PHYSIOLOGICAL CHEMISTRY.ALTHOUGH the source which usually supplies the bulk of papersdealing with biochemical subjects was cut off when the year hadrun but little more than half its course, I have not found muchrelief from the embarrassment with which a plethora of materialovertakes the writer of an annual Report. I do not think that it canbe fairly said of the contemporary output of work in biochemistry,as is sometimes said by the cynic of the general average scientificoutput, that a considerable proportion of it can be with advantageneglected. I find, on the whole, very few papers which i t is apleasure to ignore. It is, however, impossible not to feel, just nowa t any rate, how greatly current American work stands out insignificance and importance. The subject has taken firm root onthe other side of the Atlantic.It has been appreciated there, andgiven good equipment. Although a few of the more distinguishedworkers have been imported, active and successful local schools aremaking themselves greatly felt. Meanwhile, our own output isincreasing satisfactorily, and if one surveys the literature of theyear with a mind as little as possible biassed, one really feels thatmost of those papers which, having read, one wishes to discuss, arejust now written in the English language. This applies, it is true,more particularly t o the subject of metabolism, but it is studiesdealing with metabolism, those concerned with what happens inthe animal itself, that mainly justify the specialisation of physio-logical chemistry.I n the case of t h e e Reports, the pure chemistryof physiological substances is naturally often dealt with under thehead of Organic Chemistry, whilst the technical methods of thebiochemical laboratory receive a t least some attention as part ofAnalytical chemistry. The chemistry of metabolic processes maytherefore legitimately occupy a large part of the more specialisedsummary. I shall begin this year, however, with a subject which,whilst abstracted under Organic Chemistry, is of special interest tothe physical chemist, and perhaps better to be criticised by him. Itis, nevertheless, the very legitimate concern of the physiologist.18PHYSIOLOGICAL CHEMl STRY. 189Catalysis b y Enzymes.Enzyme studies, perhaps as the result of divided responsibility,have been somewhat neglected in these Reports.It would bedifficult to give here even the briefest account of the literaturethat has accumulated since the last reference to the subject; butprogress in actual fundamentals is reported in comparatively fewpapers, and it may be claimed that a large proportion of these isof English or American origin. The significance of this year’swork cannot be brought out without a brief discussion of somepapers which appeared last year. A communication by W. M.Baylissl dealing with the influence of emulsin on the equilibriumin aqueous solutions of glycerol and dextrose seems especially note-worthy, since the experimental results described remove all f ounda-tion for certain statements in the literature which have greatlycomplicated the whole subject of catalysis by ferments.Particu-larly important is Bayliss’ destructive criticism of the statementthat certain reversible reactions require distinct enzymes for cata-lysis in opposite directions; in other words, that in certain casesenzymes that synthesis0 have to be distinguished from those whichcontrol decomposition. This is a conception which, if it had asound experimental basis, would clearly remove enzymes from thecategory of ordinary catalysts. Bayliss shows, further, that, t ojudge, a t any rate, from the case studied by him, an enzyme, whencontrolling the synthesis of optically active substances, leads to theproduction of that isomeride which it also decomposes, and not, ashas been stated, of its optical antipode.He finds, in fact, thatreactions in the system studied follow in all respects the lawsdeduced from mass-action for equilibrium in a reversible systemcatalysed by a single enzyme. Closely related to the work ofBayliss is that of E. Bourquelot and his colleagues, who have pub-lished during the year many papers dealing with the synthesis ofa- and @-glucosides under the influence of appropriate enzymes.The ease with which these biological syntheses are obt-ained isremarkable. Glucosides were prepared from methy1,Z ethyl,propyl,3 and anisyl4 alcohols; also from glycol, glycero1,b and ?n-and pxylene glycols.6 Methyl 7 and ethyl galactosides 3 wereJ. Physiol, 1913, 46, 236; A . , 1913, i, 919.J.Pharm. Chim., 1914, [vii], 9, 19; A . , i, 144,Compt. rend., 1914, 158, 70; A . , i, 144.Ibid., 1377 ; A , , i, 706.lbid., 1913, 157, 1024 ; A , , i, 72.Ibid., 1914, 159, 213 ; A., i, 1080.Ibid., 158, 204 ; A., i, 253.* J. Pharm. Chim., 1914, [vii], 9, 327 ; A , , i, 498190 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.obtained under the influence of an enzyme from bottom yeast.Bourquelot and M. Bride19 have studied the equilibrium in asystem containing alcohol and dextrose, together with a- andP-glucosidases. The a-ferment induces equilibrium when the ratioof combined and free dextrose is 32.6 to 67.4, whilst with the&ferment the ratio is 23.39 t o 76.61. The authors find that eachenzyme is quite without action on the glucoside synthesised bythe other, and if both are present the equilibrium proper to eachis arrived a t independently. The French authors l o fully confirm,therefore, the observations of Bayliss.H. E.and E. F. Armstrong11 have published, in a most interest-ing paper, their views concerning the nature of enzymes andenzyme action as based mainly on the results of the long seriesof experimental researches which they and their co-workers havecontributed to the subject'. They conclude, as others, and par-ticularly Bayliss, have done, that enzymic action takes placeentirely a t the surface of colloid particles suspended in the solutionof the substrata, and not between substances which are all in truesolution. They hold that a hydroclastic enzyme has itself a doublefunction, namely, that of attracting o r holding the hydrolyte andthat of determining hydrolysis.It is a composite agent in whichthe functions of a catalyst, such as platinum-black, are combinedwith those of an acid catalyst. It differs, however, from anordinary catalyst in displaying a specific and limited activity.Using a nomenclature somewhat akin to' that of Ehrlich, theyspeak of the enzyme as containing an acceptor, together with anagent. Views as to the possible nature of the acceptor and actorgroups in special cases are developed by the authors on character-istically objective lines. With regard to the dynamics of enzyme-controlled reactions, the authors hold that their own experimentsconfirm the statement, first made by Duclaux in 1898, and sub-sequently in 1902 by Adrian Brown, and also by Horace Brownand Glendinning, that in each successive interval of time theenzyme determines the hydrolysis of the same amount of substrate.The velocity is thus a linear function of the time, and this relationis held by the Armstrongs to be fundamental, any departure from itbeing due to the influence of the products of change.The exist-ence of this relation, coupled with the fact that after a certainmaximum has been reached increase in the concentration of thehydrolyte diminishes the activity of the enzyme, is held by theauthors to show that the laws of mawaction do not apply tolo See for R general discussion of the results obtained, J. Phnrm. Chim., 1914,l1 Proc.Roy. Soc., 1913, [B], 86, 561 ;A, i, 1116.Comnpt. rend., 1914, 158, 370; A., i, 341.[vii], 9, 603 ; A., i, 1147PHYSIOLOGICAL CHEMISTRY. 191enzyme reactions. “ I n no particular,” they write, “is the change‘ a mass-action effect,’ nor are the departures such that it can besupposed that the change is primarily ‘ unimolecular,’ and sub-sequently varied owing to the occurrence of secondarychanges. . . .”In this view they differ, of course, from most writers who, whilstrecognising that the above-mentioned linear time-function is to befrequently observed, consider it as representing only a part of thecomplete mass-action velocity-curve. It corresponds with certaindefinite and limited relations between enzyme, substrate, and pro-ducts in solution.It is true that velocity-curves obtained experi-mentally have hitherto been fitted by equations derived from con-siderations of mass-action only by introducing purely empiricalconstants, but in what is perhaps the most important contributionto our knowledge of enzymecatalysis published during the presentyear, D. D. Van Slyke and G. E. Cullen12 have supplied a mass-action equation covering very exact,ly the experimental data, andcontaining no arbitrary constants whatever. The theoretical con-siderations presented in this paper are based upon an experimentalstudy of t4he enzyme urease obtained from the soja bean. Theinterest that attaches to this enzyme is clear. It hydrolyses a verysimple, neutral, and symmetrical molecule t o products of markedchemical activity.It is absolutely specific in relation t o its sub-strate, and controls a reaction of great physiological importance.Its significance has long be’en recognised by H. E. Armstrong, andas soon as its presence in the soya bean (itself a circumstance ofinterat) had been demonstrated, in 1909, by Takeuchi, he pro-ceeded to its study and, in conjunction with various co-workers,had established many interesting facts concerning it before theappearance of Van Slyke and Cullen’s publications.For the moment, however, I am concerned with the mass-actionformula presented by the latter aut’hors. The form of their equa-tion is based upon the assumption that in a reaction catalysed byan enzyme there are two phases with diff emrent velocity-constants,namely, one concerned with the rate of combination of enzyme andsubstrate, and the other with the rate of the subsequent decomposi-tion. This idea, and the view that it explains why the usual mass-action formula does not hold in zymolysis, were clearly expressedtwelve years ago, by Adrian Brown.Van Slyke and Cullen, how-ever, justly claim that the whole course of the dual reaction hasnot hitherto been formulated on these lines in such a way that theassumptions made could be fully tested experimentally.l2 J. Biol. Chem., 1914, 19, 181 ; A . , i, 1181192 ANNUAL REPOR 8 ON THE PROGRESS OF CHEMISTRP.Van Slyke's and Cullen'a equation takes the formt + "> d/ h - \ - Time for decomposi- Portion of time consumed Portion of time con-tion of 2 amount of in uniting enzyme and sumed by enzymesubstrate. substrate.in decomposingsubstrate.a represents the amount of substrate (carbamide) present per unitvolume a t the beginning of the reaction, x the amount decomposeda t time t, E the enzyme concentration, c the velocity of combina-tion of enzyme and substrate, and d the velocity a t which thecombination splits. It is Seen that the ordinary mass-actionis'only amplified by the addition of a term l a formula, t = -1ou- K ra -x'representing the velocity of the second phase of the dual reaction,but with the rmult that the equation fits the experimental datawith most remarkable accuracy. What is especially important isthe circumstance that the two constants involved, c and d, are notarbitrary, but can each be determined experimentally by sufficientlyvarying the relative velocity of the two distinct phases of the dualreaction. When the concentration of the hydrolyte is sufficientlyhigh (between 0.08 and 10 per cent.of carbamide), even widevariations in the concentration have no effect whatever on thevelocity of the reaction, a fact which experiment establishes abso-lutely. This means that, once such a concentration of substrateis reached, the time spent in uniting enzyme and substrate is1 ac a - xnegligible. The term -log-, then disappears from the equa-tion, and the value of d can therefore be directly determined. Onthe other hand, within a range of low concentrations of the hydro-lyte, and also under certain other conditions, the ratio c/d becomesrelatively small, the time consumed by the first phase of thereaction becomes measurable, and now varies with the concentra-tion of the hydrolyte.Under these conditions, the value of dbeing known, c can be determined. The experimental data agreeso exactly with the calculated values that the assumptions under-lying the formula seem to be justified. We have, therefore, anexact expression for the course of an enzyme reaction derived fromconsiderations of mass-action, and also important evidence in favourof its dual nature. Although these results are based upon thestudy of urease alone, the authors claim, from a study of theavailable data, that they may be applied to many other ferment-reactions, and probably have a general bearingPHYSIOLOGICAL CHEMISTRY.193It is interesting t o compare Van Slyke’s discussion of his resultswith the expression by the Armstrongs13 of the view that the lawsof mass-action do not apply to enzymes, although the difference isperhaps one ?f point of view only. When a catalyst forms atemporary compound with the substrate, when that compound isformed with a velocity which is great compared with that of thesubsequent decomposition, and when the number of molecules coin-bined with the catalyst (and these alone suffer change) is smallcompared with the whole number in solution, i t is clear that theenzyme-substrate system must remain practically constant inamount, and the rate of decomposition will, so long as the aboveconditions obtain, be a linear function of the time.We oughtnot, from any point of view, to expect the ordinary time relationsof either a unimolecular or a bimolecular reaction. Only if thelinear relations held under all conditions of concentration shouldwe be entitled t o wonder what had become of the influence of mass-action. Van Slyke and Cullen’s results indicate with exceptionalclearness that this is by no means the case, and would seem tojustify those who prefer to believe that the normal effects of mass-action, if exerted under somewhat special conditions, are no morein abeyance in enzymereactions than elsewhere in chemistry. Itshould bO understood, however, that Van Slyke’s clear-cut resultsand simple formula are obtained only when the influence of thedecomposition products is eliminated, a condition that may beeasily secured in the case of urease (see below).The work of VanSlyke and Cullen, being directed to other ends, doea not takeaccount of the reaction as a balanced reaction, and fails t o dealwith the question of synthesis as i t may occur under the controlof the catalyst. The Armstrongs have always given particularattention t o the influence of the products of change in their fer-ment studies. H. E. Armstrong, M. S. Benjamin, and E. Rorton14have fully investigated their effects in the case of urease, and havebrought out, among many other facts, the exceedingly interestingone that the two products of the decomposition of carbamide,namely, ammonia and carbon dioxide, show opposite effects on thecourse of the reaction, ammonia greatly retarding it, whilst carbondioxide accelerates it to an equally remarkable extent.Sinceammonium carbonate also retards, although to a less degree thanammonia, the action of urease on carbamide is self-inhibitory.Van Slyke and Cullen wholly eliminate this effect by the use ofprimary and secondary phosphate mixtures, so adjusted that thehydrion-concentration is maintained near the optimum throughoutthe course of the reaction. I n a second paper, Van Slyke, now inLOG. cit. 14 Proc. Roy. Soc., 1913, [ B ] , 86, 328 ; A . , 1913, i, 781.REP.-VOL. XI. 194 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.conjunction with G. Zacharias, gives a striking account of theinfluence of hydrion-concentration on ths activity of urease.Changes in this have absolutely different and independent effectson the two successive phases of the dual reaction.The combiningvelocity, represented by c in Van Slyke and Cullen’s equation,varies in inverse ratio to: the hydrion-concentration, whilst thevelocity of decomposition (d in the equation) is greatest a t theneutral point, and is retarded by eit,her alkalinity or acidity.Apparently ammonium carbonate inhibits because of its alkalinity,and not specifically, but one feels that the possibility of a synthesisof carbamide from this product of ita decomposition under theinfluence of the enzyme is not negatived by this fact.An important point in Van Slyke and Cullen’s work which Ihave not yet touched upon is the proof, also given by E.K.Marshal1,lb and less fully by Armstrong, that the velocity of theurease reaction is directly and in a linear sense proportional tothe enzyme-concentration, and this throughout a wide range. I nmany cases of enzyme-action the proportionality is said to be notlinear, but of a kind that suggests the formation of adsorptioncompounds between ferment and substrate as a preliminary tot theestablishment of chemical relations. I n the case of urease thereis no suggestion of ratios of this sort. The sharply defined maximalamount of carbamide which a given amount of enzyme can hydro-lyse per unit time, and the fact that the velocity of hydrolysis is soexactly proportional to the concentration of the enzyme, indicate,in Van Slyke’s opinion, the existence of definite combining propor-tions between catalyst and substrate, and point to more purelychemical relations.We have often, from time to time, been pre-sented with velocity-curves and constants for enzyme-reactions byworkers who have taken but little account of the complexity of con-ditions. Factors of an accidental, or even of an artificial, characterhave been allowed to intrude into the results. Such results haveonly been the more misleading because they have received a false andpretentious stamp of accuracy by being prwented in mathematicalform. The study of enzyme-dynamics, however, seems now t o havefound itself. Results of real accuracy and significance are beingobtained which are important t o chemists and biologists alike.Dynamical studies alone, however, will probably fail to tell us allwe want t o know about ferments.As the Armstrongs very trulyremark, in the paper already quoted, it will be difficult to arrivea t any final definition of enzymes until their specific nature hasbeen deciphered. We are not a t all justified in believing thatendeavours to isolate ferments in a pure state, or a t any rate inl5 J. Biol. Chem., 1914, 17,.351 ; A., i, 606PHYSIOLOGICAL CHEMISTRY. 195such a form as to offer evidence of constitution, must necessarilyfail, but a t present we have to be content with speculations.L. Michaelis,l6 i t is true, has advanced the view that ferments aresubstances that undergo electrolytic dissociation in solution, andthat the active agent may in one case be the undissociated mole-cule, and in others the cations or anions respectively. Thus,invertase is an acid of which the undissociated molecules are aloneactive.Erepsin and lipase are acids which are active only in theform of their anions, whilst pepsin acts proteolytically in the formof cations. These conceptions are founded chiefly upon observa-tions dealing with the effect of varying hydrion-concentrations onthe activity of the enzymes, and on the direction of their migra-tion when submitted to a potential gradient, The facts areinteresting; but i t is doubtful if the experimental evidence canbear the weight of the conclusions, and, in any case, it does notthrow much light upon the actual cheniical nature of enzymes.The Armstrongs postulate that the relation of the “acceptorsection” of the enzyme to the hydrolyte is not that of key andlock in Emil Fischer’s sense, “but that of a superposable, andtherefore identical, radicle.” The “ agent ” section which promoteshydrolysis is possibly a carboxyl group acting under exceptionallyfavourable conditions.These views are suggestive, but they re-main, of course, purely speculative. Investigations such as thoseof J. M. Nelson and S. Born 17 into the “ constitution ” of invertasewill scarcely be accepted as having arrived a t a demonstration ofthe nature of a ferment, and I imagine that few a t present willfind much sympathy with the view of Barendreclit18 that “ a nenzyme particle contains the same molecule [as that] which isliberated or acted upon by this enzyme, in some active state,” theactivity spreading through the solution of substrate as a radiation.If ever we arrive at a knowledge of the chemical constitution ofenzymes, we shall learn a great deal more than we now know aboutthe constitution of the living cell.The view that zymolysis depends upon the preliminary f ormatioiiof a compound between substrate and catalyst underlies much ofthe work of L.Michaelis. To this belief, indeed, all authors arecoming, and the conception of ‘‘ contact ” catalysis is disappearingfrom the field of enzyme chemistry. Michaelis and his co-workershave been studying the action of substances which inhibit zymo-lysis. It is assumed that inhibitors can act in one of two ways:16 Biochem.Zeitsch., 1913, 49, 333 ; A., 1913, i, 540 ; ibid., 7914, 60, 43 ; A . , i,l7 J. Amer. Chem. Soc., 1914, 36, 393 ; A . , i, 339.18 Biochem. J., 1913, 7, 549 ; A., i, 214.443 ; ,ibid., 1914, 65, 1 ; A., i, 1007.0 196: ANNUAL REPORTS ON THE YROGRESS OF CHEMISTRY.they can either ( a ) combine with the ferment, or ( b ) diminish therate of decomposition of the substrate-ferment compound.L. Michaelis and P. Rona’o have developed in theory and prac-tice a method for determining which of these alternatives may holdin a given case, and find, to take one instance, that, in the case ofyeast-maltase, sodium chloride, sodium nitrate, and glycerol actby diminishing the reaction-velocity, whilst lithium chloride anddextrose act by combining with the ferment.It may be noted, inparenthesis, that Van Slyke and Zacharias, in the paper alreadyquoted, give theoretical consideration to a similar distinctionbetween inhibitors. They suggest modifications in their funda-mental equation which take account of each type respectively.Returning once more to the work of Michaelis, a paper withH. Pechstein on the pytalin of sa1iva2O must be mentioned inillustration of another side of the subject. It is well known thatthe diastatic effect of saliva and pancreatic juice is not exerted inthe absence of electrolyt’es. The effect of salts on the activity ofthe former was first studied on modern lines by S. W. Cole,21 whoattributed the accelerative effects to the anions.It is greatest, hethought, in the case of the salts of strong acids and least in those ofweak acids. Michaelis and Pechstein now come t o the conclusionthat the diastase forms definite complexes with the anions, and i t isthese complexes, and these alone, that exert the disastatic action.The affinity of the ferment for the anion varies, being very great,for example, for NO3’, not much less for C1’ and Br’, and verysmall for SO,’, and acetate and phosphate ions. The relativeactivity of the different compounds varies greatly, the chloridebeing the most powerful, and the nitrate much less so, whilst verymuch less active are the sulphate, acetate, and phosphate com-pounds. The optimum hydrion-concentration is different andcharacteristic for each salt complex.It would appear that otherinterpretations of the experimental facts dealt with by Michaelisare possible.Many interesting papers dealing with the special properties ofindividual ferments mwt perforce be left without reference. Iwill close this section by calling attention to a case of catalysis byorganic agents which has an interest of its own, although it takesus away from the region of exact studies such as those with whichI have been dealing. Several years ago the researches of thePavlov school brought to light the fact that the proteolytic fermentof the pancreatic juice is secreted in the form of an inactive pre-cursor, trypsinogen, and that this is converted into active trypsinBiochesn.Zeitsch., 1914, 60, 62, 795 ; A., i, 444, 445.J. P?~ysiol., 1903, 30, 202 ; A., 1904, i, 131.2o Bid., 1914, 59, 77; A . , i, 340PHYSIOLOGICAL CHEMISTRY, 197by a catalyst contained in the intestinal juice, and known asenterokinase. The velocity of this activation has been studiedrecently by H. M. Vernon,22 and also by J. Medlanby and V. J.W00lley.23 I n each of these researches the fact came to light thatthe velocity of the process undergoes a positive acceleration, and,towards the end of its course, is enormously increased. Thus,in one experiment, Mellanby and Woolley found that whilst 125units of active trypsin were produced in the first ninety minutes,after an additional twenty minutes the solution contained 750units.Vernon,24 in his latest paper, states that the rate ofacceleration during the last half of the process may be a thousandtimes more rapid than the initial rate. The phenomenon suggestsautocatalysis, and Vernon at one time believed that trypsin, onceliberated by enterokinase, is itself capable of activating trypsinogen,so that a species of autocatalysis becomes established. Mellanbyand Woolley, however, bring evidence against this view, and, more-over, the velocity-curves obtained do not show the S-shaped formof ordinary autocatalysis-curves, but indicate rather a, continuedacceleration of the reaction right up to the point of its completion.Vernon’s latest research leads him to the conclusion that “thetrypsin liberated in the earlier stages of the reaction by the directaction of enterokinase gradually sets free an enzyme (calleddeuterase, to indicate that it acts secondarily t o enterokinase)from a precursor, and that the deuterase is mainly responsible forthe later stages of the activation process.’’ The temperature-coefficient of the deuterase-effect is lower than that of the entero-kinase-eff ect, and the later stages of trypsin-activation have a lowercoefficient than the earlier.Deuterase, once formed, exhibits linearo r logarithmic relations, like other ferments, but the rate of itsown liberation as i t occurs in mixed pancreatic and intestinalsecretions increases in geometrical progression. If no simpler ex-planation for the facts is forthcoming, this is certainly a remark-able case of physiological catalysis.The Specificity of Tissue-enzymes.We have long, of course, had t o recognise that chemical differ-entiation underlies morphological differentiation ; that the blood-and tissue-prpteins of one animal are, for instance, chemicallydifferent from those of another; and that within the same animalthe proteins of any one tissue are no less distinct from those ofany other.Observations made during the last year or two indicate22 J. Physiol., 1913, 47, 325 ; A., i, 214.23 B i d . , 1912, 45, 370 ; A., 1913, i, 113.z4 Biochem. J., 1914, 8, 494 ; A., i, 1205198 ANNUAL REPOHTS ON THE PROGRESS OF CHEMISTRY.that an equal specificity appertains t o the proteolytic enzymes ofindividual living tissues.E.Abderhalden began, so far back as 1906,25 to test this questionby submitting various synthetic polypeptides to the action oforganic extracts, and in succeeding years he published many papersshowing that there were marked differences to be observed in thepeptolytic powers of the various tissues-diff erences related to thestructure and configuration of the polypeptides submitted t o theiraction. Recently he has demonstrated the still more significantfa.& that the enzymes of a given tissue, whilst they can alwayshydrolyse peptones made from the proteins of the same tissue, mayfail altogether to act on peptones made from the proteins of othertissues. It was foreshadowed insome of the earliest work on autolysis; but Abderhalden has shownthat, by testing the action of the enzgnies on peptones made fromthe native proteins rat,lier than on the latter themselves, the factsbecome technically more easy to demonstrate.Observations onthese lines begun by him last year26 have now been extended, andin conjunction with G. Ewald, Ishiguro, and R. Watanabe’7 he haspublished experiments showing, for example, that extracts fromthe liver decompose peptones prepared from that organ, but nottKose prepared by similar processes from lungs, brain, kidney, orpancreas. A lung extract hydrolyses lung peptones, but not pre-parations from muscle, liver, o r kidney. All observations seem t oshow that for some reason the renal enzymes have a more generalaction. One must admit that the technique used to demonstratethese facts is evidently exceedingly difficult, and a repetition ofthe observations will not be lightly undertaken by ot.hers.I f ,however, relying upon the reputation and experience of Abder-halden, we accept them, they are certainly important and sug-gestive. The appearance of a highly specific enzyme in the cell,where alone the related and highly specific substrate is to be found,and its appearance in that cell only, are facts which bear, i t seemsto me, with some significance upon the nature and origin of theenzyme itself. It is important in connexion with what follows torecoguise that, according to Abderhalden, the blood does not9normally contain ferments which can act on tissue proteins.L. Pincussohn,Z* i t is true, in a paper published a year ago, claimedto have found in normal dogs’ blood a ferment capable of actingon peptone prepared from dogs’ muscle, although i t had no actionon similar peptones prepared in the same way from cats’ muscle,25 Zeitsch.physiol. Chem., 1906, 47, 466 ; A., 1906, ii, 464.26 B i d . : 1913, 87, 220 ; A , 1913, i, 1118.27 Ibid., 1914, 91, 96 ; A . , i, 900.28 Biockem. Zeitsch., 1913, 51, 107 ; A., 1913, i, 788.The point is not altogether newPHYSIOLOGICAL CHEMISTRY. 199or on other peptones from foreign substances. Abderhalden andG. Ewald,29 however, could not, in repeated experiments, obtainfrom normal blood any ferment capable of splitting tissue-peptones,and they claim to have had the remarkable experience of findingthat whenever the blood contained a ferment active with theproteins of some particular organ, such activity being observedseventeen times in thO course of 1000 examinations, an unsuspectedlesion of that organ always proved to be present.The discoveryof such fermentts in the blood becomes, therefore, of diagnosticimportance. This leads to the discussion of related phenomena.Defensive Ferments.We are still concerned with the work of the indefatigable in-vestigator Abderhalden. His claim t o the discovery that the entryof a foreign protein, of a disaccharide, or of a foreign fat, into thecirculation of an animal promptly leads t o the appearance in theblood of a specific ferment, not there before, capable of hydro-lysing the foreign substance, has, chiefly perhaps because of a par-ticular case with practical bearings, to which I shall referimmediately, led to no small excitement in clinical laboratorieseverywhere.Abderhalden quickly put the results of his earlierexperiments, together with his views about their significance, intoa book. The first edition of this appeared in 1912, so that thesubject is not exactly a new one, but it has hitherto received nonotice in these Reports. Ih importance, real or supposed, calls forsome reference. The current year has seen the appearance of afourth edition of the book,30 as well as many fresh papers dealingwith the subject.When proteins are injected into the vein of an animal, reachingthe circulation parentally and escaping the digestive mechanism,the blood itself rapidly develops a proteolytic power which it didnot possess before.The specificity of the enzyme or enzymes thuscalled forth, however, is not marked; the circulation of any foreignprotein causes the development of a more or less general proteo-lytic activity in the blood. Very different is the case (according t oAbderhalden) when the protein enters in special circumstances.The pregnant woman carries within her in the form, not ofthe fetus, but of the placenta, a source of proteins which areforeign to her own body. As soon as the placenta has formed, itsproteins enter the blood-stream, and the formation of a highlyspecific “defensive” enzyme is the response. The blood of thepregnant woman, and her blood alone, can digest placenta,-29 Zeitseh.p h p i o l . Chem., 1914, 91, 86 ; A . , i, 896.30 L‘ Abwehrfermente der tierischen Organismus,” 4th Auflage, 1914200 ANNUAL REPOR1‘S ON THE PROGRESS OF CHEMISTRY.proteins. No other form of tumour and no pathological conditioiisof any kind lead t o the production of this specific ferment. Thisis the particular instance of a defensive ferment t o which I referredas having caused a flutter in the clinical world. After the pheno-menon had been described, Abderhalden’s laboratory becamecrowded with ud hoe medical inquirers, all anxious to learn an easymethod of getting early information about the coming generation.Why the specificity of the ferment produced should be so muchgreater than when foreign proteins are injected is not quite clear.Artificial protein preparations seldom, of course, represent pureindividual substances, and the method of injection is more brutaland sudden, and likely, therefore, t o produce some more generalresponse than when, as in the case of the placenta-proteins,material gradually enters the blood.I have not space t o deal fullywith the technique of this work. Probably, as this reference to itis somewhat belated, i t is already more or less familiar to most.Delicate methods of detecting the digestion of the prot,eins arerequired. Either an optical method is used, or the protein (forexample, placenta-peptone) is placed, together with a sample of theblood serum, in a small dialyser.I f digestion occurs, and only then, amino-acids are to be detectedin the dialysate.For this detection, the very de1icat.e colourreaction with Ruhemann’s triketohydrindene hydrate 31 is used,this substance having been put on the market under the suggestivename of I ‘ ninhydrin.”Even more interesting, from some points of view a t least, thanthe response to foreign proteins, is the circumstance that thesimpler molecules of a disaccharide can call forth a similardefensive enzyme. Our knowledge as t o this fact is really by nomeans new, for, in 1905, E. Weinland32 showed that whilst theblood of young dogs is free from invertase, the enzyme appears ini t as the result of injections of sucrose. The significance of thisobservation was not, perhaps, sufficiently appreciated a t the time.Five years later similar observations were made in Abderhalden’slaboratory with respect to sucrose and lactose.T. Kumagai33 haspublished a long paper on the subject in which, in addition toconfirming the earlier work, he makes the statement that injectionof the monosaccharides Izvulose and galactose can call forthinvertase. This, Abderhalden and E. Bassani34 are unable t o con-firm. With regard to the influence of sucrose, i t seems that, onthe whole, the response can be got with more certainty from31 T., 1910, 97, 2025.32 Zeitsch. Biol., 1905, 47, 279 ; A . , 1905, ii, 730.Biochem. Zeitsch., 1914, 57, 380 ; A . , i, 112.34 Zeitsch. phylsiol. Chem., 1914, 90, 369 ; A., i, 765PHYSIOLOGICAL CHEMISTRY. 201rabbits than from dogs.35 Kumagai, in the paper mentioned,claimed that an independent and altogether remarkable property isacquired by the blood-serum as a result of the injection of sucrose.It is said then to act on de’xtrose and lxvulose, first converting theformer into the latter, which next suffers synthesis into a disac-charide. The observations were made in F.Rohmann’s laboratorya t Breslau. A later paper Fublished by Rohmann and EurnagaiS6con jointly, contains the supposed proof that the disaccharide islactose. That the injection of sucrose should call into the blood acatalyst capable of converting, outside the body, dextrose intolactose is a truly extraordinary event. The evidence as describedis conclusive enough, but one cannot but feel scepticism, and it isto be hoped that the experiments will be repeated.If the facts areas stated, they are of great physiological importance.It seems to be, a t any rate, of quite exceptional interest to knowthat an immunity reaction as represented by the productiond e ILOVO of a more or less specific hydrolytic ferment may be calledout by simple molecules such as tlhose of the disaccharides. I nconnexion with the immunity-phenomena of disease we are too muchaccustomed to think of the colloid nature of toxins as playing someessential part.How precisely the ferments under discussion are to be related tothe factors of immunity as recognised in other connexions is notyet clear. A. Hauptmann37 and others suggest that the defensiveferments, like other anti-substances, are probably constructed onaniboceptor-complement lines, the complement representing, asusual, their unspecific, and the aniboceptor their specific, constituent.Abderhalden protests against the intrusion of Ehrlich’s complexnomenclature into the description of phenomena that are probablysimpler than those t o which it was intended t o apply.We mayrecall here, however, the Armstrongs’ views as to the nature offermentls in general (see above), and, in any case, the hydrolyticaction of the defensive ferments and the phenomenon seen, forinstance, in hzemolysis are probably related. That the action ofthe defensive ferments is in some way related t o anaphylaxis38 isnearly certain.Many papers on the defensive ferments published during the yearin the technical medical journals have been of a critical sort,39especially in connexion with the placenta-reaction, which does not35 Abderhalden and F.Wiltlermuth, Zeitsch. physiol. CJmn., 1914, 90, 385 ; A . , i,763 ; Abderhalden and L. Grigorescu, ibid., 419 ; A . , i, 765.36 Biochem. Zeitsch., 1914, 62, 1 ; A., i, 766.37 Munch. nzed. Wochcnsch., 1914, 1167.38 Ann. Report, 1910, 201.3y Compare L. Flatow, Munch. med. Wochensch., 1914, 1168202 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.seem t o be successful in the hands of all. A. G. Hogan,lro moreover,could find no improved utilisation of sucrose or lactose as the resultof repeated intraperitoneal injections of t'hese sugars. He did notstudy the action of the serum, but holds that his results prove thatno " immunity " was developed, and that the physiological signi-ficance of Abderhalden's results is theref ore doubtful.We may haveto wait some time before the whole subject can be viewed in properper sp edive.Some Aspects of Intermediary icf etabolisrn.Carbohydrates and Fats.-The fate of sugar in the body remainsa prominent subject of research. Its secrets are stronglyentrenched, but an army of investigators is " nibbling '' a t themand not without success, although the moment f o r a decisiveadvance seems not yet t o have arrived. I gave a good deal of spaceto the subject last year, and must be somewhat briefer here.The growing conviction t'hat one aspect at least of the metabolismof sugar involves a preliminary non-oxidative cleavage resulting inthe production of lactic acid is, in a sense, greatly strengthened bythe researches of J.Pasnas and R. Wagner41 on amphibian muscle.Thzt the lactic acid of muscle takes origin from its carbohydrateshas, of course, been long suspected, but I know of no other experi-mental work that makes the relation between them so clear. Theorigin of one from the other is probably not indeed, in the strictestsense, direct, but Parnas and Wagner make it nearly certain thatthe lactic acid arises from a precursor which stands in intimategenetic relation to t'he carbohydrate.Evidence pointing, although I think with considerable lesscogency, in the same direction is advanced by 0. von Fiirth.42. Onlyif the diet of rabbit.s be rich in sugar is there, in these animals,any excretion of lactic acid as the result of phosphorus poisoning.I n the absence of carbohydrates there is none.As the increaseis, in any case, relatively small, the author suggests that his experi-ments indicate that the origin of lactic acid from sugar is throughsome intermediary substance, and not direct. There is indeed goodreason to believe that in the muscle fibre the carbohydrate issynthesised into some complex, and from this the lactic acid moredirectly arises. There is little doubt, however, that sugar suffersalso a more direct degradation in the body.I n the endeavour to discover what compounds are related to sugarin the processes of metabolism, many workers have continued to use40 J.Biol. Chem., 1914, 18, 485 ; A , , i, 1157.41 Biochem. Zeitsch., 1914, 61, 387 ; A . , i, 772,42 Ibid., 1914, 64, 131 ; A., i, 1014PHYSIOLOGICAL CHEMISTRY. 203Lusk’s method, which is based on quantitative studies of phlorid-zin diabetes. It is clear that any information regarding the natureof the substances that can yield sugar in the diabetic animal bearsalso on the fate of sugar itself in the normal animal. It is changesin chemical equilibrium in one direction or t-he other that occur.I n Lusk’s method a dog is given phloridzin under carefullystandardised conditions, and its output of sugar and nitrogen deter-mined. I f the addition to a uniform dietary of the substance understudy results in an increase of urinary sugar, without disturbancein the output of nitrogen, it is assumed that the substance isdirectly converted into sugar.I f it acts only indirectly, increasingsugar by increasing metabolism, then the nitrogen will be increasedalso. I n the former case the amount of ‘( extra ” sugar formed willbe sgme guide as to the chemical processes involved. It mayindicate, for instance, whether all, or only some, of the carbonatoms present in the molecule of the substance being studied areconcerned in the formation of sugar.Lusk’s method seems usually to give trustworthy and definiteinformation, and the more one learns about the truly remarkableaction of phloridzin in determining that everything capable ofyielding sugar in the a’nimal body shall be promptly excreted assugar, the more does one desire better information concerning theprecise mechanism of its action.The extensive use in recent yearsof the drug has helped us but little in this respect. To A.. I.Ringer and E. M. Frankel43 we owe some information as to thetime relations of the glucogenetic phenomena. They find that afterthe administration of dextrose itself the curve of sugar-eliminationreaches in two hours a point which, after the administration ofsuch a precursor as propionic acid, is only reached in four or fivehours. There is a definite time required for the processes ofconversion.With regard t o the nature of substances capable of yieldingsugar I will refer only t o the papers of the present year. Ringerand Frankel 44 find t’hat dihydroxyacetone is glucogenetic, and, inone experiment a t least, it was quantitatively converted intodextrose in the phloridzinised dog.I. Greenwald45 states that thesame is true for citric acid, and P. Schwenken46 claims to haveobtained sugar quantitatively from acrylic acid. The work of thelast few years has shown that, whilst some substances which wemight expect to yield sugar easily wholly fail to do so, positiveresults are obtained with a number of substances of very diverse43 J. Biol. Chein., 1914, 18, 81 ; A., i, 902.44 Ibid., 233 ; A . i, 1025.45 Ibid., 17, xxxiv; A., i , 629.46 Beitr. Physiol., 1914, 1, 140 ; A., i, 1156204 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.constitution, a noteworthy proportion of these being compoundswith three carbon atoms in the molecule.Every result obtainedwith this technique is of value as illustrating chemical possibilitiesin metabolism, but it is sure that some caution may be necessarybefore i t is concluded that every substance proved on these linesto be glucogenetic is really an intermediary in the normal meta-bolism of carbohydrates.Imentioned last year that whilst the claims of pyruvaldehyde(methylglyoxal) t o be a normal intermediary metabolite have muchto support them, those of pyruvic acid are less clear. A. I.Ringer, who has had much experience of the method, states thatpyruvic acid when administered to the phloridzinised animal some-times gives large quantities of dextirose and sometime8 none a t all.Either the condition of the animal becomes an important factor,or else idiosyncracy enters into the phenomenon. The resultssuggest, to my mind, that the acid is a foreign substance, and nota normal obligatory stage in sugar metabolism.P. A. Levene andG. M. Meyer 47 confirm this view, for they find that isolated animaltissues which, as we know, rapidly act on pyruvaldehyde, have,under aseptic conditions, no effect on the acid. I must not forgetindeed that I. Smedley (Mrs. MacLean) and Eva Lubrzynska48have advanced an ingenious hypothesis suggesting that pyruvicacid, formed in the body as a decomposition product of carbo-hydrate, is the starting point for the physiological synthesis offatty acids. Better evidence for the physiological nature ofpyruvic acid seems called for, however.Ringer and Frankel,49 inan endeavour to analyse the precise fate of this acid when adminis-tered, have obsarved an interesting set of facts with somewhatspecial bearings. When acetaldehyde or propaldehyde is injectedinto a phloridzinised animal there is a large absolute increase inthe dextrose eliminated, greater indeed than would occur if thewhole of the aldehyde were converted into sugar. This is associatednot with an increase, but with a decrease in the loss of nitrogento the body, and there is also a marked decrease in the excretionof acetone, P-hydroxybutyric acid, and acetoacetic acid. Clearlysomething mom complicated than direct conversion occurs. Theeflect of the aldehyde is remarkable, since neither ethyl alcohol noracetic acid has any appreciable influence on the metabolism of thediabetic dog, whilst propyl alcohol and propionic acid, althoughknown to suffer direct conversion into sugar, do not produce theSometimes the phloridzin method gives uncertain results.47 J.Biol. Chem., 1914, 18, 469 ; A . , i, 1157.49 J. Biol. Chem., 1914, 16, 563 ; A., i, 357.BiocAem. J., 1913, 7, 364 ; A., 1913, i, 1014PHYSIOLOGICAL CHEMISTRY. 205secondary effects mentioned. It would seem as though the aldehydegroup must exercise a special influence. The facts indeed haveled Ringer to consider the possibility that dextrose itself may oweits power of inhibiting the excretion of the acetone substances (its( ( antiketogenetic " action) to its aldehyde group. In specialexpesiments he found that gluconic acid, although differing fromdextrose only in the replacement of the aldehyde group bycarboxyl, has no antiketogenetic power.He advances finally atheory of diabetes and of acidosis on lines somewhat similar tothose foreshadowed in my Report last year. When P-hydroxy-butyric acid arises from the oxidation of fatty acids and amino-acids in metabolism, i t forms, under normal conditions, a glucosidewith dextrose, and only in this combination is it further meta-bolised. In diabetes (experimental or clinical) the power to formthis glucoside union is absent, and its failure is a t the bottom ofall the chemical disturbances. Such, very partly and incompletelystated, is Ringer's thesis, and in the paper quoted he supports itwith ingenious arguments.W. M.Marriott,50 however, calls attention to the fact that thediabetic, whilst unable to deal with Z-P-hydroxybutyric acid, fullyoxidises the corresponding d-acid. To explain this on Ringer'slines, he ventures upon the hypothesis that the d-acid combineswith the P-form of dextrose, whilst the Z-acid requires the a-form,in which the diabetic is presumably deficient. By this time,however, the theory has got far beyond the facts.We are still far from being able to describe fully the events ofcarbohydrate metjabohm, but it is sure that ultimate success willarise from experiments based on clear thought on chemical lines.The influence of the nervous system and the balance of glandularactivities condition events in the body, but the ultimate eventsthemselves are chemical.They are definite molecular reactionsproceeding smoothly in health whilst interrupted and diverted indisease.It is sure, of course, that apart from the effect of disturbances inthe internal secretions of the body the normal progress of thesereactions, like that of other chemical events in the body, is affectedby less specific factors, its by variation in the hydrion-concentrationin the blood and tissues. P. Rona and G. G. Wilenko have shown,in two interesting papers, how marked is the influence of thisfactor on the utilisation of sugar by the heart:' and on the glyco-lysis62 which occurs in the blood.5o J. Biol. C'hem., 1914, 18, 241.51 Biochem. Zeitsch., 1914, 59, 173 ; A., i, 350.Ibid., 1914, 62, 1 ; A., i, 766206 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.An excised heart kept active by artificial perfusioii is anadmirable object for the study of such factors quantibatively, andthe authors quoted show that extremely small departures from anormal hydrion-concentration, in the direction of acidity, in thefluid perfused prevents the consumption of sugar by the survivingorgan.Similar small departures from a normal reaction inhibitthe familiar disappearance of sugar which occurs when normalblood is kept. It is clear that thme facts are of much importancein connexion with the acidosis which is characteristic of diabetes.I have discussed in considerable detail the effects of changes inthe reaciion of the body fluids on physiological processes in theOliver-Sharpey lectures for the current year (Lancet, 1914).The Creatine Problem.-This elusive but always attractive sub-ject has been well to the front during the year.What is themeaning of the creatine of muscle; what is its origin; and whatits relation to urinary creatine and creatinine? A truly astonish-ing amount of good and quantitative work has been put into theendeavour to find answers t o these questions; but the answersare not yet complete. 0. Folin, to whose methods we owe thepossibility of quantitative study, has himself returned to the groundthis year. Before reviewing what he has written, however, I willdeal with a paper by S. R. Benedict and E. Osterberg,53 because areference to their work and views Pollows logically, as will be seen,on my discussion of the phenomena due to phloridzin injection.Creatine is known to appear in the urine of adults as the resulteither (i) of starvation, inanition, and general conditions involvingloss of body tissue, or (ii) of failure on the part of the body toobtain or make due use of carbohydrate, as in diabetes.Either ofthese may be looked upon as the essential cause of creatinuria, andbe considered as involving the other. Some believe that the originof the urinary creatine is to be sought alone in tissue-wastage,which involves an abnormal liberation of tissue (muscle)-creatine.From this point of view it is easy to indicate that excessive tissue-waste is characteristic of diabetes, and the undoubted experirnentalfact that giving carbohydrate alone, without protein, will preventthe excretion of creatine during inanition, can be explained asmerely a part of the ordinary sparing effect of carbohydrate onprotein metabolism.It spares the muscle protein structure andtherefore prevents the liberation of the associated creatine. Onthe other hand, there are those who believe, following a suggestionoriginally due to Cathcart, that creatine is a substance the func-tions and fate of which are intimately bound up with the normalprocesses of carbohydrate metabolism. When the latter are in5% J. Biol. Chem., 1914, 18, 195 ; A., i, 10%PHYSIOLOGICAL CHEMISTRY. 207abeyance, as in diabetes, the creatine, which is otherwise used,perhaps synthetically, in metabolism, appears in the urine.Fromthis point of view the effect of general starvation in inducingcreatinuria is actually the result of specific carbohydrate starva-tion, and the preventive effect of giving carbohydrate alone hasbeen held to prove it. To these two alternatives Benedict andijsterberg addressed themselves. They realised that the phlorid-zinised animal, which suffers both from an increased nitrogenousbreakdown and from inability to use carbohydrate, is an excellentexperimental objectl on which t o test them. They worked, there-fore, with animals under the influence of this drug and undervarious nutritive conditions. The dogs, when fasting, lost fleshrapidly, and their excretion of creatine was high. Benedict andOsterberg’s experiments, however, a t once established the crucialfact that if the animals were fed with creatine-free protein food,in such amounts that the loss of nitrogen to the body was coveredand even more than covered, the high elimination of creatineremained quite unaffected.The figures obtained served “ todemonstrate beyond any argument that the creatine eliminatedby these phloridzinised animals did not represent creatine pre-formed in the muscle, and that muscle ‘disintegration’ played nopart in furnishing the creatine put out.” Now when the animalsa t the close of the experiment were killed, it was found that thetotal creatine of the muscle, in spite of the prolonged hyper-ex-cretion of the substance, was by no means reduced, but was inexcess of the normal.This the observers held to be due to thecircumstance that, because utilisation of creatine is prevented bythe drug, a large amount circulates, and the tissues become morethan normally saturated with it. The fact is in any case not inharmony with the view which has been held by some that’ the totalcreatine of the muscles is depleted in proportion tot the amounteliminated in the urine. The experiments indicate, indeed, thatcreatine is normally produced, and subsequently used or destroyed,in larger amounts than is usually supposed. In simple fasting theamount excreted daily is but small, but this yields no evidenceas t o the amount actually produced, any more than a minimal gradeof glycosuria, resulting in the excretion of a few grams of sugarin the day, would be a measure of sugar-production in the body.Phloridzin makes the animal “ diabetic ” in respect of creatine,as well as in respect of sugar, and the drug, by interference withtheir normal utilisation or destruction, brings them both to lightin the urine.Benedict and Osterberg conclude that the fate ofthe one substance is related to that of tlie other, and are them-selves in agreement with the view that the power of the organis208 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.to metabolise the creatine which i t forms “is directly related t ocertain processes chief among which appears to be the utilisatioiiof carbohydrate.”0. Folin has published papers during the year dealing with thetechnical side of the subject, and has given us important improve-menix3 in the methods for estimating creatine and creatinine inthe urine, blood, and tissues, as well as supplying experimentaldata relating to the creatine in each of these.To judge, however,from a paper published conjointly with W. Denis,54 his mindhas been occupied with an aspect of the subject which, if funda-mental, is somewhat special. He is concerned for the moment, notso much with the dynamics of creatine, but with the question asto what precisely is the nature of its association with muscle sub-stance. “Creatine must,” write Folin and Denis, “be a wasteproduct or a synthetic product serving some special function, oras a synthetic product it may in fact be a part of the living activeprotoplasm. We believe that the last-named alternative representsthe facts, and in support of this hypothesis we now propose toshow that the so-called creatine of muscle is a post-mortem product,and there is very little creatine in living muscle.”The last statement implies that, when our estimations of creatineare made, the mechanical destruction of the muscle fibres liberatesthe base from a complex in which its identity had been in somesense or other submerged. It is not clear, to me a t least, whencreatine (or any other tangible tissue constituent) is spoken of asbeing ‘‘ part of the living active protoplasm,” whether i t is supposedto exist in some large molecule, from any part of which the creatinestructure is wholly absent, but whence the base emerges with itsown identity established as the result of extensive molecularrearrangements at the moment of protoplasmic death, or whethercre,atine qua creatine is supposed to be linked up in a complexand merely liberated post-mortem by such a process as hydrolysis.Folin and Denis seem clear a t any rate that the living muscle doesnot contain free creatine, nor, as I understand them, any looseadsorption compound of the base.Their proof of this is, however,by no means a direct one. They argue somewhat as follows. Muscleyields, post-mortem, from forty to fifty times as much creatine per100 grams of substance as ever blood is found t o contain. It cannot,therefore, be in ordinary equilibrium with the blood. There isevidently “some definite effective force or condition by which thecreatine is held fast in the muscle.” When fresh creatine entersthe blood it is freely taken up by the already creatine-laden tissues,a fact for which new experiments described in the paper now64 J.Biol. Chem., 1914, 17, 493; A., i, 767PHYSIOLOGICAL CHEMISTRY. 209being quoted give further evidence. If I understand the authorsrightly, it is essentially this disproportion between the concentra-tion in the blood and muscle which has led them to their very wide-reaching conclusion. Without asserting that the creatine inmuscle is simply adsorbed, I feel wholly unable to admit that thedisproportion as discussed cannot be explained by the formationof an ordinary adsorption compound with the muscle colloids.Inone of the experiments given in the paper an anmthetised dog,the muscles of which had yielded on preliminary analysis 544 mg.of creatine per 100 grams of tissue, received 3 grams of the baseby way of the small intestine. As a result the muscles yielded,after a certain interval, 689 mg. per 100 grams, showing anincrease of 26 per cent. Now the extra 145 mg. could hardlyhave come t o form part of the bioplasm during the course of theexperiment. Few a t least of those who conceive of the bioplasmas an entity, distinct from the general metmaplasmic cell contents,would admit that, as t,he result of a mere increase in the supplyof a given constituent, i t could immediately suffer so considerablea change in its constitution.If it could undergo any such con-stitutional changes as the result of variations in supply, therecognised distinction between exogenous and endogenous meta-bolism would hardly be justified. On the other hand, if i t besupposed that the extra amount of creatine found had not yethad time to be tssimilated, the data of the experiment show thatthe muscle with 145 mg. of free creatine per 100 grams was inequilibrium wit’h blood containing only 80 mg. I f this differencecan exist without the hypothetical entry of the base into the muscleprotoplasm there is no good reason to believe that larger differencescould not exist on similar terms.I do not pretend to know as much about the physiology ofcreatine as does Folin, and I owe to him most of what I do knowabout it, but I have ventured on this discussion because I feelthat certain conceptions which underlie the argument used byFolin and Denis in their paper have a general bearing on our viewsrespecting equilibrium in the living cell, and are, in my opinion,likely to confuse the issues.Many other papers which have dealt with creatine and creatinineduring the year cannot be quoted here.The physiological inde-pendence of the two substances originally demonstrated by Folinseems to be fully illustrated by the work of Benedict and Osterberg,although Schaffer seems inclined to support Morris and Fine inreturning to the older view that the latter arises within the musclefrom the former as a precursor. Folin himself now teaches that“when a tissue dies the postrmortem product, creatine, is set free,REP.-VOL. XI.210 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.whereas in the course of the normal replaceable breakdown, whichwe call tissuemetabolism, the product split off is usually (normally)not creatine but creatinine.” Whether on these lines they havea common precursor of a more definite sort than the bioplasm itselfhs does not say. It would certainly be remarkable if two meta-bolitm so intimately related chemically should have no relation atall in metabolism.The extraordinary steadiness of creatinine-output during verywide fluctuations in exogenous nitrogen which has led us, againunder Folin’s guidance, to regard its amount as the best measureof msential tissue-metabolism, is once more strikingly confirmed ina paper by A.E. Taylor and W. C. Rose.55 When, by alteringthe protein intake, the urinary nitrogen was made t o vary between7.4 grams and 30.2 grams, the creatinine only varied between0.63 and 0.68 gram.Vitamines.Interest continues to be centred in those accessory food substances of unknown nature for which Funk proposed the nowfamiliar name of vit’amines.T. B. Osborne, L. B. Mendel, and Edna L. Ferry56 find that thesubstances in milk which have a specific effect in promoting growthin young animals can be separated with the butter-fat. Theirsolubility in fats is further illustrated by the work of E. V.McCollum and Marguerite Davis,57 who find that if fresh butteris saponified and the aqueous solution of its soaps extracted witholive oil dissolved in ether, the oil left on evaporation of the etherhas acquired the property of promoting growth. This observation,unfcrtunately, does not help us in an endeavour to isolate thevitamines.It -is uncertain as yet whether the substance necessaryfor growth is the same as that which can cure the neuritis whichdevelops in fowls fed on polished rice, and the absence of whichfrom a diet is supposed to be responsible f o r the disease b e r i - h i .Funk 55 found that polished rice and a vitaminecontaining fractionfrom yeast constitate together a complete food, and decided that,as the yeast preparation used in these experiments contained nophosphorus, the importance of the phosphorised lipoids in (‘ defi-ciency diseases” is probably small.With A. B. Macallum 59 hehas published observations which do not altogether agree with55 J. Biol. Chem., 1914, 18, 519; A . , i, 1151.Ibid., 1913, 16, 423; A., i, 107.Ibid., 1914, 17, 245 ; A , , i, 1188.58 J. Physiol., 1914, 48, 228 ; A . , i, 768.59 Zeitsch. physiol. Chew&., 1914, 92, 13 ; A . , i, 1017PHYSIOLOGICAL CHEMISTHY. 211Osborne and Mendel's statements as to the vitamine content ofbutter. It was found, further, that cod-liver oil added to polishedrice prevents neuritis from developing, but does not promote growth,so here a t least we seem to have evidence for the existence of morethan cne type of vitamine. This paper contains a photographshowing an extraordinary difference in the development of chickensfed with and without the necessary vitamine supply.E.A. Cooper60 has continued some investlgations on the amountof anti-neuritic substance in certain practical food-stuff s. Thechief point that arise6 from his researches is that voIuntary muscleis poor in the vitamine, so that meat should be supplementedby other foods in the treatment of beri-beri. A hitherto unsus-pected effect of the absence of these accessory substances fromthe diet is suggested in a preliminary way by experiments madeby Funk and Count E. von Schonborn.61 They find that pigeonsfed on artificial food mixtures develop an abnormality in meta-bolism leading to a marked hyperglymmia and a diminution ofglycogen in the liver. This condition was apparently cured bygiving a vitamine fraction from yeast.No one could be more assured than I am (after a good manyyears of experiment) of the real existence and of the nutritiveimportance of these accessory food substances, but I am equallysure that as yet we know nothing of their chemical nature. J. C.Drummond and C. Funk62 have just published a paper which issignificant in its admissions. I n my opinion we have now no trust-worthy evidence for the activity as vitamines of any of the crystal-line substances which Funk has from time t o time separated anddescribed. The paper last mentioned deals with the completefractionation of a crude vitamine fraction f rorn rice polishings.From the polishings Funk63 some time ago separated aiid describeda substance, C,H,,09N,, melting at 2 3 3 O , together with nicotinicacid. The former supposititious substance, however, is nowadmitted, as the result of further study, to be also nicotinic acid,and this is not a curative material a t all. No crystalline substancewith power to cure neuritis has really been separated from ricepolishings.From yeast, on the other hand, Funk once obtained a " curative "substance to which the formula C2,H,,0,N, was ascribed. The melt-ing point of this was 2 2 5 O , but a substance prepared earlier fromthe same source by similar methods melted a t 233O, and the twowere identical in crystalline structure, solubility, and reactions.8o J. Hygiene, 1914, 14, 12 ; A , , i, 777.61 J. Physiot., 1914, 48, 328; A . , i, 1015.62 Biochem. J., 1914, 8, 598.63 J. Physiol., 1913, 46, 173; A . , 1913, i, 936.P 212 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRYNow of this last substance, when first described, it was remarked :“ Recrystallised from dilute alcohol the substance melts a t 2 3 3 O , atthe same temperature as the curative substance from rice. It givesthe same reactions, and both substances must be therefore con-sidered as identica1.’’G4 The substance from rice, melting a t thattemperature, is, however, as we now learn, nicotinic acid, and it isdifficult to avoid the suspicion that t,he first-mentioned yeast pro-duct, in spite of the elementary analysis (done by Pregl’s methodon 3 to 4 mg. of substance), was nicotinic acid also, I n any case theyeast product showed appreciable curative effects only when mixedwith what was avowedly a nicotinic acid fraction from the samesource, and the latter (although pure nicotinic acid is withoutaction) had itself some curative effect. There is not, I think, a greatdeal to be said for the substance, C24131909N5, as a crystalline vita-mine. As Drummond and Funk were quite unable t o confirm thework of Suzuki, Shimamura, and Odake,G5 who claimed to haveseparated the active substance from rice in the form of it picrate,we have evidently no certain knowledge of any pure substance withthe properties of a vitamine.F. G. HOPKINS.84 J. Phgsiol., 1912, 45, 76.65 Bioehem. Zeitseh., 1912, 43, 89 ; A . , 1912, ii, 980