BIOCHEMISTRY.IT has again been found necessary to make a, somewhat arbitrary selectionfrom the many progressive branches of Biochemistry, and in this the aimhas been to choose topics which have arrived at a fairly clear-cut stage ofdevelopment since they were last considered here. They include aspects ofmetabolism, hormones, nutrition, and chemotherapy.I. PHOSPHORYLATION MECHANISMS.Phosphate Bond Energy.The inter-relationships of phosphorylations in the transport and storageof metabolic energy l have been further clarified and their range and inter-pretation have been extended.2* 3* It is doubtful if under physiologicalconditions appreciable synthesis of phosphoric esters could occur by reversalof their hydrolytic (esterase) cleavage, and the primary introduction ofphosphoric groups falls into two main classes, though doubtless otherroutes 59 6 also exist.Phosphorolysis of the glucosidic type of linkage in poly- or di-saccharidesoccurs reversibly, as is described below, and thus provides for the introductionof phosphate into organic metabolites without the need for external energysources.The second type of phosphorylation is associated with a markedincrease of free energy, which is commonly derived from enzymic dehydro-genation accompanying the addition of phosphate to a double bond.The differences in energy level in these two types of compound arenaturally reflected in their widely different properties. The esters ofphosphoric acid with alcohols such as hexoses (including the " glucosidic ''type represented by glucose 1 -phosphate), pentoses, trioses, glycerol, choline,serine, or the 2- or 3-position in glyceric acid, are stable compounds; theirhydrolysis whether by acids or enzymes is reversible, and is accompaniedby relatively small energy change ( AF about -3000 cals.) .But the cleavageof the second type of phosphoric compound is strongly exothermic (AF about-11,000 cals.). Lipmann has therefore introduced for the latter thesymbol - P, denoting the " high energy phosphate bond " by means of whichthe high potential energy of the phosphorus linkage may be indicated:the phosphate group he writes - ph. From the biochemical standpoint thisis justified by the emphasis which it places on the ability of such highpotential groups to promote synthetic reactions.Examples of this type ofcompound include anhydrides formed from phosphoric acid, with an organicSee Ann. Reports, 1941, 38, 241 ; 1940, 37, 386, 417.H. M. Kalckar, Chem. Rev., 1941, 28, 71 ; Biol. Rev., 1941, 17, 28.I?. Lipmann, " Advances in Enzymology," Interscience Press, N.Y., 1941, 1, 99.H. M. Kalckar, Bwchem. J . , 1939, 33, 631.S. P. Colowick, M. S. Welch, and C. F. Cori, J . B i d Chem., 1940, 133, 359; 1941,* A. A. Green and S. P. Colowick, Ann. Rev. Biochem., 1944, 13, 155.137, 343DICKENS : PHOSPHORYLATION MECHANISMS. 231phosphate as in the two terminal groups of adenosine'triphosphate, or with acarboxyl as in 1 : 3-diphosphoglyceric acid or acetyl phosphate, or with anacidic enol group as in phosphoenolpyruvic acid.The amidophosphate bondis another type of high energy linkage, present in creatine phosphate andarginine phosphate, the energy reservoirs of muscle and nerve.Following the primary introduction of inorganic phosphate into meta-bolites, other enzymic reactions are known to transport these groups, eitherinter- or intra-molecularly, thus forming the wide variety of phosphorylatedintermediates. According to the energy changes involved, such reactionsmay be reversible or irreversible. Thus reversible interchange of phosphateoccurs, on the higher energy level, between the adenosine triphosphate-adenylic acid system and the creatine-creatine phosphate or phospho-glycerate-diphosphoglycerate systems. On the lower potential level it maybe between glucose 1-phosphate and glucose 6-phosphate (enzyme, phospho-glucomutase), or between 3- and 2-phosphoglyceric acids (phosphoglycero-mutase) .The phosphorylation of glucose to glucose 6-phosphate by adeno-sine triphosphate (hexokinase), i.e., a change from the high to low level, isirreversible. The concept of phosphate bond energy, and in a wider sense ofgroup potential, has many applications to biological syntheses, includingacetylations, methylations, and aminations (cf. 3). It plays an importantpart in animal phosphorylations, as will be briefly considered here.Primary Introduction of Phosphate Groups : Phosphorylase.The preparation from skeletal muscle of this enzyme, which esterifies aglucose unit of a polysaccharide, or conversely synthesises polysaccharidefrom glucose 1-phosphate, is fully described by Cori and co-worker~.~. 8i loIt has been obtained in a crystalline (a) and an amorphous (b) form. Theformer is a euglobulin of M.W.340,000400,000 and has 60-70% of itsmaximum activity without addition of adenylic acid. The more solubleb form is inactive without addition of adenylic acid, but both a and b areequally active in its presence. The optimum rate of conversion is 4 x lo4mols. of glucose l-phosphate to glycogen/mol. enzyme/min. a t 30". Glucosecompetitively inhibits the activity, while cysteine increases both activity andsolubility. Extracts of muscle and spleen contain an enzyme (" PR ") which,like trypsin, removes the prosthetic group from a, converting it into theamorphous b form. Simultaneously, pentose (0.3 pg./mg.of protein) is lost,but the substance split off is not adenylic acid. Added adenylic acid doesnot render the b form crystallisable, nor is it firmly bound as is that presentin a. Possibly this non-dissociable union of the prosthetic group is aprotection in vivo.As with vegetable phosphorylase 11 the equilibrium position varies with' A. A. Green and G. T. Cori, J . Bid. Chem., 1943,151,21.G. T. Cori and A. A. Green, ibid., p. 31.C. F. Cori, G. T. Cori, and A. A. Green, ibid., p. 39.lo G. T. Cori and C. F. Cori, ibid., p. 57.l1 C. S. Hanes and E. J. Marshall, Biochem. J . , 1942, 36, 76232 BIOCEEMISTRY .pH; polysaccharide is formed from Cori ester only when a little “ priming ”polysaccharide is added.The reaction is considered to be9 : glucose1-phosphate + terminal glucose unit maltosidic chain unit + inorganicphosphate. The terminal glucose units are supplied by the end groups ofthe highly branched glycogen or amylopectin molecule (starch amylose doesnot activate animal phosphorylase lo), polysaccharide synthesis consisting of alengthening of the side chains by addition of glucose units in 1 : 4-glucosidiclinkages g* 12* l3 to form long unbranched chains of glucopyranose units.When a supplementary enzyme from heart or liver, obtained free fromphosphorylase, accompanies crystalline phosphorylase, a branched-chaintype of polysaccharide resembling glycogen results. Presumably branchcd-chain polysaccharides such as glycogen and amylopectin arise from the jointaction of phosphorylase and another enzyme lo or factor.12 It is uncertainwhether the supplementary enzyme in Cori’s experiments is another type ofphosphorylase, able to establish 1 : 6-glucosidic linkages, or else some kindof diastase not identical with that of blood serum.1OThe yield of phosphorylase from rabbit skeletal muscle (40-80 mg./100 g.)is not altered by previous stimulation of the muscle, but the proportioncrystallisable is diminished.** l4 Phosphorylase occurs in a variety of tissues,and in embryonic tissues is related t o the activity of their glycogen meta-b01ism.l~.l5 It is contained in adipose tissue,15 which utilises glycogen,15and in cartilage the enzyme may produce phosphoric esters, yielding phos-phate needed for calcification.lGAdenylic acid is not a component of potato phosphorylase l7 or ofdisaccharide phosphorylases,18 which require no coenzyme.It is noteworthythat in muscle phosphorylase adenylic acid acts as coenzyme without anyevidence of its phoaphorylation. l9 Adenosine di- or tri-phosphates have no,and inosic acid only feeble, coenzyme a c t i ~ i t y . ~ ~ 2oAdenosine Triphosphatase.The energy liberated by hydrolysis of the final2‘ phosphoric group ofadenosine triphosphate (ATP) is believed to be directly utilised for muscularcontraction.2a Engelhardt’s important discovery in 1939 22 that myosinl2 W. N. Haworth, S . Peat, and E. J. Bourne, Nature, 1944, 154, 236.l3 W. N. Haworth, R. L. Heath, and S.Peat, J., 1942, 55; W. Z. Hassid, G. T. Cori,K. H. Meyer, ‘‘ Advances in fand R. M . MoCready, J. Biol. Chem., 1943, 148, 89;Enzymology,” 1943, 3, 109; W. Z. Hassid, Ann. Rev. Biochem., 1944,13, 59.l4 A. Mkki and E. Wertheimer, Biochem. J., 1942, 36, 221.l6 B. Shapiro and E. Wertheimer, Biochem. J., 1943, 37, 397; A. Mirski, ibid., 1942,A. B. Gutman and E. B. Gutman, Proc. SOC. Ezp, Biol. Med., 1941,48,687; A. B.36, 232 ; E. Wertheimer, Nature, 1943, 152, 565.Gutman, F. B. Warrick, and E. B. Gutman, Science, 1942, 95,461.l7 D. E. Green and P. K. Stumpf, J . Biol. Chem., 1942,142, 355.l8 M. Doudoroff, ibid., 1943,151, 351; P. H. Hidy and H . G. Day, ibid., 1944,152,l* Cf. ref. (3), p. 124.2o C. F. Cori, Cold Spring Harbor Symposia on Quantitative Biology, 1939, 7, 260.21 Cf.refs. (32), (33)) and (28).477.8’ See Ann. Reports, 1941, 38, 241DICEENS : PHOSPHORYLATION MECHANISMS. 283“ which is the contractile constituent of muscle is at the same time thecatalytic agent which promotes the chemical reaction which provides thedirect source of energy of muscular activity ” 23 has been widely accepted asprobable after several careful studies,22* 249 251 26 and so far no more activefraction has been isolated from this globulin. Nevertheless, cataphoresis 25and sedimentation 26 analyses suggest that myosin may not be quite homo-geneous, though nearly s ~ . ~ ~ The most serious challenge to the view thatmyosin itself is the enzyme is the recent demonstration by KalckeLr 28 thatan adenosine polyphosphatase, present in the soluble albumin fraction frompotato and 50-100 times more active than myosin, is strongly and apparentlysomewhat specifically adsorbed by myosin.But even if the activity ofmyosin should eventually prove to be due to adsorbed adenosine triphos-phatase, the latter might still be linked to contraction of myosin. Thispotato enzyme, like that from liver, hydrolyses ATP directly to adenylicacid without intervention of myokinase. It thus differs from the muscleenzyme, which is considered specific for the triphosphate inasmuch as it doesnot act upon adenosine diphosphttte (ADP) except through myokinase ;28but it attacks inosine triphosphate even faster than ATP.29Although iodoacetic acid does not inactivate adenosine triphosphatase,3Ooxidation does so, and SH compounds reverse the inactivati~n.~I Appar-ently the establishment of the single thioether linkages by the former reagentis to be distinguished from the cross-linked S-S bonds believed to be hereproduced by oxidants.Radioactive phosphorus has been used t o show in muscle the coupling ofoxidation with the phosphorylation of adenylic acid and creatine 32 and therapidity of resynthesis of ATP after its breakdown.33* 34Phosphokinases (Phospherases) .Myohuse.-Although Lipmann’s terminology does not differentiate them,the terminal phosphate group of ATP is more reactive than that of thediphosphate (ADP), the latter being unable to transfer phosphate directly(e.g., to glucose in presence of hexokinase), but requiring the presence of awater-soluble enzyme, myokinase, obtained from muscle and other tissues.35* 36This enzyme, which is stable to heat and to acid, catalyses the reversible23 W.A. Engelhardt, Yale J. Bid. Me&., 1942, 15, 21 (Engl. trans. from Russianorig.).24 D. M. Needham, Biochem. J . , 19-12, 36, 113.25 K. Bailey, ibid., p. 129.26 G. Schrarnm and H. H. Weber, Kolloid-Z., 1942, 100, 242 ; Brit. Chem. Physiol.27 M. Ziff and D. H. Morre, J . Biol. Chem., 1944, 153, 663.p * H. M. Kalckar, ibid., p. 355.30 D. M. Needham, ibid., p. 113.38 R. F. Furchgott and E. Bhorr, ibicl., 1943,151, 65.33 E. V. Flock and J. L. Bollman, ibid., 1944, 152, 371.34 H. M. Kalckar, J. Dehlinger, and A. Mehler, ibid., 1944, 154, 275.35 S.P. Colowick and H. M. Kalckar, ibid., 1943, 148, 117.36 H. M. Kalckar, ibid., p. 127.Abs., 1943, 111, 348.2D A. Kleinzeller, Biochem. J., 1942, 36, 729.31 M:Ziff, J. Biol. Chem., 19-14, 153, 25234 BIOCHEMISTRY.reaction : 2ADP =+= ATP + adenylic acid. Inosin diphosphate is notaffected.29 The enzyme is inactivated by oxidants and activated by SHcompounds, and it is capable of transferring 4 times its own weight ofphosphorus per min. a t 30’.Hexokinase.-The occurrence of the hexokinase reaction : hexose +ATP --+ hexose 6-phosphate + ADP, is probable in various cells whichmetabolise glucose, and from several of these this water-soluble enzyme hasbeen extracted.35* 37* 38 With yeast hexokinase direct phosphorylation ofthe 6-position of the hexose occurs with glucose or fructose,39 but it is possiblethat in aerobic liver suspensions fructose may be directly phosphorylated inposition 1, or alternatively, as has been suggested for galactose l-pho~phate,~~there may be an equilibrium between these 1-phosphates and Cori ester.39Hexokinase is of special importance in the synthesis of glycogen fromglucose, the glucose 6-phosphate being reversibly converted via the 1 -phos-phate into glycogen, by means of the enzymes phosphoglucomutase andphosphorylase (cf.37).Phosphorylation of Fructose 6- Phosphate.-This reaction proceeds by wayof an enzyme not yet isolated, sometimes called Neuberg ester phospherase.It catalyses the reaction : fructose 6-phosphate + ATP --+ fructose 1 : 6-di-phosphate + ADP.It is stated to be inhibited by oxidising agents and evenby O/R indicators of B,>0.05 v., and this sensitivity has been held to be themechanism of the Pasteur effect, by which the fermentation is checkedaerobically 41 ; but this is perhaps an over-~implification.~~Other Enzymes concerned in Reactions of Phosphoryluted Intermediates.Recent outstanding advances include the purification of phosphogluco-m~tase,~’ the isolation of aldolase (or zymohexase) of muscle,43 now crystal-lised and its distribution studied,44 and of enolase (crystalline mercury salt,) ,45It is stated that the addition of phosphate to 3-phosphoglyceraldehyde is non-enzymic, and that the unknown intermediate in the dehydrogenation of thistriose phosphate (previously considered to be 1 : 3-diphosphoglyceraldehyde 22)has the nature of a “ loose physical addition product,” analogies for whichare sugge~ted.~~Oxidative Phsphrylations.The reaction just mentioned wasthe first in which the oxidative formation of high energy phosphate bondsPreceding the formation of pyruvate.37 S .P. Colowick and E. W. Sutherland, J. Biol. Chem., 1942, 144, 423.38 I. HuzAk, Biochem. Z., 1942, 312, 315.39 C. F. Cori, Biological Symposh, 1941, 5, 131.4 0 H. W. Kosterlitz, Biochem. J., 1943, 37, 318, 321, 322.4 1 W. A. Engelhardt and N. E. Sakov, Biochirnia, 1943, 8, 9.42 Cf. E. S. G. Barron, ‘‘ Advances in Enzymology,” 1943, 3, 149 (p. 183).43 D. Herbert, H. Gordon, V. Subrahmanyan, and D. E. Green, Biochem. J ., 1940,4 4 0. Warburg and W. Christian, Biochem. Z . , 1943, 314, 149, 399.4 5 Idem, ibid., 1941-2, 310, 384.46 0. Meyerhof and R. Junowicz-Kocholaty, J. Biol. Chern., 1943, 149, 71.34, 1108DICKENS : PHOSPHORYLATION MECHANISMS. 235was clearly demonstrated, inorganic phosphate being incorporated into thecarboxyl of the final product, 1 : 3-diphosphoglyceric acid. This is the morecommon mechanism by which such bonds arise. An alternative is seen inthe action of enolase, which merely by the removal of a molecule of waterfrom 2-phosphoglyceric acid reversibly produces the high energy enolic bondin the resulting phosphoenolpyruvic acid : in this remarkable reactionthe considerable energy of dehydration is conserved within the moleculeThus in the passage from a glucose unit of glycogen to pyruvate, throughthe well-known series of phosphorylated intermediates, one externallyintroduced - ph (from ATP) is required in the formation of fructose 1 : 6-di-phosphate, and, since two mols.of pyruvate are formed, 2 - ph arise at eachof the stages resulting in 1 : 3-diphosphoglyceric and phosphoenolpyruvicacids. Thus the removal of 4 hydrogen atoms should yield a balance of3 - ph per mol. of hexose metabolised. Starting from glucose, instead ofglycogen, the primary phosphorylation by ATP and hexokinase consumes afurther - ph, and at the pyruvate stage only 2 - ph remain on balance.Possibly in intact cells, as distinct from extracts, economies are effected byunknown mechanisms (e.g., the formation of fructose 1 : 6-diphosphate couldtheoretically occur by intermolecular transfer of the 1 -phosphate from Coriester to fructose 6-phosphate).3 The synthesis in intact cells of ATP frominorganic phosphate during glucose fermentation has beel'i dem~nstrated.~'During pyruvate metabolism. The simpler conditions prevailing inbacterial extracts enabled Lipmann 4** 49p 50 to demonstrate the productionof high energy phosphate bonds during the bacterial oxidation of pyruvateto acetate and carbon dioxide, and the formation of acetyl phosphate, nowisolated as the pure silver sa1t.w The latter has been synthesised frommonosilver phosphate and acetyl chloride and the properties and determin-ation of monoacetyl phosphate are de~cribed.~~ It is assumed 3* 52 that inthe enzymatic synthesis 50 the addition of phosphoric acid to the carbonylgroup of pyruvic acid is followed by the dehydrogenation of the resulting(unknown) compound ; the analogy with bisulphite and cyanohydrin com-pounds is suggested.This mechanism finds some support in purely chemicalstudies of E. Baer.53* 54 The reaction is represented as a dehydrogenative de-carboxylation : CH3*CO*C0,H + HO*ph CH,*C(O€€)(O*ph)*CO,H -"H,CH,*CO*O - ph + CO,. The closely similar reaction occurring in cell-freeextracts of Esch. coli : pyruvic acid + H3P0, += acetyl phosphate + formic(cf. 3).4 7 D. J. O'Kane and W. W. Umbreit, J. Biol. Chem., 1942, 142, 25.4 8 Cf. Ann. Reports, 1940, 37, 417.4p F. Lipmann, J. Biol. Chem., 1940,134,463 ; Symposium on Respiratory Enzymes,50 F.Lipmann, J. Biol. Chem., 1944, 155, 55.51 F. Lipmann and L. C. Tuttle, &id., 1944, 153, 571.52 F. Lipmann, Ann. Rev. Biochem., 1943, 12, 1.53 J . Amer. Chem. SOC., 1940, 62, 1597.54 J . Biol. Chem., 1942, 146, 391.Univ. of Wisconsin Press, 1941, 145; Federation Proc., 1942, 1, 122236 BIOCELEMISTRY.acid,55 has now been shown to be reversible,56* 57 13C of radioactive formicacid appearing in the carboxyl group of the keto-acid. Since in the bacteria,though not in these cell-free extracts, carbon dioxide is normally in equi-librium with formic acid, the mechanism of a new method of carbon dioxidefixation into the carboxyl of pyruvic acid is revealed by these experiments.The synthetic reaction resulting in carbon dioxide fixation is able to proceedonly because the dehydrogenation product is acetyl phosphate, and not thefree acid : the formation of acetic acid from pyruvate would result in anenergy loss of some 15,000 ~ a l s .~ ’As yet the evidence of the formation of acetyl phosphate or homologouscompounds in animal tissues is indirect, based on reactions such as acetyl-ations in viv0,68 in isolated tissues 59 or in tissue extracts.60 However, theoxidative formation of energy-rich phosphate bonds in such material hasbeen repeatedly proved by the phosphorylation of adenylic acid, creatine, orglucose 6-phosphate7 for example.5* 32* 33* 61The oxidation by acytochrome system of a-ketoglutarate to succinate 62 causes esterificationof 3 atoms of phosphorus for each oxygen atom consumed: in this 4-Cdicarboxylic acids did not function as hydrogen carriers.Obviously anoxidative decarboxylation as formulated above could give only a 1 : 1 ratio.Even the highly speculative assumption of a diphosphate formation leaves adeficiency of one phosphorus atom esterified. Perhaps even more remark-able is the observation 63 that the whole pyruvic molecule is oxidised in heartextract with precisely the same efficiency; P/O ratio = 3. This indicatesthat no less than 15 high-energy phosphate bonds are established by theoxidation of 1 mol. of pyruvate, which at the level of 11,000 cals./bond showsthe efficiency of conversion of oxidation into phosphate bond energy to benearly 60%. If the course of pyruvate oxidation through the “ tricarboxylicacid cycle ” 64 be accepted, those of the five dehydrogenation reactionsinvolved which have been shown to be accompanied by phosphorylation areas follows : a-Keto-acid oxidation (occurring twice) could generate 2 x 3 - ph 62 ; succinate --+ fumarate not more than 1 - ph 63 ; malate to oxalo-55 M.Silverman and C. H. Werkman, Proc. Soc. Exp. Biol. Med., 1940, 43, 777;M. F. Utter and C . H. Werkman, Arch. Biochem., 1943, 2, 491.56 M. F. Utter, C. H. Werkman, and F. Lipmann, J . Biol. Chem., 1944, 154, 723.157 F. Lipmann and L. C. Tuttle, ibid., p. 725.5 8 E. A. Doisy, jun., and W. W. Westerfield, ibid., 1943, 149,229; G. J. Martin andE. H. Rennenbaum, ibid., 1943,151, 417; K. Block and D. Rittenberg, ibid., 1944,155,243.59 P. J.0. M m , M. Tennenbeum, and J. IT. Quastel, Biochem. J . , 1939, 33, 1506.60 D. Nachmansohn and A. L. Machado, J . Neurophysiol., 1943, 6, 397; D. Nach-maJlsohn, H. M. John, and H. Waelsch, J. Biol. Chem., 1943,150,485.61 V. A. Belitzer and E. I?. Tsibakowa, Biochimiu, 1939, 4, 616 (cf. footnote, p. 493,ref. 63); S. Ochoa, J. Biol. Chem., 1941, 138, 751; S. P. Colowick, H. M. Kalckar, andC. F. Cori, ibid., 1941, 137, 343.The efficiency of this process is unexpectedly high.62 S. Ochoa, ibid., 1943, 149, 577; 1944,155, S7.63 Idem, ibid., 1943, 151, 493.64 A. H. Krebs, “Advances in Enzymology,” 1943, 3, 191NEUBERGER : THE INTERMEDIARY METABOLISM OF TRYPTOPHAN. 237acefate oxidation generates phosphate bonds (phosphopyruvic acid) to anunknown extent .G5 The remaining dehydrogenation, of isocitrate toa-ketoglutarahe, has not been studied in this respect.Hence, as yet onlyabout half of the 15 ester bonds established in the oxidation of 1 mol. ofpyruvate can be accounted for experimentally, and 5 such bonds are aa manyas could be reasonably expected on the basis of known mechanisms. Itfollows that there must be yet unexplored mechanisms.which enable theenzyme equipment of the cell to tap the large energy range (up to 1.2 v.)between the potential levels of oxygen and of the metabolites, and turn asmuch as 60% of it into phosphate bond energy. F. D.2. THE INTERMEDIARY METABOLISM OF TRYPTOPHAN.The metabolic importance of tryptophan (I) was realised soon afterits discovery by F.G. Hopkins and S. W. Cole in 1901. It cannot besynthesised in the mammal and has to be supplied in the diet. The ratcan utilise d( +)-tryptophan instead of the natural I( -)-isomer for growth ; 2tthis, however, is not the case in the chick.4 The intermediary metabolismof the two isomerides in many species, including the rat, appears to bedifferent and it can be deduced that an optical inversion does not take placeto a great extent under normal dietary conditions. In man ingestion ofd(+), but not of Z(-), -tryptophan leads to the excretion of a substance,possibly indole-3-acetic acid, which can be oxidised to a red pigment.6Deficiency of (I) in the diet of the rat leads to a decrease of serum proteinsand a slight hypochromic anemia.6 Apart from these unspecific changes,which are probably common to all deficiencies of essential amino-acids,cataract of the eye and corneal lesions have been 0bserved.7.~The intermediary metabolism of (I) has yielded a number of interestingcompounds.Kynurenic acid (VI), which was discovered in 1853 byLiebig,g has been isolated from the urine of dogs (as the name implies),rabbits lo and many other species.ll. l2 It is formed from Z( -)-tryptophan,and from indolepyruvic acid, but not from d( +)-tryptophan.l3Another substance was isolated from the urine of rabbits fed on polishedg6 H. M. Kalcker, J. Bwl. Chem., 1943, 148, 127.1 J . Phy~wl., 1901,27, 418.C. P. Berg, J. Bid. Chem., 1934, 104, 373.V. du Vigneaud, R. R. Sedock, and L.van Btten, &id., 1932, 98, 565.A. A. Albanese and J. E. Frankston, J . Biol. Chem., 1944, 166, 101.4 G. I?,. Grau and H. J. Almquist, 3. Nutrit., 1944, 28, 263.6 Idem, ibid., 1943, 148, 299.7 J. R. Totter and P. L. Day, J . Nutrit., 1942, 24, 159.8 A. A. Albanese and W. H. Buechke, Science, 1942,96, 684.Annalen, 88, 125.10 A. Ellinger, 2. physiol. Chem., 1904, 43, 325.l1 \IT. G. Gordon, R. E. Kaufmann, and R. W. Yackson, J . Biol. Chem., 1936, 118,12 R. W. Jackson, ibid., 1939, 131, 469.13 R. Borchers, C. P. Berg, and N. E. Whitman, ibid., 1942, 146, 657.125238 BIOCHEMISTRY.rice and supplied with excess of (I).14 It was assigned by its Japanesediscoverers the name kynurenine and the structure (11). It was shownrecently that structure (11) is incorrect and that kynurenine is representedby (IV).15 (IV) was synthesised, though in poor yield, by condensation ofo-nitrophenacyl bromide with ethyl sodiophthalimidomalonate, followed byacid hydrolysis and reduction.The synthetic material had chemical andoptical properties identical with those of the natural product ; identificationis, however, not quite complete, since the synthetic material has not yet beenresolved. The chain of reactions leading from tryptophan (I) to kynurenicacid (VI) now becomes clear. (I) is presumably oxidised to a-hydroxy-tryptophan (111), a substance so far only found in phalloidin, a toxic peptide,obtained from Aminata phalloides; l6 (111) is then further oxidised to (IV)with loss of carbon dioxide.Under normal dietary conditions this amino-acid is further broken down, probably through the ay-diketo-acid (V), whichrearranges itself to the quinoline derivative (VI).”-- --CH2-CH(NH,)*C0,H - I 11 lloH +2 i,!INH2@\-CO-CH,*CH(NH,)*CO,H + CO,(111.) \/\/NH JStill another substance derived from (I) has been isolated from the urineof rats fed on fibrin.17 The new substance, which was called xanthurenicacid because of’ its yellow colour, was shown to be 4 : 8-dihydroxyquinoline-Z-carboxylic acid (IX). Being an 8-hydroxyquinoline derivative, it formscomplexes with metals and the intense green colour given with ferrous saltsis used for its estimation. (IX) is excreted by rats,17 rabbits,17 and swine,18l4 Y. Kotake and J. Iwao, 2. physwl. Chem., 1931,195, 139.l5 A.Hutenandt, W. Weidel, and W. von Derjugin, Naturwiss., 1942, 30, 51; 2.16 H. Wieland and B. Witkop, Annulen, 1940, 543, 171.l 7 L. Musajo, Atti R. Accad. Lincei, 1935, 21, 368; Gazzetta, 1937, 67, 165, 171, 182.l8 G. E. Cartwright, M. M. Wintrobe, P. Jones, M. Lauritsen, and S . Humphreys,physiol. Chem., 1943, 279, 27.Bull. Johns Hopkins Hosp., 1944, 75, 35NEUBERGER : THE INTERMEDIARY METABOLISM OF TRYPTOPHAN. 239but not by d ~ g s . l ~ ~ l9 d( +)-Tryptophan, indolepyruvic acid and kynurenicacid do not give rise to excretion of (IX). L. Musajo and M. Minchilli 2oclaim that kynurenine does not form (IX), but Reid et state that it does.The immediate precursor of (IX) may therefore be either a dihydroxy-tryptophan (VII) or the hydroxykynurenine (VIII).A--- CH,*CH( NH,)*CO,H /\ CO*CH,*CH(NH,)*CO,HI 11 lloH !+)kH2(VII.) OH (VIII.)\/\/OH !\)kH26 H NHOH Xanthurenic acid and kynurenine are not excreted by animals rearedon normal diets, but the exact dietary deficiency necessary to produceexcretion of these substances was not known until Lep-OH kovsky et aZ.19 showed that the green pigment found in/\/\ the urines of pyridoxine deficient rats was the iron com- ' " 'CO& plex of (IX).Later work 18# l9 established the followingfacts : Xanthurenic acid is only found in pyridoxine- OH Ndeficient animals fed on diets containing I( -)-tryptophanand the amount excreted is proportional to the trypto-phan intake. Addition of pyridoxine to or omission of tryptophan fromthe diet leads to the disappearance of (IX) from the urine.Similarly,kynurenine excretion both in rats and in swine 18,19 depends on pyridoxinedeficiency. It seems fairly certain that kynurenine a t least is a normalintermediary product of tryptophan metabolism and it appears that inpyridoxine deficiency its further breakdown is impossible. It is likely thatpyridoxine is the prosthetic group of an enzyme responsible for the furtheroxidation of kynurenine and (IV) is excreted unchanged in its absence.(IX) may possibly be a pathological product. The fact, however, thatanimals on normal diets can metabolise (IX) may indicate that the formationof this 8-hydroxyquinoline derivative is an additional normal pathway oftryptophan metabolism, at least in certain species.Kynurenine has recently acquired considerable interest in anotherdirection. E.L. Tatum 21 had found that a hormone which stimulates theformation of a brown pigment in the eyes of Drosophda and other insects wasrelated to tryptophan. The formation of this hormone is controlled by aspecific gene and can be replaced by a substance formed by bacteria fromtryptophan. This hormone was isolated by Butenandt and co-workers l5and identified as kynurenine. E. L. Tatum and A. J. Haagen-Smit 22showed in 1941 that their crystalline product was a complex of sucrose andkynurenine. The gene is apparently responsible for the ability of theorganism to convert tryptophan into kynurenine.\/\/(IX.)10 S. Lepkovsky, E. Roboz, and A.J. Haagen-Smit, J . Biol. Chem., 1943, 149, 195;*O Qazzetta, 1940, 70, 307.21 Proc. Nut. Acad. Sci., 1939, 25, 486; E. L. Tatum and G. JV. Beadle, Science,22 J . Riol. Chern., 1911, 140, 575.D. F. Reid, S. Lepkovsky, D. Bonner, and E. L. Tatum, ibid., 1944, 155,299.19.10, 91, 458240 BIOCHEMISTRY.Another interesting observation has recently been reported by E. I,.Trttum and D. Banner.% These authors found that by X-ray treatment amutant of Neurospora can be produced which requires tryptophan for growth.This amino-acid can be replaced by a combination of indole and 2( -)-serino,but not by any other possible intermediates. The growth of this deficientstrain in the presence of indole is proportional to the serine added. Theformation of tryptophan was actually demonstrated by isolation.It isassumed that a direct combination of these two compounds takes place.It is also suggested that the decomposition of tryptophan brought aboutby E . coli 24 which leads directly to indole may be a reversal of the reactionfound in Neurospora. A. N.3. HORMONES.The Thyroid Gland.Thyroid G l a d and Iodine Metabolism.-The application of radioactiveiaotopes of iodine to the study of the production of thyroxine by the thyroidgland has been very fruitful.The investigations may be divided into two main classes : (a) thoseinvolving the administration of radio-iodine to the intact animal, followedby removal of the thyroid gland and other tissues for examination somehours or days later; and ( b ) those in which the uptake of radio-iodine byrespiring slices of isolated thyroid tissue is examined.That iodine is preferentially retained bythyroid tissue has been amply confirmed in experiments in which radio-iodine, administered orally or parenterally, has been found in greater con-centration in the thyroid gland than in any other tissue of the body withina few hours of administration.l.2a 3* I. Perlman, I. L. Chaikoff, and M. E.Morton 3 distinguish between “ tracer ” doses of radio-iodine, which containtoo little iodine for detection by ordinary chemical means, and what may betermed ‘‘ physiological ” doses of radio-iodine, measured in mg., in which aminute amount of radio-active material is mixed with ordinary potassiumiodide a8 a carrier, When a relatively large dose (e.g., 2.6 mg./kg.of bodyweight) of potassium iodide containing some radio-iodine is administered toa rat, 50-60% may be excreted in the urine and faeces within the next 24hours, and only about 1% may be found in the thyroid gland. Neverthelessthe thyroid gland collects, per g. of tissue, over a hundred times as muchiodine as other tissues in the body and retains it longer, one-half still beingpresent a t the end of 24 hours.3 When such large doses are administered,the thyroid tissue may become satorated with iodine and then lose its capacityto fix this element selectively, though this power may be regained within a*‘ D. D. Woods, Biochem. J., 1935,29,640.1 S. Hertz, A. Roberts, and R. D. Evans, Proc. SOC.Exp. Biol. Med., 1938, 38, 610.2 (a) J. G. Hamilton and H. M. Soley, Amer. J. Physiol., 1939, 137, 557; ( b ) idem,(a) Studies on the intact animal,*3 Proc. Nut. Acad. Sci., 1944, 30, 30.n’hid., 1940, 181, 135.J . Biol. Ghem., 1911, 139, 433.4 C. P. Leblond and P. Sue, Amer. J. Physiol., 1941, 184, 64YOUNG : HORMONES. 241few days.4 Radio-iodine in a non-ionic form (iodate or di-iodotyrosine) isnot fixed by the thyroid t i ~ s u e . ~The administration of a tracer dose of iodine to an animal labels itscirculating iodine without significantly increasing the total amount in theblood and tissues. Such tracer doses are rapidly taken up by rat thyroidtissue, 16-20~0 being retained therein within 2 hours, and a maximum of65% 3040 hours after administration.3 Thereafter the amount in thethyroid slowly dim in is he^.^ The fact that a tissue constituting only about0.01% of the body weight and containing approximately 20% of all theiodine in the body takes up as much as 65% of the tracer dose of this elementin a relatively short time suggests that the turnover of iodine by the thyroidgland is rapid, and that circulating iodine can be removed by thyroid tissuemore rapidly than it oan come into equilibrium with tissues other than thatof the thyroid.3 The specific activity of the thyroid gland in this respect isstrikingly emphasised.When tracer doses of iodine are given to a sheep, 2-13y0 is found inthe thyroid gland 4 hours later.Of this about 9% is in the inorganic form,85% as 3 : 5-di-iodotyrosine, and 6% as thyroxine.Forty-eight hours afteradministration 3 0 4 0 ~ 0 of the tracer dose is found in the gland, about 13%of this being in the inorganic form, 78% as di-iodotyrosine and 9% asthyroxine. Thus at the end of 48 hours 3 4 % of the dose of administerediodine is found to be in the form of thyroxine.6 These fhdings, which havebeen adequately confirmed, are compatible with a rapid formation of di-iodo-tyrosine, followed by a slower conversion of the latter into thyroxine.60 7Hypophpectomy depresses the thyroid’s ability to collect radio-iodine and toform di-iodotyrosine and thyroxine, but the administration of pituitary thyro-tropin or exposure to cold both enhance these effect^.^*^ In children witha myxmdema not associated with goitre the thyroid collects less administeredradio-iodine than does the normal gland, whereas the thyroid of a child witha goitrous myxcedema collects more than norma1.O In Graves’ disease thehyperactive thyroid gland fixes as much as 80% of a relatively large (2 mg.)dose of administered radio-iodine,1° and, according to C.P. Leblond,ll thehyperplastic thyroid of iodine deficiency is also able to collect administeredradio-iodine more rapidly than octn the normal gland. It seems probablethat increased efficiency in the collection of iodine is associated with pituitaryW. T. Salter, Physiol. Rev., 1940, 20, 345.I. Perlman, M. E. Morton, and I. L. Chaikoff, J. Biol. Chem., 1941,139, 449.7 (u) W. Msnn, C. P. Leblond, and S. L. Warren, ibid., 1942, la, 905; ( b ) A.Lein,Endocrinology, 1943, 32, 429.* (a) M. E. Morton, I. Perlman, E. Anderson, and I. L. Chaikoff, ibid., 1942, 30,495; ( b ) M. E. Morton, I. Perlman, and I. L. Chaikoff, J . Biol. Citem., 1941, 140, 603;(c) C. P. Leblond, Amat. Rec., 1944, 88, 285; ( d ) C. P. Leblond, J. Gross, W. Peacockand R. D. Evans, Arner. J. PhyeioE., 1944, 140, 671.9 J. G. Hamilton, M. H. Soley, and K. B. Eichorn, Arner. J. Dis. ChiU., 1943, 66,495.10 S. Hertt, A. Roberts, and W. T. Salter, J . Clin. Inuetlt., 1942, 81, 25.l1 Rev. Canadian Bwl., 1942, 1, 402242 BIOCHEMISTRY.stimulation of thyroid activity, a phenomenon not observed in myxcedemaresulting from pituitary deficiency.The therapeutic value of radio-iodine retained by the thyroid in Graves'disease l2 and by metastases of thyroid carcinoma 13 is apparently dis-appointing, but radio-iodine is of proven value for the assessment of thecompleteness of thyroidectomy l4 and in examination of the functionalactivity of the developing thyroid gland.15 In experiments of this typefunctional thyroid tissue can be detected radioautographcially, i.e., by itspower to record its presence on a suitable photographic plate some hoursafter the administration to the animal of a tracer dose of radio-iodine.When small amounts of radio-iodine in the form of potassium iodide are added to bicarbonate-Ringer solu-tion in which are suspended surviving slices of thyroid gland, 70% of the addedradio-iodine is present as di-iodotyrosine 3 hours later, and 12 yo as thyroxine,16though the addition of excess of inorganic iodide (non-radioactive) to themedium inhibits the formation of both di-iodotyrosine and thyroxine fromadded radio-i0dine.l' Thyroid gland which has been minced is much lesseffective, and a smooth suspension of finely divided tissue is almost completelyinactive.16 These results show clearly that the process of conversion of theadded radio-iodine into the organic form in which it is found depends on theintegrity of cell function, and is not merely the result of a chemical inter-change of radio-iodine.The process is inhibited by the exclusion of air, andby addition to the medium of small amounts of cyanide, sulphide, azide andcarbon monoxide, all of which inhibit the cytochrome-cytochrome oxidasesystem.18 But 10-3~-azide completely inhibits the formation of di-iodo-tyrosine and thyroxine by the slice while permitting the collection andretention in the inorganic form of 60% of the radio-iodine of the medium.18This and other similar evidence suggests that the thyroid mechanism for thecollection of inorganic iodine can be differentiated from that responsible forthe conversion of inorganic iodine into the organic form.Non-thyroidal Production of Thyroid-active Substances.-The belief thatthyroxine-like substances may be formed from administered iodine in thetissue of an animal lacking a thyroid gland l9 has been confirmed by thedemonstration that both di-iodotyrosine and thyroxine are produced from12 (a) S.Hertz and A.Roberts, J. CEin. Invest., 1942, 21, 624; (b) J. G. Hamilton13 A. S. Keston, R. P. Ball, V. K. Franz, and W. W. Palmer, Science, 1942, 95, 362.14 W. 0. Reinhardt, Proc. SOC. Exp. BioE. Med., 1942, 50, 81.1 5 (a) A. Garbman and H. M. Evans, a i d . , 1941, 47, 103; ( 6 ) idem, Endocrinology,18 (a) M. E. Morton and I. L. Chaikoff, J. Biol. Chem., 1942, 144, 565; ( b ) idem,1 7 M. E. Morton, I. L. Chaikoff, and S. Rosenfeld, ibid., 1944, 154, 381.18 (a) H. Schachner, A. L. Franklin, and I. L. Chaikoff, ibid., 1943, 151, 191; ( b )idem, Endocrinology, 1944, 34, 159.19 (a) A. Chapman, ibid., 1941, 29, 686; (b) A. Chapman, G. M. Higgins, and F. C.Mam, J. EndocrinoE., 1944, 3, 392; ( c ) I. Perlman, M. E. Morton, and I. L. Chaikoff,Endocrinology, 3942, 30, 487.(b) Studies on slices of thyroid tissue.and J.H. Lawrence, ibid., p. 624.1943, 32, 113.ibid., 1943, 147, 1YOUNG : HORMONES. 243radio-iodine by the fully thyroidectomised rat.20 The minute amounts ofthese substances containing radio-active iodine were identified by theirconsistent behaviour when relatively large amounts of non-radioactiveauthentic substances were added as carriers during the processes of fraction-ation.2O These results are of particular interest in view of the discovery,by W. Ludwig and P. von Mutzenbecher (1939),21 that preparations ofiodinated casein containing 6-43 yo of organically bound iodine, togetherwith certain other iodinated proteins, possess the physiological activity ofthyroid protein and yield, on alkaline hydrolysis, mono-iodotyrosine (cf.22),di-iodotyrosine, and pure thyroxine (100-200 mg./100 g. of iodocasein) .21The correctness of these findings has been completely confirmed.23 Thephysiologically active iodinated proteins were prepared by the addition of alimited amount of iodine to a solution of the protein in dilute sodium bicar-bonate, followed by incubation a t 37" for some hours. For maximal thyroidactivity two atoms of iodine should be taken up for each molecule of tyrosinein the protein (Turner et P. von Mutzenbecher (1939) also showedthat incubation at 37' of 3 : 5-di-iodotyrosine in alkaline solution (pH 8-9)for 1-2 weeks resulted in the formation of thyroxine in gross yield of about0.2570,24 and this finding, too, was amply 26* 27* 28 Von Mutzen-becher also observed that casein which had been iodinated in the cold inammoniacal solution exhibited little or no biological activity, but that thedevelopment of biological activity resulted from incubation of this iodinatedprotein in alkaline solution for some days.24 Finding that the formationof thyroxine from di-iodotyrosine by alkaline incubation was accompaniedby a fall of pH (e.g., from 8-8 to 8.4) and the formation of iodide, and furtherthat the addition of sodium sulphite inhibited the formation of thyroxinewhereas the addition of sodium thiosulphate did not, von Mutzenbechersuggested that the oxidation of the di-iodotyrosine to thyroxine might beassociated with- the splitting of iodine from di-iodotyrosine in the form ofhyp~iodite,~~ a suggestion that received some independent support .26 Onthe other hand, the reaction, which is inhibited by the presence of potassiumferricyanide and of 3 : 5-di-iodo-4-hydroxybenzoic acid, requires the presenceof air,27 and C.R. Harington and R. V. Pitt Rivers 2a find that it is inhibited2o M. E. Morton, I. L. Chaikoff, W. 0. Reinhardt, and E. Anderson, J . Biol. Chem.,1943, 147, 757.21 2. physwl. Chem., 1939, 258, 195.22 C. R. Harington and R. V. Pitt Rivers, Biochem. J., 1944, 38, 320.23 ( a ) Idem, Nature, 1939,144, 205 ; (b) E. P. Reineke, J. Dairy Sci., 1942, 25, 702 ;(c) $1. P. Reineke and C. W. Turner, Univ. Missouri Agric. Exp. Stat., 1942, Res. Bull.355, 88 pp.; ( d ) E. P. Reineke, M.B. Williamson, and C. W. Turner, J . BioE. C'hem.,1943, 143, 285 ; (e) E. P. Reineke and C. W. Turner, J. Clin. Endocrinol., 1943, 3, 1 ;(f) E. P. Reineke, M. B. Williamson, and C. W. Turner, J: Biol. Chem., 1943, 147, 115.24 2. physiol. Chem., 1939, 261, 253.26 P. Block, jun., J. Biol. Chem., 1940, 135, 51.26 T. B. Johnson and L. B. Tewkesbury, jun., Proc. Nut. Acad. Sci., 1942, 28, 73.27 A. E. Barkdoll and W. F. Ross, J. Amer. Chem. SOC., 1944, 66, 898.28 ( a ) C. R. Harington, J., 1944, 193; (b) C. R. Harington and R. V. Pitt Rivers,Biochem. J., 1944, 38, PPOC. xxxiv24.4 BIOUHBIMISTRY .in conditions under which the formation of hypoiodite would occur mostreadily. The simplest explanation might be that free iodine, which convertstyrosine into di-iodotyrosine, a h brings about the oxidation of the latterto thyroxine, but von Mutzenbecher's observation that the oxidation occursin the presence of thiosulphate is not in easy agreement with this hypothesis.C. R.Harington 2k has recently obtained a net yield of 3.4% of thyroxineby directly oxidising di-iodotyrosine in alkaline solution (pH 9-10} a t 100'with hydrogen peroxide, the thyroxine formed being continually shaken outwith butyl alcohol (a solvent into which di-iodotyrosine pasees to only a,slight extent from alkaline solution) in order to protect it against decom-position under the somewhat drastic conditions employed. These experi-ments unequivocally demonstrate that thyroxine can be formed fromdi-iodotyrosine by direct oxidation.The fact that disiodotyrosine can be so easily converted into thyroxinein vitro suggests the possibility that such a conversion may also easily takeplace in the body, but pure di-iodotyrosine possesses little or no thyroxine-like activity when administered to animals.On the other hand, J. Lermanand W. T. Salter 29 claim that the physiological activity of dried thyroidgland, which contains di-iodotyrosine, is proportional to its total iodinecontent and not to its variable proportion of thyroxine iodine. It seemspossible, therefore, that administered peptide-linked di-iodotyrosine may beconvertible into thyroxine in the body.Mecha,nisrn of Production of Thyroxine in vitro and in vivo.-Haringtonand Barger (1927) pointed out that thyroxine might be formed in vivo bythe coupling of two molecules of di-iodotyrosine, and this idea has recentlybeen developed by Johnson and Tewkesbury (1942) 26 to explain the form-ation of thyroxine by the prolonged incubation of di-iodotyrosine in alkalinemedium.These investigators recall that Pummerer eb aL30 oxidised p-cresol/ Y o x&(IIIa.) (IIIb.)PV.129 J. Pharm. Exp. Thsr., 1934, 1, 298.30 (a) R. Purnmerer, D. Melamed, and H. Puttfarehen, Ber., 1922, 55, 3116; ( b )R. Pummerer, H. Puttfarchen, and P. Schopflocher, W., 1925, 58, 1808YOUNG : HORMONES. 245with potassium ferricyanide in alkaline solution and obtained two mainproducts : a ketotetrahydrodibenzofuran derivative (IIIa ; R = MeX = H) and 2 : 2tdihydroxy-5 : 5’-dimethyldiphenyl (IV; R = Me, X = H).Yummerer explained the formation of (IIIa) as resulting from the rearrange-ment of the unstable quinonoid compound (11; R = Me, X = H), which,he suggested, was formed intermediately.Johnson and Tewkesbury pointedout that, if an analogous reaction is assumed for di-iodotyrosine, rearrange-ment of the intermediate quinonoid compound [I1 ; R = CH2*CH(NH2)*C02H(alanyl), X = I] to a stable tetrahydrodibenzofuran derivative cannot takeplace, being prevented by the presence of iodine atoms ortho to the phenolichydroxyl group. They suggest that the molecule may therefore stabiliseitself by splitting off the alanyl side chain attached to the carbon carryingthe ether oxygen, with the formation of thyroxine (IIIb; R = alanyl,X = I), and claimed to be able to identify pyruvic acid and ammonia amongthe products of the alkaline incubation of di-iodotyrosine, these presumablyhaving been formed by the decomposition of the discarded alanyl side chain.Subsequently W.W. Westerfeld and C. Lowe31 showed that the two com-pounds found by Pummerer et al. to be formed by the oxidation of p-cresolwith ferricyanide were also obtained by oxidation of this substance withhorseradish peroxidase and hydrogen peroxide.I n an interesting theoretical discussion of the mechanism of the reactionspostulated by Pummerer et al., Johnson and Tewkesbury, and Westerfeldand Lowe, Harington 2h considers the implications of the assumption thatp-cresol and di-iodotyrosine are oxidised in the form of the phenoxide ionand that the oxidation consists in the removal of an electron from the ion,followed by reaction of the free radial so formed.Harington points outthat the phenoxide ion would be expected to resonate among at least threestructures (V), (VI), and (VII), and that the oxidation of a p-substitutedphenoxide ion might be assumed to consist in the removal of one electronfrom the oxygen atom of form (V), giving (VIII), and from the carbon atomspara and ortho, respectively, to the carbon carrying the oxygen atom offorms (VI) and (VII), giving the corresponding free radicals (IX) and (X).With p-cresol the interaction of (VII) and (IX) would give (11; R = Me,R R R R R R\* I/\ /\\ : - I/\ A x /\I/\ 1x XI1 llx xll I/x x(Jx XI1 v Ilx x ( y - X(/ \/ \/.II(V.1 (VI.) (VII.) (VIII.) (IX.) (X., : OII: OI : o * II: OII.. .b.- .. .. : OX = H), which would rearrange to give (IIIa). Interaction of two moleculesof (X) would give (IV). With di-iodotyrosine (R = alanyl, X = I ) , com-pound (11), formed by the interaction of (VIII) and (IX), could not stabiliseas ( I I a ) owing to the presence of the iodine atoms ortho to the phenolic31 J . BioE. Chem., 1942, 185, 463246 BIOCHEMISTRY,hydroxyl group, and would therefore give (IIIb) (thyroxine). For similarreasons (IV), formed from p-cresol, would probably not arise fromdi-iodotyrosine.Chaikoff la suggests that the formation of both di-iodotyrosine andthyroxine by the thyroid gland is linked with aerobic oxidations in whichthe cytochrome-cytochrome oxidase system is involved, but E.W. Dempsey 32believes that peroxidase is present in the thyroid follicular cells and that thisenzyme may catalyse the conversion of di-iodotyrosine into thyroxine. It ispossible that hydrogen peroxide is formed in living cells by the action offlavoprotein systems, and any peroxidase present might catalyse the oxid-ation of iodide ions to free iodine and then assist the oxidation of the di-iodotyrosine, thus formed, to thyroxine. With milk, which contains theflavoprotein system xanthine oxidase, peroxidase, and the readily iodisableprotein casein, A. S. Keston 33 finds that the addition of xanthine as substratefor the xanthine oxidase system, together with a small amount of radio-iodine in the form of iodide ion, results in the rapid formation of organicallybound radio-iodine.This may provide a model for further investigationof the mechanism for the organic incorporation of iodine in animal cells.C. R. Harington 34 supports the simplest view, namely, that " the essentialbiochemical reaction leading to the synthesis of thyroxine may be the liber-ation of iodine from iodide by an oxidising enzyme system; if this were tooccur conditions would be set up, namely, the presence of iodine in a faintlyalkaline medium, which would not only be suitable for the iodination oftyrosine but would be analogous with those which . . . will effect the formationof thyroxine from di-iodotyrosine in vitro." 34 Certainly the ease withwhich thyroxine can be formed from tyrosine in witro in the absence of enzymesbut under physiological conditions not only emphasises the possibility thatthe formation of thyroxine from tyrosine and iodine, in the thyroid glandand elsewhere, may be a non-enzymic process but also allows considerationof the simplest hypothesis concerning the r61e of the thyroid, namely, thatthe primary function of this gland is the collection of circulating iodine.Goitrogenic Substances.-For many years the existence has been realisedof substances, both naturally occurring and artificial, which are capableof inducing enlargement of the thyroid gland on experimental administrationto animals, and until recently it has been accepted that the goitrogenicaction of such substances is neutralised by the addition of iodine to the diet.In 1941 J.B. MacKenzie, C. G. MacKenzie, and E. V. McCollum 35 reportedthat sulphaguanidine, employed to combat intestinal infection, produced aremarkable enlargement of the thyroid gland in the rat, and in the followingyear J. B. MacKenzie and C. G. MacKenzie showed that this goitrogenicactivity was shared by a series of sulphonamides and t h i ~ u r e a s . ~ ~ Thethyroid hypertrophy, which was accompanied by a fall in basal metabolic32 Endocrinology, 1944, 34, 27. 33 J . Biol. Chern., 1944, 153, 335.34 Proc. Roy. SOC., 1944, B, 132, 223. 35 Science, 1941, 94, 518.36 (a) Federation. Proc., 1942, 1, 122; ( b ) Endocrinology, 1943, 32, 185; ( c ) JohnsHopkins Hosp. Bull., 1944, 74, 86YOUNG : HORMONES.247rate, was not prevented by the administration of iodine but was inhibitedby the injection of thyroxine.36 These findings were quickly c0nfirmed,~7and analogous results with substituted thioureas 3 7 v 3 8 p 39 and natural goitro-gens reported. The thyroid hyperplasia induced by these goitrogens wasaccompanied by signs of increased activity of the anterior pituitary gland,and was lacking in hypophysectomised 3 7 1 The suggestionwas then made that these substances depressed thyroid hormone production,and that the thyroid hyperplasia was secondary to increased pituitaryactivity evoked by the About the same time it was observedthat the prolonged administration of potassium thiocyanate to humanbeings could induce the appearance of thyroid goitres, associated with a fallin basal metabolic rate,41 though the development of this type of goitre couldbe prevented by the administration of dietary iodine.Acting on the assumption that thiourea interferes with the productionof thyroid hormone E.B. Astwood 42 successfully treated clinical hyper-thyroidism by the daily administration of thiourea and showed that 2-thio-uracil also was effective. The therapeutic efficacy of this new treatment ofhyperthyroidism quickly received widespread confirmati~n,~~ and it wasshown also that the administration’ of thiourea or thiouracil to experimentalanimals duplicated the effects of thyroidectomy with respect to growth,44metabolism of isolated organ morphol~gy,~~ thyrotropin-induced373 7 ( a ) E.B. Astwood, J. Sullivan, A. Bissell, and R. Tyslowitz, Endocrinology.( c ) E. W, 1943, 32, 210;Dempsey and E. B. Astwood, Endocrinology, 1944, 32, 509.( b ) E. B. Astwood, J . Pharm. Exp. Ther., 1943, 78, 79;38 C. P. Richter and K. H. Clisby, Arch. Path., 1942, 33, 46.39 T. H. Kennedy, Nature, 1942, 150, 233.40 ( a ) W. E. Greisbach and H. D. Purves, Brit. J . Exp. Path., 1943, 24, 171 ; (b)V. I. E. Whitehead, ibid., p. 192.41 ( a ) It. W. Rawson, S. Hertz, and J. H. Means, J . Clin. Invest., 1942, 21, 624;(b) J. L. Kobacker, Ohio Sta. Med. J . , 1942, 38, 541 ; (c) M. P. H. Foulger and E. Rose,J . Arner. Med. ASSOC., 1943, 122, 1072; ( d ) R. W. Rawson, S. Hertz, and J. H. Means,Ann. Int. Med., 1943, 19, 829.4 2 J .Amer. Med. ASSOC., 1943, 122, 78.4 3 ( a ) R. H. Williams and G. W. Bissell, New England J. Med., 1943, 229, 97; (b)H. P. Himsworth, Lancet, 1943, ii, 465; ( c ) R. W. Rawson, R. D. Evans, J. H. Means,W. C. Peacock, J. Lerman, and R. E. Cortell, J . Clin. Endocrinol., 1944, 4, 1 ; ( d ) P. B.Newcombe and E. W. Deane, Lancet, 1944, i, 179; (e) J. L. Gabrilove and M. J. Kert,J . Arner. Med. ASSOC., 1944, 124, 504; (f) E. C. Bartels, ibid., 1944, 125, 24; (9) K. E.Paschkis, A. Cantarow, A. E. Rakoff, A. A. Walking, and W. J. Tourish, J . Clin.Endocrinol., 1944, 4, 179; (h) R. H. Williams and H. M. Chute, New England J . Med.,1944,230,657 ; (i) E. B. Astwood, J. Clin. Endocrinol., 1944,4,229 ; (j) T. H. McOavick,A. J. Gerl, M. Vogel, and D.Schwimmer, ibid., p. 249; ( k ) F. L. Ritchie and B. L.Geddes, Med. J. Aust., 1944, 1, 381 ; ( 1 ) M. H. Sloan and E. Shorr, Endocrinology, 1944,35, 200; (m) E. B. Astwood, ibid., p. 200; ( n ) H. P. Himsworth, C. A. Joll, H. Evans,G. Melton, and S. L. Simpson, Proc. Roy. SOC. Med., 1944, 37, 693; (0) E. M. Martin,Canadian Med. Assoc. J . , 1944, 51, 39; ( p ) J. K. McGregor, ibid., p. 37; ( q ) E. M.Watson and L. D. Wilcox, ibid., p. 29.4 4 ( a ) A. M. Hughes, Endocrinology, 1944, 34, 69; ( b ) R. H. Williams, A. R. Wein-glass, G. W. Bissell, and J. B. Peters, ibid., p. 317.4 5 B. J. Jandorf and R. E. Williams, Arner. J . Physiol., 1944, 141, 91.4 6 C. P. Leblond and H. E. Hoff, Endocrinology, 1944, 35, 229248 BIOCJHEMISTRY.metamorphosis of tadpoles,47 development of fish,48 pigmentation of birdand insulin ~ensitivity.~~ Thiouracil has also been successfullyemployed in an evaluation of the amount of thyroxine secreted by the thyroidgland under different condition^.^^ These results all support the view thatthioureas and thiouracil inhibit the formation of its hormones by the thyroidgland, but do not interfere with the action of the hormone once it has beenliberated into the blood stream.Mechanism of the Action of Thiouracil and of Other Qoitrogens on theProduction of Thyroid Hormone.-The daily administration of thiouracil toyoung rats for 8 days reduces the iodine content of the thyroid gland almostto zero, though the weight of the gland may be increased nearly threef~ld.~lIf the daily administration of thyroxine is now begun, with continuation ofthiouracil treatment, the iodine content of the gland remains low but thefollicles fill with densely staining colloid 51 Similar results followthe removal of the pituitary gland during thiouracil administration.51 Itseems that under these conditions the secreted colloid material containslittle or no t h y r ~ x i n e , ~ ~ .~ ~ and that the incorporation of iodine into thismaterial has been inhibited by the thiouracil.When radio-iodine is injected into rats previously made goitrous by theadministration of thiouracil, the power of the thyroid gland to collect theadministered iodine may be only 10-20y0 of 53e 54 and the form-ation of di-iodotyrosine and of thyroxine is also inhibited.53 The capacityof thiocyanate-induced goitres to collect administered radio-iodine may besupernormal, however,52 a finding which is significant in view of the factthat thiocyanate is an iodine-inhibited goitrogen.A.L. Franklin, I. L. Chaikoff, and S. R. Lerner 55 found that the addition,to the medium in which surviving. slices of thyroid tissue were maintained,of 10-3~-thiouracil, or of a like concentration of thiourea or of potassiumthiocyanate, depressed the ability of the tissue to convert added radio-iodineinto di-iodotyrosine and thyroxine. This concentration of thiourea and ofthiouracil had little effect on the capacity of the slices to collect iodine fromthe medium, although potassium thiocyanate in a similar amount signifi-cantly diminished the collection of added radio-iodine. The latter resultsare a t variance with the data from intact animals cited above.Sulphanil-amide also depresses the formation of di-iodotyrosine and thyroxine in slices4 1 A. M. Hughes and E. B. Astwood, Endocrinology, 1944, 34, 138.48 E. D. Chldsmith, R. F. Nigrell, A. S. Gordon, H. A. Charipper, and M. Gordon,49 RI. Juhn, ibid., p. 278.60 G. J. Martin, Arch. Biochern., 1943, 3, 61.61 E. B. Astwood and A. Bissell, Endocrinology, 1944, 34, 282.52 (a) R. W. Rawson, J. F. Tannheimer, and W. Peacock, ibid., p. 245; ( 6 ) R.Larson, F. R. Keating, jun., R. W. Rawson, and W. Peacock, ibid., 1944, 35, 200; (c)R. TY. Rawson, R. E. Cortell, W. Peacock, and J. H. Means, ibid., p. 301.ibid., 1944, 55, 132.53 A.L. Franklin, S. R. Lerner, and I. L. Chaikoff, ibid., 1944, 34, 265.54 (a) E. J. Baumann, N. Metzger, and I). Marine, ibid., p. 44; (b) A. S. Keston,5 5 I b i d . , 1944, 153, 151.E. D. Goldsmith, A. S. Gordon, and H. A. Charipper, J. Biol. Chem., 1944,153, 241YOUNG : HORMONES. 249of thyroid tissue, without depressing the capacity of the slices to collectiodide from the medium.laI 66As was suggested above (p. 241), the simplest hypothesis regarding thespecific function of the thyroid is that this gland possesses special ability tocollect iodine from the circulation. Since free iodine is presumably theiodinating agent in the formation of di-iodotyrosine from tyrosine, and sinceiodide ions constitute the form in which this element is collected from theblood stream, it seems probable that the first process which the collectediodide ions undergo is enzymic oxidation to free iodine.Inhibition of thisprocess might not only inhibit the formation of di-iodotyrosine and thereforethat of thyroxine but also depress the power of the gland to collect moreiodide. D. Campbell, F. W. Landgrebe, and T. N. Morgan 67 recall E. A.Werner's observation 58 that free iodine can oxidise thiourea to formamidinedisulphide, NH:C(NH,)*S*S*C(NH,):NH, being itself reduced to iodide ionsin the process, and suggest that this may be a mechanism whereby thioureamight interfere with the synthesis of the thyroid hormone. Another possi-bility is that thiourea and other similar goitrogens inhibit the adtion of anenzyme which catalyses the formation of iodine from iodide in the thyroidgland.Thiouracil does not poison cytochrome o ~ i d a s e , ~ ~ though cytochromeoxidase inhibitors do prevent the formation of thyroxine from inorganiciodide in surviving slices of thyroid tissue.18 Thiouracil poisons per-oxidase 32e 59 and polyphenol oxidases 60 and protects p-cresol against enzymicoxidation when present molecule for molecule of substrate.60 Dempseybelieves that, although peroxidase may be concerned in the formation ofiodine &om iodide in the thyroid gland, this enzyme also catalyses the con-version of di-iodotyrosine into thyroxine.32 This belief is based on theobservation by Dempsey and Astwood 37 that di-iodotyrosine, unlikethyroxine, does not prevent the goitrogenic action of thiouracil, the assump-tion being made that thiouracil must therefore inhibit the conversion ofdi-iodotyrosine into thyroxine.Since, however, the thyroid gland appearsto be unable t o utilise administered di-iodotyrosine, for the formation ofthyroxine, in the absence of goitrogenic agents,4* 43 the assumption wouldappear on the available evidence to be of doubtful validity.It may be concluded that thiourea and thiouracil interfere with the form-ation of iodine from iodide ions, either by reducing any iodine formed baokto iodide ions, or by poisoning the enzyme system catalysing the oxidationof iodide ions t o iodine. Whether or not these goitrogens interfere in anyother way with the formation of thyroxine in the body is as yet uncertain.Nature of the Thyroid Hormone.4anzanelli et aZ.61 found that the addi-tion of thyroglobulin, but not of thyroxine, to tissues respiring in vitro'' A.L. Franklin and I. L. Chaikoff, J . Biol. Ch~m., 1943, 148, 719; 1944, 152, 295.5 7 Lancet, 1944, i, 630.J. B. Sumner and G. F. Somers, " Chemistry and Methods of Enzymes," Academics* J., 1912, 101, 2166.Press lnc., New York, 1943.6o F. Chodat and G. Duparc, Helv. Chim. Acta, 1944, 27, 334.(a) A. Canzanelli and D. Rapport, Endocrinology, 1937,2l, 779; (b) A. Canzanelli,R. Guild, and D. Rapport, &id, 1939,25, 707250 BIOCHEM.1STRY.increases the rate at which oxygen is taken up, and a stimulating action ontissue respiration in vitro has also been observed with plasma from patientswith hyperthyroidism.62 These observations suggest that thyroglobulinmight be the circulating thyroid hormone, but immunological tests fail toreveal the presence of this protein in the blood stream under a variety ofOnly under such anabnormal condition as thyroid trauma was thyroglobulin detected in theblood stream 63 and it seems probable that thyroglobulin as such does notnormally leave the thyroid follicles.Proteolytic enzymes are present inthe thyroid gland, and their activity varies under physiological conditions 65and the hydrolysis of thyroglobulin to a less complicated thyroxine-contain-ing molecule is probably a preliminary step in the secretion of the thyroidhormone. Harington, whose earlier results suggested that the secretion ofthe thyroid gland might be a thyroxine-containing peptide rather thanthyroxine itself, has recently reviewed the evidence on this point34 andconcludes that there is no satisfactory reason to abandon the simplesthypothesfs, namely, that thyroxine itself is the circulating hormone.AsHarington and his colleagues had earlier shown,66 the administration to ratsof antisera raised against thyroxyl derivatives of horse-serum aIbumin andglobulin confers resistance against the usual metabolism-increasing activityof administered thyroxine or thyroglobulin. That the administration ofthese antisera was without effect on the metabolic rate of the treated rats,though such treatment prevented the normal action of administered thyroxineand thyroglobulin, was explicable on the basis of the great power of thenormal thyroid gland to respond to a call for increased secretory activity.66Harington34 suggests that the simplest explanation of the facts is that thecirculating antibodies of the passivity immunised animal, possessing sero-logical combining sites adapted to thyroxine, interfere with the access of thelatter to its normal sites of action in the tissues, so that it is most probablefhat the injected thyroxine is present as such in the circulation.He pointsout that this simple interpretation can be avoided only by the assumptionthat injected thyroxine follows the devious route of synthesis into thyro-globulin, followed by release as such (which seems on other grounds to beunlikely) or as a peptide, which is the real hormone, and such a complicatedprocess is a t least unnecessary to account for the immunological phenomenaobserved.34 Harington concludes that thyroxine is " the true thyroidhormone as it circulates in the body." 34 J.H. Means 6' in another recentreview concludes that " the thyroid hormone travels from the thyroid to itsend-organs in a form lower than the protein level, and that it acts upon itsend-organ in a form of higher level than that of the amino-acids. It may64 including that of hyperthyroidi~m.~~W. T. Salter and F. W. Craige, J . Clin. Invest., 1938, 27, 502.J. Lerman, ibid., 1940, 19, 555.64 L. I. Stellar and H. G. Olken, Endocrinology, 19.10, 27, 614.65 A. J. Dziemian, J . Cell.Comp. Physwl., 1943, 21, 339.G6 R. F. Clutton, C. R. Harington, and M. E. Yuill, Biochem. J . , 1938,32, 11196 7 Ann. Int. Med., 1943, 19, 567YOUNG : HORMONES. 251both travel and act in the form of a polypeptide or peptone,” 67 a conclusionalso compatible with the immunological evidence provided by Harington.If one accepts as significant the observation that thyroxine fails to stimulatetissue respiration in vitro whereas thyroglobulin and plasma from patientswith hyperthyroidism are effective under these 62 the simplestexplanation of all the available evidence, including the results of the immuno-logical investigations, appears to be that thyroxine stimulates tissue respir-ation only when it is combined in peptide form, and that it is transportedfrom the thyroid tissues to the gland in this form.In the sea, the liberation of iodine from iodine ions might occur on aminute scale wherever the oxidative catalysts of respiring cells of unicellularorganisms were active.Thus the tissue proteins of a primitive protozoonmight, as the result of the oxidative capacity of its enzyme systems, come tocontain organically bound iodine, in the form of thyroxine, with the aid ofthe mechanisms reviewed above. With Means 67 we may conclude that theelaboration of the thyroid hormone preceded that of the thyroid gland inthe process of evolution, and that the gland developed as an organ specialisedfor the production and subsequent distribution of a substance which originallywas produced in the tissues in general, and which, even in higher animals,can still be made in tissues other than that of the thyroid.Thyroxine, in acombined form, may therefore be a general constituent of living protoplasm,essential for the maintenance of respiration a t the high level which is char-acteristic of the cells of the highly developed metazoon. That being so, wemight regard thyroxine not as a specific internal secretion of one ductlessgland, but as an essential amino-acid.In some respects the position with respect to choline also is analogous.Choline is an essential constituent of the normal body and the body isapparently able to manufacture all but one portion of the molecule of thisimportant substance, namely, the methyl groups. Provided that a sourceof exogenous methyl groups is available to the body, e.g., from methionine,choline can be manufactured in sufficient amount for its particular require-ments, though otherwise this substance becomes an essential food factor andqualifies for the description of vitamin.Similarly, the only portion of thethyroxine molecule that the body cannot provide from its own resources-tyrosine is not an essential amino-acid-is iodine, and once free iodine isavailable the manufacture of thyroxine can proceed. In higher animals thepresence of the specialised thyroid gland is essential if the rate of collectionof iodine and thyroxine production are to keep pace with the demand forthis amino-acid, but in lower animals the tissues in general can probablyproduce in situ all they need, provided that the essential constituent is tohand.As Means points 0 ~ t , ~ 7 man could live happily without a thyroidgland if his food proteins were properly iodinated, and it is true to say thatto the higher animals from which the thyroid gland has been removedthyroxine, or ,z thyroxine-containing iodinated protein, has become anessential constituent of the diet, and might therefore be regarded as a vitaminfor such an animal252 BIOUHEMISTRT .Thyroxine, or a compound containing it in peptide linkage, can beregarded as a hormone. But such a description does not preclude thepossibility of regarding it, from Some points of view, as an essential amino-acid, or as a vitamin or coenzyme. Once more the overriding of boundarieswhich were once thought to divide different departments of scientific activitymay be regarded as the natural concomitant of progress and development.F.G. Y.4. NUTRITION.The excretion of methylated derivatives of nicotinic acid, the relationof pyridoxine to haematopoiesis and iron metabolism, and the nutritionalvalue of “ folio acid ” and vitamin B,, are reviewed in this section.The Excretion of Nicotinic Acid.described the presence in urineof a substance with a characteristic blue fluorescence, which they called F,.Its excretion was related to the availability of nicotinic acid and increased inproportion to the nicotinic acid intake. In view of the uncertainty of theform taken by the substance in urine, the symbol F, is a convenient means ofdenoting it.F, is absent from the urine of pellagrins2 and it slowly dis-appears from the urine of dogs fed upon a nicotinic acid-deficient diet,3whereas the administration of nicotinic acid or its derivatives increases itse ~ c r e t i o n . ~ . ~ The chemical nature of F, has been elucidated and the sub-stance has been isolated as waxy, hygroscopic, needle-shaped crystals, whichin neutral or weakly alkaline conditions have a greenish-blue fluorescence,and in acidic solution a blue.6e7e A substance identical in physical andchemical properties can be obtained by the treatment of nicotinamidemethiodide with baryta. It is .well known that alkalinisation of such acompound (I) will lead to the formation of a quaternary base (11), whichsubsequently may suffer transformation into a carbinol (111) by attachmentof the hydroxyl group to one of the a-positions of the pyridine ring :/NCO*NH, jf\)CO*NH, ./\ CO *NH, /\CO*NH,In 194-0 V. A. Najjar and R. W. WoodA LN)OH A It )O*C,H, \Nf’ Y - ‘NI1 I \Nf/ I’ ,/ OH’ / /c*3 CH3 CH3 CH,w.1 (111.) (11.1 (1.1* 1 PTOC. SOC. Exp. Biol. Med., 1940, 44, 386.2 V. A. Najjar and L. E. Holt, Science, 1941, 93, 20.3 V. A. Najjar, H. J. Stein, L. E. Holt, and C. V. Kabler, J. Clin. Invest., 1942,21,263.6 P. Ellinger and R. A. Coulson, Bwchem. J., 1944, 88, 266.6 J. W. Huffand W. A. Perlzweig, Science, 1943, 97, 538; J . Biol. Chem., 1943,150,7 P. Ellinger and R. A. Coulson, Nature, 1943, 152, 583; Biochem. J., 1943, 37,* V. A. Najjar, V. White, and D.B. N. Soott, Bull. Johns Hopkins H08p., 1944, 74,V. A. Najjar and L. E. Holt, PTOC. SOC. Exp. Bwt. M d . , 1941, 48, 413.395.Proc. xvii.378O'BRIEN : NUTRITION. 253It would appear that the subetance actually isolated from urine is the car-binol. But it is uncertain 697, * whether the fluorogenic substance in urineis the quaternary base, the #-base, a pyridinium salt, or a mixture of thesedependent upon the conditions. The fluorescence observed in vitro is con-sidered' to be due to a mixture of 6-hydroxy-l-methyl-1 : 6-dihydro-pyridine-S-carboxyamide and another carbinol with an o-quinonoid structure.Atmospheric oxygen and ferricyanide oxidise an alkaline solution of F2.8This treatment, which leads to a deep violet fluorescence, might be expectedto cause the formation of a pyridone from the base.An alternative possi-bility is that the fluorescence is due to the formation of a carbinol ether (IV)from the #-base and the isobutanol used to extract F,. Either suggestionwould explain the slow increase in fluorescent intensity of isobutanol extractof F, from alkaline solutions.Upon the fluorescence of the derivative of the pyridinium salt have beenbased methods for its estimation in urine.gDIO,ll The essential feature ofsome of them is a base exchange between the substance and permutit. Theuse of these methods has shown that there is a distinct individual variationand a fluctuation throughout the day in the excretion of F,.5 The pro-portionality between the amount eliminated and the intake of nicotinamidehas led naturally to the development of load tests for gauging nutritionalstatus as regards nicotinamide.Investigation of this kind of test hasdiminished the value of trigonelline excretion as a nutritional index, sinceits determination may include the pyridinium salt, which on acid or alkalinehydrolysis is converted into trigonelline.6s 12 Nevertheless as a nutritionalindex the excretion of F, will need to be used with careful discrimination.Its dependence on the body reserves of methyl donators whose level dependsupon the diet, has been indlcated.12 This view is well attested by experi-ments upon rats, guinea-pigs, and rabbits.13n 14- l5 The feeding of unusualamounts of nicotinamide to rats adversely affected their growth and theirlivers-effects which could be remedied by choline or methionine.Inguinea-pigs and rabbits no ill effects arise from the ingestion of large amountsof nicotinamide. There is a clear difference in the response of these animalsto nicotinamide. The rat excretes the N-methylnicotinamide in the urine ;the rabbit and the guinea-pig do not. Methylation of nicotinamide has beendemonstrated in vitro with rat liver slices.16 A consequence of a high levelof nicotinamide in the diet of the rat is a depletion of its store of methyldonators, the sequels of which are retarded growth and fatty livers. Thereverse of this, diminished stores of methionine and choline from faulty dietcreating low excretion of F,, is thus conceivable.* V.A. Najjar, Bull. Johns Hopkins Hosp., 1924, 74, 392.lo R. A. Coulson, P. Ellinger, and M. Holden, Biochem. J., 1944, 38, 150.l1 J. W. Huff and W. A. Perlzweig, J . Biol. Chem., 1943, 150, 483.l2 H. P. Sarett, ibid., p. 395.l3 P. Handler and W. J. Dann, ibid., 1942,146, 357.l4 P. Handler and F. Bernheimer, J . Biol. Chem., 1943,148, 849.l6 P. Handler, ibid., 1944, 154, 203.W. A. Perlzweig, M. L. C. Bernheim, and F. Bernheim, ibid., 1943,150 401254 BIOCHEMISTRY.The Relation of Pyridoxine to Ancemia.In the last decade accumulative evidence has indicated that deficiencyof the B vitamins, particularly pyridoxine and nicotinic acid, may interruptnormal erythropoiesis. Mention in this review is confined to work of recenttimes, which attempts to define clearly the type of anzmia from blood andbone marrow investigations.The most careful work upon the effect ofpyridoxine deficiency has been done on the pig and the dog.I n dogs a hypochromic anaemia would seem to be a feature of deprivationof vitamin B6.17~18119~20 The anaemia does not respond to iron or copper;yet prompt improvement follows oral or intravenous administration ofcrystalline pyridoxine. The fact that the initial improvement is not alwaysmaintained has led to the suggestion that other factors may beWith progressing severity of the anaemia the plasma iron rises, and rapidlyfalls with the administration of pyridoxine and with the initial bloodregeneration.22I n 1938 Chick and her collaborators 23 showed that omission of the eluatefraction of a liver concentrate, a source of pyridoxine, from a synthetic dietled to a microcytic anaemia and epileptic fits in young pigs.I n pigs lackingthe filtrate fraction a normocytic anaemia developed. Wintrobe and hisco-workers 24v 2 5 9 26 found that pigs fed on a diet supplemented with vitamins Aand D and all the known crystalline B vitamins except pyridoxine developa severe microcytic anaemia which is most clearly hypochromic at its height.As the anaemia progresses, anisocytosis becomes more marked ; large poly-chromatophilic corpuscles and cells containing blue-staining granules makean appearance. An irregular reticulocytosis may also occur. The anzmiais associated with hyperplasia of the bone marrow and an irregular reticulo-cytosis.The ansmia is not haemolytic in type ; no significant changes occurin the serum bilirubin or in the excretion of urobilinogen or urinary porphyrin.Fatty infiltration of the central portion of the hepatic lobules also occurs.Epileptiform convulsions are seen in the majority of B6-deficient pigs. Anoutstanding feature was haemosiderosis of the spleen, liver, and bone marrowand an increase in the serum iron, which is apparently chiefly in the ferric197.l7 P. J. Fouts, 0. M. Helmer, S. Lepkovsky, and J. H. Jukes, J . Nutrition, 1938,16,l8 Idem, Amer. J . Med. Sci., 1943, 199, 163.l9 H. J. Borson and S. R. Mettier, Proc. SOC. Exp. Biol. Med., 1940, 43, 429.2o H. R. Street, G. R. Cowgill, and H. M. Zimmerman, J . Nutrition, 1941, 51, 275.21 Idem, ibid., p.275.J. M. McKibbin, A. E. Schaeffer, D. V. Frost, and C. A. Elvehjem, J . Biol. Chem.,23 H. Chick, J. F. Macrae, A. J. P. Martin, and C. P. Martin, Biochenb. J., 1938, 32,24 M. M. Wintrobe, M. Samter, and H. Lisco, Bull. Johns Hopkins Hosp., 1939, 64,25 M. M. Wintrobe, R. H. Pollis, M. H. Miller, H. J. Stein, R. Alcayago,26 G. E. Cartwright, M. M. Wintrobe, and S. Hymphreys, J . Biol. Chem., 1944,1942, 142, 77.2207.399.S. Hymphreys, A. Suksta, and G. E. Cartwright, ibid., 1943, 72, 1.153, 171O’BRIEN : NUTRITION. 255state. Administration of pyridoxine produced a rapid regeneration of bloodwith a return of the red cells to normal size. This response was accompaniedby a niobilisation of iron, which was indicated by the disappeafance of thehaemosiderosis and a fall in the serum iron.These interesting results clearlyimply a r81e for pyridoxine in iron metabolism. From the fact that in com-bined pyridoxine and iron deficiency no hsmosiderosis or elevated serumiron occurs despite the development of anaemia, it would appear that thedisturbances in iron metabolism are due to increased absorption or decreasedexcretion. This is an interesting possibility, since it is contrary to the ideathat the animal absorption of iron is dependent upon its needs. In manyrespects-the ferrsmia, haemosiderosis, hyperplastic bone marrow andneurological disturbances-the pyridoxine anaemia is similar to perniciousansmia, although it differs in being characterised by a microcytosis and lackof response to liver extract.IYevertheless the study of the mechanism ofBs ansmia may provide some help towards the solution of perniciousanBmia.The possible relationship of the ansmia and kindred symptoms ofvitamin B,-deficiency with tryptophan metabolism has been referred to inDr. Ncuberger’s Report (p. 237).Folk Acid and Vitamin B,.During the last four years it has become evident that certain micro-organisms need for their growth one or more factors distinct from any of theknown vitamins. It has also become apparent that these factors have a r6lein animal nutrition which consists, in the main, in promoting growth, counter-ing the effect of sulphonamides, and in stimulating the formation of the cellsof the blood.I n 1940 E. E.Snell and W. H. Peterson 27 described a factor of acidicnature needed by Lactobacihs msei E ; to it they gave the name norit eluatefactor. From spinach concentrates another acidic factor, named folic acid,was prepared,2* defined as the material necessary for the growth of Xtrepto-COCCUS Zuctis R on a given medium. This nutrilite is abundant in green leavesand occurs in animal tissues and yeast.Williams and his co-workers 29,30131 have now obtained folic acid inamorphous form from spinach. It’is a substance of M.W. about 400, noteasily soluble in organic compounds and extremely labile. Esterification,acylation and methylation destroy its biological activity. It is also sensitiveto oxidation and reduction, and is none too stable in acid or alkaline solution.From analysis it has an approximate empirical formula of Cl,Hl,08N5 andabsorption spectra indicate that it may contain a structural unit similar toxant hop terin .322 7 J .Bact., 1940, 39, 273.29 Idem, ibid., 1944, 66, 267.30 E. H. Frieden, H. K. Mitchell, and R. J. Williams, ibid., p. 269.3 1 H. K. Mitchell, and R. J. Williams, ibid., p. 271.32 H. K. Mitchell: ibid., p. 275.H. K. Mitchell, E. E. SneII, and R. J. WilIiams,J. Amer. Chem. SOC., 1941,63,2288.1256 BIOUHEMISTRY.From most of the work on concentrates it would appear that the noriteluate factor and folic acid are either the same substance or closely similarcompounds: Concentrates of folic acid are active in stimulating the growthof yeast and other organisms, including Lactobacillus casei, and B, L.Hutch-ings, N. Bohonos, and W. H. Peterson have concluded33 that the eluatefactor was similarly of general nutritional significance for the lactic acidbacteria and the growth of Streptococcus Zuctis, From descriptions 31n34 ofconcentrates of folic acid it would appear that, together with folio acid, othersubstances of biological importance are present ; these include p-amino-benzoic acid and xanthopterin, which are capable of counteracting theinhibitory effect of sulphonamides upon the growth of bacteria and rats.The fact that concentrates prepared from different sources stimulate thegrowth of Lactobacillus casei and Streptococcus Zuctis probably led to theinterchangeable use of the terms folic acid and eluate factor.The term" folic acid " may therefore be used to indicate this group of growthstimulants .On animals, concentrates of the eluate factor and folic acid exert effectswhich may be attributable to similar groups of substances. In the chick,SSeluate factor has been found to promote growth; in the rat, folic acid.Z8Concentrates of both factors share with p-aminobenzoic acid the property ofantagonising the noxious effects of sulphonamides, sulphaguanidine andsulphathiazole, which are poorly absorbed from the intestine. Besides pro-ducing a reduction in growth ,86 sulphonamides may cause agranulocytopenia,leucopenia, and often ana?mia and other pathological conditions when theyare incorporated in synthetic diets adequately supplied with ~itamins.~'Their action may be partly due to an interference with enzyme sptems ofthe body or to suppression within the intestine of bacterial synthesis ofessential factors ; folic acid 38 and biotin are synthesised by intestinalbacteria.Both biotin and concentrates of the eluate factor and folk acidcounteract the growth inhibition which is produced by sulph~namides.~~Biotin and folic acid also appear to influence the utilisation of pantothenicacid by the rat. On diets abundantly supplemented with pantothenate andcontaining succinyl sulphathiazole, rats developed the characteristicsymptoms associated with deficiency of this vitamin.40 This change wascorrected by the administration of folic acid and crystalline biotin. Althoughagranulocytopenia and leucopenia, produced in rats by feeding sulphon-amides, respond to crystalline folic acid from different sources,41 the effectof concentrates upon growth and blood formation may not be due solely to33 J .Bid. Chem., 1941,141, 521.35 B. L. Hutchings, N. Bohonos, D. M. Hegsted, C. A. Elvehjem, and W. H. Peterson,36 S. Black, R. S. Overman. C. A. Elvehjem, and K. P. Link, ibid., 1942, 146, 137.37 F. S. Daft, S. S. Ashburn, and H. H. Sebrell, Science, 1942, 96, 322.38 H. K. Mitchell and E. R. Isbell, Univ. Texas Pub. No. 4327, 1942, 125.39 E. Nielsen and C. A. Elvehjem, J. Bid. Chem., 1942, 145;. 713.40 L. D. Wright and A. D. Welch, Science, 1943, 97, 423.4 1 F. S. Daft and W. H. Sebrell, Pub. Health Reps. U.S.A., 1943, 68, 1542.3d H.K. Mitchell, Science, 1943, 97, 442.J . Biol. Chem., 1940, 140, 647O’BRIEN : NUTRITION. 267their folic acid content. I n concentrates obtained from liver, Elvehjem andhis co-workers 42 claimed to have identified a growth factor, vitamin Bll,and a faotor necessary for good feathering in chicks, vitamin Blo, in additionto folic acid. In several respects the properties of vitamins KO and Bllare akin to those of folic acid.In 1940 A. G. Hogan and E. M. Parrott 43 observed that on simplifiedrations chicks developed a macrocytic hypochromic ansmia whioh wasattributed to the lack of a dietary factor, vitamin B,, present in aqueousextracts of liver. A greater incidence of anzemia in chicks is produced byfeeding sulphaguanidine.u Vitamin B, is insoluble in organic solvents,more stable in alkali than in acids, adsorbable on fuller's earth and super-filtrol, and precipitable with metallic salts and phosphotungstic acid 44-properties, in fact, similar to those of folic acid and the eluate fwtor.Itsantianzmic action could not be reproduced by xanthopterin or by the anti-pernicious anaemia factor. Vitamin Bc has now been obtained in the crystal-line form both as the free acid and as the methyl ester.45 Incorporated in asynthetic diet amply supplemented with all the known vitamins, the crystal-line substance prevented retardation in growth (both body weight andfeathering) and the development of anzmia and leucopeniaj6 Givenparenterally, it produced the same effects.46 This observation has beentaken to indicate that vitamin B, produces those effects which have beenclaimed for folic acid and vitamins B,, and Bll.Furthermore vitamin B,was highly active as a growth stimulant for Lactobacillus casei E. This ledto the suggestion that vitamin B,, the norit eluate factor and folic acid arethe same substance.The isolation of other crystalline substances has complicated rather thanclarified the relationship among the microbial and the animal factors. Twocrystalline compounds have been obtained; one from yeast and the otherfrom liver.47 Both are acids with similar absorption spectra and highlyactive towards Lactobacillus casei. There is a striking difference in theiractivities ; towards Lactobacillus casei they are equally active, towardsStreptococcus lactus R the yeast product is half as active as the liver one.Contrary to the behaviour of these crystalline acids, certain concentratesshow activities greater towards Streptococcus than Lactobacilhs. Thesefacts can be harmonised by assuming the existence either of two or moresubstances or of different forms of oneSubstance.In milk and in yeast folicacid may be present in a combined form, inactive to the two micro-organism%.Whole milk is more effective in inhibiting the harmful action of sulphon-4a G. M. Briggs, T. D. Luckey, C. A. Elvehjem, and E. B. Hart, J. BWZ. Ohm., 1943,148, 163; 1944,153, 423.43 Ibid., 1940, 132, 507.I4 B. L. O’Dell and A. G. Hogan, ibid., 1943, 149, 323.J. J. Pfiffner, S. B. Binkley, E. S. Bloom, R.A. Brown, 0. D. Bird, A. D. Emmett,46 C. J. Campbell, R. A. Brown, and A. D. Emmett, J . BWZ. Chem., 1944, 152, 483;47 E. L. R. Stokstad, ibid., 1943, 149, 573.A. G. Hogan, and B. L. O’Dell, Science, 1940, 97, 404.164, 721.REP. VOL. XLI. 258 BIOCHEMISTRY.amides upon rats than would be expected from its low folic acid content.48Yeast extracts have a high vitamin B, activity and low microbiologicalactivity. When submitted to enzymatic hydrolysis, they stimulate thegrowth of the micro-organisms. From such extracts by the same method asthat used in the isolation of the antianzcmic factor, a crystalline compoundhas been obtained which contains the same percentage amount of carbon,hydrogen, and nitrogen as vitamin B,.4g The individualisation of combinedforms of this vitamin and the bacterial growth factors will be an importantstep towards an explanation of the discrepancies which have been observedin the microbial activities of different materials.It may also elucidate therelation of folic acid and vitamin R, to vitamins B,, and Bll.It is too early to say how important the pterins may be in animal nutritionand lack of space prohibits their inclusion here. J. R. P. O’B.5. THE ASSAY OF VITAMINS B, WITH SPECIAL REFERENCE TOMICROBIOLOGICAL METHODS.The necessity of establishing nutritional requirements and levels forvitamins of group B has stimulated investigations of assay methods, and greatstrides have been made in recent years. These methods are of three maintypes : biological, microbiological, and chemical ; and each has its difficultiesand objections.Ideally, the three methods should be so developed thateach furnishes an accurate check on the others. The development of assaymethods provides an interesting example of modern collaborative work ;a number of teams in this country and the United States are engaged in thismanner, and some examples are quoted later.Aneurin.-The stimulatory effect of aneurin on fermentation by livingyeast has been shown to be highly .specific and formed the basis of one ofthe first microbiological methods for the assay of the vitamin as describedby A. S. Schultz, L. Atkin, and C. N. Frey.1 have foundthat by sulphite treatment the fermentation activity of aneurin is completely(99%) destroyed, whilst interfering substances are unaffected.The authorsdescribe a differential method, employing a new fermentometer, in which themeasurement of fermentation activity is determined before and after sulphitetreatment.H. H. Bunzel13 employs the same principle, but a different type ofapparatus. Results are obtained more rapidly and are accurate for amountsof the vitamin as small as 0.01 pg. A modified Warburg technique isdescribed by E. S. Josephson and R. S. Harris.*There is in general good agreement between results by the microbiologicaland the chemical methods, but sometimes differences are observed whenbiological assays are compared. reported48 A. D. Welch and L. D. Wright, Science, 1944, 100, 153.‘ 9 S. B. Binkley, 0. D. Bird, E.S. Bloom, R. A. Brown, D. G. Calkins, C. J. Campbell,a Ind. Eng. Chem. Anal., 1942,14,35.These authorsJ. C. Moyer and D. K. TresslerA. D. Emmett, and J. J. Pfiffner, ibid., p. 36.J . Amer. Chem. SOC., 1937,59,948,3547.Ibid., p. 279. Ibid., p. 755. Ibid., p. 788NORRIS : THE ASSAY OF VITAMINS B. 259assays on a number of frozen vegetables in which they used fermentationand thiochrome methods successfully. in astudy of aneurin in the materials and process of brewing found good agree-ment between results by the fermentation method and the thiochromemethod as described by R. G. Booth.7 Similar satisfactory comparisonswere made in a collaborative study by the Accessory Food Factors Com-mittee of the Medical Research Council8 in which flours and bread wereassayed by several biological methods, the fermentation method, the thio-chrome method and an azo method elaborated by B.S. Platt and G . E.A number of workers have reported assays using other organisms,including Phycomyces,lOI l1 Phycomyces bhkesleeanus,l2- l4 and Staphylococcusa u ~ e u s . ~ ~ These methods, however, have been criticised by C. F. Niven andK. L. Smiley l6 on various grounds. The authors claim that Streptococcussalivarius (Strain S20B) is more suitable. The growth response is deter-mined turbidimetrically, and owing to the extreme sensitivity of the organismno difficulty is experienced due to incidental turbidity of added food extracts.Co-carboxylase is some 40% more active than aneurin, a fact which has notyet found explanation, and which renders enzymatic hydrolysis necessaryfor precise determinations in some foods.The stability of aneurin to heat has been studied by B.W. Beadle, D. A.Greenwood, and H. R. Kraybill.17 Stability is a function not only of thehydrogen-ion concentration of the solution, but also of the particularelectrolyte system employed. Results were obtained by chemical andspectrographic examination and indicated that for a heating period of onehour at pH 5.4, there was 100% destruction a t the boiling temperature inthe presence of borates, as compared with 57% in unbuffered solution, 10%in the presence of acetates, and 3% where phosphates were used. R. G .Booth l8 confirms many of these findings and has extended his observationsto co-carboxylase, which he finds very much less stable than aneurin a t thesame pH.Destruction of aneurin is not primarily an oxidation effect,although, as copper can catalyse destruction, oxidation may be involved.An everyday application of work of this type concerns the losses of thevitamin which occur in cooking. Booth found that his estimate of lossagreed reasonably well with published figures.Ribo$avin.-Although both the microbiological and the fluorimetricmethods for assay of riboflavin have yielded results in reasonable accordR. H. Hopkins and S. Wiener~10cls.gJ . Inst. Brew., 1944, 41, 124.Biochem. J . , 1943, 37, 433.7 J . SOC. Chein. Ind., 1940, 59, 181.Ibid., p. 439.lo M. Malm and H. Lundeen, Svensk Kem. Tid., 1941, 55, 246.l 1 J.Lehmann and H. E. Nielsen, Acta Med. Skand., Suppl., 1941, 123, 374.l2 W. H. Schopfer and A. Jung, Compt. rend., 1937, 204, 1500.l3 J. Bonner and J. Erickson, Amer. J . Bot., 1938, 25, 685.l5 P. M. West and P. W. Wilson, Science, 1938, 88, 334.l o J . Biol. Chem., 1913, 150, 1.l 7 Ibid., 1943, 149, 339, 349.J. Meiklejohn, Biochem. J., 1943, 37, 349.Biochein. J . , 1943, 37, 518280 BIOCHEMISTRY.with those obtained by biological methods, much evidence has accumulatedthat interfering substances may be present in natural producfs. It isnecessary that each type of product should be treated in relation to its ownpecubrities and the problems arising therefrom. I n the fluorimetric method,originally developed by A. Z. Hodson and L.C. Norris,lg later modified byV. A. Naj jar,2o pigments and non-flavin fluorescent aubstances must eitherbe removed or allowed for.The original microbiological assay method of E. E. Snell and F. M.Strong 21 used Lactobacillus cusei-c as test organism. Henceforth this organismwill be denoted by its more convenient synonym, Lactobacillus helveticus.In a study of assay methods for cereals, J. S. Andrem, H. M. Boyd, andD. E. Terry 22 found that the method of extraction is of great importance ifaatisfactory results are to be obtained. Extraction with taka-diastase wasnecessary in order to eliminate the effects of undesirable impurities. I n thismmner agreement was obtained between results by the microbiologicalmethod and the fluorimetric method in the case of patent and whole wheatfloura, but there were discrepancies in the case of other cereal products.Onthe other hand, M. I. Wegner, A. R. Kcmmerer, and G . S. Fraps23 foundtaka-diastase (and also papain) treatment unsatisfactory in microbiologicalwork on similar products, nor could the difficulty be obviated by addingphotolysed extracts to the basal medium.J. C. Bauernfeind, A. L. Sotier, and C. S. Boruff 24 found that the effectof additional growth substances in some foodstuffs was observable in assaysusing L. helveticus, especially when the amounts of riboflavin were below theoptimum. The authors described methods for countering these effects, andsuggested that the interfering substances were of the nature of fatty acids.This suggestion was followed up in an important paper by F.M. Strong andL. E. Car~enter,~5 who examined the effects of added fatty acids, to whichthe organism was sensitive, and showed that the difficulty did in fact arisefrom their presence. If they are removed by suitable preliminary treatment,reliable values for riboflavin may be obtained.Satisfactory concordance in results by the microbiological method, whichwas modified by E. C. Barton-Wright and R. G. Booth,26 and the fluori-metric method, as adapted by V. A. Najjar,*O has been achieved by theseauthors in the assay of many cereals and cereal products. D. W. Kent-Jones and M. Meiklejohn 27 also have obtained satisfactory results by thesemerthods,give figures for riboflavin in brewingmaterials by the microbiological method, but indicate that additionalinvestigation of the fluorimetric method is necessary owing to disturbingfactors in such materials as hops.R.H. Hopkins and S. Wiener19 J . Bwl. Ciaem., 1939, 131, 621.21 Ind. Eng. Chem. Anal., 1939, 11, 346.23 J . Biol. Chem., 1942, 144, 731.26 Ibid., p. 909.27 Analyd, 1944, 69, 330.20 Ibid., 1941, 141, 366.24 Ind. Eng. Chem. Awl., 1942, 14, 666.2 6 Biochem. J.,1943, 37, 26.Ibid., 1942, 14, 271NORRIS: THE ASSAY OF VITAMINS B. 261Pinally, a collaborative study of the riboflavin content of meals servedin R.A.F. messes may be mentioned. In this instance good agreement wasobtained between the biological and the microbiological methods and it isconcluded by T. F. Macrae, E. C.Barton-Wright, and A. M. Copping 28 thatthe adult riboflavin requirement does not exceed 2 mg. per day.Nicotinic Acid.-An excellent review on nicotinic acid is contributed byC. A. Elvehjem and L. J. T e p l e ~ . ~ ~There are a large number of chemical methods and their modificationsfor the estimation of nicotinic acid. All depend on the reaction withcyanogen bromide, followed by colour production with an amine.30 Probablythe most extensive study has been made by E. K0dicek,~1 who later modifiedthe procedure in collaboration with Y. L. Wang.32 The colour-producingbase employed in both methods is paminoacetophenone ; other baaesproposed include orthoform (orthocaine) ,33 p-phenylenediamine dihydro -and procaine.35 The last gave good results with animal productssuch as meat extract and meat juice ; but in general it may be said that thechemical methods are unreliable for plant products.The method of E.X. Snell and L. D. Wright 36 was modified by W. A.Krehl, F. M. Strong, and C. A. El~ehjem,~' who employed LactobuciEZusarabinosus 17/6 and a synthetic medium.In a study of methods of extraction V. H. Cheldelin and R. R. Williams 38find that many materials yield their nicotinic acid completely under digestionwith taka-diastase and papain, and that similar values in the case of meatsand milk are obtained whether hydrolysis is enzymatic or by acid or alkali.On the other hand, acid or alkaline extracts of cereals give higher valuesthan those prepared by enzyme action.Comparison of results by microbiological and chemical methods of assayhas shown that higker results by microbiological assays are obtained whenplant products, particularly cereals, are treated in the preliminary stage withacid.R. D. Greene, A. Black, and F. 0. Howland39 employed a methodsimilar to that of Snell and Wright 36 for microbiological assays, and amodified cyanogen bromide method due to W. S. Jones.40 With someproducts, good agreement was found between the two types of method,although the authors prefer the microbiological method where small quanti-ties of nicotinic acid are present. J. A. Andrews, H. &I. Boyd, and W. A.Gortner 41 have studied the nicotinic acid content of cereals and cereal28 Biochenz. J . , 1944, 38, 132.30 W. Kbnig, J .pr. Chem., 1904, 69, 105.3 1 Riochem. J . , 1940, 34, 724.33 R. G. Martinek, E. R. Kirch, and G. L. Webster, J . Biol. Chern., 1943, 149, 245.3 4 A. E. Teeri and S. R. Shimer, ibid., 1944, 153, 307.36 E. C. Barton-Wright and R. G. Booth, Lancet, 1944, 565.36 J . Biol. Chem., 1941, 139, 675.37 Ind. Eng. Chem. Anal., 1943, 15, 471.3 8 Ibid., 1942, 14, 671.40 J . Amer. Pham. ASSOC., Sci. Ed., 1941, 30, 272.4 1 Ind. Eng. Chem. Anal., 1942, 14, 663.29 Chem. Reviews, 1943, 33, 185.32 Ibid., 1943, 37, 630.39 Ibid., 1943, 15, 77262 BIOCHEMISTRY.products, and also conclude that the microbiological assay is influenced bythe type of hydrolysis procedure employed.Nevertheless, the method of Krehl, Strong, and Elvehjem 37 is provingmost valuable, and has recently reached a high level of accuracy as modifiedby E.C. Bart~n-Wright,~~ who has applied it to a wide range of materials,which are extracted under pressure with N-hydrochloric acid. Fats andfatty acids do not appear to have any effect on the organism. D. W. Kent,-Jones and M. Meiklejohn 27 have applied the method with success.Pyridoxine.-Colorimetric methods for assay of pyridoxine have beenproposed by M. Swaninathan 43 and by J. V. S ~ u d i . ~ ~ Modifications of thelatter method have been suggested by 0. D. Bird, J. M. Vanderbelt, and A. D.Emmett,45 and by A. F. Bina, J. M. Thomas, and E. B. Br0wn.~6 The mostrecent reference to such methods is probably that by A. C. B ~ t t o m l e y . ~ ~A yeast growth method originally presented by L.Atkin, A S. Schultz,and C. N. Prey** has been modified by these authors together with W. L.Williams.49 The organism used is a yeast strain (No. 4228) which is char-acterised by a specific response to pyridoxine. Extracts of the materialsfor assay are prepared by acid treatment, and yeast growth is estimatedturbidimetrically. Satisfactory assays on a large number of substances arereported. Bound pyridoxine is liberated also by acid treatment underpressure by L. Siegel, D. Melnick, and B. L. O~ler.~O Their results for anumber of natural materials agreed well with those obtained by biologicalmethods.It was shown by E. E. Snell, B. M. Guirard, and R. J. Williams 51 thatStreptococcus Zuctis R would grow on a medium if in addition to the usualconstituents pyridoxine were present.Growth on such a medium, however,was many times as great as could be accounted for on the basis of actualcontent of pyridoxine. The indications were that pyridoxine is convertedinto a more highly active metabolite, called +pyridoxine for the present,prior to utilisation by the organism, and that +pyridoxine exists in naturalproducts. The original presence or derivation of pyridoxine renders micro-biological assays for pyridoxine invalid, and the case is complicated by thefact that the effect varies with different organisms ; e.g., very high values areobtained as indicated with Streptococcus Zactis R, but low values are obtainedwith Sacchuromyces cerevisice as test organism.In a later communication, E.E. Snell 52 advances suggestions as to thenature of +pyridoxine, and shows that mixtures having enhanced growth-promoting properties for Lactobacilli may be formed from pyridoxine byprocesses involving (a) possible amination and (b) partial oxidation. Thelatter change had also been noted by L. E. Carpenter and F. M. Strong.534 2 Biochem. J., 1944, 38, 314.44 J . Biol. Chem., 1941,139, 707.4 6 Ibid., 1943, 148, 111.4 8 ,J. Amer. Chem. Soc., 1939, 61, 193150 J . Biol. Chem., 1943, 149, 361.62 Ibid., 1944, 154, 313.4 3 Indian J . Med. Res., 1941, 29, 561.4 5 Ibid., 1942, 142, 317.4 7 Biochem. J . , 1945 (in the-press).49 Ind. Eng. Chem. Anal., 1943, 15, 141.5 1 Ibid., 1942, 143, 519.63 Arch. Biochem., 1944, 3, 375NORRIS : THE ASSAY OF VITAMINS B.263An amine (IV) and an aldehyde (11), “ pyridoxamine ” and “ pyridoxal ”respectively, have been synthesised,54 and there is much evidence that thesecompounds or their higher combinations are responsible for the +pyridoxineactivity of natural materials.N J NThe use of-biochemical mutants in the mould Neurospora induced by meansof ultra-violet and X-rays is an interesting development in microbiologicalmethods of assay of vitamins of the B group. The production of thesemutants has been described by G. W. Beadle and E. L. T a t ~ m , ~ ~ and theyare characterised by an inability to carry out specific syntheses which canbe effected by the normal unmutated strain.Ari X-ray-induced mutant of Neurospora sitqhila, produced by Beadleand Tatum is utilised as test organism by J.L. Stokes, A. Larsen, C. R.Woodward, and J. W. Foster 56 in a microbiological method for pyridoxine.Growth response is determined by actual dry weight of the mould, and themethod is thus free from some objections which arise in turbidimetric assays.Under the conditions employed, the organism exhibits a specific response topyridoxine, but none to +pyridoxine. The results obtained are in goodagreement with those obtained by biological assay.Biotin.-The elucidation of the structure of biotin has been discussedin detai1.57 The importance of this needs no stressing, since, apart fromscientific interest in the substance itself, it is a valuable tool in much modernmicrobiological work.It may be some time before a chemical test for biotin of the requireddelicacy and specificity is forthcoming.In the meantime, the micro-biological methods are being intensively studied, one of the more importantproblems centring on the question of free and bound biotin. Earlier methodsof extraction included treatment merely with hot water,58 but it was laterfound that much larger amounts of biotin were yielded by autolysis oftissues such as liver.59 Later still,60B61 a combination of autolysis and acidhydrolysis was resorted to, and in 1941, after a series of tests of all types oftreatment, R. C. Thompson, R. E. Eakin, and R. J. Williams 62 came to theconclusion that the best method for many types of material consists in drastic64 S. A. Harris, D. Heyl, K.Folkers, and E. E. Snell, J. Biol. Chem., 1944, 154, 315.5 5 Proc. Nut. Acaca. Sci., 1941, 27, 499; 1942, 28, 234.5 6 J . Biol. Chem., 1943, 150, 17.5 8 F. Kogl and W. van Hrtsselt, 2. physwl. Chem., 1936, 243, 189.5s E. E. Snell, R. E. Eakin, and R. J. Williams, J. Arner. Chem. SOC., 1940, 62, 175.6o R. E. Eakin, W. A. McKinley, and R. J. Williams, Science, 1940, 92, 224.61 Univ. Texas Publication, 1941, No. 4137.62 Science, 1941, 94, 589.6 7 Ann. Reports, 1943, 40, l i 2 264 BIOCHEMISTRY.aoid treatment. Some destruction of the biotin occurs, but it is remarkablystrtble in acid solution. The problem is complicated by the fact that biotinappears to exist in different combinations which are broken down with vary-ing degrees of ease, each type of product requiring individual treatment.I n earlier methods for the assay of biotin, the growth of yeast was usuallymeasured turbidimetri~ally,~~$ 64 a procedure which involved serious difficultywith solutions which were already cloudy or highly coloured.Similarly,P. M. West and P. W. Wilson 65 used Rhixobium trifolii as test organism.I n order to overcome inherent difficulties in these methods, G. M. Shull,B. L. Hutchings, and W. H. Peterson 66 proposed the use of Lactobacillushelveticus as test organism, and measured the effect of added biotin by theincrease in titratable acidity. An added advantage of this method lies inthe fact that the same organism may be used for assay of pantothenic acidand riboflavin, thus obviating additional cultures. G.M. Shull and W. H.Peterson 67 later suggested two modifications in the assay. The eluatefactor level in the yeast supplement in the basal medium is increased so thatoptimal growth of the organism is obtained. A procedure whereby theinoculum is independent of drop size is described.The chemistry and biochemistry of biotin is reviewed by K. Hofmann.68A detailed account of methods and results of microbiological assay of allvitamins in the B group is provided by R. J. Williams and his collaborators.69Pantothenic Acid.-No satisfactory chemical method of assay of panto-thenic acid has as yet been devised. The earlier microbiological methodsbased on stimulation of yeast growth have largely given place to methodsin which L.helveticus is used as test organism.70-75The method of Pennington et aE. employed autoclaving with or withoutprevious autolysis under benzene in order to free pantothenic acid from testmaterials. Various enzymatic methods have been ernpl~yed.~~I 76- ’*In more recent studies on the microbiological assay, A. L. Neal and F. M.Strong v9 have endeavoured to overcome some of the difficulties previously63 F. K6gl and B. Tonnis, 2. physiol. Chem., 1936, 242, 43.64 E. E. Snell, R. E. Eakin, and R. J. Williams, J . Amer. Chem. Soc., 1940, 62, 176.6 6 Enzymologia, 1940, 8, 152.6 7 Ibid., 1943, 151, 201.6* “ Advances in EnzymoIogy,” 1943, 3, 289.e9 Uniu. Texas Publications, 1941, No. 4137; 1942, No. 4237.70 E. E. Snell, F. M. Strong, and W. H. Peterson, Biochem.J., 1937, 31, 1789.71 Idem, J . Amer. Chern. SOC., 1938, 60, 2825.72 Idem, J . Bact., 1939, 38, 293.73 D. Pennington, E. E. Snell, and R. J. Williams, J. Biol. Chem., 1940, 135, 213.74 F. M. Strong, R. E. Feeney, and A. Earle, I n d . Eng. Chem. Anal., 1941,13,666.75 D. Ppnnington, E. E. Snell, H. K. Mitchell, 5. R. McMahan, and R. J. Williams,76 H. A. Waisman, L. M. Henderson, J. M. McIntire, and C. A. Elvehjem, J .7 7 A. H. Buskirk and R. A. Delor, J . Biol. Chern., 1942,145, 707. ’* E. Willerton and W. H. Cromwell, Ind. Eng. Chem. Anal., 1942,14, 603.6 6 J . Biol. Chem., 1942, 142, 913.Interscience Publishers, New York.Univ. Texm Publication, 1941, No. 4137, 14.Nutrition, 1942, 23, 239.Ibid., 1943, 15, 654NORRIS: THE ASSAY OF VITAMINS B.265encountered by modifying the medium employed and improving the methodof growing the inoculum. Enzymatic methods of liberating “bound ”pantothenic acid were studied until satisfactory results were obtained andsteps were taken to eliminate interfering fat-soluble substance^.^^^ 25 Theeffect of water-soluble substances, present particularly in brans, was mini-mised by modifications in the basal medium. The authors claim that themodified method gives concordant results at increasing levels of dosage, andthat very small amounts of the vitamin may be estimated with accuracy.There appears to be an additional growth factor or factors for L. helveticusin the concentrate of rice polishings according to M. F. Clarke, M. Lechycka,and A.E. Light.8O Notable increases in acid production were observed overand above those normally experienced with pure calcium pantothenete.The high values obtained by these workers may not, however, necessarilybe due to a supplementary growth stimulator. J. L. Stokes and B. B.Martin81 report that high acid production may be obtained merely byincreasing the amounts of glucose and sodium acetate in the medium. Witha view to increasing acid production and hence the titration range, A. E.Light and M. F. Clarke 82 propose a modification in the medium.Other test organisms have been employed, among which Streptococcuslac ti^,^^ Streptobacterium plantarum,84 Proteus rnorg~nii,~~a and L. arabin-osus may’be mentioned.A useful review of pantothenic acid is contributed by R.J. Williams.85p- Aminobenzoic Acid.-Chemical methods are not greatly in evideiira asyet, but are being developed. Colorimetric methods are described by E. R.Kirch and 0. Bergeim 86 and by H. W. E ~ k e r t . ~ ’Acetobacter suboxydans is recommended as test organism for p-amino-benzoic acid by M. Landy and D. M. Dicken,*8 who describe a suitable basalmedium. Related or derived compounds of p-aminobenzoic acid havelittle or no biological activity, and the method has high specificity.A mutant strain of Neurospora crassa of G. W. Beadle and E. L. Tatum 55is used by R. C. Thompson, E. R. Isbell, and H. K. Mitchell.89 Additionsof graded amounts of p-aminobenzoic acid to a synthetic medium stimulatea specific growth response in the mould which is determined by measurementof the growth produced..The extraction of p-aminobenzoic acid by waterand by acid hydrolysis is compared. The latter treatment involves a certainloss of the vitamin, but this loss is not significant in comparison with theenhanced yield of “ bound ” p-aminobenzoic acid. The same authorshave later shown that complete extraction is effected only by acid hydrolysis83 H. K. Mitchell, H. H. Weinstock, E. 33. Snell, S. R. Stanbury, and R. J. Williams,8 4 R. Kuhn and T. Wioland, Ber., 1940, 73, 962.84a M. J. Pelczar and J. R. Porter, J . Biol. Chem., 1941, 139, 075.84b H. R. Skeggs and L. D. Wright, ibid., 1944,156, 21.J . Biol. Chern., 1942, 142, 957.J . Amer. Chem. SOC., 1940, 62, 1776.Ibid., 1943, 147, 483.82 Ibid., p . 739.“ Advances in Enzymology,” 1943, 3, 253.J . Biol. Chem., 1943, 148, 445.Ibid., 1948, 146, 109.Interscience Publishers, New York.*O Ibid., 1943, 147, 485.Ibid., p . 197.88 Ibid., 1943, 148, 281266 BIOCHEMISTRYunder pressure. They suggest that the method of Landy and Dicken 88responds to only a fraction of the total yielded by acid hydrolysis.Quantitative response to p-aminobenzoic acid is evinced by ClostridiumacetobutyZicum Strain S9, which attains maximal growth in 24 hours on asuitable medium proposed by J. 0. Lampen and W. H. Peterson.g1 Theseauthors claim that the vitamin is rapidly destroyed by acid hydrolysis, andprefer to hydrolyse with alkali under pressure. This method of extractionis also favoured by J.C. who uses L. urubinosw as test organism.Reference should not be omitted to the synthetic medium of M. Landyand D. M. Dicken 93 for use with L. heEveticus and applicable to assay of eachmember of the group. Whilst this ideal has not perhaps been realised, themedium or modifications of it have proved useful to many workers.The family of B vitamins is ever-increasing and it is too early to discussassay methods for new members. It may be mentioned, however, thatmethods for “ folic acid ” are a ~ a i l a b l e . ~ ~ F. W. N.6. ACTIONS OF CHEMOTHERAPEUTIC AGENTS AND RELATED COMPOUNDS.Chemotherapy concerns interactions of drug, parasite and host, but themajority of investigations of chemotherapeutic agents during the periodreviewed have been of their effects upon bacteria.The present account ismainly limited to such effects and is arranged according to their type.Factors involved in the comparison of in vivo and in vitro actions of drugshave been examined,l and their relations to other interactions in thecomplete chemotherapeutic system have been reviewed elsewhere.2I. Biological Eflects.(a) Mor23hologicaZ.-Abnormal size or shape in bacterial cells is inducedby many agents ; 3* by sulphanilamide, andfrequently by compounds without known chemotherapeutic a ~ t i o n . ~ Theiroccurrence in response to changes in media has been ascribed to independenteffects of the change upon chemical factors conditioning cell elongation anddivision.5*(b) Upon Growth.--Sulphonamides increase the mean generation timeduring the logarithmic phase, and the length of the lag phase, of Bact.lactiscerogenes ; 7 pantoyltaurine, in concentrations active in vivo against Strepto-sometimes but not alwaysS1 J . Biol. Ghem., 1944, 153, 193.S3 J. Lab. Glin. Med., 1942, 27, 1086.B2 Ibid., 1942, 148, 441.1 Symposia, Trans. Paraday Soc., 1943, 39, 319; Ann. N . Y . Acad. Sci., 1943, 44,445.H. McIlwain, Riol. Rev., 1944, 19, 135.E.g., J. W. Foster and H. B. Woodruff, Arch. Biochem., 1943, 3, 241.G. H. Spray and R. M. Lodge, Trans. Paraday Soc., 1943, 39, 424.C. N. Hinshelwood and R. M. Lodge, Proc. Roy. SOC., 19-14, B, 132, 47.6 R. M. Lodge and C. N. Hinshelwood, Trans. FaracEay A’oc., 1943, 39, 420.D. S. Davies and C. N. Hinshelwood, ibid., p.431MCILWAIN : ACTIONS OF CHEMOTHERAPEUTIC AGENTS. 267coccus hcemolyticus, has effects upon that organism which are similar andwhich, like the in vivo activity, are annulled by pantothenate.8 The effectsof many other metabolite-analogues upon overall growth have been reported.Pyrithiamine, in which a pyridine ring replaces the thiazole ring of aneurin,inhibits several bacteria 9 ~ 1 0 and has greatest effects on those most exactingin their requirements for aneurin,g which antagonises its action. Inhibitionby benziminazole is counteracted by some aminopurines.11 Dethiobiotin,12biotin sulphone,13 and an analogous imidazolidone derivative l4 competitivelyinhibit certain organisms but to others may be indifferent or in some casesact as source of biotin ; or they may make available to bacteria, biotin whichis inactivated by avidin. 6 : 7-Dichloro-9-ribitylisoalloxazine l5 and phen-azine analogues of riboflavine l6 inhibit bacterial growth and this may berestored by riboflavine.New analogues of p-aminobenzoate l7 and panto-thenate, l8 some of which are antibacterial, have been reported. Inhibitorysubstances designed in this way can act upon strains of organisms resistantto the agent used as model,lg though cross-resistance can be developed toagents apparently different in type.20 Orthanilamide does not inhibit anorganism to which anthranilic acid is a growth-factor.2lConsidering existing chemotherapeuticals, the inhibition of growth ofEscherichia wli caused by atebrin 22 and of a lactobacillus and streptococcuscaused by diamidines 23 are antagonised by spermidine and polyamines.The interaction of p-aminobenzoate and sulphonamides has been investigatedunder various conditions of aeration 24 and temperat~re.~~ The latterfactor influences also the mutual interaction of p-aminobenzoate, sulphon-amides and urea ; 26 joint action of the last two can be additive, as urea issometimes bacteri~static.~~ Effects of sulphonamides on certain micro-H.McIlwain, Biochem. J . , 1944, 38, 97.9 D. W. Woolley and A. G. C. White, J . Exp. Med., 1943, 78, 489.10 0. U'yss, J . Bact., 1943, 46, 483.11 D. W. Woolley, J . Biol. Chem., 1944, 152, 225.12 K. Dittmer, D. B. Melville, and V. du Vigneaud, Science, 1944, 99, 203; V. G.13 K.Dittrner, V. du Vigneaud, P. Gyorgi, and C. S . Rose, Arch. Biochem., 1944, 4,l4 K. Dittmer and V. du Vigneaud, Science, 1944,100, 129.l5 R. Kuhn, F. Weygand, and E. F. Moller, Ber., 1943, 76, 1044.16 D. W. Woolley, J . Biol. Chem., 1944, 154, 31.1' 0. H. Johnson, D. E. Green, and R. Pauli, ibid., 1944, 153, 37.la J. Barnett, J., 1944, 5; J. Barnett, D. J. Dupr6, B. J. Holloway, and F. A.Lilly and L. H. Leonian, ibid., p. 205.229.Robinson, ibid., p. 94.H. McIlwain, Brit. J . Exp. Path., 1943, 24, 203.2O J. McIntosh and F. R. Selbie, ibid., p. 246.21 E. E. Snell, Arch. Biochem., 1943, 2, 389.22 JI. Silverman and E. A. Evans, jun., J . Biol. Chem.., 1943,150,265; 1944,154,521.23 E. E. Snell, ibid., 1943, 152, 475.24 J. W. McLeod, A. 'Mayr-Harting, and N.Walker, J . Path. Bact., 1944, 56, 377.2 5 S. W. Lee and E. J. Foley, Proc. SOC. Exp. Biol. Med., 1943, 53, 243.26 S. W. Lee, J. A. Epstein, and E. J. Foley, ibid., p. 245.27 W. M. M. Kirby, ibid., p. 109268 BIOCHEMISTRY.organisms differ from the compounds' normal antibacterial effects in notbeing antagonised by p-aminobenzoate.28 Lack of such antagonism is avaluable feature in the homosulphonamides, of which new members areactive in v ~ u o . ~ ~(c) Upon ViabiZity.-An outstanding finding of the period under reviewis of the unusual action of penicillin. At concentrations approximating tothose attained during therapy, penicillin has little effect upon the viabilityof staphyloco~ci,~~ hmmolytic streptococci 31 and meningococci 32 underconditions which do not permit growth of the organisms; e.g., in salt solu-tions or in very dilute broth, in rich media in the cold or in rich media whengrowth is inhibited by sulphonamides 33 or by boric acid.30 Under conditionsotherwise permitting growth, an extremely small concentration of peni-cillin is bacteriostatic, 0.0009 unit/ml. (c.0.0005 vg./ml.) having aneffect comparable with that of 100 pg./rnl. of sulphadiazine ; concentrationscomparable with those used therapeutically (e.g., of 1 /24 unit/ml.) are,however, bactericidal. Factors which normally increase the rate of growthof streptococci, in the presence of penicillin increase their rate of death.A proportion of organisms in staphylococcal cultures is not susceptible tobeing killed by penicillin ; such " persistent " organisms are considered to bein a particular cultural phase.The proportions of organisms of a culturewhich are persistent can be altered by manipulation of the culture; 30 theyincrease on chilling. Recommendations in the clinical use of penicillin havebeen made on the basis of the new findings.30 Varying susceptibility ofbacteria at different phases of the culture-cycle has frequently been observed 34and a further example has appeared recently in the greater sensitivity toacriflavine of B. salmonicida while it is in its logarithmic pha~e.3~Of agents already known to be bactericidal, the relations between con-centration and action 36 and time of exposure and action 37 of phenol havebeen further studied.The significance of rates of death has been discussed.3sSurface-active cations such as benzylalkylammonium chlorides are bacteri-cidal, but their toxic action upon bacteria can be prevented, and when inprogress halted, by anions of large molecular weight such as sodium dodecyl~ulphate.~g This shows two phases in the action of the cation : a pre-2 8 J. T. Tamura, J . Bact., 1944. 47, 529; F. Hawking, Brit. J. Exp. Path., 1944, 25,63.29 D. M. Hamre, H. A. Walker, W. B. Dunham, H. B. van Dyke, and G. Rake,Proc. SOC. Ezp. BioZ., N. Y., 1944, 55, 170; D. G. Evans, A. T. Fuller, and J. U'alker,Lancet, 1944, 247, 523.30 J. W. Bigger, ibid., p. 497.31 G. L. Hobby and M. H. Dawson, PTOC. SOC. Exp. BioZ. N.Y., 1944, 56, 178.32 C. P.Miller and A. Z. Foster, ibid., p. 205.33 G. L. Hobby and M. H. Dawson, ibid., p. 181.34 C.-E. A. Winslow and H. H. Walker, Bact. Rev., 1938, 3, 147.35 W. W. Smith. Proc. SOC. Exp. BioZ. N.Y., 1944, 56, 238.36 D. P. Evans and A. G. Fishburn, Quart. J . Pharm., 1943,16, 201.37 R. C. Jordan and S. E. Jacobs, J. Hygiene, 1944, 43, 275.38 0. Rahn, Bwdynamica, 1943, 4, 81.30 E. I. Valko and A. S. DuBois, J . Bact., 1944, 47, 16MCILWAIN : ACTIONS OF CHEMOTEERAPEUTIC AGENTS. 269liminary reversible one and later irreversible changes assooiated with death.The reversible one is considered to be the attachment of the agent to the oelland was shown to have some of the characters of ionic exchange; the actionwas reduced by the additional presence of less toxic cations.Suah anfagon-ism was effeutive against only limited concentrations of toxie cation. Gimilctrphases in the action of other bactericides have been proposed; 36 here alsothe second phase was considered to be fundamentally diffefent and to uonshtin denaturation or precipitation of the bacterial protein. The aotivitiea ofantiseptics a t different pH have been related to the conoentratima of ionisedand undissociated molecules ; undissociated and not ionised benzoic, salioylic,and sulphurous auids were found antisepti~.~~ CEstrogens and related com-pounds are bectericidalY41 42 but optimal antibacterial activity is not shownby members of greatest oestrogenic a~tivity.~3 Propamidine is bactericiddas well as bacteriostatic to staphylococci 44 and to Escherichia wli 45 and botheffects are antagonised by lecithin.46II. Biochemical Efsects.(a) Upon Energy- yieldijzg Processes.-Evidence has been collected 46suggesting a correlation of the inhibitions of bacterial respiration or anaerobiccarbon dioxide production, with inhibition of growth, by sulphonamides.The respiratory inhibition is only partial (and by some investigators has beenreported absent) at concentrations of aulphonamides which are compleklybaoteriostatic.To affect glycolysis or respiration of streptococci in thepresence of glucose and a few other substrates, pantoyltaurine is required inmuch greater preponderance over pantothenate than is required for if toinhibit growth ; these metabolic inhibitions also are relatively small ormay be absent. Oxidation of amino-acids by Escherichia coli is inhibitedby low concentrations of propamidine and is more sensitive to the compoundthan is oxidation of glucose.47 The inhibitions are markedIy increased byadding the inhibitor before the substrate, and by inorease in PH.~* Theaotivity of antimalarials in inhibiting oxygen uptake of malarial parasitesis correlated with their therapeutic efficacy.49(b) Upon Metabolism of Vitamin-like Compounds.-The system a t whiahsulpbonamides and p-aminobepzoate are believed to interaot has pot y0$been specified biochemioally, but further interpretations of actions ofaulpbonamides in terms of their competing with p-aminobenzoate for enzymes4 0 0. Rahn and J. E. Conn, I n d . Eng. Chem., 1944, 86, 185.4 1 G. H. Faulkner, Lancet, 1943, 245, 38.4 2 B. Heinemm, J . Lab. Clin. Med., 1944, 29, 254.*3 0. Brownlee, F. C. Copp, W. M. Duffi, and I. M. Tonkin, Biochem. J., 1943, 37,4 4 W. R. Thrower and F. C. 0. Valentine, Lancet, 1943, 244, 133.4 5 W. 0. Elson, J. Biol. Chem., 1944, 154, 717.4 6 R. J. Henry, Bact. Rev., 1943, 7 , 175.47 F. Bernheim, Science, 1943, 98, 223.4 8 F. Bernheim, J . Pharm. Exp. Ther., 1944, 80, 199.49 S. R. Christophers, Trans. Paraday Xoc., 1943, 39, 333.572270 BIOCHEMISTRY.have been 51 Increased synthesis of p-aminobenzoate haq beenfound to be associated with development of sulphonamide-resistance instaphyloc~cci.~~ By training certain strains of Corynebacterium diphtherimto synthesise pantothenate, strains resistant to pantoyltaurine were pro-duced in the absence of that compound and of any other inhibitor ; 53 butnot all drug resistance is by synthesis of specific- antag0nists.1~ The systemthrough which pantoyltaurine inhibits streptococcal growth has to someextent been characterised 54 and its functioning is associated with panto-thenate-inactivation. In the preparations studied, the pantothenatemetabolism required a concurrent energy- yielding process such as glycolysis.The pantothenate metabolism, but not glycolysis, was inhibited by con-centrations of pantoyltaurine even lower than those affecting growth andthe activities of a series of pantothenate analogues in inhibiting growth werecorrelated with their activities in inhibiting pantothenate-inactivation. Abacterial degradation of riboflavine is inhibited by structurally related com-pounds but occurs independently of a process such as glycolysis and itsinhibition does not affect growth.55111. Chemical or Physical Effects.Analyses of sulphonamide action, the effect of pH upon it, and itsantagonism, base these processes upon reversible combination of the drug,antagonist, or their ions with enzymes in accordance with the law of massaction.a* 519 56 The bulk of the p-aminobenzoate of preformed organisms isnot, however, displaced by bacteriostatic concentrations of sulphanilamide ;67the equilibria may obtain during or before paminobenzoate assimilation.Similar lack of displacement of pantothenate by pantoyltaurine has beenobserved.57 Correlation of the action of drugs with properties which theyexhibit apart from biological systems has been reported 36* 58 andrevie~ed,~ge 60IV. Chemotherapeutic Mechanism.The year’s findings have shown the multiplicity of types of antibacteria1action exhibited by chemotherapeuticals. The connection of these withchemotherapeutic activity in vivo is established only in certain cases and inothers would not be expected to be very close. Discussion of such con-nections (chemotherapeutic “ mechanisms ”) is beyond the scope of thepresent account, but it will be seen that evidence for such connections is,50 W. D. Kumler and T. C. Daniells, J . Amer. Chem. SOC., 1943, 65, 2190.5 1 I. M. Klotz, ibid., 1944, 66, 459.62 M. Lrsndy, N. W. Larkum, E. J. Oswald, and F. Streightoff, Science, 1943, 97,295.63 H. McIlwain, Brit. J . Exp. Path., 1943, 24, 212.54 H. McIlwain and D. E. Hughes, Biochem. J . , 1944, 38, 187.55 J. W. Foster, J . Bact., 1944, 48, 97.56 F. H. Johnson, H. Eyring, and W. Kearns, Arch. Biochem., 1943, 3, 1.57 H. McIlwain, Proc. Biochem. SOC., 1924, 38, viii.5* A. Albert and R. Goldacre, J . , 1943, 454.59 A. Albert, Australian J . Sci., 1944, 6, 137.60 W. S. Gledhill, ibid., p. 170MCILWAIN : ACTIONS OF CHEMOTHERAPEUTIC AGENTS. 27 1most fully provided in the cases of pantoyltaurine and the sulphonamides, byobservations in vivo and of categories I ( b ) , I1 (a), I1 ( b ) , and 111. Othercompounds are of radically different mode of action and one compound mayact in more than one way.g1 As the normal life of organisms involves a,working together of processes which include all the above categories, manyother means can be envisaged for their disturbance. H. McI.F. DICKENS.H. MCILWAIN.A. NEUBERQER.F. W. NORRIS.J. R. P. O'BRIEN.F. G. YOUNG.61 C. E. Hoffmann and 0. Rahn, J . Bact., 1944, 47, 177