PHYSIOLOGICAL CHEMISTRY.SINCE writing last year’s Report the deaths of the following haveoccurred: G. von Bunge, Sir Thomas Fraser, Armand Gautier,Wilhelm Pfeffer, Max Siegfried, and Nathan Zuntz. Von Bungewas Professor of Physiological Chemistry a t Basle, and the authorof a well-known text-book. His best known research was con-cerned with the mineral constituents of blood and milk. Fraserwas for many years professor of materia medica at Edinburgh; heintroduced Calabar beans into medicine in the early sixties, buti t is chiefly during the last decade that physostigmine has at-tracted much attention from organic chemists. A study of arrowpoisons led him to the therapeutic application of strophanthin ;he made also an extensive study of snake venoms, and his nameis associated with that of Crum Brown in the generalisation thatquaternary salts of organic bases have a curare-like action.Gautier, the veteran of French biochemistry, originally receiveda medical training, but became later assistant to Wurtz and wasfor many years Professor of Medical Chemistry a t Paris.He isknown for his studies on ptomaines and on the occurrence of thebiologically rare elements (fluorine, arsenic, etc.). He also workedon arsenical drugs and questions of hygiene, food and generalchemistry.Pfeffer’s Osmotische Untersuchungen,” published in 1877,became known five years later to Van’t Hoff, through the latter’sbotanical colleague, de Vries, and thus became one of the mostfruitful stimuli ever given by biology to physical science.Doubt-less Pfeffer owed some of his success to his early training as achemist ; his doctor’s dissertation related to an organic chemicalproblem. Siegfried also began as an organic chemist; as assistantt o Drechsel he was diverted to physiological chemistry. He be-came extraordinary and (1919) ordinary professor of this subjecta t Leipzig. His best known work is on the extractives of muscIeand the kyrines. Zuntz, on the other hand, was by training aphysiologist, and an early appointment a t the Agricultural Collegeof Poppelsdorf, near Bonn, determined his career. He was mainly15PHYSIOLOGICAL CHEMISTRY. 153concerned with nutrition and gaseous interchange, for which heworked out exact methods of gas analysis. At Berlin he wouldrejoice in later years in showing visitors his respiration chambercapable of accgmmodating an ox.I n July, 1920, an international congress of Physiology met a tParis; the next meeting is to take place a t Edinburgh in 1922.I n France the SociM de Chimie Biologique,’founded in 1914, hasresumed the publication of its Bulletin.After two pre-war num-bers, the third followed in October, 1919, and others during thepresent year. Besides original papers there have been occasionalr6sum6s of current questions, valuable on account of their lucidity.During the year a new periodical of biochemical interest hasbegun under the title of the British Journal of ExperimentalI n America a series of monographs has now also been started,‘ I on experimental biology and general physiology.” Some of thoseannounced are on more or less chemical subjects. I n the series of“ Monographien aus dem Gesamtgebiet der Physiologie derPflanzen und der Tiere,” there appeared last year I f Die Narkose inihrer Bedeutung fur die allgemeine Physiologie,” by H.Winter-stein, and this year, “Die biogenen Amine,” by M. Guggenheim,The latter is an excellent up-to-date account of the chemical andphysiological properties of the simpler bases of biological interest,written by a well-known worker on the subject. The productionof large hand-books has been resumed in Germany to some extent.A new ‘‘ Handbuch der biologischen Arbeitsmethoden ” is appear-ing under the general editorship of E. Abderhalden. It willreplace the (‘ Biochemische Arbeitsmethoden,” now out of print,and is on a very extensive scale; there will be 13 parts, of whichthe first, dealing with purely chemical methods, is of primaryinterest to us here.By the publication, towards the end of 1920,of the second half of his Lehrbuch der physiologischen Chemie.”E. Abderhalden has completed the fourth edition. The first halfappeared more than a year ago. The second half (which actuallybears the date 1921) has been almost entirely rewritten. It is afew pages shorter than Vol. 11. of the 1915 edition, in spite of anew chapter on vitamins. I n 1920 there has also appeared Part11. of the first. volume of a “Handbuch der experimentellen Phar-niakologie,” edited by A. Heffter. This, the first instalment ofthe whole work, deals with many important alkaloids, for example,cinchona alkaloids and derivatives, colchicine, cocaine, ipecacuanhaalkaloids, strychnine.The manuscript was prepared before thewar and although some articles have apparently been revised to1918, others do not extend beyond 1913, and do not take intoPathology 154 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.account the therapeutic discoveries of the war. The book will,however, doubtless interest some chemists. Two outstandingFrench books on bacteriology should also be mentioned here, boththe result of scientific isolation during the war: “Trait6 del’immunit6 dans les maladies inf ectueuses,” by J. Bordet, and(‘L’infection bacillaire et la tuberculose chez l’homme et chez lesanimaux,” by A.Calmette. The former book especially shouldappeal to anyone seeking a chemical basis for immunologicalphenomena.Me t a b o lism of Car b o hydra t es .The hypothesis of Chauveau, that fats only become a source ofmuscular energy after they have been transformed into carbo-hydrates, was shown to be untenable some twenty years ago by thework of Zuntz and his pupils, who drew the conclusion that fatsand carbohydrates are isodynamic, that is, that in both theseclasses of foodstuffs the same fraction of the heat of combustioncan be converted into work. The researches of Fletcher, Hopkins,A. V. Hill and others have, however, made it pretty certain thatthe process of muscular contraction is associated with chemicalreactions of substances closely related to the carbohydrates, andthen it becomes difficult to understand that the utilisation of fatsis not attended by some loss of energy.These considerations haveled A. Krogh and K . G. Lindhard to an important reinvestigationof the relative value of fat and carbohydrate as sources of muscu-lar energy.’’ Their method involved the determination of therespiratory quotient of a subject working an ergometer in a respira-tion chamber, and they determined this quotient within 0.005 byvery accurate analysis of the air passing through the chamber. Inconnexion with this A. Krogh has published subsidiary papers2 ona gas analysis apparatus accurate to 0*001 per cent., and on thecalibration, accuracy and use of gas meters, which papers may be ofuse to non-biological chemists. Krogh and Lindhard find that thenet expenditure of energy (after deducting the standard meta-bolism) necessary to perform the equivalent of one calorie oftechnical work on the ergometer varied from 4-5-5 calories.Inthe three best series of experiments it was 4.6 cal. when fat alonewas catabolised (R.Q. =0-71) and 4.1 cal. for carbohydrate alone(R.Q. = 1.0). This shows a waste of energy from fat of 0-5 cal., or11 per cent. of its heat of combustion. The authors suggest as aworking hypothesis that both during rest and work the proportionof fat to carbohydrate katabolised is a function of the available1 Biochem. J . , 1920,14, 290 ; A., i, 692.a Bid., 267, 282 ; A., ii, 553, 630PHYSIOLOGICAL CHEMISTRY.155supply of these substances. With the respiratory quotient below0.8 carbohydrate is formed from fat and provisionally stored; thereverse transformation takes place with a respiratory quotientabove 0.9.A novel aspect of the metabolism of reducing sugars is dealt withby J. A. Hewitt and J. Pryde,3 who find that solutions of dglucose introduced into the intestine of the living animal undergorapid downward mutarotation from + 52-5O to below + 1 9 O , and inone experiment the solution even became Ibevorotatory. Onwithdrawal from the intestine the reverse change takes place moreslowly, until the original rotation of a- and P-glucoses in equili-brium is reached. This change is not due to preferential absorp-tion of the a-form or t o disaccharide formation, but probably tothe formation of y-glucose in excess of any amount normallypresent in glucose solution which has reached an equilibrium.Pro t eins .Various classifications of proteins have been discussed by P.Thoinas.4 The two principal methods for determining the degree ofhydrolysis of a protein are that of Van Slyke, who determines thefree amino-groups, and that of Sorensen, who titrates the carboxylgroups. The former method has been elaborated into an indirectanalysis of amino-acids in groups, of which the largest is that withamino-nitrogen, comprising glycine, alanine, serine, phenylalanine,tyrosine, valine, the three leucines, aspartic and glutamic acids.This large group A.C . Andersen5 subdivides further by neutralis-ing the solution with sodium hydroxide in the way indicated bySorensen for his formol titration.Under these conditions onlyaspartic and glutamic acids combine with one equivalent of sodiumhydroxide, and the monocarboxylic acids remain in the free state.On ashing such a solution the amount of sodium carbonate in theresidue is equivalent to the monamino-dicarboxylic acids present.Amino-A cids.H. D. Dakin 6 has synthesised racemic /3-hydroxyglutamic acid,of which an active modification was discovered by him in casein.'The synthesis presented unexpected difficulties and severalattempted methods failed or gave only minute yields. The bestBiochem. J . , 1920, 14, 395; A., i, 508.Bull. SOC. Chim. biol., 1920, 2, 112 ; A., i, 644.Kong. Vet.oy Landboh6jskole Aarslcrift, 1917, 308 ; A., ii, 647.Biochem. J., 1919, 13, 398 ; A., i, 294.Ann. Reports, 1919, 16, 153; A., 1919, i, 150156 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.results were obtained by the following method. Glutamic acidwas converted by potassium cyanate into a-carbamidoglutaric andthen into hydantoinpropionic acid (I). The action of bromine onYO-NH 70-NH YO-NH YO,HCH-NH CH*NH >” CH-NH CH*NH,(33, 6HBr 6H CH-OH60,H 60,H CO,H 60,H..CH, 6H2 CH 6H2(1.1 (11.) (111.) (IV.)the latter is complex; after bromine enters the molecule it issplit off again as hydrogen bromide, introducing a double bond inthe By-position, but when the bromination is carried out in glacialacetic acid saturated with hydrogen bromide, the latter is addedon again and i-hydantoin-P-bromopropionic acid (11) results.Onboiling this with water hydantoinacrylic acid (111) is formed withthe double bond in the required ab-position. This acid is boiledwith baryta solution until half the nitrogen has been evolved asammonia, indicating the complete opening of the hydantoin ring.At the same time a molecule of water is added and inactive 0-hydroxyglutamic acid (IV) results, in a yield of 20 per cent. ofthe hydantoinacrylic acid or 2 per cent. of the glutamic acid em-ployed. The original paper should be consulted by organic chemistsdesirous of effecting a smoother synthesis. A secondary result ofDakin’s experiments was the preparation of malic semi-aldehydeCHO-CH(OH)*CH2*C02H, which, however, does not lend itself tothe application of the Strecker synthesis.Apart from casein Dakin has now also found his new amino-acidin glutenin (2.4 per cent.) and in gliadin (1.8 per cent.); D.B.Jones and C. 0. Johns8 have also isolated it from stizolobin, theglobulin of the Chinese velvet bean (yield 2.8 per cent.). In dogsrendered diabetic by phloridzin, 0-hydroxyglutamic acid appearsas (‘extra glucose,” as is the case with glutamic acid, proline andornithine, and the’ amount was found to correspond closely withthat derivable from three of the five carbon atoms. Dakin con-siders that P-hydroxyglutamic acid very likely arises in the bodyfrom glutamic acid and would then constitute an example of‘(P-oxidation” such as is known to occur in fatty acids, but hasnot yet been observed in amino-acids.Thus proline would beconverted to glutamic acid via pyrrolidon&arboxylic acid, andornithine would also be converted into glutamic acid. The latterwould then be changed into dextrose by successive conversion into8 J. Biol. Chem., 1919, 4Q, 435 ; A., i, 191PHYSIOLOGICAL CHEMISTRY. 157P-hydroxyglutamic, malic and lactic acids, two molecules of thelatter forming the hexose. In support of this view Dakin hasoxidised 6-hydroxyglutamic to malic acid in vitro and the bio-chemical conversion of the latter into sugars has been broughtabout in several ways.A new method for preparing esters of amino-acids has beenpublished by F. W. Foreman.9 It consists in converting theamino-acids into their dry lead salts, which are suspended inabsolute alcohol and esterified by saturating with hydrogen chloride.After removal of the free hydrochloric acid and the alcohol, theester hydrochlorides are dissolved in dry chloroform and the freeesters liberated by shaking with anhydrous barium hydroxide.Thus the considerable loss of esters by hydrolysis is avoided, whichoccurs in aqueous solution.The process has been applied tocaseinogen and some of the deficit has been accounted for, withouthowever taking Dakin’s above-mentioned hydroxyglutarnic acidinto account. Although amino-acids are usually formulated withtervalent nitrogen and free carboxyl, they might also be repre-sented as internal anhydrides of an acid with an ammoniumhydroxide.This “ betaine ” formula, originally suggested by R.Willstatter, was supported experimentally by A. Geake and M.Nierenstein,lo who found that amino-acids are not methylated inethereal suspension by diazomethane. J. Herzig and K. Land-steiner 11 have confirmed this observation as regards glycine andalanine, but find that in other amino-acids the carboxyl groupis slowly and a t least partly esterified, so that in them there appearsto be an equilibrium between the two forms thus:-NH2*CH,-C0,€I and hH3*CH2*CO*0.An unstable variety of glycine, differing from the ordinary onein crystalline form, has been described recently by H . King andA. D. Palmer.12 Presumably this is the variety correspondingwith the former of the above two formulae, with a free carboxylgroup, and it might be made to react with diazomethane.Eingand Palmer consider it probably identical with the fine needleswhich Emil Fischer obtained on precipitating an aqueous glycinesolution with alcohol; it is the only variety of glycine that reactswith phosphorus pentachloride, which reaction also postulates a freecarboxyl group. King and Palmer are mainly concerned with con-firming the existence of compound of glycine with neutral salts,lo Zeitsch. physiol. Chem., 1914, 92, 149 ; A., 1914, i, 1057.l2 Biochem. J., 1920, 14, 576 ; A , i, 823.-IBiochem. J., 1919, 13, 378 ; A., i, 338.Biochem. Zeitsch., 1920, 105, 111 ; A , i, 719158 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.described by Pfeiffer and his co-workers and called into questionby Bayliss.Hydroxyproline contains two asymmetric carbon atoms andin addition t o the natural acid three stereoisomerides shouldexist.All have now been obtained by H. Leuchs and K. Bor-mann.13 The puzzling transformation of tryptophan in theorganism of the dog into kynurenic acid has now been almostcertainly elucidated by A. Ellinger and Z. Matsuoka 14; they syn-thesised indole-3-pyruvic acid and' found that, like tryptophan, itis converted into kynurenic acid. The mechanism of the formationof this quinoline derivative would therefore presumably be asfollows : -"-C*CH2*CH(NH,)*C0,H ()-,C* CH,* CO CO,H+ \/\/NH KEcoI Q",\ I ,NH*C02HC-OHHere indolepyruvic acid figures as the first transformation pro-duct of tryptophan, which is in accordance with the known beha-viour of other amino-acids.15 As Ellinger and Matsuoka point out,the only criticism which can be urged is, that indolepyruvic acidis first transformed to tryptophan and that the amino-acid isconverted into kynurenic acid by some other reaction, for it hasbeen shown that, for instance, pyruvic acid itself may yieldalanine, and phenylpyruvic acid phenylalanine, when perfusedthrough the surviving liver.The hydroxyl group of kynurenicacid must be represented in its precursor, for quinoline-a-carboxylicacid is not oxidised to kynurenic acid in the dog, but is excretedpartly unchanged, partly combined with glycine. The Hopkins-Cole test for tryptophan has been examined by W.R. Fearon l6in a suggestive paper. Although the conclusions are based onIs Ber., 1919, 52, [B], 2086; A., i, 185.I4 Zeitsch. physiol. Chem., 1920, 101, 259 ; A., i, 696.Is For example, F. Knoop and E. Kertess, ibid., 1911, 71, 252 ; A., 1911,l6 Biochem. J . , 1920, 14, 548 ; A., ii, 786. ii, 514PHYSIOLOGICAL CHEMISTRY. 159molecular weight and nitrogen determinations only of amorphouspigments, and are theref ore somewhat speculative, a considerableadvance has been made, which ought to be useful in dealing withother substances (for example, alkaloids) related to tryptophan.Two molecules of indole, scatole, tryptophan or carbazole, werecondensed with one molecule of an aldehyde (formaldehyde,glyoxylic acid, benzaldehyde) in pure glacial acetic acid bymeans of hydrogen chloride too leuco-compounds, which are oxidisedto pigments, for example, for scatole.H RCRC \/II II I I II II\/\/ \\A/A/\/\--\/\/ \\A/+OMell 11 I I I II Me-/\/\A-Me --fNH NH NH NIt will be seen that they are regarded as related to diphenyl-methane dyes.With tryptophan and formaldehyde or glyoxylicacid in the above-mentioned proportions the compounds are red, butwith three molecules of the aldehyde to two of tryptophan bluecompounds result, which are considered to arise from the condensa-tion of the two additional aldehyde molecules with the two trypto-phan side chains to form carboline derivatives of the type foundin harman.17 It is thus clear that blue compounds cannot be ob-tained from tryptophan combined as peptide, but red compoundsmight perhaps be expected in this case.As usually carried out,the Hopkins-Cole test results in a mixture of tryptophan redand blue.Various colorimetric methods for estimating tryptophan havebeen examined by P. ThomasY18 who prefers the use ofp-dimethylaminobenzaldehyde as advocated by E . Herzfeld .19Thomas finds 1.7-1.8 per cent. in caseinogen, and as much as 2.3per cent. in cerevisin, a protein from yeast, which is evidentlyable to form tryptophan from simpler compounds.Similarly, L. Hugounenq and G . Florence20 find that Aspergillusforms tryptophan when it has as only source of nitrogen any oneof a series of natural amino-acids (it does not grow on phenyl-glycine) . These authors also prefer p-dimethylaminobenzaldehydefor17115,181920detecting tryptophan.Ann.Reports, 1919, 16, 156.967.Bull. SOC. Chim. biol., 1914, 1, 67 ; A., i, 266.Biochem. Zeitsch., 1913, 56, 258 ; A., 1913, ii, 1088.Bull. SOC. Chim. biol., 1920, 2, 13 ; A., i, 466.The synthesis of tryptophan has alsoW. H. Perkin and R. Robinson, T., 1919160 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.been investigated by W. J. Logie21 in various bacteria, for example,B. Coli, which can form it from straight-chain compounds suchas ammonium lactate and sodium aspartate, and also from freeindole; the appearance of indole in some cultures may be attri-buted both to increased production and diminished consumption.The action of bacteria on amino-acids has been further studied inJapan by T.Sasaki 22 and his pupils. Thus K. Hirai 23 has foundthat B. Zactis aerogenes, like yeast, forms tyrosol from Z-tyrosine,but only in minute quantity. The same author 2* finds that astrain of Yroteus vulgaris, capable of converting Z-tyrosine intocl-p-hydroxyphenyl-lactic acid, also converts histidine intod-/3-iminazolyl-lactic acid. This new degradation of histidineshould be compared with the production of urocanic (iminazolyl-acrylic) acid by bacilli of the Coli group, observed by H. Raistrick.25The curious difference in the stereo-chemical behaviour of B.proteus and B. subtilis originally observed by Sasaki for tyrosine,has been also demonstrated for d-Z-phenylalanine by H. Amatsu andM. Tsudji.26With Henderson’s phosphate mixture phenyl-lactic acid and verylittle phenylethylamine is formed, 23.proteus forming the dextro-and B. subtilis the laevo-variety of the acid. If instead of Hen-derson’s mixture, uranyl phosphate and milk sugar are used asnutrient medium the amine is formed to the exclusion of the acid.The Gases of the Blood.This subject, more than any other, continues to occupy Englishphysiologists, four out of the five papers in the current numberof the Journal of Physiology are concerned with it. R. Wert-heimer 27 has confirmed the observation hitherto only madeaccurately by R. A. Peters28 that oxygen and hzmoglobin combinein molecular proportions as originally suggested by Hufner. Itshould be noted, however, that Peters’ values apply to hzmoglobinin the presence of dilute ammonia, and that Wertheimer onlyobtained agreement with these values in the presence of sodiumcarbonate; hzemoglobin dissolved in pure water had an oxygen21 J .Path. Bact., 1920, 23, 224 ; A., i, 912.22 Compare Ann. Reports, 1917, 14, 190, 191.23 Acta Scholae Med. Univ. Kyoto, 1918, 2, 425 ; A., i, 581.24 Ibid., 1919, 3, 49 ; A., 1919, i, 612.26 Ann. Reports, 1917, 14, 191 ; A., 1917, i, 499; Biochem. J., 1919, 13,446 ; A., i, 348.Acta Scholae Med. Univ. Kyoto, 1918, 2, 447 ; A., i, 581.27 Biochem. Zeitsch., 1920, 106, 12 ; A., i, 773.28 J . Physiol., 1912, 44, 131 ; A., 1912, i, 519PHY SIOLOQICAL CHEMISTRY. 161capacity 7 per cent. smaller. It is, however, the carbon dioxide inblood rather than the oxygen which stimulates work and con-troversy.We may first consider those authors who attempt toutilise the laws of mass action and of electrolytic dissociation tothe full, without perhaps always considering sufficiently whetherthese laws apply to a colloidal solution like plasma or to a grosslyheterogeneous system like whole blood.T. R. Parsons29 has attempted to calculate the carbon dioxidedissociation curve of blood on the assumption that a fixed amountof sodium is shared between two weak acids, namely, carbonic acidand haemoglobin (with plasma proteins). The changes of reactionin blood due t o increase of carbon dioxide pressure are much moregradual than in a sodium hydrogen carbonate solution because theproteins, and especially hzemoglobin, act as ‘‘ buffers.” HEmo-globin would therefore have as important a function in carbondioxide transport and maintenance of normal hydrogen ion concen-tration as in the transport of oxygen.The share of the plasmaproteins (as distinct from haemoglobin) in this buffer action is stilla matter of dispute. I f they act as buffers at all they must formionised salts. A. R. Cushny30 denies this; he filtered serumthrough collodion and found all the crystalloid constituents t o bepresent in the same concentration in the filtrate as in the serum(with the exception of calcium and probably of magnesium).F. G. Eopkins,31 on the other hand, evidently believes in theexistence of ion-proteins. W. M. Bayliss32 finds that the plasmaproteins play no perceptible part in the maintenance of neutralitybetween limits of hydrogen ion concentration possible in the livingorganism.Parsons, in his second paper quoted above, considersthat the weak acid competing with carbonic for the sodium ismainly, if not entirely, hmnoglobin. Using a similar conception,L. J. Henderson 33 has attempted t o explain the simultaneousreaction of hzemoglobin with oxygen and with carbon dioxide. Heis led to the assumption that a certain acid radicle of reduced hzemo-globin has a dissociation constant of 2.3 x 10-8, which is increasedin oxyhaemoglobin t o 2.0 x 10-7, thus expressing quantitatively anidea put forward by J. Christiansen, C. G. Douglas, and J. S.Haldane.34 When the hmnoglobin thus becomes more electro-lytically dissociated owing to its taking up oxygen, it also takes up2 9 J .PhyeioZ., 1919, 53, 42, 340 ; A., i, 508.30 Ibid., 1920, 53, 391 ; A., i, 508.81 Brit. Med. J., 1920, ii, 70.8% J. Physiol., 1920, 53, 162 ; A., i, 607.53 J . Biol. Chem., 1920, 41, 401 ; A., i, 403.34 J . Physiol., 1914,48, 244; A., 1914, i, 1012.REP.-VOL. XVn. 162 A"U& REPORTS ON THE PROGRESS OF CHEMISTRY.more base (sodium) and carbonic acid is set free. (This would bethe process in the lungs; the reverse would occur in the tissues.)It is impossible to do more here than indicate the fundamentalnotion of Henderson's paper.I n such theoretical speculations it is important to rememberthe observation made 30 years ago by Hamburger that red bloodcorpuscles are permeable t o chloride and phosphate ions; hispupil S.de Boer 35 more recently showed the same t o be the casefor sulphate ions. This ionic interchange between plasma andcorpuscles has lately attracted the attention of a number ofworkers. L. S. Fridericia36 finds that the increased chlorinecontent of the corpuscle can be demonstrated a t low pressures ofcarbon dioxide in the plasma, for example, 0.1 atmosphere. Theamount of chlorine gained by the corpuscles on increasing thecarbon dioxide pressure from 0.08 t o 162 mm. almost completelyaccounts for the increased carbon dioxide-combining power coinci-dently gained by the plasma. The hydrogen ion concentrationsof plasma and corpuscles remain fairly constant. This is anextension of K.A. Hasselbalch's37 attempt t o explain thatwhereas a sodium hydrogen carbonate solution contains the sameamount of carbon dioxide a t all (except very low) pressures ofthe gas, in blood, on the other hand, the carbon dioxide-combin-ing power increases with the pressure (as if bicarbonate had beenadded). Hasselbalch invokes the ampholyte character of haemo-globin, which he conceives as having an acid character a t lowcarbon dioxide pressures so that it displaces carbon dioxide frombicarbonate, and alkaline qualities a t high carbon dioxide pres-sures, so that it combines with increasing amounts of carbondioxide. The obvious question remained, how can bmoglobinin the corpuscles influence bicarbonate in the plasma ? Fridericiainsists that the necessary link between the two phases is to befound in the wandering of the anions.13. Straub and E. Meier 38have approached the same question by subjecting corpuscles inphysiological saline to various concentrations of carbon dioxide.The corpuscles themselves act as buffers, their P, being 7.00 whenthat of the solution was 6-67, The authors connect this effectwith the partial permeability and the colloidal properties of thecorpuscles. Their work has been discussed by L. Mi~haelis.~~Working on somewhat similar lines, J. M. H. Campbell and E. P.86 J . PAySiol., 1917, 51, 211 ; A., 1917, i, 671.86 J . Biol. Chem., 1920, 42, 245 ; A,, i, 648.87 Biochem. Zeitsch., 1916, 78, 112 ; A., 1917, i, 490.s8 Ibid., 1918,89, 156 ; 90, 305 ; 1919, 98, 205, 228.A., 1918, ii, 467 ;1919, i, 63 ; 1920, i, 200. aa Ibid., 1920,103, 53 ; A., i, 679PHYSIOLOGICAL CHEMISTRY. 163Poulton40 also find the isoelectric point of haemoglobin a t P, 6.98Under physiological conditions the blood proteins act as acids anddo not themselves combine with carbon dioxide until the blood ismuch more acid than ever happens in the body. The partitionof carbon dioxide between corpuscles and plasma a t differentcarbon dioxide pressures has also been recently investigated byJ. Joffe and E. P. Poulton 41 with results similar t o those ofStraub and Meier. The former authors criticise D. D. van Slykeand G. E. Cullen’s method,42 for the determination of the alkalireserve, because the venous plasma, after the separation of thecorpuscles, is brought into equilibrium with alveolar carbon dioxide,so that an ionic interchange with the corpuscles can no longer takeplace.In most of the above papers carbon dioxide is not consideredto be combined with the blood proteins, but t o be entirely presentas bicarbonate (at least under physiological conditions).Thisview is opposed by G . A. Buckmaster,43 who revived Bohr’s con-ception of a direct combination between carbon dioxide and h m o -globin. A third view has recently been put forward by J.Mellanby and C. J. Thomas44 in a paper which contains some novelexperiments and views running counter to the current conception.These authors studied more particularly the ash associated withthe blood proteins under various conditions.For instance, theprotein precipitated from serum by adding an equal volume ofalcohol a t -loo is associated with a large amount of inorganicsalt. When redissolved in water this protein combines with morecarbon dioxide than can be calculated as existing in combinationwith the alkaline salt ; from this and other experiments Mellanbyand Thomas conclude that the carbon dioxide is adsorbed, chieflyon the fibrinogen, and that the proteins effect the transport ofcarbon dioxide. Since a 0.2 per cent. solution of sodium hydrogencarbonate is not precipitated by an equal volume of alcohol a t-loo, it is incidentally concluded that this salt does not exist freein serum. The authors consider the bicarbonate hypothesis inad-missible (according to which the sodium is shared between car-bonic and another weak acid).Sodium hydrogen carbonate withprotein constitut,es the alkali reserve. In shed blood lactic acidis produced from the corpuscles and that is why the carbon dioxideof shed blood falls steadily, and why this gas can be extractedcompletely from blood in a vacuum, but not from serum.It seems t o the writer that if the bicarbonate is adsorbed by theq o J . Physiol., 1920, a, 152.4 2 Ann. Report8, 1917, 14, 173.44 J . Physiol., 1920, 541, 178.q1 Ibid., 129.q3 Ibicl., 1918, 15, 147.a 164 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.serum proteins, the latter still may transport carbon dioxide assalt, in accordance with the view more generally accepted.Mellanbyand Thomas lay great stress on the colloidal properties of serumand support the doubt, expressed above, that the laws of equili-brium in homogeneous systems are not immediately applicable tothis case.The estimation of carbon monoxide in blood and of the latter’sCO-capacity has been described by M. Nicloux.46An interesting contribution to the technique of determining thehydrogen-ion concentration of blood has been made by H. H. Daleand C. L. Evans.46 They dialyse about 5 C.C. of oxalated bloodinto 1 C.C. of saline, avoiding loss of carbon dioxide. The PH ofthe dialysate is then found colorimetrically, by mixing phosphatesolutions until the mixture gives with neutral-red the same d o u ras the dialysate. The method appears to be very convenient andaccurate, and may well be applicable t o fluids other than blood.The regulation of the blood’s alkalinity has been investigated byH.W. Davies, J. B. S. Haldane, and E. L. Eennaway,47 who finda t high carbon dioxide-pressures a considerable deviation fromParsons’ theoretical dissociation curve, ref erred to above. Theystudied the effect of eating large quantities of sodium hydrogencarbonate, which produces increased carbon dioxidecapacity of theblood, increase in alveolar carbon dioxide, rapid excretion of bicar-bonate in the urine, disappearance or great decrease of urinaryammonia and sometimes appearance of acetone substances. Theseeffects illustrate the way in which the organism compensates foralkalosis by changed respiration and metabolism.The ammonianormally excreted in the urine would ultimately appear as neutralurea and the acid normally combined with it becomes available forneutralising the alkali ingested. For the related subject ofacidosis reference can here only be made t o papers by H. W.Haggard and Y. Henderson.4*Accessory Food A% bstancca.The name vitamines is all but established, in spite of the factthat it suggests a relationship t o amines, of which there is no proof.J. C. Drummond49 suggests a compromise by dropping the final“ e,” so as not t o suggest basic properties (to those familiar with theChemical Society’s nomenclature). He further calls f at-soluble-ABull. SOC. Chim. biol., 1920, 2, 171.46 J . Physiol., 1920, a, 167.48 J .Biol. Chem., 1920, 43, 3, 15.4g Biochem. J., 1920,14, 660 ; A., i, 908.4 7 Ibid., 32PHYSIOLOGIUAL CHEMISTRY. 166and water-soluble-B, simply vitamin-A , vitamin-23, etc. Workon accessory food substances is going on with unabated vigour inEngland and in America; there are also signs of increased interestin and recognition of the subject in France and in Germany. Onaccount of its great value, we may once more refer here to theSpecial Report No. 38 of the Medical Research Committee (nowResearch Council), which survey was mentioned in last year’sReport.It seems that the resistance of some accessory food factors tohigh temperatures has been somewhat underestimated. Thus R.Steenbock and P. W. Boutwell50 now report that the fat-solublevitamin-A of yellow maize is unaffected by heating for three hoursunder 7 kilos pressure, nor does this treatment appreciably diminishthe same factor in chard, carrots, sweet potatoes and squash.Therelative stability of vitamin-A is also insisted on by T. B. Osborne,L. B. Mendel and A. J. Wakeman,sl who cannot confirm thegreat thermolability formerly attributed to this substance fromanimal sources by H. Steenbock, P. W. Boutwell and H. E. Kent,6%and by J. C. Drummond.63 The explanation of this discrepancyis probably found in an experiment described by F. G. Hopkinsin opening a discussion on vitamins in clinical medicine a t theannual meeting of the British Medical Association last July. But-ter fat heated for four hours to 120° without aeration remainsactive, but when a stream of air is bubbled through it during theheating, it becomes inactive.It seems that the fat-soluble vitaminpossesses considerable heat-stability but is easily oxidised. Thatvitamins are readily destroyed by oxidation seems also to resultfrom the fact, related by A. F. Hess,55 that milk or neutralisedcanned tomato juice loses much of its activity by being shakenwith air for half an hour. The deleterious effect sometimes ob-served-in pasteurised milk may be the result of exposure to warmair rather than a simple temperature effect. G . F. Still56 pointsout that so-called “buddized milk,” sterilised by being warmedwith hydrogen peroxide to 50°, was found clinically to have lost itsanti-scorbutic properties. Laboratory experiments on the effect ofsuitable oxidising agents on the various vitamins a t roomtemperature now seem desirable.Similarly, E. M. Delf 57 findsthat the anti-scorbutic vitamin4 of orange and swede juice has6o J . Biol. Chem., 1920, 41, 163; A., i, 358.61 IbicE., 549 ; A., i, 457.62 J . Biochem., 1918, 35, 517 ; A., 1918, i, 513.Biochem. J., 1919, 13, 81 ; A., 1919, i, 363.54 Bm’t. Med. J., 1920, ii, 147.55 Ibid., ii, 154.57 Biochem. J., 1920,14, 211 ; A., i, 460.66 Ibid., 5, 166166 ANNUAL REPORTS ON THIE PROGRESS OF UHEMISTRY.an unexpec€edly great stability above looo if the heating is con-ducted in the absence of air. The practical bearing of these experi-ments on canning and other methods of food-preservation is obviousand the same applies to those of A.Harden and R. Robison,58 whofind that with suitable precautions orange juice may be evaporatedto dryness without loss of activity and that the dry residue retainsmuch of its activity after two years’ storage.A few further attempts have been made to isolate the water-soluble antineuritic vitamin-B, which for various reasons seemsto hold out more hope than vitamins-A or -C. The most interest-ing of these attempts is that made by F. Hofmeister and M.Tanaka,5Q who during the war isolated from rice polishings a baseoridine, C,H,,O,N, which as crude hydrochloride cured poly-neuritis of pigeons in small doses, but became inert when purifiedfor analysis. Either the antineuritic vitamin was a mere impurityin the crude crystals and therefore extraordinarily active, or theactive substance underwent chemical transformation during theprocess of purification (regeneration from aurichloride and recry-stallisation of the hydrochloride so obtained).Although theempirical formula of oridine is the same as that of valine, the sub-stance appears to be more closely related to pyridine and is possiblya dihydroxypiperidine. The result reminds one of C. Funk’searlier investigations when the antineuritic substance was asso-ciated with nicotinic acid. C . N. Myers and C . Voegtlin,GO usingmethods for the isolation of bases somewhat similar to thoseemployed by Funk, obtained from dried yeast a crystalline anti-neuritic substance which became inactive on drying. The methodof extracting vitamin-B from rice bran has been studied byB.C . P. Jansen,61 who states that an alcoholic extract of ricepolishings is now used in Java against beri-beri. He used 0.3per cent. aqueous hydrochloric acid, or 70 per cent. alco]hol, or96 per cent. alcohol with & volume of concentrated hydrochloricacid, and found that with each solvent the vitamin is completelyextracted in two days. He criticises curative experiments withpigeons as being uncertain unless much time is expended on them;it is better, and not necessarily slower, to find the minimum pre-ventive dose which must be added to a diet of polished rice.Much time may be saved by using a small species of Indian bird(Musica maja), which is far more sensitive than the pigeon.The results obtained in the laboratory with animals are now513 Biochem.J., 1920, 14, 171 ; A., i, 460.69 Biochem. Zeitsch., 1920,103, 218 ; A., i, 686.6olJ. Biot. Chem., 1920,42, 199 ; A., i, 500.Mededeelingen Geneeak. &ah. Webvreden, 1920, [iii], A, 23PHYSIOLOQIOAL CHEMISTRY. 167more and more being put to clinical use, most of all in Vienna.@Charts63 giving the effect of butter or cod liver oil and fresh turnipjuice, added to the diet of nursing mothers or of infants, demon-strate the same remarkable effect on the body weight of the chil-dren as has been studied in animals. Turnip juice contains theantiscorbutic vitamin and is a cheap substitute for lemon juice;its clinical value has now also been emphasised in Germany.MVarious gastro-intestinal disorders in adults are now attributed byR.McCarrison65 to vitamin deficiency and he has found, for in-stance, that healthy monkeys, carriers of Entamaba cysts, developdysentery when placed on devitaminised food.Evidence is accumulating that vitamins are not only necessaryfor animals, but also for some fungi. R. J. Williams’66 F. M.Bachmann,67 W. H. Eddy, and H. C. Stevenson6* estimate thestrength of vitamin ( B ? ) solutions by growing yeast cells in them.In his second paper Williams has made the method gravimetricby weighing the yeast. The method has been adversely criticisedby G. de P. Souza and E. V. McCollum,6g who find that manysubstances stimulate the growth of yeast. Pasteur already failedto grow yeast from a single cell in synthetic media, and Wildier70postulated a special substance, “ bios,” necessary for the growthof yeast cells.A similar relationship seems to hold for ScZerotkiacinerea, the fungus causing brown r o t in peaches and plums; ac-cording to J. J. Willaman,71 it does not grow on synthetic mediasufficing for AspergiZZu.s, for example, but it will grow when aI‘ vitamin ” preparation is added, obtained by means of adsorptionby fullers’ earth from a variety of animal and vegetable sources.This substance, like “ bios,” is thought to be identical with water-soluble vitamin-B, but such a speculation is of course incapable ofexact verification as long as vitamins have not been isolated.Indeed, the identity of the growth-promoting water-soluble vitaminwith the antineuritic, now generally presumed, is denied by A.D.Emmett in conjunction with G. 0. Lures" and with M. Stock-holm.73 That vitamins are necessary for fungi is also denied; A.LumiBre73Q finds that yeast heated to 135O and no longer capable62 (Miss) H. Chick, Brit. Med. J., 1920, ii, 131.I34 H. Aron and S . Samelson, Deutsch. med. Woch., 1920, 4.8, 772.65 Brit. Med. J., 1920, i, 822.66 J . Biol. Chem., 1919, 38, 465; 1920,42, 59; A., 1919, i, 463.67 Ibid., 1919, 39, 235 ; A., 1919, i, 613.68 Ibid., 1920, 43, 295 ; A., ii, 716.6f) Ibid., 1920, M, 113; A., i, 919.71 J . Amer. Chem. SOC., 1920, 42, 649 ; A., i, 412.72 J . Biol. Chem., 1920, 43, 266 ; A., i, 698.7s Ibid., 287 ; A., i, 701.asE. J . Ddyell, ibid., ii, 132.7 O La oellule, 1901, 18, 313.Cmpt. rend., 1920,171, 271 ; A., i, 663168 ANNUAL REPORTS ON W E PROGRESS OF CHEMISTRY.of curing polyneuritis gives a bouillon which greatly improves thedevelopment of fungi.From what has been said above it will beseen that Lumibe’s experiments are not necessarily in conflictwith those of Williams and of Bachmann. Heating to 135O maynot have left enough vitamin to cure pigeons; yet there might beenough to have a favourable effect on the growth of yeast. It iseven more difficult t o judge of the vitamin-nature of the crudenucleic acid derivatives in bacterised peat, which according toW. B. Bottomleg 74 favour the growth of Lemna in water culture.Experiments on the growth-promoting substances in various organicmanurial composts by F.A. Mockeridge76 seem to depend moredefinitely on the presence of purine and pyrimidine bases in thesemanures.Pellagra, a #disease occurring in countries (Italy, Caro-lina) where maize is the principal article of diet, has of late yearsbeen more and more considered due to a dietary deficiency, and ithas been suggested that the cause lies in the absence of tryptophanand perhaps also of lysine from zein, the chief protein of maize.The metabolic importance of the former amino-acid was estab-lished by E. G. Willcock and F. G. Hopkins 76 in the case of youngmice, that of the latter by T. B. Osborne and L. B. Mende1.77Occasional outbreaks of pellagra in institutions, camps, etc. ,have always cleared up on the inclusion of more milk, meat, eggsor cheese in the dietary, but whether the cure was due to trypto-phan in caseinogen is not thoroughly established.H. Chickand E. M. Hume78 describe experiments with monkeys on a dietrich in all known vitamins, but with zein as its principal protein.Symptoms closely resembling those of pellagra were produced andwere undoubtedly of dietary origin. In one case they cleared uprapidly when caseinogen was administered as well, but the crucialpoint, whether it was tryptophan which made the difference, couldnot be established with certainty. This amino-acid appeared tohave a beneficial effect, but no cure was effected with it alone.Ferments.The discussion on the diastase-like properties of formaldehyde,which has been carried on in Germany during the last few years,may be mentioned here, not so much as a contribution to our know-Proc.Roy. SOC., 1920, [B], 91, 83 ; A., i, 265.76 Biochern. J., 1920, 14, 432 ; A., i, 704.7 6 J . Physiol., 1906, 35, 88 ; A., 1907, ii, 88.7 7 J . Biol. Chern., 1914, 17, 325; A., 1914, i, 620.‘8 Biochern. J., 1920, 14, 135PHYSIOLOGICAL CHEMISTRY. 169ledge of enzymes, but rather as a curious example of scientificcontroversy. G. Woker and H. Maggi have repeatedly asserted,both separately and together,79 that formaldehyde has the power ofhydrolysing starch. They have been attacked by various criticsin a number of separate papers, and finally these critics havebanded themselves together in a final onslaught.80 The explana-tion of the supposed diastatic action of formaldehyde lies in thefact that the latter forms a loose additive compound with staroh(which compound does not give a blue cdour with iodine), andthat formaldehyde also modifies the physical properties of thecolloid.This view is shared by E. Herzfeld and R. Klinger,alwho hold similar views as to the action of formaldehyde and for-mulate, in addition, somewhat revolutionary ideas on starch hydro-lysis, according to which the formation of dextrins (includingachroodextrins) may be a purely physical change in the degreeof dispersion, without any hydrolysis. The hydrolysis of starch(by amylase) is also discussed in a suggestive, largely theoreticalpaper by L. Ambard, E. Pelbois and M. Bricka,82 who considerit to be just as much a unimolecular reaction as the hydrolysis ofsucrose by acids.The action of neutral salts is similar, accelerat-ing the latter, and making possible the former action (dialysedsaliva is without action on starch). In either case the action ofthe neutral salt is on the substrate, not on the catalyst. For starchthe action depends on the anion and is greatest for chlorides, a tP, 6.45, which gives also the reaction of a solution in which theamylase is most stable. Under these conditions the optimum con-centration of sodium chloride is 0.006 molar, but in this neigh-bourhood considerable changes in the salt concentration do notvery much affect the rate of hydrolysis.Potato tyrosinase has been separated by H. Haehn83 into twocomponents by means of a Bechhold ultra-filter.The residue isthermolabile " a-tyrosinase," the filtrate is an " activator " whichretains its activity after incineration. In the case of a successfulseparation (which is not always possible), the two components areseparately inactive on tyrosine, but become so when mixed.It is natural that attempts have been made to demonstrate thereversibility of hydrolytic action in such a simple and specific caseas that of urease, and H. P. Barendrecht,s4 in developing a radia-7 9 For example, Ber., 1919, 52, [B], 1594; A., i, 10.8 0 M. Jecoby, W. von Kaufmenn, A. Lewite, and H. Sallinger, ibid., 1920,Biochem. Zeitsch., 1920, 107, 268 ; A., i, 713.Bull. SOC. Chim. biol., 1920, 2, 42.Ber., 1919, 52, [B], 2029 ; A., i, 102 ; Biochem.Zeihch., 1920,105, 169 ;63, [B], 681 ; A., i, 424.hi., i, 777.a4 Proc. R. Akad. Wetewch. A m s t e r h , 1919, 22, 29, 126; A,, i, 102, 196.a170 ANNUAL REPORTS ON THE PROGRESS OF UHEMISTRY.tion theory of enzyme actions, claims that urease under certainconditions can transform ammonium carbonate into urea. Thisis denied by T. J. F. Mattaar.g5 I n any case urease undergoesmore or less rapid destruction in a solution of ammonium carbonate.E. Yamasakis6 has investigated the kinetics of urease and con-siders that the hydrolysis of urea is a simple catalytic action carriedon in the substrate phase and does not consist in the decompositionwith measurable velocity of an intermediate compound formedinstantaneously.Nor .do the enzyme and substrate form an inter-mediate compound with measurable velocity. The addition ofelectrolytes diminishes the activity of the enzyme owing to adsorp-tion by the latter, and the effect may in various cases be expressedaccording to Freundlich’s adsorption formula. Perhaps it is theelectrolyte nature of ammonium carbonate which prevents thereversibility of the urease action from being demonstrated.The same author 87 has compared the temperature-coefficientsfor the destruction of catalase from bamboo shoots, germinatedsoja beans and blood. As the coefficient is different in each case,he concludes that the enzymes are also different. The effect off‘poisons’’ is considered to be due to adsorption by and coagula-tion of the enzyme.C. G . Santesson8* also considers that theeffect of electrolytes on the rate of the catalase action is due toadsorption and finds that the anions can be arranged in Hof-meister’s lyotzopic series, SO, having the smallest and CN thegreatest inhibitory effect. C. Neuberg and F. F. NordB9 have ex-tended the reduction, by yeast, of the carbonyl group from alde-hydes to ketones. In the latter case they get optically activesecondary alcohols in yields of about 10 per cent. Diacetyl yieldsE-By-butylene glycol, whereas Harden and Walpole found thatbacteria produce from carbohydrates a mixture of the racemic andmeso-forms. Another interesting product of ferment action isthe crystalline specimen of sucrose obtained by E. Bourquelot andM.Bride1,go by the action of emulsin on gentianose. Previouslythis trisaccharide had only been hydrolysed to fructose and gentio-biose by invertase, and then the latter sugar could be split intotwo molecules of dextrose by “ gentiobiase ” of bitter almonds.The authors were, however, led t o attempt a different degradationby the simultaneous occurrence of sucrose and gentiobiose in fresh86 Rec. trav. chim., 1920, 39, 495 ; A., i, 649.8 6 Sci. Rep. TGhoku Imp. Univ., 1920, 9, 97 ; A., i, 577.87 Ibid., 1920, 9, 13, 69, 75, 89 ; A., i, 194, 453, 574, 576.8 8 Skand. Arch. Physiol., 1920, 39, 236 ; A., i, 576.89 Ber., 1919, 52, [B], 2237, 2248 ; A., i, 135.Bull. SOC. Chim. biol., 1920, 2, 160; Compt. rend., 1920, 171, 11 ; A.,i, 630PHYSIOLOGICAL CHEWSTRY.111gentian root. They finally succeeded by using a specimen ofbitter almonds as free as possible from invertase, and controllingthe length of the reaction polarimetrically .Estimation and Formation of Urea.Hypobromite does not liberate the whole of the nitrogen fromurea and the addition of substances like dextrose has been shownby M. Krogh91 t o give illusory results, since carbon monoxide isgiven off. L. Ambard92 now criticises Krogh's high results becauseoxygen is also evolved. After absorption of this gas by sodiumhyposulphite the nitrogen corresponds with 90 per cent. of thetheoretical. Apart from the use of urease, a more elegant methodof estimating urea is that given by R. F o s s ~ , ~ ~ which does notappear t o have received in this country the attention it deserves.It is based on the fact that xanthhydrol precipitates urea a t dilu-tions as high as 1 : 1,000,000.The method has been recently criti-cally examined and favourably reported on by Prenkel.94 Withits aid Fosse has lately studied the formation of urea by oxidisihgproteins with permanganate, first observed by BBchamp in 1856,but afterwards denied. Ordinarily only small amounts are pro-duced, but if the oxidised solution is subsequently heated withammonium chloride, much larger quantities of urea result,Q5 becausethe solution contains cyanic acid.96 When dextrose is added dur-ing oxidation the yield of urea is also much increased and smallquantities of dextrose alone, in the presence of concentrated am-monium hydroxide, may yield 70 per cent.of the sugar as urea.Q7The reaction is considered to proceed through the stages: formaldehyde, hydrocyanic acid, cyanic acid, ammonium cyanate, and itis suggested that this explains the formation of urea in plants.Under certain conditions a little oxamide may also be formed.9891 Zeitsch. phyriol. Chem., 1913, 84, 379 ; A., 1913, ii, 641.92 Bull. SOC. China. biol., 1920, 2, 205 ; A., 1921.93 Compt. rend., 1914, 158, 1076, 1588; 159, 250; A., 1914, ii, 506, 693,756 ; Ann. Reports, 1914,11, 178.94 Ann. China. anal., 1920, rii], 2, 234 ; A . , ii, 646. Compare also P. Carnot,P. GBrard and S . Moissonnier, Compt. rend SOC. Biol., 1919, 82, 1136 ; M.Laudat, ibid., 1920, 83, 730; A., ii, 645 ; W.Mestrezat and M. Janet, ibid.,1920, 83, 763 ; A . , ii, 645, 779.95 Compt. vend., 1919, 168, 320 ; A., 1919, i, 152.96 Ibid., 1919, 169, 91 ; A., 1919, i, 459.97 Ibid., 1919, 168, 1164; A . , 1919, i, 313.98 Compt. rend., 1920,171, 398; A., i, 664.a" 172 BNNUBL REPORTS ON THE PROGRESS OF CHEMISTRY.Hormones.During the current year several attempts have been made t oisolate the physiologically active principles of the pituitary body,but the position seems to be rather less hopeful than last yearwhen J. J. Abel and S. Kubota 99 suggested that the plain musclestimulant of pituitary might be identical with histamine. Thissuggestion was promptly rejected by D. Cow 1 and a t once reaffirmedby J. J. Abel and D. I. Macht.2 The attack and defence weremade on purely pharmacological grounds, and may be cited as anillustration of the difficulties involved in settling the identity ornon-identity of substances exclusively by their physiological action,without the chemical isolation of both.The differences in thephysiological behaviour of the two substances, used by Cow in sup-port of his argument, were considered by Abel and Macht to bethe result of differences in dosage. The notion that the pituitaryuterine stimulant (oxytocic principle) is merely histamine wasalso rejected on more chemical evidence by H. W. Dudley? whoextracted the dry powdered gland with acidulated water andpurified the extract with colloidal ferric hydroxide, which leavesthe active principle entirely in solution. It can then be removedcompletely without loss by continuous extraction with butyl alcohol(under reduced pressure, so as to lower the temperature). Thesubstance from pituitary is not identical with histamine, because,unlike this amine, it is destroyed a t room temperature byN-sodium hydroxide, and further, because it is destroyed bytrypsin, is extracted from acid solution by butyl alcohol, and isinsoluble in boiling chloroform.As the result of Dudley's experi-ments and later ones of their own, J. J. Abel and T. Nagayama4have had to abandon the hope that the pituitary uterine stimulantis histamine. They nevertheless claim that inf undibular extractsfrom fresh glands contain a little histamine, but much less thanextracts of commercial dried gland previously examined by them,or than the extracts commonly employed in therapeutics. Theysuggest that the specific active principle, on boiling and sterilisa-tion, partly breaks down t o histamine.The fact that they ob-tained an impure substance which is many times more oxytocicthan histamine itself, is in itself sufficient to dispose of theirprevious suggestion that the two are identical. It also makes thechances of isolation much smaller-evidently this pituitary prin-Ann. Rpports, 1919, 16, 158.Ibid., 279.lbid., 1920, 15, 347.J . Pham. Expt. Ther., 1919,14, 276.a Ibid., 296 ; A., i, 344PHYSIOLO~ICAL CHEMISTRY. 173ciple is a substance of quite extraordinary potency and present invery small amount.M. T. Hanke and K. K.Koessler5 go further than Abel andNagayama, and deny that histamine is present a t all in freshpituitary; they partly rely on a colorimetric method for estimatinghistamine,sa and they incidentally cite a number of chemical andphysiological differences between the amine and the pituitary prin-ciple. With regard to the latter, F. Fenger and M. Hull6 statethat in the fresh gland it is united t o a protein complex and isinsoluble in 95 per cent. alcohol, which, however, on boiling splitsoff a highly active, hygroscopic substance, more readily decomposedthan its precursor. Of late most writers, for instance, Dudley3and C. Crawford,7 consider the uterine and the pressor principle ofpituitary t o be distinc€. According to the latter, the pressorprinciple gives no Millon reaction and only a very doubtful Paulyreaction, but on keeping an aqueous solution it becomes reactivet o Pauly’s reagent. Abel and Macht find that the Pauly reactionis always given by active preparations. In spite of the greattherapeutical importance of the pituitary, the prospects of isolat-ing any specific active principle from it do not appear to be verybright.L. Stern and E. Rothlin 8 have prepared an impure substancefrom the spleen which they call ‘‘ li6nine”; it acts on smoothmuscle very much like histamine, and has some of the chemicalproperties of the latter substance. The chief difference appears t obe that lienine is destroyed by 1 per cent. sodium hydroxide, andhistamine is not. Of a number of organ extracts examined asregards their action on smooth muscle fibre, that of the spleen isby far the most potent and the active substance is stated to bepresent in the blood of the splenic vein. Further chemical workis required to prove or disprove its identity with histamine.Thyroxine was referred t o a t some length in last year’sReport. Since then a long paper by E. C. Rendall and A. E.Osterberg has come to hand, containing numerous microphobgraphs of crystals. Many analyses of thyroxine and its derivativesare now given for the first time, and the conditions governing thetransformation of the ketonic or lactam into the enolic or lactimform and into the open chain hydrate are discussed. It is statedthat the position of the three iodine atoms is determined byJ. Biol. Chem., 1920, 43, 557. Ba Ibid., 543 ; A., 784.F, Ibid., 1920, 42, 153.7 J. Pham Expt. Ther., 1920,.65, 81 ; A . , i, 468.* J. Phy8kl. Path. gdn., 1920, 18, 753 ; A., i, 649.a J. Biol. Chem., 1919,40, 265 ; A , i, 180174 m u & REPORTS ON THE PROGRESS OF CHEMISTRY.synthesis, a description of which is promised later. MeanwhileE. C. Kendall has further developed the estimation of iodine inthe thyroid; he thinks it best t o use only 0-5 gram of the gland.E. C. Kendall and F. S. Richardson10 find that there is 0.013 mg.of iodine in 100 C.C. of normal blood.GEORGE BARGER.10 J . Biol. Chem., 1920,43, 161 : A., ii, 631.PTOC. Iowa A c d . Sci., 1918,25, 495 ; A., ii, 445.Compare also S. B. Kuzirian