first of these lecture courses has been publishedin B condensed form in Science Progress, 1908, April and July230 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.manifests what we still may call “vital properties,” namely, thenervous tissues, and it is in the brain and similar structures wherethey have been most studied. Thudichum’s great work on brainchemistry, published in 1874, was very much neglected, probablybecause of the polemical nature of most of his writings. It was,however, valuable pioneer work, and the research of the last fewyears has done much to demonstrate its correctness.Many state-ments, attributed in text-books to more recent investigators, aremerely confirmations of what Thudichum discovered. Confusionhas been introduced into a subject already sufficiently complex bythe coining of many names for the same substances. I shallendeavour here to retain, as far as possible, Thudichum’s originalnomenclature, and although Thudichum did not employ the termlipoid, his classification of them is still the one which is adopted.The lipoids are practically all contained mixed with fat, fattyacids, and lipochromes in the ether-alcohol extract of tissues andorgans.Their separation from the fats, and from each other, isusually difficult, involving troublesome processes of fractional preci-pitation by various solvents, into a description of which I do notintend to enter. Selective extraction, however, is sometimespossible, and gives much better results; for instance, if the residueof the alcohol-ether extract is treated with cold acetone, cholesterolonly passes into solution; if extraction with hot acetone is thenperformed, the mixture known as protagon is extracted; andprotagon may be separated into its constituents by pyridine andso forth.The lipoids may be classified in the following way:1.Those which are free from both nitrogen and phosphorus.The most important member of this group is cholesterol.2. Those which are free from phosphorus, but contain nitrogen.These yield galactose on cleavage, and were termed cerebro-galactosides or cerebrosides (for short) by Thudichum. Phrenosinand kerasin are the best known members of this group.3. Those which contain both phosphorus and nitrogen, andwhich are best known by Thudichum’s name of phosphatides.They are grouped by Erlandsen52 according to the proportion ofnitrogen and phosphorus in their molecules as follows :.a. Monoamino-monophosphatides, N : P = 1 : 1, for instance,lecithin and kephalin.6 . Diamino-monophosphatides, N : P= 2 : 1, for instance,sphingo-myelin and amido-my elin.c. Monoamino-diphosphatides, N : P = 1 : 2, for instance, cuorinof heart muscle.52 Ann.Beport, 1907, 251PHYSIOLOGICAL CHEMISTRY. 231d. Diamino-diphosphatides, N : P=2: 2. One of these wasseparated from brain by Thudichum, but has not since beenexamined.e. Triamino-monophosphatides, N : P = 3 : 1. One of these(neottin) is present in egg-yolk. It differs from other phosphatidesin yielding no unsaturated fatty acids.This system of classification is obviously capable of extension,as phosphatides are discovered in which the N : P ratio is differentfrom those enumerated above.We may now take these various substances in order, mentioningin relation to each the new facts which have been made outconcerning them during t'he last year.Cholesterol.-This is found in small quantities in all forms ofprotoplasm; until within the last month it was stated to be absent,however, in heart muscle.J. A. Gardner 52 has reinvestiga ted thispoint, and finds it present there in about the same proportion asin other forms of muscular tissue. It is a specially abundantconstituent of nervous . tissues, particularly in the white sheathof nerve fibres. It occurs there in the free state, and is readilyextracted by cold acetone.Until a few years ago all that was known of its chemistry wasthat it has the formula C,,H,,O (or C,,H,,O), and that it is anunsaturated monatomic alcohol.Recent research has shown that it belongs to the terpene family,a group of substances previously known only in plants, and thefollowing formula has been tentatively put forward to indicateit's constitution :C*3 /\/\ ,'\"'\G3H7.1 1 1 1 1 J \/\/-\/\/\=/ UH3that is, five reduced benzene rings are linked together, and it isimportant to note that in order that cholesterol may exercise itsphysiological action, the double linking shown as well as thehydroxyl group must be intact."Cholesterol compounds also exhibit a physical phenomenon53 Communication made to the Physiological Society, December, 1908 : not yetpublished.C. Dorde (Proc. physiol. Soc., 1908, lviii-Iix ; J. Physiol., 37 ; A., ii,769) has also found cholesterol in ccelenterate animals.54 The function of cholesterol in enabling the body cells to withstand the actionof toxins was indicated in last year's Report (pp.252-253). A very useful-summaryof the chemistry, distribution, and biological importance of the cholesterols andphytosterols (vegetable cholesterols), with bibliographical references, is given in anarticle by W. Glikin (Biochem. Zentr., 1908, 7, 289, 351)232 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.recently studied by Lehmann, namely, the formation of liquidcrystals, which is also given by several other lipoids. It is notwithin the province of this article to enter into the many interest-ing data which the study of liquid crystals has brought out; itwill be sufficient for my present purpose to point out two onlyof these, both having a biological bearing.The first of these relates to what Virchow termed I f myelin-forms’’ in 1855.If brain substance is mixed with water, threadsare observed shooting out and twisting into fantastic shapes; thesehave a superficial resemblance to nerve fibres, and the movementsoften simulate protoplasmic or amceboid movements. The term“ myelin-forms ” applied to them is somewhat unfortunate, for theword myelin has been applied to different chemical substances, and ithas now no precise chemical meaning ; it is most frequently employedsynonymously with the white sheath of nerve fibres, and no doubtthe earlier observers thought that myelin-forms were associatedin some way with the behaviour of the lecithin-like substances whichare present in white matter. Myelin-forms can also be obtainedwith cholesterol mixtures, and some have gone so far as to say,“ without cholesterol no myelin-forms.” This is not correct, forother lipoids and also certain oleates show the same phenomenon.Various theories have been advanced to account for myelin-forms,and some of these imply that in the investigation of this remarkableappearance will be found the explanation of living movement. Ithas now, however, been conclusively shown that myelin-forms aremerely distorted liquid crystals, due to the presence of cholesteroland other lipoids.The second biological outcome of a study of liquid crystalsrelates to the fat globules seen in the cortex of the suprarenal body,during cell-proliferation in cancer, and in the liver and other organsduring so-called fatty degeneration.These are not composed offat, for the polarisation microscope shows them to be anisotropic,and further investigation has shown them to be lipoids in the fluidcrystalline condition.55 There is no .doubt that cholesterol formsa very considerable constituent of these globules, but it was foundthat pure cholesterol, or cholesterol ethers, do not exhibit thephenomenon, nor do they give the characteristic colour reactionswhich are given by the white matter of nerve fibres. Whatappears to be necessary is a mixture of cholesterol a.nd fatty acid,and it has been suggested that in such mixtures the acid is incor-porated as “ acid of crystallisation,” analogous to the “ water of55 C. P. White, J. Path. Buct., 1908, 13, 3, 11 ; J. L. Sniith and others ibid.14 ; A., ii, 966, 968, 972 ; T.Panzer, Zeitsch. physio2. Chem., 1907, 54, 239 ; A . ,ii,122PHYSIOLOGICAL CHEMISTRY. 233crystallisation ” in many other crystals. It is quite probable thatthe ethers of cholesterol described by Hurthle as present in theblood are not true esters, but similar mixtures of cholesterol andfatty acid. This is rendered all the more probable if the view thatcholesterol is a protective agent against toxins is upheld; for wehave already seen that, in order that it may exercise this function,the double linking and the hydroxyl group must be intact, whichwould not be the case in an ether. We have already noted thatcholesterol occurs free in the brain, and Salkowski has shown thatthe same is true for the cholesterol of the bile.56The globules referred to, however, do not consist altogether ofcholesterol mixtures.Rosenheim and Miss Tebb have preparedfrom the suprarenal cortex a substance analogous to the sphingo-myelin of brain which shows the same appearance^.^^These investigations throw light on the possible function of thecortex of the snprarenal gland; it may be that the cells thereare engaged in the secretion of cholesterol and other lipoids, andthat this has some connexion with the regulation of growth anddevelopment; from his observations on the liquid crystals oftumours, C. P. White 58 suggests that cholesterol is associated ratherwith cell-proliferation than cell-degeneration ; and Mendel,59 inhis studies on growth in embryos, arrives a t much the same con-clusions; in the chick embryo, cholesterol disappears like the otherlipoids of the yolk, being sources of energy in growth.Before passing from cholesterol to the consideration of otherlipoids, there is one more piece of work which deserves a passingreference; I refer to that by C.Dor6e and J. A. Gardner.60 onthe excretion of cholesterol. A t an earlier date, when cholesterolwas supposed to be a mere waste material excreted by the bile,Austin Flint found in the faeces a substance which he namedstercorin, and which, as he rightly surmised, is a cholesterolderivative. This material, which was re-named coprost’erol byBondzynski, is a saturated alcohol with the formula C2,H4*0; itspresence in the faxes is, however, not constant; it was found byDor6e and Gardner in dogs’ fxces after feeding on raw brain,but in dogs fed on cooked vegetables or meat, cholesterol is presentas such.Whether this cholesterol originates from the food orfrom the bile is very uncertain, for in horses it is entirely absent;if these animals excrete cholesterol in their bile, it must thereforeeither be destroyed or re-absorbed. The substance called hippo-.56 Zeitsch. phyiol. Chem., 1908, 57, 521 ; A., ii, 1055.57 J. Physio?., 1908, 37, 348 ; A . , ii, 879.O9 Amcr. J. Physiol., 1908, 21, 77 ; A . , ii, 208.J. Path. Bact., 1908, 13, 3 ; A . , ii, 972.Proc. Xoy. Soc., 1908, 80, B, 212, 227 ; A , , ii, 514234 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.coprosterol in horses’ faxes (which was formerly supposed to beanalogous to coprosterol of human or dogs’ faeces, and considered tobe derived from the bile by bacterial reduction in the intestine) isonly present after feeding on grass, and is undoubtedly a phytosterolor cholesterol of vegetable origin.Following the usual custom oflabelling phytosterols by their plants of origin, Dor6e and Gardnerpropose to term it chortosterol to indicate that it is derived fromgrass; its formula is C27H540, and it gives none of the usual colourreactions of cholesterol.The only paper I shall allude to on the protective action ofcholesterol confirms those I referred to in last year’s Report. Itis by Minz,G1 and deals with cobra and viper venoms. Cobravenom, as is well known, contains two toxins, one which dissolvesblood-corpuscles (hzmolysin), and the other which attacks nerve-cells (neurotoxin); the hzmolytic action of the venom is aloneinhibited by cholesterol; the latter is believed to remove fromsolution the lecithide, the prolecithide, and to a less degree lecithinitself.It will be seen from this that the author accepts thestatements of Kyes and others concerning the part lecithin andlecithides play in the anchoring of toxins on to cells, statementswhich we shall presently see have been subjected to criticism.The action of the neurotoxin is not influenced by cholesterol.Viper poison also contains two toxins, a hEmolysin, and a poisonwhich leads to the occurrence of hzemorrhages (hcemorrhagin).The latter is not inhibited by cholesterol, the former is.Thehzemorrhagin, however, is destroyed by hydrochloric acid, thehzemolysin not.The Cere6rosides.-A warm alcoholic extract of brain depositsa white precipitate on cooling; if the cholesterol contained in thisdeposit is extracted with ether, the residue may still be calledprotagon, not as implying that it is a definite chemical individual,but as a convenient expression, employed in much the same wayas the term peptone is still used for a mixture of protein cleavageproducts. Protagon was originally called c&rQbrote by Couerbe ;the word protagon we owe to Liebreich, who regarded it not onlyas a definite compound, but the mother substance of the otherphosphorised and non-phosphorised constituents of nervous tissue.It has now been conclusively proved in confirmation of whatThudichum stated in 1874, that protagon is a mixture of phos-phorised and non-phosphorised substances, in such propor-tions that it usually contains about 1 per cent.of phosphorus.6261 Bi0che.m. Zeitsch,., 1908, 9, 357 ; A . , ii, 415.62 The attempted resuscitation of protagon by Cramer alluded to in last year’sReport (pp. 247-249) has led to further writing of a somewhat polemical naturPHYSIOLOGICAL CHEMISTRY. 235By treatment with appropriate reagents and recrystallisation,protagon can be separated into its constituents; the best methodis to dissolve ‘‘ protagon ” in pyridine; on allowing this solutionto stand, the constituent rich in phosphorus separates out in theform of anisotropic globules (fluid sphzro-crystals), and thosewhich are free from phosphorus and comprise about 70 per cent.of the original protagon reniain in solution.The phosphorus-richconstituent is a phosphatide sphingo-myelin which we shall dealwith under its appropriate heading, and the phosphorus-free con-stituents are the cerebrosides. Although these have received manynames, the total number of known cerebrosides is two. These arenamed, to employ Thudichum’s original terminology, phrenosinand kerasin. The former is a crystalline product, the latter ofwaxy consistency.Phrenosin yields on cleavage three substances : -(1) A reducing sugar, galactose.(2) A base termed sphingosine, about which little or nothingchemically is yet known.(3) A fatty’acid, termed neuro-stearic acid by Thudichum; ithas a higher molecular weight than stearic acid, but has not beenyet definitely identified.Kerasin also yields galactose and sphingosine, but its third con-stituent, the fatty acid, is not neuro-stearic, but another acid,which has also not been identified as yet.The only noteworthy piece of work during the last year relatingt o these substances is that by K.Takaki63 in connexion withphrenosin, which he speaks of under Thierfelder’s name as cerebron.He finds that it is one of the substances in the brain which uniteswith tetanus toxin; it is apSparently not the only brain constituentwhich acts in this way, for more of the tetanus toxin disappearswhen mixed with brain substance than can be accounted for bythat which combines with the phrenosin.It is apparently theneuro-stearic acid constituent of phrenosin which is responsible forthis action.The Phosphatides.We are now free to pass to a consideration of the phosphatides,and will deal with them under the headings already given in ourclassification a few pages back. I n the first group, the mono-(see Wilsoii and Cramer, Quart. J. exp. PhysioZ., 1908, 1, 97 ; A., i, 234 ; Rosenheimand Bliss Tebb, ibid., 297 ; also J. Physiol., 1908, 37, 341, 348.; A., ii, 879). Theadditional facts brought out by the last-named observers will, it is to be hoped, besuccessful in ‘ ‘ laying ” protagon beyond hope of further resurrection.63 Beitr. chern. Physiol. Path., 1908, 11, 288 ; A ., ii, 521236 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.amino-monophosphatides, we have to deal with lecithin andkephalin.Lecithin.-Although the empirical, and probably also the con-stitutional, formula of this substance are fairly well known, suchknowledge is largely based on conjecture, for lecithin is a mostlabile material, as recent work on its cadmium and other saltshas shown.64 We know, however, that on decomposition it yieldsthe base choline, glycerophosphoric acid, and two fatty acidradicles. The nature of the fatty acids may vary, but in thelecithins found in the body, oleic acid is always one; the fattyacid radicles are linked to glycerol as they are in ordinary fats.The place of the third fatty acid radicle of an ordinary fat istaken by the phosphoric acid radicle, and this in its turn is inethereal combination with choline.Lecithin forms compounds with many substances, with metals,with alkaloids, with proteins (lecitho-proteins), and with carbo-hydrates. Possibly many of these are adsorption rather than truecompounds.The main interest of lecithin to the biologist is the part it issupposed to play as an amboceptor in linking poisonous proteinsto cell protoplasm, as was first pointed out by Kyes in his work onthe hzmolysin of cobra venom.65 I accepted this view with con-siderable reserve in my Report last year, and my cautiousness hasbeen justified by much of the work that has been issued duringthe current twelve months.I f teleological argument is permissible,it is difficult to see the advantage in the struggle for existencewhich lecithin would confer upon living organisms.It may, ofcourse, be that the assimilation of a food protein is on all fourswith that of a toxic protein, and the nutritive value of lecithin,which has been asserted so frequently, may possibly rest upon thisamong other factors; but even this falls to the ground if theassimilation of protein matter is usually accomplished, not by theincorporation of ready-made protein, but by that of its simple(amino-acid) cleavage products.I. Bang, who stands in the front rank of modern investigators,in his attempts to repeat the work of Kyes, has entirely failed tosubstantiate his main premises.66 He finds that the existence ofcobra lecithide is unproved, and that Kyes’s lecithides are mixturesof fats, soaps, and decomposition products of lecithin.Lecithinitself is wholly inactive as an activator; the same is true forcuorin. He found the kephalin fraction to possess some activityG4 W. HeuEner, Arch. exp. Path. Phnrm., 1908, 59, 420 ; A . , 1909, i, 5 .65 Ann. Xeport, 1907, 251.GG Biochem. Zeitsch., 1908, 11, 520 ; A . , ii, 721YHYSIOLOG ICAL CHEMISTRY. 237in this direction, but there was no guarantee even here that hewas dealing with a pure substance, and he found that Kossel’sprotagon (which he speaks of incorrectly as consisting largely oflrephalin) is also inactive. What does seem to be certain is tha’cthe hzemolysis produced by snake venom depends on the existenceof a lipolytic enzyme; this view is accentuated by the careful workof v.Dungern and Coca.67 It is the scission products liberatedby such an enzyme which act hzmolytically, especially de-oleo-lecithin (th?t is, lecithin minus its oleic acid) and oleic acid itself.These observers agree with Bang that compounds of lecithin andcobra toxin do not exist, and that Kyes’s preparations are mixturesof numerous substances. Cobra poison contains no amboceptor,and the hemolysis produced by a combination of cobra poison andthe complement of serum -is due to a complex serum hEmolysinwhich acts only in certain circumstances, of which the mostimportant is that the blood-corpuscles must have taken up acertain quantity of lipase.I n this connexion it should also be noted that the Wassermannreaction of the cerebro-spinal fluid so much employed to-day forthe detection of syphilis is also due to lipolytic activity, probablyproduced by the agency of the syphilis parasite (spirochzte).Two papers deal with the estimation of lecithin in animaltissues; one of these is by W.GlikinY6* who finds a specially highpercentage of this substance in the bone-marrow of new-bornanimals, especially in those which are born in an immature con-dition. The other is by J. NerkingYG9 who has estimated the totalyield in the bodies of certain animals, as well as in their individualorgans. I n the rabbit the quantity works out as 0.4 per cent.of the body-weight. I n the hedgehog the yield is especially high,particularly in bone-marrow and suprarenal.Nerking somewhatinconsequently concludes that this may explain the comparativeimmunity the hedgehog possesses against sna ke-bite ; for if lecithinfavours the safe anchorage of snake poison, it can hardly beexpected to act also as a protection against the venom. Thehedgehog, if immune against snakes, much more probably owesits freedom from attack to its protective coating of spines.ChoZine.-This basic product of lecithin cleavage possesses a gooddeal of physiological interest ; its presence in the circulating fluidsis an exact chemical proof of the breakdown of lecithin, and soof nervous material. The methods of detecting this substanceI need not go into again, but will refer readers interested in the67 Biochorn.Zeitseh., 1908, 12, 407 ; A., ii, 866.63 lbid., 1907, 7, 286 ; A., ii, 120.69 lbid., 1908, 10, 193; A., ii, 608238 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.subject once more to my Report of last year. Choline does notpass as such into the urine; therefore if this test for nervousdegeneration is employed, the patient must be prepared to sacrificea small quantity either of his blood or cerebro-spinal fluid. Theexamination of the urine does, however, give some indication ofnervous breakdown (in the actual, not the figurative, sense), forBauer70 has shown that the trimethylamine of the urine is con-siderably increased in such circumstances. This product of lecithindegradation is also increased by the administration of foods richin lecithin.It is not increased if the nervous disease is functional(that is, figurative rather than actual).A few years ago Donath stated that choline is discoverable,at any rate, in one disease, usually reckoned as functional, namely,epilepsy. Kajiura71 in my laboratory has shown that this is notso, and has pointed out how Donath made his mistake.H. MacLean72 finds that only 52 per cent. of the nitrogen oflecithin is recoverable in the choline. He therefore suggests thatanother nitrogenous base may be present in lecithin; or, of course,it is possible that his method for the collection of the choline wasnot sufficiently accurate.C. Schwarz and R. Lederer 73 identify the substance that lowersblood-pressure in extracts of the thymus, spleen, and lymphaticglands, as choline; v.Fiirth and Schwarz74 find the same truefor the depressor substancs in thyroid extracts; and the sameobservers 75 have also stated that the secretin of Bayliss andStarling contains considerable quantities of choline. Cholineproduces a flow of pancreatic juice, but it is not identical withsecretin, the action of secretin on the pancreatic flow being onlypartly neutralised by atropine, whilst that of choline is whollyinhibited by the same alkaloid.Speaking of the antagonisms of choline leads me next to mentionan investigation by A. Lohmann,76 who finds that adrenaline andcholine are antagonistic so far as relates to blood-pressure, cardiacactivity, and intestinal peristalsis, but not in relation to thediabetic condition which adrenaline sets up.The foregoing mention of secretin leads me into anotherparenthesis, and a reference to a polemical paper by L.Popielski.77He still considers that pancreatic secretion is largely influenced7o Bcitr. CJLWL PhyslsioZ. Path., 1908, 10, 502 ; A . , ii, 717.71 Qiia;'t. J. cxp. PhysioZ., 1908, 1, 291 ; A . , 1909, ii, 71.7'L Zeitsch. physiob. Cheyn., 1908, 57, 296 ; A . , ii, 967.78 PJEuver's Archiv, 1908, 124, 353 ; A . , ii, 968.i4 Ibid., 361 ; A., ii, 968.i6 Ibid., 1908, 122, 203 ; A . , ii, 407.77 f b i d . , 1907, 120, 451 ; A., ii, 119.75 Ibid., 427 ; A . , ii, 963PHYSIOLOGICAL CHEMISTRY. 239by nervous reflexes; he finds that extracts of all parts of thegastro-intestinal mucous membrane produce the effects of " so-called secretin," and is not limited to the upper portion of theintestines.He also calls Bayliss and Starling to book for labelling,with a chemical name, a substance of which they kriow nothingchemically. This is a, piece of good advice which he unfortunatelyforgot to apply to his own work a few months later, when henamed the unknown substance in Witte's peptone which lowersblood-pressure, vaso-dilatin.78A somewhat lengthy communication on the physiological actionof choline by G. Modrakowski 79 must next be referred to ; he findsthat this substance, prepared synthetically, does not, when abso-lutely pure, produce lowering of blood-pressure, as all previousobservers have found. On the contrary, it raises blood-pressure.This, however, does not invalidate the work of those who haveused the depressor effect usually seen as a physiological test forcholine, for extraordinary precautions have to be taken to preventthe pure choline from undergoing that change which leads to thedevelopment of a depressor modification.The impurity or modi-fication which causes the fall of blood-pressure is also consideredto be responsible for some other physiological effects previouslyascribed to choline; it is neutralised by atropine, and that,according to Modrakowski's view, is the reason why even impurecholine will produce a rise in blood-pressure after an animal hasbeen atropinised.KephaEin.-This is a monoamino-monophosphatide, concerningwhich we know much less than we do of lecithin. I f an etherealextract of brain is evaporated and the residue treated with alcohol,lecithin enters into solution, but kephalin remains undissolved. Itsname indicates the waxy nature of this compound.On decomposi-tion, it yields phosphoric acid, and fatty acids which are lesssaturated than oleic and probably belong to the linoleic series. It isquestionable whether the base it contains is choline. It is foundalso in egg-yolk, and appears to be the most abundant phosphatidein nerve fibres, and that this is contained not merely in the medul-lary sheath is seen by comparing the figures for medullated and non-medullated fibres. F. Falk 8O gives the following numbers :Medullated nerve. Non-medullated nerve.Cholesterol .....................25.0 pcr ctnt. 47'0 per cent.G e phalin ........................ 12-4 , , 23'7 ,,Cerebrosides .................. 18 -2 , , 6-0 ,,Lecithin ........................ 2.9 ,, 9.8 ..78 Arch. exp. Path. Pharrn., ,SuppZ., 1908, 435 ; A . , ii, 1059.79 PJluger's Archiv, 1908, 124, 601 ; A . , ii, 974.go Biochem. Zeitsch,, 1908, 13, 153 ; A., ii, 966240 ANNUAL REPOltTS ON THE PROGRESS OF CHEMISTRY.Under the heading of the second group of phosphatides, in whichthe N : P ratio is 2 : 1, we have to consider two substances, namely,sphingo-myelin and amido-myelin.Sphingo-myelin was the name well selected by Thudichum forthis material, on account of its sphinx-like character. It is theconstituent of so-called protagon which contains the phosphorus,and it is the one which is slowly deposited from a solution ofprotagon in pyridine.It resembles lecithin in yielding choline oncleavage, but differs, among other points, from the phosphatidesalready mentioned in yielding no glycerol on decomposition. Thenature of the alcohol which takes the place of glycerol is uncertain.It may also be prepared from the cortex of the suprarenal body,and exhibits a physical phenomenon which has hitherto not beendescribed in connexion with any other substance; this was dis-covered by Rosenheim and Miss Tebb81 during their work on theoptical activity of protagon." Protagon," dissolved in pyridine, possesses a t 30° a slightdextrorotatory power, which changes to optical inactivity a t higheror lower temperatures, showing a maximum laevorotation of - 2 4 2 Oand a final constant laevorotation of [a]: - 1 3 . 3 O .Wilson andCramer had also noticed the constancy of this figure, althoughthey omitted to note the change of sign, and they took thisconstant as one of their proofs for the chemical entity of protagon.The explanation of the change is as follows: When a solution of'' protagon '' in pyridine is kept a t 20°, sphingo-myelin is precipi-tated, and it is the appearance of this precipitate of fluid sphzero-crystals which gives rise to the high lzvorotation; as the pre-cipitate settles, the laevorotation decreases, and the final lzevorota-tion is due to a minute quantity of the precipitate which doesnot settle. But if the precipitate is removed by filtration orcentrifugation, the fluid (which then contains the cerebrosidesonly) is optically inactive. I f the precipitate is once more shakenup with the fluid, high laevorotation is again obtained, whichlessens as the precipitate once more settles. This is the first timeoptical action of this nature has been observed in substances notactually in solution, and the term sphaerorotation is proposed forthe phenomenon. Although these observers express the highlaevorotation in the usual way, the optical activity of the pre-cipitated material does not follow Biot's laws.A mido-my elin.-This is another monoamino-diphosphatide de-scribed by Thudichum which has not been examined since histime. It possesses the protein-like character of being coagulableby heat.81 J. Phpiol., 1908, 37, 348; A . , ii, 879PHYSIOLOGICAL CHEMISTRY. 241The third group of the phosphatides contains those in whichthe N : P ratio is 1 : 2. Thissubstance received its name from Erlandsen,82 who first found itamong the phosphatides of heart muscle, but it has since beenfound in liver and other organs, and also (or a correspondingmonoamino-diphosphatide) in egg-yolk.83 On decomposition ityields glycerol, fatty acids, and a base, but the nature of thesela,&-named constituents has not yet been made out.The remaining groups of the phosphatides we know still lessabout, and beyond their enumeration already given in the classi-fication on p. 230, there is practically nothing to say about them.There is, however, one more substance which we must mentionin order to make our survey complete, and this is jecorin. Witha brief description of this substance, our account of the lipoidsmay be brought to a conclusion.Jecorim-This material was originally so named by its discoverer,Drechsel, who found it first in the liver, and later in other organs.Much doubt has ,been expressed concerning its chemical indi-viduality; the yield of sugar from it was found to be inconstant,and it has therefore been very generally regarded as one ofnumerous adsorption or similar compounds of lecithin and sugar,the proportion between which varies with the amount of sugarin the organ it is obtained from, The recent work of Baskoff B4has, however, shown that by a careful method of preparation it ispossible to obtain a product of constant composition yielding e l 4 percent. of sugar and a considerable quantity of incorporated ash.He has further shown that the phosphatide in combination withthe sugar is not lecithin, but a member of the diamino-monophos-phatide group.It was my intention, on starting this Report, to conclude it withan account of recent work on the pituitary body. I find nowthat I have already overstepped the limits of the space allotted tome, and so I propose to postpone the consideration of this interest-ing gland to my next Report. The work in relation to this subjectis still unfinished, so there may be a more complete story to tellthis time next year.The best known of these is cuorin.W. D. HALLIBURTON.82 d., 1907, i, 371.83 H. MacLean, Zeitsch. physiol. Chem., 1908, 57, 304 ; A . , ii, 963.84 Zeitsch. yhysiol. Chwz., 1908, 57, 395 ; A., i, 1029.REP.-VOL. V.


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