PHYSIOLOGICAL CHEMISTRY.BEFORE reviewing the year’s work, we may record the deaths ofIvar Bang, Ludwig Brieger, Adrian Brown, Emil Fischer, andFranz Rohmann. Bang, who diea suddenly on December llth,1918, in the prime of life, was a Norwegian by birth, a pupil ofHammarsten, and Professor of Medical Chemistry in the Universityof Lund since 1904. He worked on nucleic acids, histones, andimmuno-chemistry, but is best known for his micro-methods forthe determination of sugar and fat in the blood. His publishedworks include ‘‘ Chemie und Biochemie der Lipoide ” (1911), ‘‘ DerBlutzucker ” (19 13), “ Methoden zur Mikrobestimmung einigerBlutbestandteile ’’ (1916), and “ Lehrbuch der Harnanalyse ”(1918). “ Die Nukleinsauren und ihre Verbindungen ’’ was in pre-paration for the series of German biochemical monographs.Brieger, known among biochemists for his work on ptomaines morethan thirty years ago, was since 1900 Director of the Institute ofHydrotherapy of Berlin University.Adrian Brown was Professorof Brewing at Birmingham. To Emil Fischer biochemistry owesa debt greater than that to any other organic chemist of ourgeneration. Rohmann was for many years Ext,raordinary Professorof Physiological Chemistry at Breslau.Among new publications, we may mention “ Medical Science :Abstracts and Reviews,” a monthly journal published by theOxford University Press for the Medical Research Committee inplace of the Medical Supplement t o the “Review of the ForeignPress,” which was issued from January, 1918, to April, 1919.“Medical Science,” started in October, 1919, aims at providing acritical and selective survey of the medical publications of allcountries, and should therefore be of interest t o physiologioalchemists also.During the war, the eighth and ninth volumes ofAbderhalden’s Handbuch der biochemischen Arbeibmethoden ”have appeared. The earlier volumes are now partly out of print,and a second edition is about to appear. The seriea “Die Bio-chemie in Einzeldarstellungen” was continued at the end of 1918by No. 4, ‘(Die Einwirkung von Mikro-organismen auf die Eiweiss-14148 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.korper” by Paul Hirsch. This very complete, if somewhat un-critical, compilation deals with acidic as well as with basic decom-position products of proteins ; in particular, the physiologicallyactive arnines, like tyramine and histamine, are fully dealt with.The third monograph of this series, (‘ Uber kunstliche Ernahrungund Vitamine,” by F.Rohmann, was noticed two years ag0.1 Amost valuable “Report on the present state of knowledge concern-ing accessory food factors (vitamines) ” has appeared in the specialreport series of the Medical Research Committee. It is by a com-mittee consisting of F. G. Hopkins (chairman), H. Chick, J. C.Drummond, A. Harden, and E. Mellanby. Second editions ofW. M. Bayliss’s ‘‘ General Physiology ” and of H. Bechhold’s ‘‘ DieKolloide in Biologie und Medizin ” have appeared. (‘ The Natureof Enzyme Actions,” by the former author, has reached its fourthedition, and ‘‘ Practical Physiological Chemistry,” by S.W. Cole,its fifth. The latter book was originally written for students only,but has now been so much revised and enlarged by the addition ofwell-chosen recent methods that it will be found useful in researchlaboratories.Reviewing the year’s work as a whole, we must recognise that,at least as regards publications, we are still in a period of transi-tion. Probably nearly all war problems have1 been abandoned bynow, but in the matter of publication there is a hysteresis. Forinstance, it is only during the present year that the fermentationprocess of manufacturing glycerol from sugar has come to light.As one of its effects on biological chemistry, the war has left anincreased interest in food problems, especially those concerned withaccessory food substances and deficiency diseases.The Radioactivity and Biological Importance of Potassium.The fact that of the dozen or fifteen elements essential to life,potassium2 is the only one which possesses a distinct, if minute,radioactivity, induced H.Zwaardemaker 3 to replace it in Ringer’ssolution by other radioactive elements. Ringer discovered longago that, in order to keep the isolated frog’s heart beating normallyfor a prolonged period, the perfusion fluid must contain potassiumas well as calcium and sodium salts; he also showed that thepotassium may be replaced by an equivalent amount of rubidiumor of cesium, but if these salts were entirely omitted, the heart1 Ann.Report, 1917, 184.3 See ibid., 1909, 266 for references.For a &sum6 of Zwsardemaker’s researches and those of his pupils, seeP f i e r ’ s Archiu, 1918, 173, 28PHYSIOLOGICAL CHEMISTRY. 149soon stops beating. Now rubidium 4 also has a distinctive &activityfar less penetrating than that of potassium. The &radiation ofceesium of very low penetrating power seems to be absorbed soreadily as to escape detection altogether, but it is postulated byZwaardemaker on biological grounds.5 As a preliminary, he andT. P. Feenstra6 calculated the amount of other elements radio-actively equivalent to the potassium in Ringer’s solution.Zwaardemaker and his pupils showed that the frog’s heart con-tinues to beat equally well if a litre of the perfusion fluid con-tains, instead of 100 mg.of potassium chloride, 25 mg. of uranylnitrate, 50 mg. of thorium nitrate, and 0.000005 mg. of radiumbromide, or a minute quantity of niton (about 100 Mache units).This means that a heart which has stopped beating owing to per-fusion with potassium-free Ringer’s solution begins to beat againwhen an equi-radioactive amount of another element is added tothe perfusion fluid. Moreover, a heart may be made to resumepulsation by exposure to &radiation from mesothorium or fromradium a t a distance of 1-2 cm.7The amounts of radioactive elements quoted above refer to frogsin winter; in summer, smaller amounts suffice, and a reduction inthe necessary amount may also be brought about by addingfluorescein or eosin to the perfusion fluid; in eit.her case, the reduc-tion seems to be due to improved adsorption of the radioactiveelement by the endothelium.Ths adsorption of electricallycharged particlm seems also to be the explanat?ion of the followingparadox. The various means of keeping a heart pulsating, orrestoring its beat when it has stopped, may be arranged in twogroups, namely,potassium uraniumrubidiumcaesium&radiationA heart beating under the influence of any one agent will continueto do so if we, switch over to a perfusion fluid containing theappropriate amount of another member of the same group, butis at once stopped by a member of the other group. Thus,rubidium-Ringer’s solution or &radiation will re-start a heartstopped by a Ringer’s solution free from a radioactive element, andsuch a heart will continue to beat if w0 switch over to msium-4 N.Campbell, Proc. Camb. Phil. SOC., 1909, 15, 11 ; A., 1909, ii, 288.Proc. R. Akad. Wetensch. Amsterdam, 1917, 20, 773.Ibid., 1916,19, 99, 341, 633; A., 1917, i, 70, 105, 241.H. Zwaardemaker and J. W. Lely, Arch. Nderbnd. Physiol, 1917, 1, 746150 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY,Ringer’s solution, for instance, but will stop if we next perfuse itwith a solution containing uranium or any other member of thes m n d group. Conversely, a heart beating under the influence ofa solution containing radium is stopped by ordinary Ringer’ssolution containing potassium. I n order to change over from onegroup t o the other, it is necessary first to wash out the heart witha solution free from radioactive elements ; accordingly, a mixturefrom both groups stops pulsation.Zwaardemaker finds the ex-planation of these paradoxical rmults in the fact that in the firstgroup we are concerned with negatively charged &radiation, inthe second with positively charged a-particles. (In the oase ofradium, which sends out both kinds, a-particles predominate.) Theadsorption of either kind of particle gives the heart the electriccharge which appears to be nece-ssary, but in a mixture of bothkinds the particles with opposite charge neutralise, each other, sothat the requisite electrical condition of the heart does not result,The radioactive ‘( equilibrium,” due to electrical neutralisation,between potassium and uranium is different in summer and inwinter, and is also modified by fluorescein? because the adsorp-tion of these two elements is affected unequally (see above).Zwaardemaker 9 has furnished a most interesting botanical analogyto the above-mentioned antagonism between a- and &radiation.The centres of plate cultures of luminous bacteria were exposed to&radiation from mesothorium and to a-radiation from polonium,and the cultures were subsequently photographed by their ownlight, when in both cases the, centre was found to be black, onaccount of the local death of the organism as a reeult of radiation.When, however, part of the mesothorium field was simultaneouslyexposed to polonium radiation, colonies developed in this area,where the two kinds of radiation apparently neutralised each other.This is an example of the antagonism, it should bel noted, betweentwo radiations, whereas in the experiments on the frog’s heart atleast one side of the antithesis is concerned with ordinary matter.These experimentkj with minute unicellular organisms are furtherof considerable interest in showing that both kinds of radiationaffect the protoplasm of one and the same cell; obviously we arehere concerned with a general phenomenon, and it is not surprisingthat other examples have been found, relating to vagus inhibitiontoskeletal muscle, endothelium of the blood vessels, and to the per-meability of the kidney epithelium.H. J.Hamburger andH. Zwaardemaker, Proc. K . Akad. Wetenach. Amsterdam, 1918, 20, 768;Ned. Tijdschr. t’. Geneeak, 1919, i, 260.A., 1918, ii, 182.l o H. Zwaardemaker and J. W. Lely, Arch. Nderbd. Phgsiol., 1917,1,746PHYSIOLOGICAL CHEMISTRY. 151R. Brinkmanll showed that when the frog’s kidney is perfusedfrom the abdominal aorta with a solution free from potassium, thekidney ‘becomes abnormally permeable to dextrose. A t Zwaarde-maker’s suggestion, these authors replaced the potassium chlorideby a small quantity of uranyl nitrate, and found this equally activein restraining the dextrose from passing the kidney, but a mixtsureof potassium and uranium was found to be inactive, in accordancewith what was said above.Inorganic.E. Winterstein l2 has detected iodine in the beetroot, potato,celery, lettuce, and carrot, but failed to find it in thirty-six otherplants; he also failed to find it in milk, cheese, and cow’s urine.The method allows of the detection of 0.04 mg.of iodine added to10 grams of spinach. The presence of selenium in animals, especi-ally in bones and teeth, and in plants has been asserted by T. Gass-mann,l3 but R. Fritsch14 has lately failed to confirm this observa-tion, especially as regards plants; his method allows of the detectionof 0.5-2 mg. in 30-50 grams of plant material. Zinc has beenfound to be constantly present in animal cells, and occurs moreparticularly in the venom of serpents, t o the extent of 0-11-0.56per cent.l5 It is here combined with organic constituenta (perhapsa proteose rich in sulphur), so that it cannot be precipitated byhydrogen sulphide, nor does it dialyse.The zinc contents ofvenoms are in inverse order of their proteolytic and coagulatingpowers, but in the same order as their nucleolytic and.diastaticactivities Traces of zinc have also been found in various foods.16Thus hen’s eggs contain about 1 mg., nearly all in the yolk; cow’smilk contains, on an average, 4.2 mg. per kilo., human milk dis-tinctly more. The same metal is invariably present in oystersgrown in Atlantic waters; the amount could not be correlated withthat of the water in which the oysters grew. Probably copper isalways present also?Pro t eim .The work of various investigators has made it probable thateuglobulin and pseudoglobulin of serum are very closely related.l1 Proc.K . Akad. Wetensch. Amsterdam, 1918, 20, 944.la Zeitsch. physiol. Chem, 1918, 1041. 54; A., i, 190.Ibid., 1916, 97, 307 ; 1917, 100, 182 ; A., 1916, i, 772 ; 1917, ii, 540.Ib!id., 1918, 104, 59 ; +A, i, 191.l6 C. Delezenne, Ann. Inst. Pasteur, 1919, 33, 68; A., 1917, i, 187.l6 V. Birckner, J . Biol. Ohern., 1919, 38, 191 ; A., i, 420.R. S. Hiltner and H. J. Wichmann, ibid., 206 ; d., i, 421152 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.Thus H. Chick1* described an artificial euglobulin which sheregarded as a mechanical complex originating from the mutualprecipitation of pseudoglobulin and a lipoid substance.P. Hartley l9 could find no difference on analysis of the two proteinsby Van Slyke’s method, and, using the same method, C.Crowtherand H. Raistrick20 failed to find any difference between theeuglobulin and pseudoglobulin of cow’s colostrum and betweenthese proteins and the corresponding ones in ox serum. H. W.Dudley and H. E. Woodman21 have examined the colostrumglobulins by the racemisation method of Dakin and Dudley.22The optical properties of the amino-acids obtained after racemisa-tion and hydrolysis, as well as the rate a t which racemisationproceeds, are identical in agreement with the supposed identity ofthe two globulins of colostrum. A comparative study of thecaseinogens of the cow and sheep, however, by H. W. Dudley andH. E. W0odman,~3 who used the delicate method of Dakin andDudley, referred to above, revealed at least a difference in the“ make up ” of these caseinogens from closely related species; theirconstituent amino-acids are arranged differently.Now H. D.Dakin and H. H. Dale24 have supplied a second example of struetural difference in the corresponding proteins of allied species, andham added additional interest to the result by showing that thetwo proteins are also antigenically different, thus suggesting achemical basis for at least some case of antigenic specificity. Theychose, the crystalline egg-albumins of the hen and the duck. Afterpartial racemisation by N / 2-alkali a t 3 7 O and subsequent hydro-lysis by sulphuria acid, definite differences were found in the opticalproperties of the leucine, the aspartic acid, and the histidine.Thequantities of these and of the other amino-acids obtained were verysimilar in the two casw, so that the difference between hen’s andduck’s albumin seems to be a difference in the arrangement of thesame constituent amino-acids. This difference is, however, sufficientto give an antigenic specificity, for the two crystalline prot’einsbehave as distinct antigens for the anaphylactic reaction. Mostof the experiments were made by sensitising virgin guinea-pigs t oone protein and examining successively the effects of both proteinson the surviving isolated uterus. I n order to give an idea of theextreme delicacy of this reaction, i t may be noted that in one case18 Biochern J., 1914, 8, 404 ; A., 1914, i, 1145.19 Ibid., 541 ; A., 1914, i, 1206.2o Ibid., 1916, 10, 434 ; A., 1916, i, 864.21 Ibid., 1918, 12, 339 ; A., i, 178.22 J . Bi02. Chern., 1913, 15, 263, 271 ; A., 1913, i, 1249 ; Ann. Report,23 Biochem. J., 1915, 9, 97 ; A., 1915, i, 468.1913, 192.24 Ibidea 1919, 13, 248PHYSIOLOGICAL CHEMISTRY. 1530.0001 mg. of albumin in a bath containing 50 C.C. of Ringer’ssolution produced a very distinct contraction of the sensitiseduterus, that is, a concentration of the specific antigen of1 : 500,000,000.Amino-acids.The int’roduction of Emil Fischer’s method of separating mono-amino-acids by the fractional distillation of their esters led tonumerous invmtigations on the amino-acid content of variousproteins. Naturally, these investigations were of very unequalvalue.After the first decade, when the technique had beenthoroughly worked out, we find investigators carefully consideringthe sources of loss involved in the process, for even in the mostfavourable cases the amino-acids isolated do not represent evenapproximately the whole of the protein hydrolysed. The experi-mental losses affect almost entirely the monamino-acids, and aredifficult to overcome. I n the case of zein, the amino-acids isolatedamounted to 85 per cent., but this includes the water taken up inhydrolysis; the results of this investigation led Osborne to a carefulconsideration of the sources of loss. Apart from experimentalerrors, one of these sources might be the presence of unknowncleavage products which had entirely escaped detection.Subse-quently, norleucine, and probably also a-aminobutyric acid,25 wererecognised as possible constituents of protein, but during ,the seconddecade, after the introduction of the ester method, there has beena great falling off in its application, and interest has shifted toD. D. Van Slyke’s methods 26 for finding the distribution of nitrogenin various groups, according to its mode of combination, withoutisolating individual amino-acids.It is all the more interesting, therefore, that H. D. Dakin27 hasfound a new method for separating amino-acids which promises tobe of great use, and has already revealed the presence in casein ofa new cleavage product in an amount of more than 10 per cent.As is so often the case, progress depended on the introduction ofa new technique.Dakin found that, contrary to expectation,certain amineacids can be extracted from aqueous solution bypartly miscible solvents, in particular by butyl alcohol, which isnow available as a by-product in the fermentation process of acetonemanufacture. He hydrolyses the protein with sulphuric acid,removes the latter quantitatively with barium hydroxide, con-centrates, and allows the tyrosine t o crystallise out. The filtratefrom the tyrosine is further concentrated, made approximatelyAnn. Reports, 1913, 198.e7 Biochern. J., 1918, 12, 290; A,, i, 150.as Ibid., 1911, 179164 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.neutral to litmus, and then extracted in a continuous apparatuswith butyl alcohol, preferably at 60-80O.Of course, the processis not very rapid, but the surprising thing is that any amino-acidsshould be extracted a t all. The coefficient of partition is entirelyin favour of the aqueous solution, but soon after the extractionhas been started amino-acids begin to crystallise from the butylalcohol in the boiling flask. With reference to this (‘apparentparadox,” Dakin observes that the presence of water is a condition-ing factor, and that the passage of a certain proportion of waterfrom the fluid undergoing extraction to the butyl alcohol mediumis essential. An excess of a salt, such as calcium chloride, in theaqueous phase almost entirely prevents the extraction of amino-acids. I refer to these practical details because I feel, with Dakin,that the use of butyl alcohol and similar solvents will be found ofvalue for many other purposes in biochemistry.Now as regards the kind of amino-acids extracted, it is foundthat proline coma out most readily, as was to be expeoted, sincethis is the only amino-acid appreciably soluble in ethyl alcohol.lDakin obtains the proline in a fairly pure solution without race-misatim in a yield corresponding with 8 per cent.of the caseinogenhydrolysed. D. D. Van Slyke28 found that 7.13 per cent. of thenitrogen in caseinogen is in the non-amino-form, correspondingwith 9.2 per cent. of free proline (if we exclude hydroxyproline).Yet Abderbalden by the ester method could only isolate 3.1 percent. of proline, and that partly racemised.I n addition to proline, butyl alcohol extracts all the mono-basicmonoaminoracids (alanine, valine, leucine, etc.), and this mixtureis at once obtained in a form suitable for separation by the estermethod.Tryptophan is also extracted by butyl alcohol from asolution after one precipitation by Hopkins’ reagent.The more strongly ionised diaminclacids and the dicarboxylicmonoamino-acids are not extracted under the conditions describedby Dakin. The former may be precipitated by phosphotungsticacid, leaving the latter (aspartic and glutamic acids) in a relativelypure state. It was owing tqo this that Dakin could obtain 21 percent. of the caseinogen as glutamia acid hydrochloride, again muchmore than the yield previously obtained by the ester method,and the yield of aspartic acid (as crystalline copper salt) was twoor three times as large as that previously recorded, but the chiefloss occurring in this fraction in the older method is due to a newdibasic aminohydroxy-acid, of which Dakin isolated 10.5 gramsfrom 100 grams of caseinogen.This new acid is almost certainly a-amino-8-hydroxyglutaric orJ .BioL Chem., 1911, 9, 205 ; A., 1911, ii, 780PHYSIOLOGIUAL CHEMISTRY. 1558-hydroxyglutamic acid, CO,H*CH (NH,) CH (OH) *CH2*CO2H.For the evidence for its constitution, Dakin’s paper should beconsulted.It is probable that P-hydroxyglutamic acid had already beenpartly separated by F. W. Fore,man,29 who was unable to pursuehis investigations. The reason why such a relatively abundantconstituent should have escaped the notice of the numerousinvestigators of caseinogen would appear t o be that the free acid isextremely readily soluble in water and crystallises only slowly fromits syrupy solution.The diethyl ester does not distil Withoutdecomposition, and, on heat’ing, the acid is rapidly converted intohydroxypyrrolidonecarboxylic acid. It seems conceivable thathydroxyglutamic acid is the precursor of the base carnitine(novaine) occurring in meat extract.The Origin of Alkaloids from Amino-acids.After the chief amino-acids of protein had become known, variouschemists, who saw in them the precursors of the vegetable alkaloids,began to speculate on the manner in which the alkaloids might bederived from the aminocacids.The first serious speculation of thiskind is due to A. Pictet,30 who laid great stress on the methylationof hydroxy- and imino-groups by means of formaldehyde. H enaturally regarded alkaloids containing a pyrrole or indole ring asresulting from proline, hydroxyproline, and tryptophan, but he alsoderived from the same source piperidine, pyridine, and quinolinerings, which do not occur in protein. He1 imagined that methylatedpyrroles and indoles undergo isomeric change to their ring homo-logues pyridine and quinoline, because 1 -methylpyrrole when dis-tilled through a red-hot tube, yields a small quantity of pyridine.31The next extensive speculation on the phytochemical synthesis ofalkaloids was by E. Winterstein and G. Trier:, who adopted someof Pictet’s ideas, but did not consider the latter’s pyridine synthesissufficiently biochemical, and instead derived pyridine from lysine,which Drechsel had already considered as a possible parent sub-stance, of alkaloids, when he discovered it as the first basic degrada-tion product of protein.The latest and most elaborate paper onthis subject is due to R . Robinson33; theoretically it marks aa0 Biochem. J , , 1914, 8, 463 ; A., 1914, ii, 826.Arch. Sci. phgs. nat., 1905, [iv], 19, 329 ; Arch. Pharrn., 1906, M,389; A., 1905, i, 641. See also F. Czapek, “Biochemie der Pflasleen,”vol. 11, p. 267. A. Windaus and F. Knoop, Ber., 1905, 38, 1166; A., 1905,i, 381.*l Bet“., 1904, 37, 2792 ; A., L904, i, 771.“ Die Alkaloide,” 1910, pp. 263-317.88 Ann. R e w , 1917, 136156 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.great advance, owing to the simplicity of the reactions postulated.From another point of view, however, such speculations may beregarded as mere jeux d'esprit. The chief value of a theory is inthe experiments which i t suggests, and, judged by this test, Robin-son's theory is seen to be of outstanding importance and on adifferent plane from the speculations of his predecessors. Notonly did it suggest to him a simple new synthesis of tropinone,5*which Willstatter had already regarded as the precursor of atropineand of cocaine in plants, but it has now led to the elucidation ofthe constitution of the alkaloids harmine and harmaline,35 and, attlhe same time, of the mysterious base, C,,H,,N2, obtained by F.G .Hopkins and S. W. Cole36 in the oxidation of tryptophan, for thelatter base is identical with harman, a derivative of both alkaloids.Harman, moreover, occurs in nature, for recently E. Spath37 hasshown that it is identical with the alkaloid aribine from Arraribarubru, Mart; the old formula, C,H,,N,, previously assigned to thisalkaloid is erroneous. Harman, aribine, and the oxidation productof tryptophan all have almost certainly the constitution I.(1.1 (11.)Harmine would then have the constitution 11, and harmalinetwo additional hydrogen atoms in the pyridine ring. Perkin andRobinson discuss the synthesis of the two alkaloids in the plant4.They consider that the starting point is a hydroxytryptophan (111),(111.)which is decarboxylated and condensed with aldehyde (IV),probably derived from alanine.The condensation product wouldbe methylated to harmaline by formaldehyde on the lines sug-gested by Pictet (above), and the harmaline oxidised to harmine.Perkin and Robinson are sufficiently bold to predid that thiss4 Ann. Report, 1917, 134.s5 W. H. Perkin, jun., andR. Robinson, T., 1919, 115, 967.86 J. PhysioE., 1903, 29, 451 ; A,, 1903, i, 690.37 Chem. Zeit., 1919, 4& 665PHYSIOLOGICAL CHEMISTRY. 157hydroxytryptophan will be found among the amino-acids fromvegetable proteins. E. Abderhalden and L. Baumann 38 have,indeed, stated that a hydroxytryptophan accompanies tryptophan,but beyond a few analyses nothing further has been publishedabout this substance.Perhaps it would be more hopeful to searchfor it in plants ; thus, a hydroxytyrosine (3 : 4-dihydroxyphenyl-alanine) has been isolated from T7icia faba by M. G~ggenheim,3~although it has never been obtained from protein. Perkin andRobinson also discuss how the base, C,,H,,N, ( = harman = aribine)might arise from tryptophan, C,,H,,O,N,. As the amino-acid hasbeen decarboxylated, not one, but two carbon atoms must havebeen added, which might come from accompanying alanine or fromalcohol or other impurity in the reagents eimployed, or even fromanother molecule of tryptophan. The experimental conditions forobtaining the base are not well uiiderstood. Professor Hopkinsinforms me that on several occasions a yield of 30 per cent.wasobtained, on other occasions nothing. It is furnished by puretryptophan, so that alanine probably plays no part in its form-ation. A. Ellinger 40 failed to isolate it in numerous experimenb ;the mechanism of its formation remains a mystery.Other plant alkaloids which are obviously derived from amino-acids are tetramethylputrescine, from argiizine via ornithine, andhordenine from tyrosine. Hordenine# has acquired additionalinterest, since E. Spath41 has shown it to be identical withanhaline, an alkaloid from Mezcal buttons (Cactaceae). The chiefalkaloid, mezcaline, which has a peculiar physiological action, isfound by Spath to have the somewhat similar constitution:Me0An N-methylhydroxyproline (4-hydroxyhygric acid) has been dis-covered in the bark of Croton gubouga, S.Moore.42 This substancehas two optically active carbon atoms, and probably the same con-€LO*QH-$.!H,CH, CH*CO,H\/NMe88 Zeilsch. phy&ol. Chem., 1908, 55, 412 ; A., 1908, i, 488.80 Ibid., 1913,88, 276; A., 1914, i, 49,40 Ber., 1906, 39, 2615 ; A., 1906, i, 696.U Mortcrtsh., 1919,40, 129 ; A., i, 648.a J. A. Goodson and H. W. B. Clewer, T., 1919,115, 923158 ANNUAL REPORTS ON THE PROURESS OF (JHEMISTRY.figuration as Z-hydroxyproline from proteins. On methyldion ityielded a mixture of the two stereoisomeric betakes, betonicine andturicine, from Betonicn 0ficinuZis.~3 According to H. Leuchs andK. Bormann,44 turicine is very likely formed from betonicine byracemisation (of the a-carbon atom only) during extraction andduring the methylation of Z-hydroxyproline.Histamine.Histamine (P-iminazolylethylamine) continues to a t t r a h atten-tion on account of its powerful physiological action and Pyman’ssynthesis has been repeated and elaborated.45 There are so manysuccessive reactions involved that the yield of histamine is only4.2 per cent.of that theoretically possible from the citric acid em-ployed. It would seem preferable, therefore, to prepare cyano-methylglyoxaline from histidine by Dakin’s method 46 (yield, 80 percent.), and reduce this nitrile to histamine, thus eliminating allstages but the last of Pyman’s synthesis.The investigation of the physiological action of histamine has beencontinued, and it has been shown that the shock-like conditionwhich is brought about in cats by injection of 1 mg.and upwardsper kilo. depends on an action on the capillary endothelium, ofsuch a nature that a large part of the blood collects in dilatedcapillaries; whilst part of the plasma escapes from the vessels intothe tissues.” The significance of histamine in surgical shock isdoubtful, and the same may be said of anaphylactic shock, wherethe similarity of symptoms to histamine poisoning is the only reasonfor postulating its presence. It is still a question whether histamineis only a product of bacteria and fungi or whether it is also fordedby the tissues of the higher animals. Dale and I found it, indeed,in small quantity in the intestinal mucous membrane of the 0 ~ , 4 8but it seemed likely that its presence could be explained by theaction of intestinal bacteria. Recently, however, J.J. Abel andS. Kubota 49 have concluded that histamine is ‘‘ a widely distributedconstituent of animal tissues, organ extracts, and enzymatic pro-as A. Kiing and G. Trier, Zeitsch. phpioF. Chern., 1913, 85, 209; A., 1913,44 Ber., 1919, 52, [BJ, 2089; A., 1920, i, 85.45 K. I(. Koessler and M. T. Hanke, J. Amer. Chem. SOC., 1918, 40, 1706;413 Bioc7wrn. J., 1916, 10, 319 ; A., 1916, i, 698.47 H. H. Dale, P. P. Laidlaw, and A. N. Richards, J. PhykoZ., 1919, 52,Is G. Barger and H. H. Dale, ibid., 1911, 41, 499 ; A., 1911, ii, 217.49 J. PhaTrn. Expt. Ther., 1919, 13;243 ; A., i, 606.i, 708.A., i, 4.110, 355PHYSIOLOGICAL CHEMISTRY.159ducts, such as Witte’s peptone,” and that its occurrence here is‘‘ entirely independent of micro-organisms.” They also state thatit is formed early in the hydrolysis of pure proteins, and that it isidentical with that constituent of the posterior lobe of the pituitarybody which stimulates plain muscle. These results, if confirmed,will have great physiological significance. Abel and Kubota isolatedhistamine from the intestinal mucosa of the dog by a process which1ef.t; no chance for post-mortem changes. From other organs, such asdog’s liver and striated muscle, boiled immediately after death,they- did not actually isolate histamine, but they extracted by asimilar chemical procedure a substance having a closely similarpharmacological action.It is, however, their identification of hist-amine with a pituitary active principle which will arouse mostinterest. There is no doubt that they isolated histamine dipicrate insmall quantity from commercial, dry, entire pituitary gland, butthe exact significance to be attached to this finding is not yet quiteclear. Whether histamine occurs as such in the posterior lobe ofthe flesh g!and or whether it is formed by autolysis during dryingis doubtful, and in any case there is a good deal of evidence, bothchemical and pharmacological, which prevents the identification ofhistamine with the specific plain muscle stimulant, contained inthe posterior lobe. Incidentally, it should be noted that the claimof the Farbwerke vorm. Meister, Luoius & Briining 60 to haveisolated t-he active principles in a crystalline form is unfounded.According to J.J. Abel and M. C. Pincoffs,51 tihe “hypophysin” ofthe Hoechst works is a mixture of albumoses with varying andunknown amounts of aotive and inactive constituents.Hormones.A pituitary hormone has been discussed in the previous section.E, C. Kendall52 has now published a full paper on the isolation ofthyroxin, the thyroid hormone, but this communication is a con-solidation of previous publications, rather than an extension thereof ;the formula :HI:H NHgiven in last year’s Report53 is retained, but no indication is as yetCompare H. Fiihner, Z&&. f. d. D.R.-P. 268841 ; A., 1914, i, 756.ges. exp. Medixin., 1913, 1, 397.6’ PTOC. Nat. A d . &i., 1917, 3, 507.s2 J . Biol. Chem., 1919, 39, 125 ; A., i, 496.63 Ann. Report, 1918, 170180 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.given of the manner in which it was arrived at. The synthesis byOsterberg, first carried out in 1917, has been repeated in 1919, butno hint is given as to the method employed. The isolation of thenatural substance is, however, fully described and illustrated, andit is stated that some three tons of fresh thyroid glands, chieflypigs’, have been used in the research, which has been in progresssince 1910. Thyroxin may exist in three forms, a ketonic or lactamform, as represented above, a tautomeric enolic or lactim form, anda hydrated form, in which the ring is opened. This opening of thepyrrole ring seems also to occur on acetylation, for the N-acetylderivative yields a disilver salt.Perhaps the method of publicationis not unconnected with a desire to protect the synthesis by patent.Organic and biological chemists will await with the greatest interestthe evidence for the constitutional formula and the method of syn-thesis, particularly with regard to the position of the three iodineatoms. Even the elementary analysis must present considerabledifficulty, for the above compound contains about 67 per cent. ofiodine and only 1.60 per cent. of hydrogen. If it were not hydratedin the benzene ring and contained two hydrogen atoms less, thiswould only lower its hydrogen content by 0.35 per cent. Kendallhas also published further observations on the remarkable physio-logical action of thyroxin.54 A dose of 1 milligram increased themetabolic rate of an adult by 2 per cent., but when the imino-hydrogen is displaced the substance becomes inactive.Both thyr-oxin and N-isubstituted derivatives accelerate the metamorphosis ofthe tadpole. This acceleration is also brought about by other iodinecompounds and by iodine itself, and is therefore not nearly asspecific as the great effect on metabolism. It was shown previouslythat thyroxin produces in minute doses all the therapeutic effectsof the entire gland in cretinism and in myxoedema.A hormone of a different type, non-specific and less active, hasbeen isolated by J. W. Le Heux55 in Magnus’s laboratory. It hadbeen shown previously 56 that washed portions of the alimentarycanal (of rabbits, dogs, and cats), suspended in water or Tyrodesolution, give off a substance capable of stimulating the survivingsmall intestine to increased movement. Le Heux has shown thissubstance to be choline.The surviving small intestine of a rabbitmay give off 1-3 mg. per hour. The base was not identified untilit wits found by chance that the use of glacial acetio acid in an54 Proc. Amer. PhyGol Soc., Amer. J . Physiol., 1919, 48, 136.6s P w e r ’ e Archiv, 1919, 173, 8. Compare Proc. K. Aka&. WetewcA.68 W. Weiland, &id., 1912, 147, 171.Amsterdam, 1918, 20, 806; A., 1918, i, 323PHYSIOLOGICAL CHEMISTRY. 161attempted purification greatly increased its activity, owing toacetylation.Acdylcholine is about 400-2500 times as active ascholine in stimulating the movements of the rabbits’ intestine. Itwas also known that the acetyl derivative is very much more potentthan choline itself in lowering blood pressure ; acetylation hereincreases the activity 5000--10,000 fold, and the same ratio appliesto its action on the isolated frog’s heart. Le Heux finally isolatedthe minute quantity of choline given off from the rabbit’s intestineas crystalline platini- and auri-chlorides, and considers that cholineis a natural hormone and the chief cause of the automatic move-ments of the intestine.Glycerol and some of its Derivatives.The industrial production of glycerol by the action of yeast ondextrose is referred to in a subsequent section on fermentation.This process was a direct result of the war shortage of glycerol; thesame cause led to experiments on the utilisation of fatty acids asfood by a committee of the Royal Society,j7 who have confirmedolder experiments that such acids can be utilised by the organismta a very high extent.show that this applies also to fattyacids from “ hardened ” oils, for example, whale oil. Vitamines are,however, complet6ly destroyed in the process of hardening. As thefatty acids ars somewhat unpalatable, feeding experiments havebeen made before with their ethyl esters, which are also very wellutilised. A closer approximation to the natural fats has beenobtained by A. Lapworth and Mrs. L. K. Pearsoq59 who distilledolive oil.and stearin with mannitol and 1.5-2 per cent. of drysodium ethoxide. Glycerol passes over in a yield of nearly 80 percent., and the residue in the flask consists of a complex mixture ofoleates or stearates (of mahnitol, mannitan, and isomannide 1).According to W. D. Halliburton, J. C. Drummond, and R. K.Cannan,59 the “ mannitol ” olive oil is utilised by the animal organ-ism to practically the same extent as olive oil itself. As theseauthors remark, the importance that was att,ached to the investiga-tion during the war is fortunately now no longer so great.With a view to studying the action of lipolytic ferments,E. Abderhalden and E. Eichwald,GO some five years ago, prepared anumber of optically active fats from optically active epibromo-hydrins, but the specific rotation of the fats was very small.WithTheyb7 J . Physiol., 1919, 52, 328.b8 Biochem. J . , 1919, 13, 296; A., i, 570.69 Ibid., 301.6 o Ber., 1914, 47, 1856; A., 1914, i, 801.REP.- VOI. xvr. 162 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.the same end in view, these authors have more recently preparedoptically active propylene glycol 61 by the resolution of /3-chloro-n-propylamine which was converted successively into optically active@-chloro-a-propanol, propylene oxide, propylene glycol, and itsdibutyrin. Incidentally, this work led to the synthesis of the opti-cally active, biologically important E-hydroxybutyric acid, which waspxepared from d-propylene oxide, and is found to have the sameconfiguration as E-alanine.The same authors,@ by treating&a-bromohydrin, previously prepared by them, in dry pyridine solu-tion with phosphoryl chloride, have also obtained optically activeglycerophosphoric acid, which must be the first step in the synthesisof a phosphatide, since natural glycerophosphoric acid is opticallyactive.A curious biochemical degradation of glycerol has been discoveredby E. Voisenet,63 who has described a new organism in bitter wines,Bacillzis amaracrylus, which has the characteristic reaction ofdehydrating glycerol t,o acraldehyde.Some Tissue Constituents.Cholesterol and the bile acids are the only representatives in theanimal organism of polycyclic hydroaromat,ic compounds ; bothcholesterol and cholic acid have four reduced rings, and a relation-ship between these subst'ances has been suspected for a long time,all the more because cholic acid and some of its derivatives givecolour reactions similar to those of cholesterol.Thus, H. Wielandand F. J. Weil G4 showed that cholatrienecarboxylic acid, formedby the distillation of cholic acid under diminished pressure, givesthe Liebermann-Burchard cholesterol reaction, and I. Lifschiitz Gconcluded that cholic acid is derived from cholesterol on accountof a remarkable colour reaction, given in identical manner by bothsubstances. A definite proof of the relationship has, however, onlybeen furnished recently.66 The difference in the number of carbonatoms of cholesterol (27) and of cholic acid (24) is due to the presenceof an additional isopropyl group in the former compound; onoxidation with chromium trioxide; these three carbon atoms areremoved as acetone, which cholic acid does not yield under theseconditions. Owing to the further presence of an alcoholic hydroxylgroup and of a double bond, it is impossible to remove the isopropyl61 Rer., 1918, 51, 1312 ; A., i, 2.e3 Ann.Inst. Pastew, 1918, 32, 476; A., i, 55.64 Zeitsch. physiol. Chern., 1912, 80, 287: A., 1912, i. 830.6 5 Rer., 1914, 47, 1459; A., 1914, i, 657.6e A. Windaus and K. Neukirchen, ibid., 1919, 52, [B], 1915 : A . , 1920.62 Ibid., 1308 ; A., i, 3.i, 41PHYSIOLOGICAL CHEMISTRY. 163group from cholesterol itself without destroying the rest of themolecule. The displacement of the hydroxyl by hydrogen and thereduction of the double bond furnishes cholestane, C27H48, and whenthis hydrocarbon is oxidised with chromium trioxide, there resultsthe acid, C24H4002, which is isomeric with cholania (cholanecarb-oxylic) acid, a reduction product of the cholatrienecarboxylic acid,CWHS402, of Wieland and Weil, referred to above.The acids,C,H4,0, from cholesterol and from cholic acid are, indeed, verysimilar ; they are not identical, however. Their close similarityreminded Windaus and Neukirchen of that existing betweendihydrocholesterol obtained from cholesterol by ordinary chemicalreduction and coprosterol, its reduction product formed by intes-tinal bacteria. The latter alcohol is derived, not from cholestane,but from a diastereomeric hydrocarbon, $-cholestane (coprostane) .67Now the cholanic acid of Wieland and Weil is derived from+-cholestane.On oxidation, the latter hydrocarbon yields acetoneand an acid, C24H4,02, isomeric with that from cholestane andidentical with the reduction product of cholatrienecarboxylic acid.Hence this is the bridge between cholesterol and the bile acids.The relationship may be summarised as follows:CHMe,*CH2*C2,H3,*OH + CHMe,*CH,*C,,H,, @02H*C23H33Cholatriene-Cholesterol. Cholestane. carboxylic acid.I I++2H 4'""CH Me2*CH ,*C,,H3,*OH -+ CHMe,-C H,*C,,H3, 2-2 CO,H *C23H39Coprosterol. J. - Cholestane. Cholanic acid.I t is note'worthy that the two naturally occurring derivatives ofcholesterol, namely, coprostsrol and cholic acid, are both derivedfrom $-cholestane, and not from cholestane, t,he laboratory reduc-tion product of cholesterol.The chemistry of the lipoids is being simplified by Levene andhis collaborators, and there are indications that the number ofindividuals in this group is smaller than has been supposed.Thus,cuorin, the name given to the lecithin-like substance from heartand other muscles,6* is, according to P. A. Levene and S. K o r n a t ~ ~ u , ~ ~not an individual, but an impure kephalin, in agreement with the67 Compare A. Windaus and C. Uibrig, Ber., 1915, 48, 857; A., 1915,68 A. Erlmdsen, Zeitsch. phpiol. Chem., 1907, 51, 71 ; A., 1907, i, 371.69 J . Biol. Chem., 1919, 39, 83, 91 ; A., i, 466.G 2i, 678164 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.view already expressed by H.Maclean.70 The so-called lecit<hillfrom heart muscle is a mixture of lecithin and kephalin; on reduc-tion with hydrogen and palladium, the substance has all the proper-ties of crude hydrolecithin of egg-yolk,71 and can be fractionatedinto hydrolecithin and hydrokephalin. According to Levene andKomatsu, the opinion of Fraiikel and Linnert,72 that thereare specific phosphatides in the individual organs of the sameanimal, is unfounded. Perhaps the number may be reduced t otwo, namely, lecithin and kephalin. As usually prepared, kephalinis mixed with its decomposition products, chiefly arising throughloss of a fatty acid group, either by enzymes or by chemicalmanipulation.The existence of methylguanidine in normal urine had alreadybeen rendered very doubtful by A.J. Ewins,73 who suggested thati t is formed by oxidation from creatinine during the precipitationwith silver salts in alkaline solution. T. Greenwald74 now deniesthat i t ‘occurs in muscle. Even when mercuric acetate is employedas precipitant in the presence of sodium carbonate, creatine isoxidised to a-methylguanidinoglyoxylic acid,75NH,*C( :NH)*NMwCO-CO,H,and this substance(, on evaporation with hydrochloric acid, yieldsmethylguanidine.Even if methylguanidine is absent from normal muscle and urine,this enhances rather than diminishes the importance t o be attachedto its undoubted excretion after parathyroidectomy, which was dealtwith in last year’s Report.76 For a further discussion of methyl-guanidine, as well as its relationship to creatJne and arginine, thePresidential Address to the Physiological Section of the BritishAssociation, 1919, by D.N. Paton, should be consulted.I have here only space to direct attention to interesting indirect,support of the view77 that arginine and histidine are largelycapable of replacing one another in metabolism. This support hasunexpectedly come from the purely chemical side, for R. G. Fargher70 “ Lecithin and Allied Substances,” 1918, p. 52 (Longmans).71 P. A. Levene and C. J. West, J. BioE. Chem., 1918, 33, 111; 34, 175;72 Biochem. Beitsch., 1910, 24, 268 ; A., 1910, i, 295.73 Biochem. J . , 1916, 10, 103 ; A., 1916, i, 528.74 J . Amer. Chem. SOC., 1919, 41, 1109; A., i, 562.7 6 L.Baumann and T. Ingvaldsen, J. BioE. Chem., 1918, 35, 277; A.,35, 285 ; A., i, 98, 288, 421.1918, i, 423.Ann. Report, 1918, 152.77 H. Ackroyd and F. G. Hopkins, Biochem. J . , 1916, 10, 551 ; A,, 1917,i, 237. Compare also Ann. Report, 1918, 155PHYSIOLOGICAL CHEMISTRY. 165and F. L. Pyman 78 have shown that 2-benzeneazoglyoxaline yieldson reduction aniline and glycocyamidine.flK*”H CH,*NHCH--” CO--NH >C*N:NPh -+ I >C:NH + NH,Ph.It should also! be remembered in this connexion that creatinine isN-meth ylgl y cocyamidine.On the other hand, H. B. Lewis and E. A. Doisy 79 have assailedAckroyd and Hopkins’s conclusion that arginine and histidine arepre-eminently the raw material for the synthesis of purines. Theyfound no difference in the uric acid output of two men maintainedfor successive periods on purinefree high protein diets containing(a) much arginine and histidine, ( b ) little of these amino-acids.A ccesso~y Food S~cbstccn cesThe1 present state of our knowledge on these substances (mis-named “vitamines”) has been admirably summarised by a com-mittee consisting of F.G. Hopkins (chairman), H. Chick, J. C.Drummond, A. Harden, and E. Mellanby,so appointed jointly bythe Lister Institute and Medical Research Committee. This reportconstitutm a veritable monograph of 107 pages with 18 illustrationsand more than 200 references t o the literature, extending to thefirst few months of the current year. It deals with accessory factorsand growth, beri-beri, scurvy, ricket.s, pellagra, and practicalproblems relating to the diets of adults and infants. On accountof the appearance of this compilation and the fact that the interestin accessory food substances is still almost entirely physiological, Ipropose to limit myself here to very few references.E. M. Delf g1compared, necessarily in a very crude manner, the rate of destrua-tion of the antiscorbutic substance in cabbage on heating tot 60° andto 90-looo. From the relatively low temperature-coefficient ofthis reaction (1.3 for loo rise of temperature), she1 concludes thatthe destruction does not consist in the heat denaturation of aprotein or enzyme. T. B. Osborne and L. B. Mendel82 find thatcertain green vegetables, for example, spinach, are rich in fat-soluble A even after drying a t 60°, and they have succeeded inextracting the substance from dried leaves by means of ether.83The green extract was evaporated on starch, and gave a very potent78 T., 1919, 115, 221.7 0 J .Biol. Chem, 1918, 36, 1 ; A., 1918, i, 559.‘‘ Special Report Series of the Medical Research Committee,” No. 38 ;H.M. Stationery Office, 1919.81 Biochem. J., 1918, 12, 416.83 Proc. SOC. Exp. Biol. Med., 1919, 16, 98.82 J . Biol. Chem., 1919, 37, 187166 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.preparation. The fact that this may be described as a distinctadvance will illustrate the very rudimentary nature of our know-ledge concerning this substance. Water-soluble B is much morestable, and is not destroyed completely a t 120° in three hours unlessalkali has been added .*4 The antiscorbutic substance is readilydestroyed by heat, but nevertheless cow’s milk, rapidly dried a t ahigh temperature, retains some of its antiscorbutic properties.Onthis important practical question a recent, careful paper by R. E.Barnes and E. M. Hum085 should be consulted.Enzymes and Fe rnz e nt a t ion.The importance of adsorption as a preliminary to enzyme actionmay provide the excuse for a brief reference to papers by I. Lang-muir,86 who, for example, suggests that the spreading of oil 0.11water is due t o the residual valencies in the carboxyl group beingheld by the residual valencies of the water, so that the oil mole-cules lie in a single layer with their hydrocarbon chains verticallyupwards.The layer is also one molecule thick in the adsorption ofgasm by plane surfaces of platinum, mica, and glass, Langmuirobjects that in the porous substance usually worked with the surfaceis not known. A. M. Williams87 has deduced a theory of gaseousadsorption which, over a long range, agrees very closely with ex-perimental observations ; it also enables one to calculate the surfaceof the adsorbent, and it supports the views of Langmuir and othersas to the smallness of the range of molecular attraction.Because specificity in enzyme action is closely related to’ theasymmetric structure of the substrate and adsorption is a necessarypreliminary, C. W. Portelr and C . T. Hirst88 have prepared(raeemic) dyes with an asymmetric carbon atom; these dyes arepartly resolved when wool is dyed with them.The result isspecially significant if we consider the dyeing process to be one ofadsorption rather than of chemical action.Sucroseis hydrolysed by dialysed colloidal silicic acid ; 89 concentrated solu-tions of the acid soon change their degree of dispersion, becomeless active, and finally coagulate. Sucrose is also hydrolysed to as4 Compare C. Voegtlin and G. C. Lake, Arner. J . Physiol., 1919, 47, 558.86 J . Amzr. Chem. Soc., 1916, 38, 2221 ; 1917, 39, 1848 ; 1918, 4, 1361 ;87 Proc. Roy. Soc., 1919, 96, [A], 287, 298; A., ii, 496.s8 J. Amer. Chem. SOC., 1919, 41, 1264; A., i. 658.89 Albert Mary and Alexandre Mary, Compt. rend., 1918, 167, 644:;The action of invertase has been simulated in t>wo ways.Biochem. J., 1919, 13, 306.A ., 1917, ii, 19, 525; 1918, ii, 430.A.,ii, 14PHYSIOLOGICAL CHEMISTRY. 167slight extent (about 1 per cent.) when a solution is passed five timesthrough a Richardsoii pulveriser,gO which ionises the watermechanically; the biological interest lies in the fact that theinversion is increased in Ringer’s solution, and particularly bytraces of zinc or manganese, but is prevented by enzyme “poisons,”such as potassium cyanide. The favourable effect of some inorganicsalts and the inhibitory effect of antiseptics on the growth ofL48pergiZZu8, first studied by Raulin, runs also parallel to the effectsof these salts and antiseptics on the inversion of sucrose, as studiedby Abelous and Aloy.Pepsin has lately been purified by L.Davis and H . M. Merker,glwho consider that the pure enzyme might be a gluco-protein. A tall stages of purification, the rennetic activity corresponded closelywitch the proteolytie. A simple method of purifying trypsin andother enzymes has been indicated by J. T. WOO^,^^ who allows aconcentrated solution to soak into filter paper, which is then rapidlydried a t a low temperature. On extracting with water, the enzymedissolves more rapidly than some impurities, and an active solutioncontaining very little protein can be obtained.The numerous fermentlative changes which can be brought aboutby fungi and by yeast continue to be studied intensively. ,4sper-9iZZzi.s niger is found93 to ferment large quantities of sucrose, withthe production of 60-70 per cent.of acids, mostly fumaric with alitkle citric; the solution becomes acid to Congo-red. It hadalready been observed that Rhizopus niy/ricans (Mucor Stololtifer)will do the same.94 The various stages involved in the productionof succinic acid from glutamic acid by yeast are, according toC. Neuberg and M. Ringer,gs the following:C0,H*CH2*CH,-CH(NH,).C0,H +C0,H*CH,*CH2* CO- C0,H -+CO,H*CH,*CH,*CHO + CO,H*CH,*CH,-CO,H.The last reaction, conversion of aldehydopropionic into succinicacid, may be brought about, by maceration juice in the absence ofair, and all the stages have now been shown to be purely enzymaticexcept the first, which is only known to occur in the living cell.The discovery of a ferment! which can convert an amino-acid intothe corresponding keto-acid would indeed be interesting.Succinic acid is but one of the by-products of alcoholic ferment.91 J .Amcr. Chem. Roc., 1919, 41, 222; A., i, 180.92 J . Soc. Chem. Ind., 1918, 37, 3 1 3 ~ ; A., i, 102.93 C. Wehmer, Ber., 1918, 51, 1663; A., i, 58.94 F. Ehrlich, ibid.. 1911, a, 3737 ; A., 1912, ii, 192.Biochm. Zeitsc7~., 1918, 91, 131 ; A . . i, 56.J. E. Abelous and J . Aloy, Compt. rend., 1919, 168, 1125; A., i, 310168 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.ation; glycerol is another, and a more important one, for it isformed from the sugar itself. During the war, this circumst'ancehas assumed enormous importance in Germany, for it made possiblethe production of glycerol from sugar on an industrial scale; i t wasdiscovered that, under special conditions, the ordinary yield ofglycerol of about' 3 per cent.can be increased a t least tenfold.Although it was known that Germany possessed a new biochemicalsource of glycerol, the process was kept, a close secret until itspublication by K. Schweizer96 and by W. Connstein andK. Ludecke.97 The latter were the real autlhors of the industrialpr0cess,~8 the essential feature of which is the employment of con-centrated sugar solutions containing a large quantity of sodiumsulphite. Crude sugar, or even molasses, may be used, and neitherthe race of yead nor the1 temperature are of much influence on theyield of glycerol. The monthly output in Germany finallyamounted to 1000 tons, 100 parts of sugar yielding 20 parts ofpurified glycerol, 27 of alcohol, and 3 of aldehyde.The process isbased on the work of Neuberg and his pupils, and he has alsolately furnished a theoretical explanation in a paper,99 which shouldbe consulted by all interested in the theory of alcoholic ferment-ation. I n 1913 C. Neuberg and J. Kerb1 put forward the hypo-thesis that dextrose, by loss of two molecules of waker, furnishesthe aldol of methylglyoxal, CsH804, which breaks down to twomolecules of this keto-aldehyde, C,H,02, one of which is reducedto glycerol, whilst the other is oxidised t o pyruvic acid:CH,:C(OH)-CHO + H,O H, CH,(OH)*CH(OH)*CH,*OH+ II = +CH,:C(OH)*CHO 0 CH,-CO*CO,HThe pyruvic acid is decarboxylated by carboxylase t o acetaldehyde,CH,=CO*CO,H = CO, + CH3:CH0,and the latter is reduced to alcohol, whilst from a further moleculeof methylglyoxal pyruvic acid is regenerated.CH;CO*CHO 0 CFT,*CO*CO,H+ II =CH,*CHO H2 + CH,*CH,*ORs6 Helv.chim. Acta, 1919, 2, 167 ; A., i, 239.97 Ber., 1919, 52, [B], 1385; A., i, 463.s8 Compare also J . SOC. Chem. Ind., 1919, 38, 2 8 7 ~ .ss C. Neuberg and E. Reinfurth, Ber., 1919, 52, [B], 1677 ; A., 1920, i, 124.Biochem. Zeitsch., 1913, 58, 158 ; A., 1914, i, 118PHY SIOLOQICAL CHEMISTRY. 169Hence methylglyosal and pyruvic acid would be intermediatestages, glycerol and acetaldehyde necessarily by-products ; as amatter of fact, the latter are always both present in alcoholicfermentation; the circumstance that the only known form ofmethylglyoxal does not ferment is no fatal objection, since it isprobably the most stable of the many possible forms. (At leasttwenty-two are conceivable.)Next it was found 2 that slightly alkaline salts do not suppressthe fermentation, but increase the yield of the by-products at theexpense of tlhe main products, and then i t was shown3 that by theuse of sodium sulphite the acetaldehyde may be fixed in a yieldof 70 per cent. of the theoretical as the additive compoundCH,*CH(OH)*O* S0,Na.The similar additive product of pyruvic acid undergoes decarboxyl-ation. As the acetaldehyde is now no longer reduced, the(' hydrogen of fermentation " is used up in forming more glycerol.Since the aldehyde-sulphite compound dissociates, its yield, andthat OF the glycerol, should depend on t,he concentration of thesodium sulphite employed, but not be proportional to i t (massaction). The theory further demands that glycerol and acetcaldehyde should be formed in molecular proportions. Both thesepostulates are fulfilled; thus, f o r 100 grams of dext'rose and vary-ing amounts of sulphite, the following yields were obtained :Na,SO, used.Grams.Yield of Yieldac eta1 deh y de .33 11-90 23-3750 12-52 24-8675 13.89 27-61150 18-65 36-96of gl yc er 01.Grams. Grams.The molecular ratio acetaldehyde : glycerol is therefore 0.94-0.95instead of 1. The highest yield of glycerol corresponds with 35.06per cent. of hexose, or 70 per cent.. of the moiety which couldfurnish glycerol. For a 100 per cent. conversion, the fermentationwould have to proceed completely according to the equationC,H,,O, + Na,SO, + H,O =C3H803 + CH,*CH(OH)*O-SO,Na + NaHCO,.The shortage of 30 per cent. is due to unsuppressed dissociation ofaldehyde-sulphit'e. With the same relative quantities of sugarC. Neuberg and E. Fiirber, Biochem. Zeitsch., 1916, 78, 238 ; A., 1917,i, 502.C. Neu1)erg and E. Reinfurth, ibid., 1918, 89, 365 ; A., 1918, i , 517 ;-4nn. Report, 1918, 166.Q170 ANNUAL REPORTS ON THE PROGRESS OF CHEMISTRY.and sulphite in dilute solution, the dissociation is much greaterand the yield of glycerol falls off considerably. I n their mostrecent paper, Neuberg and Reinfurth state that insoluble calciumsulphite suspended in the fermenting solution has advantages overthe sodium salt. As calcium sulphite has a neutral reaction, itseems that alkalinity is not an essential condition for an increasedproduction of glycerol. GEORGE BARGER