BIOCHEMISTRY.1. THE INTEGRATION OF THE INTERMEDIARY METABOLISM OFCARBOHYDRATES, FATS, AND AMINO-ACIDS.THE existence of a clearing-house common to intermediates in the oxidativemetabolism of carbohydrates, fats, and proteins has long been suspected,but its demonstration appears to have been brought substantially nearer byrecent work. Although many gaps remain and some stages are still highlycontroversial, a provisional description on the basis of known mechanism isnow possible.The Metabolism of Pyruvate.F. Knoop predicted 15 years ago that pyruvate “ represented thebridge across which the most varied classes of foodstuffs could be inter-converted.” If the various products arising from the multiple metabolicreactivity of pyruvate are included, this statement still holds good.Pyruvatearises on either of the main pathways of carbohydrate breakdown hithertodescribed, on the one hand through the well-known phosphorylated inter-mediates of the Embdcn-Meyerhof series, or alternatively aerobically throughthe presumably stepwise oxidation and decarboxylation of hexose 6-phosphateby a coenzyme II-catalysed system,2 independent of fermentation 39 andrelatively insensitive to poisons such as iodoacetate and f l ~ o r i d e . ~ ~ Anon-phosphorylating mode of carbohydrate breakdown originally advancedfor brain and embryonic tissues 7 now seems to have been insecurelyfounded, and typical phosphorylative mechanisms have since been clearlydemonstrated in these tissues.8 But in moulds and some bacteria loapparently non-phosphorylating pathways of hexose degradation exist,which may lead to the formation of p y r ~ v a t e .~The great reactivity of pyruvate in cells and tissues is illustrated by thelist of such reactions compiled by E. S. G. Barron l1 which includes 151 Ahrens Vortrage, 1931, 9, 18.2 0. Warburg and W. Christian, Biochem. Z., 1937, 292, 287 ; F. Lipmann, Nature,1936, 138, 588; F. Dickens, ibid., p. 1057; Biochem. J., 1938, 32, 1626, 1645.S. B. Barker, E. Shorr, and M. J. Malam, J . Biol. Chem., 1939, 129, 33.S. Spiegelman and M. Nozawa, Arch. Biochem., 1945, 6, 303.E. Stetz, “Advances in Enzymology,” Interscience Press, N.Y., 1945, 5, 129.C. A. Ashford, Biochem. J., 1934, 28, 2229.7 J. Needham and H. Lehmann, ibid., 1937,31,1210,1913 ; J.Needham and W. W.Nowinski, ibid., p. 1165.8 M. F. Utter, H. G. Wood, and J. M. Reiner, J. Biol. Chem., 1945, 161, 197;0. Meyerhof and E. Perdigon, Enzymologiu, 1940, 8, 353; J. R. Klein, J . Biol. Chem.,1944, 153, 295 ; Fed. Proc., 1945, 4, 94.F. F. Nord and R. P. Mull, “Advances in Enzymology,” Interscience Press, N.Y.,1945, 5, 165.lo C. R. Brewer and C . H. Werkman, Enzymologiu, 1940, 8, 325.11 “Advances in Enzymology,” Interscience Press, N.Y., 1943, 3, 149198 BIOCHEMISTRY.different enzyme systems yielding a wide variety of products from pyruvatein animal tissues, yeast, bacteria, fungi, and plants. Some of the morerecently investigated pathways will be considered later in this review, afterthe primary oxidation of pyruvate has been discussed.Oxidation of Pyruvate in Bacteria.Acetone preparations of Lactobacillus delbrueckii oxidise pyruvate toacetyl phosphate and carbon dioxide in presence of inorganic phosphate,diphosphoaneurin, a bivalent metal (Mn, Mg, Co), and alloxazine dinu-cleotide.12 In gonococci, however, oxidation of pyruvate requires thecytochrome system,13 and there is now evidence that, in some systems whichoxidise pyruvate, phosphate is not a component: E.S. G. Barron (un-published ; cited in 11) found that thoroughly washed M . piltonemis requiredno added phosphate, and in careful studies P. K. Stumf l4 has recently shownthat, although Lipmann’s findings with preparations of L. delbrueckii couldbe fully confirmed, similar extracts of Proteus vulgaris and Esch.wli do notappear to require inorganic phosphate for their activity. The productsformed are in both these cases acetic acid and carbon dioxide, and the lack ofevidence of acetyl phosphate formation could not be explained by thebreakdown of this substance by hydrolysis as soon as it was formed. Theextracts contained acetyl phosphatase, the enzyme specifically responsiblefor this hydrolysis which has also been found to occur in skeletal and heartmuscle ;I5 but, although this dephosphorylation is considerably inhibitedby O.lM-phosphate, even in presence of this concentration of inorganicphosphate no acetyl phosphate accumulated in Stumpf’s experiments,and in any case the activity of the acetyl phosphatase was too low to be theexplanation of the non-occurrence of acetyl phosphate as the primary product.The new enzyme system therefore differs from the pyruvate oxidase studiedby Lipmann in that it appears to catalyse a non-phosphorylative oxidation;it resembles Lipmann’s enzyme in that it requires diphosphoaneurin and abivalent metal (in this case, Mn, Mg, Fe, Ni, Zn, or Co), and it is specific forpyruvate among the keto-acids tested.Nature of the Primary Product in Pyruvate Oxidation.The existence of these two types of bacterial pyruvate oxidase may beconsidered in relation to the mechanism of pyruvate oxidation in general.Many attempts to explain the metabolic reactions of pyruvate, acetate, andacetoacetate appear to necessitate the assumption of the existence of a high-energy C, intermediate, which in the past has frequently been referred to as“ nascent acetic acid,” although the unsatisfactory nature of such a term hasl2 Ann.Reports, 1940,37,417; 1944,41, 235; F. Lipmann and L. C. Tuttle, J . Biol.l3 E. S . G . Barron, ibid., 1936, 113, 695.14 Ibid., 1945, 159, 529.15 F. Lipmmn, Proc. Div. BWE. Chern., 108th Meeting, h e r . Chem. SOC. (1944).Cham., 1945,158, 505; M. F. Utter, F. Lipmann, and C. H. Werkman, ibid., p. 521DICEENS : THE INTERMEDIARY METABOLISM OF CARBOHYDRATES, ETC. 199long been admitted. The discovery by Lipmann of acetyl phosphate as aprimary stage in pyruvate oxidation led a t first to the rather uncriticalassumption by several workers that acetyl phosphate, with its high-energyphosphate bond,l6 was always the reactive intermediate. While this maybe true in some systems, evidence is accumulating of examples in which theproperties of acetyl phosphate are not necessarily adequate to account forthe experimental findings.I n the first place, evidence of acetyl phosphateformation in animal tissues is still lacking. Acetylations in which pyruvatemay be a primary source of acetyl groups occur with choline l7 and sul-phanilamide.18 Acetylation in the former system has been shown l9 to bebrought about by cell-free extracts of choline acetylase (the synthesisingenzyme system prepared from nervous tissue, which requires adenosinetriphosphate as primary energy source) and potassium ions, and is furtheractivated by I ( -)-glutamate.However, the system still appea.rs to becomplex, and the intermediate mechanism remains undecided. Acetylationof sulphonamides occurs aerobically in liver slices 2O and in liver homo-genates and extracts.z1 It also proceeds anaerobically a t a similar rateprovided adenosine triphosphate is supplied ; the amounts formed aredoubled by the addition of acetate to the system ; acetoacetate and pyruvateare about half as active in this respect as acetate, while acetoin causes someincrease over the acetate formation without added substrate. I n this systemacetyl phosphate was not active as an acetylating agent and it is assumedthat what is necessary is a complex of the acetyl donator, amino-compound,and adenosine triphosphate a t one and the same enzyme, rather than anintermediate formation of acetyl phosphate.21Though S.Ochoa, L. A. Stocken, and R. A. Petersz2 were unable todemonstrate in brain preparations the utilisation of acetyl phosphate or itsability to phosphorylate adenylic acid, the rate of breakdown by acetylphosphatase in such preparations is not known. There is, however, someevidence (vide infra) that acyl phosphates of fatty acids may be concerned infat metabolism in animal tissues.Since inorganic phosphate is indispensable for the oxidation of pyruvateby Lipmann’s preparation, the assumption has been made that the primaryreaction is an additive reaction of pyruvate and phosphate.23 If this is so,the enzyme concerned in the oxidation of this addition compound is pre-sumably different from that of Stumpf’s bacterial non-phosphorylating oxidasesystem.The finer points of these reactions remain to be investigated : forl6 F. Lipmaan, “Advances in Enzymology,” 1941, 1, 99.l7 P. J. G. Mann and J. H. Quastel, Nature, 1940, 145, 856; cf. ref. 18.l8 P. Handler and W. A. Perlzweig, Ann. Rev. Biochem., 1945, 14, 618.lS D. Nachmansohn and H. M. John, J . Biol. Chem., 1945, 158, 157; Fed. Proc.2o J. R. Klein and J. S. Harris, J . Biol. Chem., 1938,124, 613.21a F. Lipmann, Fed. PTOC., 1945, 4, 97; J . BioZ. Chem., 1945, 160, 173.21b B. Shapiro and E. Wertheimer, Nature, 1945, 156, 690.22 Ibid., 1939,144, 760; cf. Ann. Reporb, 1940,37,417.t 8 See Ann. Reporb, 1944,41, 236.1945, 4, 93200example the nature offormulated by equationTOi HBIOCHEMISTRY.the actual dehydrogenation, which Lipmann 24(I) might perhaps be represented by some suchc o 2I ' - 2H +I IMe*C*4H --+ MeCOHypotheticaladditioncompound.enol-Pyruvic acid.reaction as is shown in equation (11) in the non-phosphorylating oxidation,in which it is assumed that dehydrogenation of enol-pyruvate might yield amolecule of carbon dioxide plus one of keten or some other similar highlyreactive C, compound.This type of oxidative decarboxylation was originallysuggested ten years ago by H. Weil-Malherbe 25u to explain the similar reactionof a-ketoglutaric acid and the decarboxylative r61e of dipho~phoaneurin.~~~C. Martius 26 has quite recently adopted a view very similar to that outlinedabove in which the primary product of the dehydrogenation of pyruvate iswritten as a radical, -CH2-CO-.It is evident that such hypotheses mightexplain the remarkable reactivity of pyruvate in biological systems alreadymentioned ; e.g., the keten-like intermediate could be hydrolysed orphosphorylysed to acetic acid or acetyl phosphate; by the addition of 2Hit could yield acetaldehyde; diacetyl could be formed by condensation withacetaldehyde, and reduction of the product might be a source of acetoin;"amino-compounds could be acetylated ; acetoacetate formation from acetateor fatty acid oxidation could proceed via keten formation. The incorpor-ation of acetate into glycogen, higher fatty acids, or cholesterol, could beexplained by a similar mechanism. Two molecules might unite to formsuccinate.Keten could be the reactant with oxaloacetate in the formationof components of the tricarboxylic acid cycle. In the discussion of some ofthese reactions which follows, the term '' reactive C, compound " will be usedsince a more precise description appears unjustified at present. Alternativesto keten which have been suggested include, besides acetyl phosphate, acetyldiphosphoaneurin 26 and glyoxylic a~id,~'a but the last forms oxalate withtissue enzymes.27bOxidative Metabolism of Pyruvate in Animal Tissues.It is generally considered that in animal tissues simple decarboxylationof pyruvate to acetaldehyde is not on the pathway of pyruvate metabolism,I4 F. Lipmann, Cold Harbor Xymposia on Quant.Biol., 1937, 7 , 248.25a Nature, 1936, 138, 551.2 627a R. H. Barnes and A. Lerner, Proc. SOC. Exp. B i d . Med., 1943,52, 216.276 S. Ratner, V. Nocite, and D. E. Green, J . Biol. Chem., 1944, 152, 119.* Cf. references 27a, b.Zsb Idem, ibid., 1940, 145, 106.2. physiol. Chem., 1943, 279, 96DICKENS : THE INTERMEDIARY METABOLISM OF CARBOHYDRATES, ETC. 201since acetaldehyde is apparently not vigorously metabolised by skeletalmuscle 28 or some other animal tissues which metabolise pyruvate. On theother hand, decarboxylation of this kind occurs readily in a number ofbacteria, yeasts, plants, moulds, and protozoa, some of which have beenstated not to oxidise pyruvate.ll An enzyme from sheep’s heart which iscatalysed by diphosphoaneurin and apparently decarboxylates pyruvate toacetaldehyde (or a-ketoglutarate to succinic semialdehyde), with simultaneouscondensation to acetoin, has been described by Green and co-workers.292CH3*CO*C0,H + CH,*CO*CH(OH)*CH, + 2C02Acetaldehyde is believed to be the primary product of ethanol metabolismin animals, and acetaldehyde injected into rats is rapidly metabolised and,a t least in part, is converted into acetoin ; yet injected dl-acetoin disappearsonly slowly.30 The reason for the apparently different behaviour of theacetoin formed in vivo is not at all understood a t present, but it may beconnected with its optical configuration.* Homogenised brain tissue meta-bolises added acetaldehyde almost quantitatively to acetoin, the rate beinglowered in brain tissue from aneurin-deficient animals (rats and pigeons) ; inthis case the acetoin was not further o ~ i d i s e d .~ ~However, S. Ochoa 31 has shown that his purified preparation from cat’sheart of a-ketoglutarate dehydrogenase failed completely to catalyse theanaerobic decarboxylation of E-ketoglutarate, or to cause a t a sufficient ratethe aerobic oxidation of succinic semialdehyde; nor did the latter competewith a-ketoglutarate as substrate for the oxidation. The balance of evidenceis likewise against a two stage process (decarboxylation followed by oxidation)in the oxidation of pyruvate by animal tissues, but the matter is stilluncertain.From the pioneer work of Peters and co-workers the essential r61e ofdiphosphoaneurin in pyruvate oxidation by animal tissues has been madeabundantly clear, this being the first case in which the action of a vitaminin vitro was dem~nstrated.~~ Pyruvate oxidase of brain tissue has acomponent requiring an essential SH group and is readily inactivated byhalogenoacetates, dichlorodiethyl sulphone, and arsenicals,33, 34 or by exposure. 2 8 H.A. Krebs, “Advances in Enzymology,” Interscience Press, N.Y., 1943, 3, 191.2Q D. E. Green, W. W. Westerfeld, B. Vennesland, and W. E. Knox, J. Biol. Chem.,1941, 140, 683; W. W Westerfeld, E. Stotz, and R. L. Berg, ibicl., 1942, 144, 657;R. L. Berg and W. W. Westerfeld, ibid., 1944, 152, 113.30 E. Stotz, W. W. Westerfeld, and R. L. Berg, ibid., 1944, 152, 41; R. L. Berg,E. Stotz, and W. W. Westerfeld, ibid., p.51.31 Ibid., 1944,155, 87.32 See Ann. Reports, 1940, 37, 417; 386; 1939, 36, 339.33 R. A. Peters, H. Rydin,,and R. H. S. Thompson, Biochem. J., 1936, 29, 63 ; R. A.Peters, Nature, 1936, 138, 327; Current Science, 1936, 5, 212; “Perspectives inBiochemistry,” Cambridge, 1937, p. 41; R. A. Peters and R. W. Wakelin, reportprivately circ. in U.K. and U.S., 1941; R. A. Peters, L. A. Stocken, and R. H. s.Thompson, Nature, 1945, 156, 616.34 E. S. G. Barron and T. P. Singer, J. Biol. Chem., 1944,157 221.* Cf. reference 6.Q 202 BIOUHEMISTRY.to high pressures of oxygen.35 Barron l3 had earlier shown that gonococcalpyruvate oxidase was inhibited by oxygen. There is reason to believe that thisstage in carbohydrate metabolism is one of the most susceptible of all to inter-ference by toxic agents of the type mentioned, and is no doubt the seat ofaction of a variety of metabolic poisons, some of which may be importantpharmacologically. hf.Rlichaelis and J. H. Quastel 36 had earlier pointed outthe susceptibility of brain pyruvate oxidation to narcotics which probablyreacted with the flavoprotein component. This system in brain requires,like the bacterial systems, a bivalent metal (Mn or Mg) in addition to diphos-phoaneurin and inorganic ph~sphate,~’ but it is not yet possible to sayexactly what enzymes are involved, and in several animal tissues the evidencefavours a complicated mechanism of the type of the tricarboxylic acid cycle.The Tricarboxylic (isoCitrate) Cycle.The importance now widely assigned to this cycle, not only in the oxid-ation of pyruvate (or triose) arising in carbohydrate metabolism, but also inintermediary metabolism of fatty acids and amino-acids, warrants somedescription additional to those previously given in these Reports,3* althoughInter-relationship of metabolism of carbohydrate, fat, and protein through thetricarboxylic acid cycle.(Carbohydrate) + Phosphopyruvate .1 (Fatty acid)/ .1 + Acetate Acetoacetatev JI(Amino-acid) +Oxalocitraconate ( 1 ) +--/ loxaloacetate Malate J cis- Aconitate11(Amino-acid)11Fumarate11 11isoCitrate + Citrate Succinate11 + cozOxalosuccinate a-Ketoglutarate +co: Succinicsemialdehyde 11(Amiw-acid)for a full consideration of the literature up to 1943 reference should be madeto the review of H.A. Krebs.28 The cycle, slightly modified to accord with35 F. Dickens, Biochem. J., 1946, 40, 145, 171; P. 3. G. Mann and J. H. Quaatel,ibid., p. 139.36 Ibi&., 1941, 35, 518.37 S. Ochoa, Nature, 1939,144, 834; I. Banga, S. Ochoa, and R. A. Peters, Biochem.38 Ann. Reports, 1937, 34, 416-9; 1941, 38, 260-1.J . , 1939, 33, 1980; C. Long, ibid., 1943, 37, 215; 1945, 39, 143DICKENS : THE INTERMEDIARY METABOLISM OF CARBOHYDRATES, ETC. 203recent work, is shown in the scheme on p. 202. This system has the greatmerit of explaining how, for each complete course of one cycle, a molecule ofpyruvate could be completely oxidised to carbon dioxide and water by asystem of reactions almost all of which (at least in pigeon muscle tissue, themain material used in working out the cycle) have been shown to occur a t asufficiently rapid rate.It further explains how, in presence of malonate toinhibit direct reduction via succinic dehydrogenase, oxaloacetate might beconverted to succinate by an oxidising route, thus explaining the observedformation of succinate and the oxidative regeneration of C, catalyticallyactive dicarboxylic acids. Fortunately carbon dioxide fixation is consideredto be slight or absent in minced pigeon mu~cle,3~ so that for this material theinterpretation of the experimental evidence is not complicated by this factor.In pigeon-liver preparations, on the other hand, use has been made of theability to fix carbon dioxide in the formation of a-ketoglutarate from pyruvatein presence of bicarbonate containing isotopic 41 thus establishing,from the fact that the fixed C is located in the C0,H adjacent to the COgroup of the a-ketoglutarate, that a symmetrical intermediate such as citratecould not be involved in the primary condensation of pyruvate andoxaloacetate.Nature of the Interaction of Pyruvate and Oxaloacetate.Originally Wood et aL40b suggested that oxalomesaconic acid might be theprimary condensation product, but it is more probable41 that the cis-isomeride, oxalocitraconic acid, conforming to the cis-aconitate, which is thenext stage, would be the primary product.It is not known whether oxid-ation is subsequent to this stage or precedes it.* In fact this stage is theleast understood of the whole cycle.C.Martius, who with F. Knoop42 first described the smooth chemicalconversion of oxaloacetate and pyruvate in weakly alkaline solution to aproduct which on oxidation with hydrogen peroxide gave citric acid, hasmade a detailed study of this reaction.43 The simplest assumption, an aldolcondensation which may be catalysed by an enzyme similar to aldolase,would yield oxalocitramalic acid,H02C*CH2*C( OH) (CO,H)*CH,*CO*CO,Hwhich has been purified as the la~tone.~* However, this substance in spiteof the ease of its formation cannot be the intermediate in the citric acidsynthesis since (a) it is not oxidised nor does it yield citric acid in the presence39 E. A. Evans, junr., Harvey Lectures, 1944, 39, 273.406 E. A.Evans, junr., and L. Slotin, J . B w l . Chem., 1940,136, 301; 1941,141, 439,406 H. G. Wood, C. H. Werkman, A. Hemingway, and A. R. Nier, ibid., 1941, 139,4Oe See Ann. Reports, 1941, 38, 257-61.,il H. A. Krebs, Biochem. J., 1942, 36, Proc. ix.4a 8. physwl. Chem., 1936, 242, 1.44 Idem, Habilitation Dissertation, Tubingen, 1937.* Cf. reference 28.377; 1942,142, 31.43 C. Martius, ibicE., 1943, 279, 96204 BIOCHEMISTRY.of suitable enzymes, arid (b) its accumulation cannot be demonstrated underenzymic conditions which should favour its detection. The alkali-labilecondensation product obtained by F. L. Breusch 45 was probably identicalwith oxalocitramalate, and thus is not connected with citric acid synthesisin V ~ V O . ~ It is concluded that oxidation of the pyruvate is probably coupledwith condensation to a C, compound. .Since neither acetate, acetaldehyde,nor monoacetyl phosphate could be substituted for pyruvate in this synthesis,the formation was assumed of an active C, compound resembling the ketenradica1,43 as has been discussed above.The oxolocitraconic acid is assumed to be oxidatively decarboxylated tocis-aconitic acid, this substance being readilyand isocitrate through the action of theaconitase :isocitrate cis-aconitate- H,O7- + HZO(1.1According to K.P. Jacobson 46 reaction (I) isinterconvertible with citratewidely distributed enzyme,+ Ha0 citrate7 - HZO(11.1catalysed by a-aconitase andreaction (11) by p-aconitase, these two enzymes always -being associated,though in variable proportions, in animal and vegetable tissues.However,C. MartiusY4’ who showed that the cis-isomeride was that concerned in thesereactions, considered that a single enzyme catalysed both equilibria, and theevidence to the contrary is not yet convincing.The dehydrogenation of isocitrate to a-ketoglutarate has recently beenshown48ajb to be a two-stage reaction; the primary reversible dehydro-genation, by a coenzyme I1 system present in dialysed extract of an acetone-precipitated pig’s heart preparation, requires no manganese and yieldsoxalosuccinic acid. The secondary stage, the decarboxylation by oxalo-succinic carboxylase, a specific carboxylase requiring manganese ions andpresent in similar extracts of pig’s heart, is also reversible in its action.Thereaction constants are :Stage I : (isocitrate) (Co. II-oxidized)(oxalosuccinate) (Co. II-reduced) = 0.3.( oxalosuccinate)(a-ketoglutarate) (CO,) Stage I1 : = 0.5 x lo3.or for the overall reaction :(isocit.) (Co. II-oxid.)/(CO,) (a-ketoglut.) (Co. 11-red.) = 1.3 x 10-4Thus a new system for the fixation of carbon dioxide which yields isocitricacid is revealed by these experiments. The rate of carbon dioxide fixation46 8. physiol. Chem., 1937,250,262; Biochem. Z . , 1937,295, 101; Biochem. J., 1939,33, 1757 ; Enzymologia, 1942,10, 165.F. L. Breusch and P. Kaza, ibid., 1944, 11, 165.4 6 Ibid., 1940, 8, 327. 47 2. physwl. Chem., 1938, 257, 29.484 S. Ochoa, J . Biol. Chem., 1945, 159, 243; O E b S.Ochoa and E. Weisz-Tabori,ibid., p. 246DICKENS : THE INTERMED-Y METABOLISM OF CARBOHYDRATES, ETC. 205is increased : (a) by removal of the oxidised Co. I1 as it is formed, which maybe accomplished by its re-reduction by the simultaneous presence of thehexose monophosphate-dehydrogenase system, ( b ) by the removal of iso-citrate by addition of aconitase.Since the isocitrate dehydrogenase is part of a coenzyme I1 catalysedsystem, the reoxidation by a cytochrome system of reduced coenzyme pre-sumbably requires the cytochrome c reductase of E. Haas et ~ 1 . ~ ~ or a similarflavin intermediate carrier. Very recently this reaction has also been shownto occur in extracts of pig’s heart.50 It would appear therefore that acoupling of coenzyme I systems with this coenzyme I1 system might beconceived as proceeding through the cytochrome system and suitable cyto-chrome c reductases :Co.II-H, + cyt. c + Co. I1 + cyt. c-H,Cyt. c-H, + Co. I -+ Co. I-H, + cyt. c.This is a point of some importance in the tricarboxylic acid cycle, since, ashas been frequently pointed out,cf- l1 Krebs’s scheme of transfer of electronsfrom isocitrate to oxsloacetate involves two systems of which the former isbelieved to be catalysed by Co. I1 and the latter by Co. I, so that a directinteraction of the two systems does not seem likely. Possibly Ochoa’sobser.vation may supply a way out of this difficulty. In this connexionE. S. G. Barron l1 has pointed out that the isocitrate dehydrogenase systemis not widely distributed in nature, and the introduction of this system limitsthe tissues to which the cycle could be applicable.F. L.Breusch,46 throughout a severe critic of the importance of this cyclein carbohydrate metabolism, has stated that only in kidney tissue doescitrate formation from pyruvate and oxaloacetate occur with sufficientrapidity, and there only when an excess of the two reactants is present. Heregards the cycle as being mainly concerned in the metabolism of P-keto-acidsarising in fatty acid oxidation (see below). Earlier criticism 459 51 that addedcitrate did not increase the respiration of minced muscle has been answeredby H. A. Krebs 28 as being due to the inhibition of respiration by excess ofcitrate which suppresses the ionisation, and therefore the catalytic effect, ofmagnesium.It is still uncertain how far the cycle could explain the carbohydrateoxidation in tissues other than pigeon muscle, the tissue mainly used inKrebs’s experiments, and probably also in heart muscle.52 Usually only afew of the component reactions have been studied in other tissues.Inaddition to “deionising ” effects of the kind just mentioned, there aredifficulties in interpretation due to the impermeability of cells to some of thecomponents of the cycle. For example, malonate probably penetrates49 E. Haas, B. L. Horecker, and T. R. Hogneas, J . BioZ. Chem., 1940,136, 747.50 S. Ochoa, ibid., 1945,160, 273.51 F. J. Stare, M. A. Lipton, and J. M. Goldinger, ibid., 1941,141, 981 ;68 D.H. Smyth, Bbchem. J., 1940, 34, 1046.Q. Thomag,Entymologia, 1939, 7, 231206 BIOCHEMTSTRY.intact animal tissues with difficulty;63 and, although yeast cells are almostimpermeable to succinate and citrate,S4 the free succinic acid has been foundto penetrate the cell quite readily; hence intact yeast cells are readily ableto oxidise the free acid but not its salts.55 Malonate has virtually no effecteither on the whole respiration or on the oxidation of acetate in yeast cells,but malonic acid is inhibitory.55 Since with animal tissues the use of freeacids is generally impossible, it is important that these considerations shouldbe kept in mind when the applicability of metabolic cycles to intact cells ofanimal tissues is being discussed. This factor has often been overlooked inin the past, and indeed presents a formidable problem in practice.The Metabolism of Acetate in Animal Tissues : Earlier Views.Although in the intact 56 or eviscerated 57 animal, as in the isolatedperfused heart,58 acetate is rapidly metabolised, it is remarkable that slicesor homogenates of most animal tissues oxidise added acetate rather feebly.An exception is kidney cortex, in which acetate disappearance may reachalmost the same rate as pyruvate oxidation.59$ 6o I n liver 60 and brain 609it is very slow.Various condensative mechanisms have been sug-gested e2, 59, 63, 64$ 6o none of which can yet be said to be convincing as amechanism of acetate metabolism. More recently, F. Lynen 557 65 fromexperiments with impoverished yeast suggested that acetate condenses withoxaloacetate and is then metabolised via the tricarboxylic acid cycle.Thiswould explain the inhibition of acetate oxidation by m a l ~ n a f e , ~ ~ , 60 andmight be capable of application to animal tissues a t least in a modified form.28There is now experimental support for the view that in some animaltissues acetate may be converted into acetoacetate as a first stage in itsaerobic metabolism, and it will be necessary to discuss this before returningthe tricarboxylic route of fatty acid oxidation.Interconversion of Acetate and Acetoacetate.Liver slices fairly readily convert added acetate into acetoacetate andp-hydroxybutyrate 66, 67 in a ratio determined largely by the effect of the53 G.D. Greville, Biochem. J., 1936, 30, 877.54 F. Lynen and N. Neciullah, Annulen, 1939, 541, 203.55 F. Lynen, ibid., 1943, 554, 40.566 T. B. McManus, C. B. Bender, and 0. F. Garrett, J . Dairy Sci., 1943, 20, 13.57 J. A. Dye and R. W. Marstens, Fed. PTOC., 1943, 2, 11.58 J. Barcroft, R. McNally, and A. Phillipson, Nature, 1943, 151, 304.59 K. A. C. Elliott, h1. B. Benoy, and Z. Baker, Biochem. J., 1935,29, 1936; I(. A. C.60 A. Kleinzeller, ibid., 1943, 3'9, 674.61 K. A. C. Elliott, D. B. M. Scott, and B. Libet, J . Biol. Chem., 1942, 146, 251.62 E. Toenniessen and E. Brinkmann, 2. physiol. Chem., 1930, 187, 137; 1938,63 H. Weil-Malherbe, Biochem. J., 1937, 31, 299.64 H. A. Krebs and W. A. Johnson, ibid., p. 772.65 Annulen, 1942, 552, 270.67 M. Jowett and J.H. Quastel, &id., 2143, 2169.66a G. Lusk, J . Biol. Chern., 1921, 49, 452.Elliott, M. E. Greig, and M. B. Benoy, ibid., 1937, 31, 1003.252, 169.86 N. L. Edson, Biochern. J., 1935, 29, 2082DICKENS : THE INTERMEDIARY METABOLISM OF CARBOHYDRATES, ETC. 207state of oxygenation on the cozymase-catalysed P-hydroxybutyrate dehydro-genase system of Green et aE.68 Until recently, opinion was divided as towhether acetoacetate was hydrolysed to acetate before being oxidised.Among those who originally supported this view was A. L. Lehninger,69as bwho demonstrated the chemical and enzymic cleavage to two molecules ofacetate. The yields were small and irregular with animal tissues (kidney,muscle), but in Esch. coli the activity was higher.The isolation fromliver tissue of acetic acid as the 2 : 4-dinitrophenylhydrazide 7O was con-firmed.71 However, W. C. Staclie et aL7, did not observe this hydrolysis inliver slices; it seems too slow in animal tissues to be intermediary inoxidation.Tracer studies with acetate containing isotopic C in the carboxyl haveclearly established the transfer of this C to the acetone compounds formed.73-76Fasted rats fed with this isotopic acetate contained excess 13C in the carboxylof the acetoacetate, while NaH13C0, was not fixed in the acetone compounds.73But the most convincing evidence was provided by similar experiments withcarboxyl-labelled acetate added to tissue slices.74* 75 I n liver it was foundthat 41-45% of the acetoacetate arose from the labelled acetate, the re-mainder being endogenous and accompanied by considerable formation ofacetate from tissue constituents.The respiratory carbon dioxide producedhad approximately the same 13C content as the acetoacetate, indicating thatprobably the whole of the acetate utilized by liver tissue passed through theintermediate stage of acetoacetate. It seems likely that in such reactions" active " acetate, arising oxidatively from acetate, is involved.76 The factthat more of the l3C found its way into the CO,H than into the CO groupof the acetoacetate suggests that the union of two different C, moleculesmight be concerned, one of ordinary acetate and one more reactive, possiblyacetyl phosphate. I n kidney and heart tissue, intermediates did notaccumulate during the oxidation of acetate.In kidney tissue some 13Cpasses into the non-volatile ether-soluble fraction, in conformity with theassumption that the further metabolism of acetate in this tissue passesthrough the tricarboxylic acid cycle, as will now be considered. In hearttissue ketonicsubstances do not appear to be intermediates in acetateoxidation. Evidence is given to show that the results cited above werenot due to fixation of liberated 13C0,.74, 756 8 D. E. Green, J. G. Dewan, and L. F. Leloir, Biochem. J., 1937, 31, 934.6Da J . Biol. Chem., 1941, 140, lxxvi.696 Idem, ibid., 1942, 143, 147.70 R. P. Cook and K. Harrison, Biochem. J., 1936, 30, 1640.7 1 A. L. Lehninger, J. Biol. Chem., 1943, 149, 43.72 W.C. Stadie, J. A. Zapp, junr., and F. D. M'. Lukens, ibid., 1941, 137, 75.73 M. E. Swendseid, R. H. Barnes, A. Hemingway, and A. 0. Nier, ibid., 1942, 142,74 S. Weinhouse, G. Medes, and N. F. Floyd, ibid., 1945, 158, 411.76 G. Medes, S. Weinhouse, and N. F. Floyd, Fed. Proc., 1945,4, 98.78 S. Weinhouse and G. Medes, Abstracts 108th Meeting, Amer. Chem. SOC., p. 47B47.(New York, 1944)208 BIOCHEMISTRY.Oxidative Mechanism of Acetate and Acetoacetate MetabolGm.F. L. Breusch 76a9b and H. Wieland and C. Rosenthal77 have recentlyindependently suggested that acetoacetate condenses with oxaloacetate toform citrate :CH,*CO*CH,*CO,H + H0,C*CO*CH2*C02H + H,O + citric acid + aceticacid (I).CH,*CO*CH,*CO,H + 2H02C*CO*CH2*C02H + H20 -+ 2 citric acid (11).These authors based their theory on the higher yields of citrate found whenthe two keto-acids were added to tissues than with either keto-acid alone.Breusch earlier favoured reaction (I) but later,78 because of the higher yieldsof citrate then obtained, agreed with Wieland and Rosenthal that reaction(11) more correctly represented the course of the condensation, at least foracetoacetate; with higher p-keto-acids the yields were smaller, and areaction of the type; R*CO*CH,*CO,H + H0,CCOGH2*C02H + H,O --+R*C02H + citric acid (111) represents the course of degradation of ap-keto-acid to a fatty acid with two less C-atoms.This would have greatimportance in explaining the fact that acetic acid has not been shown toarise during oxidation of higher fatty acids a t anything approaching the levelexpected from the original p-oxidation theory of K n o ~ p .~ ~ Unfortunately,the evidence presented, particularly by Breusch, is very incomplete, beingbased almost entirely on the yields of citrate obtained by incubation ofmuscle, kidney, heart, or brain (lung or pancreas did not react) withthe various p-keto-acids and oxaloacetate. Nevertheless Breusch hasnamed the condensing enzyme " citrogenase " ; a suggestion rightly con-sidered to be premature by Marti~s,4~ who has, however, been able toconfirm that acetoacetate and oxaloacetate together gave regularly about50% more citrate than either substance alone in presence of ox or pigheart .43Breusch reports the enzyme system to be stable in solution and ex-tractable from tissues by dilute sodium bicarbonate, though destroyed byheat.It is also inactivated by 0*002~-As,0,, but is unaffected by 0.005 M-iodoacetate or fluoride. The pH optimum is " in the alkaline region " 76bor pH 7-5,78 and cat and pigeon tissues form up to 6 mg. citric acid/g. moisttissue/hr. from added p-keto-acid and oxaloacetate. A number of keto-acidsand other substrates were tested in presence of oxaloacetate : acetoacetate,benzoylacetate, and acetonedicarboxylate yielded citrate ; butyrate,crotonate, and p-phenylpropionate did not. a- and y-Keto-acids are stated76@ Science, 1943, 97, 490; 76b Enzymologia, 1944, 11, 169.7 7 Annalen., 1943, 554, 241.7 8 F. L. Breusch and H . Keskin, Enzymologia, 1944, 11, 243.70 W.H. Hurtley, Qua.rt. J . Med., 1915, 9, 301 ; H. D. Dakin, J. Biol. Chem., 1909,6, 373. See also refs. 67, 72DICKENS : THE INTERMEDIARY METABOLISM OF CARBOHYDRATES, ETC. 209to condense with only about one tenth of the rapidity of p-keto-acids. Of alarge number of p-keto-acids tested, some did not react. The kidney tissueof the cat is, according to Breusch, the only tissue with which any considerablecondensation of pyruvate with oxaloacetate occurs, but this is a t variancewith the work of others,sO and like many of Breusch’s statements is notsupported by adequate published experimental evidence.H. Wieland and C. Rosenthal 77 found, in experiments with rabbitkidney, that the oxygen uptake was increased by about 6--12% by theseparate addition of acetoacetate or oxaloacetate, but by more than 40%when both substances were added together.By addition of barium ions 81(excess of magnesium ions has the same effect S2) the further metabolism ofcitrate is inhibited, and Wieland and Rosenthal found amounts of citrate upto 80% of those calculated according to reaction (11) from the amount ofacetoacetate added. These experiments were all performed aerobically withmechanically sliced rabbit kidney. Only a small yield of citrate was obtainedwith either substrate alone. Acetate incubated with kidney and oxaloacetategave only about half the yield obtained from acetoacetate plus oxaloacetate ;hence it was concluded that the metabolism of acetate probably proceeds viaacetoacetate, and not vice versa.Somewhat similar results were obtainedwith ox heart, but the reaction failed in liver tissue. The authors considerthat it cannot be excluded that the whole carbohydrate metabolism, at leastin kidney and heart, may pass through acetoacetate, rather than wiapyruvate, as the stage which condenses with oxaloacetate to form citrate.They suggest (without ezperimental investigation) that if this were so theappearance of acetonic compounds in diabetes might be connected with thelack of the enzyme responsible for the condensation to “ pro-citric acid ” theprecursor of the citrate formed. H. A. Krebs and L. V. Eggleston *& pre-viously suggested that insulin may act catalytically in the tricarboxylicacid cycle, In this connexion it may be pointed out that very recently astriking effect has been reported 83 of hormones in vivo and in vitro on thehexokinase reaction : glucose + adenosine triphosphate --+ glucose 6-phosphate + adenosine diphosphate.This primary reaction in the break-down of glucose or its synthesis to glycogen is inhibited by anterior pituitaryextract, and the resulting inhibition is counteracted by insulin. Yeasthexokinase, unlike that of muscle, liver, kidney, heart, and brain, is notinhibited by pituitary extract. The full publication of these importantobservations is awaited with great interest.H. Wieland and C. Rosenthal 77 assume, on the basis of earlier work 653 81on yeast, that a preliminary condensation of acetoacetate with oxalo-acetate occurs, to form a “pro-citric acid ” (I or 11), which may80 Hallman, Acta physiol.Scund., 1940, 2, Suppl. IV.8 1 R. Sonderhoff and M. Diffner, Annalen, 1938,536,41.82 A. I. Virtanen and J. Sundman, Bwchern. Z., 1942,313, 236.P2fl Biochern. .J., 1938, 32, 913.83 W. H. Price C . F. Cori, and S. P. Colowick, J . Biol. Chem., 1945,160, 633210 BIOCHEMISTRY.possibly itself condense with a second molecule of acetoacetate to yield(111) :HO,C*CH, *C ( OH) CO,H(I.) (" citroylacetic acid ")RO,C.CH,*CO*CH, I---" H0,C*CH2*CO*C0,H+ 1 \ HO,C*CH,*C( OH)*CO,HH0,C*CH,*CO*CH3 % H0,C *CH *C O*CH,(11.) (" a-acetylcitric acid ")HO,C*CH,*CO*CH, -+ (I) --+HO,C*CH,*C( OH)(C02H)*CH2*CO~CH(C0,H)*CH(C0,H)*CH2*C0,Compound (111) might be split as shown by the broken line to give two mols.of citric acid.This would explain why acetate formation was not observed;alternatively the C,-fraction split off from (I) or (11) on hydrolysis might be" active " acetic acid, which would then react differently from the freesubstance.In yeast, possibly a similar condensation of acetate with aldehyde mayoccur, the CH,*CO*CHR*CHO which results corresponding to acetoacetatein the above reactions."The combination of oxaloacetate and acetoacetate is apparently a simpleadditive reaction : yet Wieland and Rosenthal 77 found that oxygen wasnecessary for the formation of citrate in kidney tissue. In nitrogen virtuallyno synthesis occurred. They concluded, therefore, that the enzymicsynthesis was coupled with an aerobic process which is dependent on theoxygen pressure but is otherwise of an unknown nature.Similarly in yeastno citrate was formed from oxaloacetate and acetate except in presence ofoxygen.H. A. Krebs and L. V. Eggleston 84 made almost complete balance sheetsof the metabolite interchange occurring in sheep heart muscle during incuba-tion with acetoacetate. The rate of acetoacetate removal (Qac. ac.) was about15 aerobically and 6 in nitrogen; only the aerobic removal was inhibited bymalonate, indicating that succinic dehydrogenase plays a part in the aerobicprocess only. When fumarate, oxaloacetate, or a-ketoglutarate was addedboth the aerobic and the anaerobic removal of acetoacetate were increased toa similar level, and the malonate inhibition was largely negatived.Virtuallyall the metabolised acetoacetate was recovered as p-hydroxybutyrate,resulting mainly from the dismutations :Acetoacetate + a-Ketoglutarate = p-Hydroxyglutarate + Succinate + CO,Acetoacetate + Malate = p-Hydroxybutyrate + Oxaloacetate.It was concluded that in the experiments of Breusch and of Wieland andRosenthal, described above, the citrate formed arose from oxaloacetate,which added alone is in part reduced and in part oxidised to citrate and othercompounds. When acetoacetate is added, it is partly reduced to (3-hydroxy-(111.) (" citroylcitric acid ")134 Biochem. J . , 1945, 39, 408DICKENS : THE INTERMEDIARY METABOLISM OF CARBOHYDRATES, ETC. 211butyrate at the expense of some reoxidation of malate to oxaloacetate, thusmaking more oxaloacetate available for citrate formation.84I n considering these opposed interpretations, it should be rememberedthat Wieland and Rosenthal specifically stated that their reaction did notoccur anaerobically.Hence it remains to be seen if in oxygen there mightnot be quite a different outcome of experiments of the type made by Krebsand Eggleston. It is true that the latter authors have far more compre-hensive data, and a similar application of the “ balance sheet ” principle toWieland and Rosenthal’s experiments would be highly desirable. Never-theless, the failure of Krebs and Eggleston a43 a5 and of H. Weil-Malherbea6to observe the condensation of oxaloacetate and acetoacetate may prove tohave been due to their adoption of anaerobic conditions and to the use byWeil-Malherbe of hand-cut (and therefore presumably less damaged) kidneyslices, in which material acetoacetate consumption is probably nearlymaximal *7 even without added C,-acids.I n support of the oxidative nature of the condensation of oxaloacetatewith acetoacetate (or a product derived from it oxidatively), are two recentstudies.F. E. Hunter and L. F. Leloir88 found that dog kidney tissueparticles, which did not oxidise citrate, yielded very little citrate upon theaddition of acetoacetate + oxaloacetate, .unless some a-ketoglutarate or asubstance which yields a-ketoglutarate (glutamate or glutathione) wasadded. Citrate then appeared in good yield (about 2 mols. of citrate foreach mol.of acetoacetate metabolised) and the reduction of acetoacetate tohydroxybutyrate accounted for only a small part of the acetoacetate whichdisappeared during the synthesis. I n this system, the simultaneous oxid-ation of a-ketoglutarate is necessary for citrate formation, but the oxidationcan here be either aerobic or dismutative. It was thought that the oxidationof a-ketoglutarate was in some way coupled with the formation of an activeC, intermediate from the acetoacetate, but acetyl phosphate addition didnot yield any citrate.Finally, J. M. Buchanan et u Z . , ~ ~ who used CH3J3C0,H andCH,*13CO*CH2*13C02H to study the intermediate metabolism of thesesubstances in homogenised guinea pig kidney, were able to show that notonly did the components of the tricarboxylic acid cycle greatly stimulate theconsumption of acetoacetate, but the isolated acids (a-ketoghtaric, succinic,fumaric) contained excess of 13C which could not be accounted for by assimi-lation of 13C0,. The amounts recovered showed that the tricarboxylic acidcycle is in fact an important metabolic pathway for the oxidative metabolismof acetoacetate as well as of acetate.These results seem to prove that areaction similar t o that proposed by Wieland and Rosenthal undoubtedlydoes account for a considerable part of the metabolism of these two acidsunder the conditions adopted.Nature, 1944, 154, 209. 86 Ibid., 1944, 153, 435.87 J. M. Buchanan, W. Sakami, S. Gurin, and D. W. Wilson, J. Bwl. Chem., 1946,159, 695.88 Ibid., 1946, 169, 2962 12 BIOCHEMISTRY.In addition, the fact that 90% of the 13C excess is present in the carboxylof a-ketoglutarate which is remote from the keto-group suggests that cis-aconitate or isocitrate, or both, are intermediates, and not a symmetricalmolecule such as citrate.The last therefore arises by a side-reaction,presumably catalysed by aconitase. The authors favour the view thatacetoacetate provides a reservoir of available reactive C, compound,according to the theories already discussed.The Metabolism of Higher Fatty Acids.During the past year important new evidence of the essential correctnessof Knoop’s p-oxidation theory has been obtained by the use of fatty acidscontaining isotopic carbon. At the same time an explanation has beenprovided of many of the diEculties which led to the assumption of alternativepaths,*9 such as multiple alternate o ~ i d a t i o n , ~ ~ ~ 679 72 and w-oxidation,w oracetopyruvate formationYs4 so that at present these theories seem no longeressential at least for liver slices.The puzzling facts that no acetate accumu-lation is demonstrable during oxidation of the higher fatty acids, and thatthe yield of acetonic compounds exceeds that predicted by the classical Knooptheory, are now accounted for by the demonstration that p-oxidation isfollowed by condensation of acetate, or other C, fraction, to form aceto-acetate ; a suggestion previously advan~ed,~la* b but only recently proveddirectly.S. Weinhouse, G. Medes, and N. F. Floyd 92* found that when liver slicesderived from fasting rats were incubated with n-octanoic acid, containingl3C in the carboxyl, the resulting acetoacetate contained excess of 13C equallydistributed between the keto- and the carboxyl group.As the authors state,this seems to be unequivocal evidence that the ketonic compounds areformed by random condensation of pairs of similar C, units arising from thep-oxidation of the fatty acid :CH3*[CH,]4*CO*CH2*C*02H -+ CH3*[CH2],*C02H + CH3*C*0,H2 CH3*C*02H --+ CH,*C*O*CH,*C*O,H.The above experiments do not decide the fate of the n-butyric acid whichremains after splitting off two acetate residues from n-octanoic acid; thiscould be either (A) cleavage into two molecules of acetate followed by theirresynthesis to acetoacetate or (B) direct P-oxidation to acetoacetate.Thiswas settled 93 by similar experiments with carboxyl-labelled n- butyratewhich showed that mainly route (A) was followed, but that to a lesser extentdirect p-oxidation did occur. The butyrate was not diluted by endogenousbutyrate, which is therefore either not formed or else completely oxidised89 See Ann. Reports, 1935, 32, 4 1 4 4 1 7 .90 E. J. Witzemann, “Advances in Enzymology,” Interscience Press, N.Y ., 1942,910 E. M. MacKay, R. H. Barnes, H. 0. Came, and A. N. Wick, J . BioZ. Chem., 1940,916 E. M. MacKay, A. N. Wick, H. 0. C m e , and C. P. Barnes, ibid., 1941,138,63.Ibid., 1944, 153, 689.O3 G. Medes, S.’Weinhouse, and N, F. Floyd, ibid., 1945, 157, 36.2, 266.135, 157.oab Ibid., 1944, 155, 143DICEENS : THE INTERMEDIARY METABOLISM OF CARBOHYDRATES, ETC.213under these conditions. All the excess 13C of the respiratory carbon dioxidecould be accounted for as having been metabolised via the ketonic compounds.To a slight extent butyrate may be rnetabolised by an unknown reactionresulting in the presence of 13.8y0 of the utilised 13C in the residual non-volatilecarbon compounds. Since the two routes of butyrate oxidation discussedabove lead to different distributions of isotopic C, it may be calculated from thedistribution actually observed that about 64-78y0 of the acetoacetate arisesby route (A) (disruptive p-oxidation and resynthesis) and about 22-36% byroute (B) (direct p-oxidation). A less likely possibility is that all the butyratemight first pass through the stage of acetoacetate which then splits into C,fractions, but the interconversion of these into acetoacetate would have to beslow, otherwise all the latter substance would be brought to the same isotopicdistribution ?3When fed together with glucose to rats, propionate and butyrate 94 werestated to yield liver glycogen, while acetate was not, except in so far as itgave rise to carbon dioxide which was assimilated into carb~hydrate.~~The last conclusion is denied by V.Lorber, N. Lifson, and H. G. Wood, whofound after preliminary experiments g6 that by pathways other than carbondioxide fixation the 13C of carboxyl-labelled acetate,97 p r o ~ i o n a t e , ~ ~ orbutyrate 98 enters the glucose molecule of liver glycogen in positions 3 and 4.The same positions are those occupied by fixed carbon of carbon dioxide.99Space does not allow discussion of the synthesis of higher fatty acids looor cholesterol lo1 from acetate, nor can details be included of enzyme systemswhich oxidise fatty acids lo2 possibly viu the acyl phosphate.lo3Similarly, the developments linking the intermediate acids of the tri-carboxylic acid cycle with amino-acid metabolism, by means of transamin-ations and oxidative deamination,l" can only be mentioned here.Butevidence is steadily accumulating to show the participation of these reactionsin the interconversion of amino-acids by animal tissues.F. D.94 But see H. J. Deuel, junr., C. Johnston, M. G. Morehouse, H. S.Rollman, and95 J. M. Buchanan, A. B. Hastings, and R. B. Nesbett, ibid., 1943,150, 413.96 N. Lifson, V. Lorber, and H. G. Wood, Fed. Proc., 1945, 4, 47.O 7 V. Lorber, N. Lifson, and H. G. Wood, J . Biol. Chem., 1945,161,411.98 H. G. Wood, N. Lifson, and V. Lorber, unpublished, cited in ref. 97.g9 Idem, J . Biol. Chem., 1945, 159, 475.loo D. Rittenberg and K. Bloch, ibid., 1944, 154, 311 ; Arch. Biochem., 1945, 4, 101.Io1 K. Bloch, E. Borek, and D. Rittenberg, Fed. Proc., 1945, 4, 84.lo2 See J. M. Muiioz and L. F. Leloir, J . Biol. Chem., 1943,147, 355; 1944,153,53;-4. L. Lehninger, ibid., 1944, 154, 309; 1945, 157, 363; also E. L. Cosby, and J. B .Sumner, Arch. Biochem., 1945, 8, 259.R. J. Winzler, J . Biol. Chem., 1945, 157, 135.lo3 A.L. Lehninger, Zoc. cit., ref. 102.lo4 See P. P. Cohen, Fed. Proc., 1942, 1, 73; A. E. Braunstein and S. M. Bychkov,Nature, 1939, 144, 751 ; Biochimia, 1940, 5, 261 ; A. E. Braunstein and R. M. Asarkh,J. Biol. Chem., 1945,157,421. Cf. M. Blanchard, D. E. Green, V. Nocito, and S . Ratner,ibid., 1944, 155, 421; L. F. Leloir and D. E. Green, Arch. Biochem., 1945, 4, 96; F.Schlenk and A. Fisher, ibid., 8, 337; H. C. Lichstein and W. W. Umbreit, J. Biol.Chem., 1946,161, 311 ; F. Schlenk and E. E. Snell, ibid., 157, 425214 BIOCHEMISTRY,2. BIOCHEMISTRY OF THE ADRENAL CORTEX.In the biochemical section of the Annual Reports the last review con-cerning the adrenal glands appeared in 1936.l It is characteristic of thedevelopment of biochemistry that since that date three reviews of theorganic chemistry of adrenal steroids have appeared 2s 39 and only now doesthe subject return, for review of functional aspects, to the biochemicalsection.The Results of Removal of the Adrenal Glands.For adequate consideration of the action of adrenal steroids, the chiefeffects of experimental removal of the adrenal glands must be briefly reviewed.References to the original literature will be found in recent publica-t i o n ~ .~ ~ 6, 8, 9 3 lo, l 1 3 l2 Although species variations are encountered thefollowing summary of the results of adrenalectomy, based on observationswith the dog, cat, and rat, may be taken as relevant to the more commonlyinvestigated species, though some elasmobranch fishes, and the opossum,exhibit interesting variations from the normal picture.llUnless the contrary is indicated the influence of removal of the wholeadrenal gland (medulla plus cortex) may be attributed largely or solely t oabsence of the cortical portion.Among the most striking effects of adrenalectomy is diminution or dis-appearance of appetite (anorexia).In its turn anorexia may significantlyinfluence metabolic functions, and hi studies on the effects of adrenalectomyit is important to appreciate the possible complications introduced in this way.For instance, restriction in food intake can depress the rate of absorption ofmaterial from the gut and can also reduce the width of the proximal epiphy-seal cartilage in the tibia of young rats, both of which effects also followadrenalectomy.Since the anorexia of adrenalectomy can often be combattedby the administration of sodium chloride, attempts may sometimes thusbe made to dissect the direct from the indirect effects of removal of adrenalhormones.(a) The Metabolism of Electrolyte and Water.-In the dog, death mayfollow adrenalectomy in little more than a week, and the development of thecondition of adrenal deficiency thus induced is associated with important1 C. P. Stewart and J. Stewart, Ann. Reports, 1936,33, 395.2 R. K. Callow, ibid., 1938, 35, 281.3 F. S. Spring, ibid., 1940, 37, 332.5 (a) R. F. Loeb, Bull. N . Y . Acud. Med., 1940,16, 347; (b) idem, Harvey Lectures,1942, 37, 100; (c) idem, “ Glandular Physiology and Therapy,” American Medical Ass.,Chicago, 1942, p.287.6 (a) E. C . Kendall, Arch. Path, 1941, 32, 474; (b) idem, “ Glandular Physiology andTherapy,” h e r . Med. Ass., Chicago, 1942, p. 273 ; (c) idem, Endocrinology, 1942,30,853.7 J. J. P f f i e r , Advances in Enzymology, 1942, 2, 325.13 F. A. Hartman, Endocrinology, 1942, 30, 861.9 (a) D. J. Ingle, ibid., 1942, 31, 419; ( b ) a m , in Amer. Ass. Advancement of10 T. Reichstein and C. W. Shoppee, Vitamins and Hormones, 1943,1,346.11 W. W. Swingle and J. W. Remington, Physiol. Rev., 1944 24,89.l2 L. J. Soffer, J . Mount Sinai Hospital, 1946,11,263.4 Idem, ibid., 1943, 40, 147.Science, Washington, D.C., 1944, p. 83YOUNG : BIOCHEMISTRY OF THE ADRENAL CORTEX. 216changes in the concentration in the body of Na+, a characteristic inorganicconstituent of extracellular fluids, and of K+, a prominent inorganic constitu-ent of the interior of muscle and other cells.Na+ (together with C1-) is lostin excessive amounts in the urine, and the concentration of these ions in thebody fluids (and also in the tissues) falls to a subnormal level. Water thenpasses from the blood into the cells of the body, with the result that the contentof dry matter of the blood rises. At the same time the urinary excretion ofK+ is greatly diminished and K+ therefore accumulates to an abnormaldegree in the tissue fluids. Kidney function is depressed generally and,although Na+ and C1- constitute an exception, the excretion of most urinaryelectrolytes, and often of nitrogenous substances also, is subnormal. Theabnormalities in electrolyte metabolism are initially associated with diuresis ;nevertheless the ability of the animal to excrete administered water may laterbe greatly depressed, and there is usually an exaggerated susceptibility towater intoxication.Oliguria may be a terminal symptom of fatal adrenalinsufficiency.The fall in plasma volume, associated with a passage of water from theblood into the tissues, contributes to a lowering of the blood pressure and adiminution in the rate of blood flow. This in its turn may enhance the failureof the kidney to excrete K', an effect which thus may become exacerbated.Nevertheless the accumulation of K+ cannot be regarded as the sole cause ofdeath, because a rise in blood K+ equal to that found in adrenalectomisedanimals is compatible with life in normal or in treated adrenalectomisedanimals.ll Likewise the depletion of sodium cannot be regarded as the solecause of death from adrenal failure.ll(b) Carbohydrate MetaboEism.-In the adrenalectomised rat the glycogenstores, which may not be unduly low in a well-fed animal, disappear at anabnormally fast rate during a short period of starvation.Anorexia, whichmay be a prominent feature of adrenal insufficiency, contributes greatly tothe depression of glycogen storage, while a diminished formation of sugar fromprotein (see below) conduces to the same end. Glycogen formation fromadministered carbohydrate becomes slow, and subnormal in amount, whilean excessively high respiratory quotient may indicate that an unusuallylarge proportion of the available carbohydrate is undergoing oxidation.Experimental diabetes may be reduced in intensity as the result of removalof the adrenal glands.The hypoglyczmic action of a small dose of administered insulin becomesgreatly exaggerated, and, particularly in the terminal stages of adrenalinsufficiency, a spontaneous fall of blood sugar may occur.Despite theprofound influence of adrenalectomy on the metabolism of glucose andglycogen it is not possible to ascribe the resulting death solely to the abnormali-ties in carbohydrate metabolism.(c) Protein Metabolism.-In adrenal insufficiency protein catabolism isdiminished and consequently there is decreased production of carbohydratefrom protein.It is of particular interest that this is associated with afall in liver arginase activity. Since urinary excretion may be subnormal216 BIOCHEMISTRY.particularly during starvation , the concentration of non-protein nitrogen inthe blood may rise despite the diminished protein catabolism in the tissues.Plasma protein concentration may rise in association with the decrease inblood volume, but the plasma albumin fraction is diminished and the rise intotal plasma protein content is to be ascribed to a predominating increase inthe globulin fraction.(d) Fat MetaboEisrn.-Adrenalectomy has a less clear-cut influence on fatmetabolism than on the metabolisms of carbohydrate and protein.13 Lessfat is stored in the livers of adrenalectomised animals than is usual, and thedevelopment of fatty livers which, in normal animals, follows the adminis-tration of-a high-fat diet and of certain poisonous substances, does not occurin the absence of the adrenal cortex.It is possible that the rate of oxidationof fat is depressed in adrenal insufficiency, but it is difficult to obtain un-equivocal evidence of this. Experimental ketonuria may be diminished inintensity as the result of removal of the adrenal glands, but this effect mayin part be the result of a rise in the kidney threshold for ketonic substances.(e) Resistance to Stress.-The adrenalectomised animal is abnormallysensitive to alterations in environmental conditions, and may die as theresult of changes (rise or fall) in environmental temperature or pressure whichare not fatal to intact animals.Likeyise adrenalectomised animals areabnormally easily killed by many toxic substances, by numerous sorts ofdietary deficiencies, and by many types of experimental “ shock,” e.g.,haemorrhage, surgical trauma, and bacterial infection.The muscles of adrenalectomised rats are particularly easily fatiguedwhen stimulated to activity, this fatigue being associated to some extentwith the development of hypoglyczmia, the excessive diminution in thestores of muscle glycogen, and possibly with circulatory changes, all ofwhich are found in adrenalectomised animals.(f) Sex GZands.-The recognition of a clinical condition in which anadrenal tumour or adrenal cortical hyperplasia is associated with the appear-ance of masculinising features in female patients and, according to somethough not all clinicians, the occasional development of female traits in themale, emphasised the possibility that experimental adrenslectomy mightexert an outstanding influence on the sex glands.Although testicular andovarian degeneration have been described as sequels of adrenalectomy themost striking observations have been in human patients with Addison’sdisease-the condition, in the human being, of adrenal hypofunction. InAddison’s disease the excretion by female patients of androgens (neutral17-keto-steroids) may be subnormal or even nil,149 15, 16, 17 while axillary1s D. J. Ingle, J. Clin. Endocrinol., 1943, 3, 603.14 R. K. Callow, Proc.Roy. SOC. Med., 1938, 31, 841.16 (a) R. W. Fraser, A. P. Forbes, F. Albright, H. Sulkowitch, and E. C. Reifenstein,J . Clin. Endocrinol., 1941, 1, 234; (b) F. Albright, P. H. Smith, and R. W. Fraser,Amer. J. Med. Sci., 1942, 204, 625; (c) F. Albright, Harvey Lectures, 1943, 38, 123.16 0. Wintersteiner, “ Glandular Physiology and Therapy,” Amer. Med. Ass., Chicago,1942, p. 327.17 E. J. Kepler, G. A. Peters, and H. L. Mason, J . Clin. Endocrinol., 1943, 3, 497YOUNG: BIOCHEMISTRY OF THE ADRENAL CORTEX. 217hair, the existence of which is believed to depend on androgens secreted bythe adrenal cortex, is usually lackirig.l5, 16( g ) Miscellaneous Observations.-In adrenalectomised rats the rate ofabsorption of glucose from the gut is depressed; that of long-chain fattyacids is also diminished, but not that of short-chain fatty acids such as butyricacid.The theory of Verzar, that the secretions of the adrenal cortex areessentially concerned in the processes leading to the phosphorylation ofcarbohydrate or fat, was originally based on observations concerning thesubnormal rate of intestinal absorption (presumably through phosphorylatedintermediates) in adrenalectomised animals, but the theory suffered seriousembarrassment with the demoiistration that treatment with sodium chloriderestores the rate of glucose absorption to normal in the adrenalectomisedrat.ll9 l8 Verzar, however, believes that disturbances in the phosphorylationof glycogen in vitro by preparations of muscle from adrenalectomised ratscan be restored by the addition of adrenal cortical steroids, and he maintainshis viewpoint regarding the particular importance of the adrenal cortex inphosphorylation mechanisms.l9 This idea has, however, not gained generalacceptance.20The basal metabolic rate may be initially unchanged but may laterbe diminished in adrenalectomised animals. In rats, cytochrome oxidaseactivity and cytochrome-c concentration may both fall as a result of removalof the adrenal glands.21Chronic adrenal insufficiency inhibits normal growth in the young ratand prevents the normal regression of the thymus gland. Adrenalectomisedmice show a lyrnphocytosis with a decrease in polymorphonuclear lym-phocytes22 and, when exposed to conditions of stress, do not show thelymphocytopenia exhibited by normal mice.23Conditions which modify the Survival of Adrenalectomised Animals.Since the classical work of Swingle and Pfiffner it has been possible toprepare extracts of the adrenal cortex, the frequent parenteral administrationof which allows the adrenalectomised animal to survive indefinitely.It ispossible, however, to prolong the survival of adrenalectomised animals nottreated with adrenal extracts under some conditions, notably by the adminis-tration of a diet high in Na+ and low in K+. Untreated dogs may also surviveadrenalectomy for the period of pregnancy or of pseudopregnancy, while inhibernating animals the season of the year influences the length of survivalafter adrenalectomy, the animals generally surviving the period of torpor.Evelyn Anderson, “ Essays in Biology in Honor of Herbert M.Evans,” California,U.S.A. : Univ. Calif. Press, 1943, p. 33.l9 C. Montigel and F. Verzar, Helv. Physiol. Pharrn. Acta, 1943, 1, 115.*O N. Stillman, C. Entenman, E. Anderson, and I. L. Chaikoff, Endocrinology, 1942,21 S. R. Tipton, ibid., 1944, 34, 181.22 A. White and T. F. Dougherty, ibid., 1945, 36, 16.2s F. Elmadjian and G. Pincus, ibid., 1945, 37, 47.31, 481218 BIOCHEMISTRY.In adrenalectomised cats the administration of certain anterior pituitaryextracts may prolong survival.The Preparation and Properties of Physiologically Active Adrenal Extracts.(a) Assay of Adrenal Cortical Extrack-The above considerationsemphasize the importance of maintaining a strict control of dietary andenvironmental conditions in animals employed for the assay of adrenalcortical substances capable of maintaining the life of the fully adrenalec-tomised animal.The maintenance of the adrenalectomised dog 24 and rat 25in good health have both been employed as a criterion of activity of adrenalextracts, while the maintenance of a normal electrolyte balance in adrenal-ectomised dog has also been utilised.26Since the diversity of the effects of adrenalectomy are paralleled by thevariety of the qualitatively different actions of the substances isolated fromthe gland it is not surprising to find that methods of assay based on criteriaother than that of life maintenance in adrenalectomised animals often fail toyield concordant results. Nevertheless such tests have been of great valuein elucidating the nature of the complex action of crude adrenal corticalextracts.Two such methods of assay of particular importance have been the" Everse-de Fremery " work test,27 based on the height of the contractiveresponse in the stimulated calf muscles of the extract-treated adrenalec-tomised rat, and the " Ingle " work test, which utilises the total amount ofwork the muscle of the trea.ted adrenalectomised rat is capable of yielding onstimulation to exhaustion.2* As will be seen later these two methods of assaydetermine different types of adrenal cortical substances.A widely used method is that based on the ability of adrenal preparationsto protect the adrenalectomised rat against the otherwise lethal effects of alow environmental temperature ; 29 this method is proving of particular valuefor the assay of adrenal substances in urine.Another useful method utilizesthe ability of adrenal extracts to raise the liver glycogen content of fastingadrenalectomised or normal rats.30 The polarographic estimation of adrenalsteroids, in which no biological test is necessary, may become of especialimportance,31 while colorimetric methods of assay are also being developed.32J. J. Pfiffner, W. W. Swingle, and H. M. Vass, J . BWZ. Chem., 1934,104, 701.26 G. F. Cartland and M. H. Kuizenga, Anzer. J . Physiol., 1936,117, 678.28 (a) G. W. Thorn, L. L. Engel, and H. Eisenberg, J. Exp. Med., 1938, 68, 161;21 J. W.R. Everse and P. de Fremery, Acta Brev. Need., 1932, 2, 152.28 (a) D. J. Ingle, Amer. J . Physiol., 1936, 116, 633; ( b ) idem, Endocrinology, 1944,*@ (a) G. Widstrom, Acta Med. Scund., 1935, 87, 1; ( b ) H. Selye and V. Schenker,Proc. SOC. Exp. BWZ. Med., 1938, 39, 518.(a) R. M. Reinecke and E. C. Kendall, Endocrinology, 1942,31, 573 ; (b) idem, ;bid.,1943, 32, 605; (c) H. V. Bergman and D. Klein, ibid., 1943, 33, 174; (d) R. E. Olson,S. A. Thayer, and L. J. Kopp, ibid., 1944, 35, 464; ( e ) R. E. Olson, F. A. Jacobs,D. Richert, S. A. Thayer, L. J. Kopp, and N. J. Wade, ibid., 1944, 35, 430.( b ) G. W. Thorn and L. L. Engel, ibid., 1938,68, 299.34, 191.s1 J. K. Wolfe, E. B. Hershberg, and L. F. Fieser, J . Bbl. Chem., 1940, 136, 653.32 N.C. Talbot, A. H. Saltzman, R. L. Wixom, and J. K. Wolfe, ibid., 1946,160, 635YOUNG: BIOCHEMISTRY OF THE ADRENAL CORTEX. 219Among the tests for adrenal cortical substances which have been adaptedfor the purpose of assay are the inhibition of the hypoglycmnic action ofadministered insulin in normal or adrenalectomised starving animals,33 theexacerbation of an existing diabetes in partially depancreatisedthe production of glycosuria in the normal intact rat,35 and the enhancementof the depressed glycosuria in adrenalectomised-phloridzinised rats.36(b) The Isolation of “ Life Maintaining ” Adrenal Cortical Ster-oids.’, 37, 38-In the review the term “ life maintaining,” applied toadrenal substances, connotes ability of the substance, on repeated parenteraladministration in suitable dosage, t o prolong indefinitely and in good healththe life of an adrenalectomised animal.Although the methods employed inthe preparation of such active adrenal steroids vary substantially, certaincommon principles underlie most of the methods in use. Preliminaryextraction of fresh whole adrenal gland with ethanol or acetone is followedby removal of the solvent by low-temperature distillation. The activematerial is then extracted from the aqueous residue by benzene, chloroform,or other fat solvent. Further purification often depends on the fact that inthe distribution of an active extract between ether, benzene, or light petro-leum on the one hand, and aqueous solvents on the other, the distributioncoefficient of the active material is 1 to 3 or 4 in favour of the aqueousphase. Traces of adrenaline or of adrenaline decomposition products areremoved by fractionation processes employing weakly acidic or alkalineextractants. The neutral water-soluble material obtained as the result ofthese manipulations constitutes the whole adrenal cortical extract oftenemployed clinically.Further fractionation with neutral organic solvents such as ethyl acetateor benzene yields a variety of crystalline products.Interaction with sub-stances which condense with ketones (e.g., Girard’s reagent) yields productsfrom which active crystalline substances may be obtained by fractionalhydrolysis with acid. Chromatographic fractionation of acetyl derivatives,followed by hydrolysis under mild conditions (e.g., aqueous methanolicpotassium bicarbonate at 20’) of the separated acetyl derivatives has beenextensively and successfully employed by Reichstein and his colleagues.1°33 ( a ) H. Selye and C.Dosne, Proc. SOC. Exp. Biol. Med., 1939, 42, 680; ( b ) J. F.Grattan and H. Jensen, J . Biol. Chem., 1940,135, 511 ; (c) J. F . Grattan, H. Jensen andD. J. Ingle, Amer. J . Physiol., 1941, 134, 8.34 (a) C. N. H. Long, E. G. Fry, and K. W. Thompson, ibid., 1938,123, 130; ( b ) D. J.Ingle, Proc. SOC. Exp Biol. Med., 1940,44, 176; idem, Amer. J . Physiol., 1941,132,670;( c ) E. C. Kendall, Endocrinology, 1943, 30, 853.35 ( a ) D. J. Ingle, ibid., 1941, 29, 649; (b) idem, Amer. J . Physiol., 1941, 133, 337P;( c ) D. J. Ingle, R.Sheppard, J. F. Evans and M . H. Kuizenga, Endocrinology,1945, 37, 341.36 B. B. Wells and A. Chapman, Proc. Staff Meetings Mayo Clin., 1940, 15, 493.37 ( a ) M . H . Kuizenga and G. F. Cartland, Endrocrinology, 1939, 24, 526; ( b ) M. H.Kuizenga in “ The Chemistry and Physiology of Hormones” Amer. A s s . Advancerncnt ofScience, Washington, D.C., 1944, p. 57.38 M. H. Kuizenga, J. W. Nelson, S. C. Lyster, and D. J. Ingle, J . Biol. Chem., 1945,160, 16220 BIOCHEMISTRY.Whatever methods of fractionation are employed there is obtained aseries of crystalline physiologically active steroids, numerous inactivecrystalline substances, and a syrup (" amorphous fraction ") which maypossess much of the total physiological activity present at this stage butwhich has so far failed to yield crystalline material.Altogether 27 crystalline steroids of known constitution have beenobtained from adrenal tissues.listed 21 which had beenisolated by 1938.R. K. CallowTable I gives an additional six.TABLE I.Steroids Isolated from the Adrenal Cortex since 1939.Alphabeticaldesignationby ReichsteinSubstance. et aE.l0 Isolators. Remarks.Ad-Pregnene-20 : 21-diol-3 : 1 l-dione T T. Reichstein andJ. von EuwssA4-Pregnene-17(p) : 20 : 21-triol- U T. Reichstein andJ. von Euw 40A4-Androstene-3 : 17-dione I J. von Euw and Androgenic. Pos-T. Reichstein 41 sibly a decom-position pro-duct of (IV).aEZoPregnane-3(p) : 11(p) : 17(/l) : 21- V J. von Euw and Stereoisomer at C,tetrol-20-one T.Reichatein 42 of Reichstein'sA substance.1°Oestrone - D. Beall O3 Oestrogenic.A4-Pregnene- 17( 8) -01- 3 : 20-dione - J. J. Pfiffner and Weak androgen.(1 7-hydroxyprogesterone, VIII) H. B. North44 No progesta-3 : 11 -dionetional activity.(c) Physiologically Active Adrenal Steroids.-Since a t least six crystallinesteroids extracted from the adrenal gland (seven if progesterone be included)are active in prolonging the life of the adrenalectomised animal, and, sinceno single known substance combines all the recognised types of physiologicalactivity possessed by adrenal extract, a term such as " the adrenal corticalhormone " is at present otiose.The chemistry of the six adrenal steroids active in prolonging life (I-VI)has recently been adequately re~iewed.~, *, lo, 37 Three of them have beenprepared artificially (" partially synthesised ") from other naturallyoccurring steroids by T.Reichstein and his colleagues. 1 l-Deoxycorti-costerone (V), in the form of its C,, acetate, has been a commercial productfor 7-8 years 2, 3, 41 lo7 3' while recently the partial syntheses of ll-de-hydrocorticosterone (111) 45 and of corticosterone (I) have also beenaccomplished.T. Reichstein and J. von Euw, Helv. Chim. Acla, 1939, 22, 1222.40 Idem, ibid., 1941, 24, 2473.I1 5. von Euw and T. Reichstein, ibid., 1941, 24, 879.p 2 Idem, ibicl., 1942, 25, 988.43 D. Beall, Nature, 1939, 144, 76; J. Endocrinol., 1940, 2, 81.44 J. 5. Pfiffner and H. B. North, J. Biol. Chem., 1940, 132, 459; 1941, 139, 855.45 A.Lardon and T. Reichstein, Helv. Chim. Acta, 1943, 26, 747.4 6 J. von Euw, A. Lardon and T. Reichstein, ibid., 1944, 27, 1287YOUNG : BIOCHEMISTRY OF THE ADRENAL CORTEX. 221Because of its high activity the amorphous fraction is of particular interest.I n physiological activity it more closely resembles (V) and (VI) than (I) andother active steroids carrying an oxygen atom at C,,, but its life-maintainingactivity is much greater than that of (V), though the latter is the most active of(1.)Corticosterone(Ad-Pregnene-ll( 9 ) : 21-diol-5: 20 dione)(111.)1 1 -Dehydrocorticosterone(Ad-Pregnene-21-01-3 : 11 : 20-trione)CH2.0H(V-)1 1-Deoxycorticosterone(A4-Pregnene-21-ol-3 : 20-dione)(11.)17-Hydroxycorticosterone(A4-Pregnene-11(p) : 17@) : 21-triol-3 : 20-dione)7H2.DHW.)17-Hydroxy- 11 -dehydrocorticosterone(A4-Pregnene-17(,9) : 21-diol-3 : 11 : 20trione)7H2*OH(VI.)17-Hydroxy - 1 1 -deoxycorticosterone(Aq-Pregnene-l7(B) : 21-diol-3 : 20-dione)the isolated crystalline compounds in this respect.Elementary analysis ofthe amorphous fraction suggests the presence of C2,0, steroids [cf. CZlO, for(I) and (VI) and C210, for (V)]. In keeping with its higher oxygen contentthis fraction is more soluble in water, but unlike (V) it loses activity ontreatment with acidic or alkaline reagents, being unstable even towardspotassium bicarbonate. Moreover, its physiological activity differs signi-ficantly in some respects from that of both (V) and (I), and there can be n222 BIOCHEMISTRY.doubt that this fraction contains active substances of hitherto unrecognizedconstitution.Adrenal glands from different species of animal yield different pro-portions of the various active fractions.Table I1 gives the approximateyields of the active crude and crystalline fractions from ox, pig, and sheepadrenal glands.TABLE 11.Approximate Yields from 1000 kg. of Whole Adrenal Gland.7, 10,37,38Yield (mg., =% x 10’) fromadrenals of-Fraction or substance.A. Soluble in benzene : little soluble in water1. Crude crystalline mixture of (I + 111) ......2. Crystalline (I) .......................................3. Crystalline (111) ....................................4. Crystalline (V) ....................................5.Crystalline (VI) ....................................B. Soluble in water; little soluble in benzene(I1 + IV + amorphous fraction) ...............1. Crude crystalline mixture of (I1 + IV) ......2. Crystalline (11) ....................................3. Crystalline (IV) ....................................4. Amorphous fraction ..............................Neutral fraction soluble in ethyl acetate .........(I + I11 + v + VI) ..............................ox.21,1005,8001,570672336271310,6501,3401684502,460Pig.28,90016,8003,360Sheep.16,6006,0001,57011,5203,8001,3704901,8009,6401,79009001,340Administration of Adrenal Steroids.For experimental and clinical use the active steroids are usually con-verted into esters, which are more active on parenteral administration thanare the free substances.Thus 11-deoxycorticosterone (V) is now employedalmost without exception as its C21-acetate. With corticosterone thediethylacetate (presumably a t C21) is four times as active as the freesubstance.4’Corticosterone (I) and 11 -dehydrocorticosterone (111) are as effectiveorally in the rat as they are by parenteral administration; 48 this is also trueof the amorphous fraction, but 1 l-deoxycorticosterone is much less effectiveby mouth than it is by subcutaneous injection.489 49 There is evidence that11-deoxycorticosterone is destroyed in the gut of the rat.49The most economical way of using 1 1-deoxycorticosterone acetateclinically is by the subcutaneous implantation of tablets of the crystallinecompound.50Physiological Action of Adrenal Steroids.Five of the methods described above (p.218) which have been developedfor the assay of adrenal preparations have also been applied extensively to47 M. H. Kuizenga and G. F. Cartland, Endocrinology, 1940, 27, 647.M. H. Kuizenga, J. W. Nelson and G. F. Cartland, Amer. J. PhysioE., 1940,130, 1.4B (a) H. Fraenkel-Conrat, Proc. SOC. Exp. Biol. Med., 1942, 51, 300; (b) R. A. c l e ghorn, A. P. W. Clarke, and W. F. Greenwood, Endocrinology, 1943, 32, 170.so (a) G. W. Thorn, S. S. Dorrance, and E. Day, Ann. I n t . Med., 1942,16, 1053; (b)D. M. Dunlop, Brit. Med. J., 1943, i, 557; (c) E. P. McCullagh, L. A. Lewis and%’. L.Shively, J.Clin. Endocrinol., 1943, 3, 493YOUNG : BIOCHEMISTRY OF THE ADRENAL CORTEX. 223individual crystalline adrenal steroids and their derivatives. The fivemethods are based on (a) ability to maintain the adrenalectomised dog orrat in good health (“ life-maintenance ” test) ; 24 25 26 (b) the “ Everse-deFremery ” work test ; 27 (c) the “ Ingle ” work test ; 28 (d) ability to influencecarbohydrate metabolism (i.e., to raise the liver-glycogen content 30 ordiminish the insulin sensitivity 33 of starving rats, or to exert a “ diabeto-genic ” action in partially depancreatised 34 or normal 35 animals) ; ( e )activity in influencing the retention of Na+ and C1- in the normal animal.51In Table 111, which is based on the mean of the results in the literature, theactivities of six crystalline adrenal steroids, and of the amorphous fraction,are compared with respect to results with these five methods of assay.FromTable I11 the following conclusions may be drawn : (1) the data with the “ life-TABLE 111.Comparative Physiological Activities of Adrenal Preparations.(The figures, which are based on the mean of the data in the literature, indicatethe order which the seven preparations occupy with respect to activity in any giventest, the most active substance being designated 1, and the least active 7.)“ Life-main-tenance ”Substance. test.(a) Corticosterone group.Corticosterone (I) 317-Hydroxycortico- 41 l-Dehydrocortico- 4sterone (11)sterone (111)corticosterone (IV)sterone group.corticosterone (VI)17-Hydroxy- 1 1 -dehydro- 4(b) 11 -Deoqcortico-11 -Deoxycorticosterone ( V ) 217-Hydroxy- 1J -deoxy- 7(c) Amorphous fraction I“ Everse-deFremery ”work test.36?5132‘ Ingle ”worktest.3141665Influence Naf and C1-on carbo- retentionhydrate in normalmetabolism.dog.3 31 nil(excretion)31 nil(excretion)5 16 ?? 2maintenance ” test approximately parallel those with the (‘ Everse-deFremery ” work test; (2) results with the “ Ingle ” work test parallel thoseconcerning influence on carbohydrate metabolism; (3) the presence of anoxygen atom (hydroxyl or ketonic oxygen) a t C,, of the steroid nucleusgreatly increases activity both with respect to the “ Ingle ” work test andwith respect to influence on carbohydrate metabolism ; (4) the presence of atertiary hydroxyl group a t C,, diminishes activity in the “ life-maintenance ”test and in the Na+ and C1- retaining tests.In (11) and (IV) potency toinduce retention of Na+ and C1- is not only lost but is replaced by ability tofacilitate the excretion of these ions by the kidney of the intact dog.61 (IX)and (X), produced artificially by the removal of the elements of water from6 1 (a) G . W. Thorn, L. L. Engel, and R. A. Lewis, Science, 1941, 94, 348; ( b ) M. J.Clinton and G. W. Thorn, ibid., 1942, 96, 343; ( c ) M. Clinton, jun., G. W. Thorn,H. Eisenberg, and K. E. Stein, Endocrinology, 1942, 31, 678224 BIOCHEMISTRY.cortic~sterone,~~ are active in the " Everse-de Fremery " test, (X) beingcomparable in activity with (V), while (IX) is only 4-i as active as (V).52Another artificial product (XI), in which the C,, hydroxyl of corticosteronehas been replaced by a hydroxyl group at Cl2,= has less potency in the" Everse-de Fremery " test than corticosterone, and exerts no obviousinfluence on carbohydrate metabolism.63Me Me I ICO(VII.) (VIII.)Progesterone 17-Hydroxyprogesterone( A4-Pregnene-3 : 20-dione) (A4-Pregnene- 17(p)-ol-3 : 20-dione)7H2*OH vH2.0Hco coMe :\?$/--0 A//\/ OH\/\(IX.) (X-1Anhydrocorticosterone (1 ) Anhydrocorticosterone (2)( A4: 9-Pregnadiene-2 l-ol- 3 : 20-dione) ( A4:l1-Pregnadiene-21-01-3 : 20-dione)(XI.1(A4-Pregnene-12(p) : 21-diol-3 : 20-dione)Progesterone (VII) is active in maintaining the life of the adrenal-and induces retention of Naf52 C.W. Shoppee and T. Reichstein, Helv. Chim. Acta, 1943, 26, 1316.6t H. G. Fuchs and T. Reichstein, ibid., 1943, 26, 511.5' R. Gaunt, W. 0. Nelson, and E. Loomus, Proc. SOC. Ezp. Biol. Med., 1938,39, 319.56 (a) J. A. Wells and R. R. Greene, Endocrinology, 1939, 25, 183 ; (b) F. E. Emeryectomised ferret,54 rat,55 mouse,s6 andand P. A. Greco, ibid., 1940, 27, 473.C. A. Pfeiffer and C. W. Hooke, Amer. J. Physwl., 1940,131,441.6 7 (a) E. L. Corey, ibid., 1940, 129, P.340; (b) idem, ibid., 1941, 132, 446YOUNG : BIOCHEMISTRY OF THE ADRENAL CORTEX. 225and C1- in the adrenalectomised rat.58 It has, however, no obvious activityin the “ Ingle ” test (rat) s9 and in the anti-insulin test in the same anima1,32bbut raises the blood-sugar level and liver-glycogen content of the fastingnormal ferret,60 of the young rabbit,61 and of the adrenalectomised ~at.~’bThus in some species progesterone reacts physiologically like those adrenalsteroids possessing an oxygen atom a t Cll.17-Hydroxyprogesterone (VIII)exerts no obvious effect in the (‘ Ingle ” work test lo and fails to exhibitactivity also in the “ life-maintenance ’’ test in rats.lO Here again theintroduction of the tertiary hydroxyl group a t C1, diminishes “ life-main-tenance ” activity.It is not possible to give here details of the active doses of all the sub-stances in all the various tests, but average figures from the literature % 379 48, 62may be mentioned.E’or maintenance in good health the adrenalectomiseddog requires, by the subcutaneous route, about 0.15 mg./kg./day of corti-costerone, about 0.015 mg. /kg./day of 11 -deoxycorticosterone, and about0-005 mg. /kg./day of 11-deoxycorticosterone acetate. For the rat thecorresponding figures are about 3-5 mg. /kg./day, 0.80 mg./kg./day, and 0.55mg./kg./day respectively, although there is no good agreement in theliterature concerning such data for the rat.On the basis of the results discussed above it is convenient to divide theactive adrenal steroids into two groups; the first (the “ corticosteronegroup ”) having an oxygen atom at Cll, and the second (the “ deoxycorti-costerone group ”) having no oxygen atom in this position. The membersof the first group are particularly potent in the “ Ingle ” work test and ininfluencing carbohydrate metabolism generally, while those of the secondexert little influence on carbohydrate metabolism but are highly active in the“ life-maintenance ” test and in bringing about retention of Na+ and C1-.The amorphous fraction falls physiologically into the “ deoxycorticosteronegroup .”Since most, if not all, of the many recognised effects which follow removalof the adrenal cortical tissues are neutralised by the administration of adrenalextracts, it is possible here t o consider only a few of the more outstandingfeatures of the results of administration of excess of adrenal steroids toadrenalectomised or to normal animals.M-Substances of the deoxycorti-costerone group, together with the amorphous fraction, are the most activein this connexion.The retention of water and of Na+ and C1- under theinfluence of adrenal substances, together with the excretion of K+, are prob-ably the result of an action on the kidney tubules.65 The loss of K+ from(a) Water and EZectroZyteI8 G. W. Thorn and L. L. Engel, J . E x p . Med., 1938, 68, 299.sv D. J. Ingle, Proc. SOC. E x p . Biol. Med., 1940, 44, 450.61 A. B. Corkill and J. F. Nelson, Aust. J. Exp. Biol. Med. Sci., 1941, 19, 211.62 H. L. Mmon, Endocrinology, 1939, 25, 405.R. Gaunt, J. W. Remington, and E. Edelmann, ibid., 1939, 41, 429.G . W. Thorn, J. Mount S i n a i Hosp., 1942, 8, 1177.J. A. Anderson, J. Clirt. Endocrinol., 1943, 3, 615.86 R.Chambers and G. Cameron, Amer. J. Physiol., 1944,141, 138.REP .-VOL . XLII. 226 BIOCHEMISTRY.the body after repeated large doses of deoxycorticosterone acetate (1-4 mg .in the rat) may be sufficient to bring about cardiac lesions.66 The retentionof water produced initially by adrenal steroid administration is associatedwith an increase in plasma v0lume,~7 and the blood pressure may rise.68Prolonged treatment may result in p ~ l y u r i a , ~ ~ which appears to be a com-pensatory mechanism for the excretion of the retained electrolytes and water.Posterior pituitary extract and adrenal preparations to some extent appear toact antagonistically with respect to water elimination by the kidney.70(b) Metabolism. 71-Substances of the corticosterone series are here mostactive.The administration of suitable adrenal extracts leads to a rise in theliver glycogen content of starving normal rats,30, 71, 72 of starving hypophy-sectomised rats,73 and in the isolated perfused liver.7* Likewise a diabeticcondition is induced or exacerbated in a partially depancreatised rat,34 in anormal rat with an adequate food and in a hypophysectomised-phloridzinised rat.36 The control of glycosuria in the diabetic conditioninduced by treatment with adrenal steroids may require the administrationof large doses of in~ulin.~, 35When the fasting rat (normal or adrenalectomised) is treated withadrenal steroids it is primarily the glycogen stores of the liver which rise,although the muscle glycogen may secondarily increase.71 The control ofmuscle glycogen level appears to be directly influenced by anterior pituitaryextracts 75s 76 and only indirectly by the secretion of the adrenal cortex, thelatter acting with the intermediation of liver glycogen.When adrenal steroids induce a rise in the glycogen stores, or induce orexacerbate a diabetic condition,34, 35, 36* 71, 72, 73 the nitrogen excretion of thetreated animal rises, and it is to be assumed that glyconeogenesis fromprotein is a contributary factor in the accumulation of the extra glycogen or66 (a) D.C. Darrow and H. C. Miller, J . Clin. Invest., 1942,21,601; ( b ) D. C. Damow,e7 M. Clinton, jun., and G. W. Thorn, Johns Hopkins Hosp. Bull., 1943,72, 255.68 ( a ) G. A. Perera, A. I. Knowlton, A.Lowell, and R. F. Loeb, J. Amer. Med. ASSOC.,1944, 125, 1030; (b) J. R. Leatham and V. A. Drill, Endocrinology, 1944, 35, 112; (c)G. A. Perera, J. Amer. Med. Assoc., 1945, 129,537; ( d ) N. M. Gaudino, Rev. SOC. argent.Biol., 1944, 20, 470.69 (a) M. G. Mulinos, C. L. Spingarn, and M. E. Lojkin, Amer. J. Physiol., 1941,135,102; ( b ) J. W. Ferrebee, D. Parker, W. H. Carnes, M. K. Gerity, D. W. Atchley, andR. F. Loeb, A m r . J . Physiol., 1941,135, 230; ( c ) C. A. Winter and W. R. Ingram, ibid.,1943,139, 710; (d) A. S. Harned and W. 0. Nelson, Fed. Proc., 1943, 2, 19.70 ( a ) J . A. Anderson and W. R. Murlin, J. Pedkzt., 1942, 21, 326; (b) R. Gaunt,Trans. N.Y. A d . Sci., 1944, 6, 179; ( c ) ident, Endocrinology, 1944, 34, 400.7 1 (a) C.N. H. Long, B. Katzin, and E. G. Fry, ibid., 1940, 26, 309; (b) C. N. €I.Long, Cold Spring Harb. Symp. Qwtnt. Biol., 1942, 10, 91 ; ( c ) idem, Endocrinology, 1942,30, 870; ( d ) J. Tepperman, F. L. Engel, and C. N. H. Long, {bid., 1943,32,373.73 R. G. Sprague, Proc. Stafs Meet. Mayo Clin., 1940, 15, 291.78 C. N. H. Long and B. Katzin, Proc. SOC. Exp. Biol. Med., 1938, 38, 516.74 ( a ) E. L. Corey and S. W. Britten, Amer. J. Physiol., 1941, 131, 783; ( b ) S. W.76 L. L. Bennett and R. Z . Perkins, Endocrinology, 1945,36, 24.' 6 W. H. Price, C. F. Cori, and S. P. Colowick, J. Bbl. Chem., 1945,160, 633.Proc. SOC. Exp. BWE. Med., 1944,55, 13.Britten and E. L. Corey, ibid., p. 790YOUNG: BIOCHEMISTRY OF THE ADRENAL CORTEX. 227glucose. Nevertheless in carefully controlled experiments it has becomeclear that if the classical data relating to the conversion of protein to carbo-hydrate (100 g.of protein yield 58 g. of carbohydrate) be adopted, it is notpossible to account for all of the extra carbohydrate which appears as theresult of adrenal treatment.6, 9, 3% ~9 359 7' It appears probable that theadrenal steroids diminish the rate of utilization of carbohydrate by theperipheral tissues % 34, 7 8 9 79 and that this is an important factor in the antag-onism to the hypoglycaemic action of insulin exerted by many adrenalsteroids. Since the respiratory quotient is depressed as the result of theadministration of adrenal steroids 9, 34) 35, 71, 78, 79 it appears probable thatthe depression of carbohydrate oxidation thus induced is associated with anincreased combustion of fat.C.N. H. Long 71 has suggested that adrenal substances may stimulatethe conversion of tissue proteins to amino-acids, the hepatic deamination ofthe latter giving rise to the accumulated glycogen. It is perhaps of particularsignificance that D. J. Ingle,gl 35 has recently found that when an adrenal steroiddiabetes is induced in normal rats the administration of large doses of insulin,sufficient to bring the hyperglycaxnia and glycoauria under control, does notreduce urinary nitrogen excretion to the normal level. It therefore seemsunlikely that the enhanced rate of formation of carbohydrate from protein,induced by pituitary substances, can be secondary to inhibition of carbo-hydrate oxidation brought about by such treatment.If the adrenal cortical secretions directly stimulate the breakdown ofintracellular protein it is apparently paradoxical that the presence of theadrenal cortex or of its secretions is necessary for the manifestation of thefull growth-promoting activity of anterior pituitary extract,*O particularlyin view of the fact that the administration of excess of adrenal steroids (Iand IV) inhibits the growth of young *l It may be pointed out, how-ever, that the incorporation of exogenous amino-acids into the tissues may beassisted by a limited stimulation of the catabolism of tissue protein,B2although undue enhancement of protein catabolism would obviously preventor depress growth.C.N.H. Long 71 has pointed out that, if the primary action of adrenalsteroids is to catalyse the breakdown of protein in the cell, the action of thesesteroids on electrolyte balance might follow as secondary effects, sinceintracellular proteolysis would liberate K+ previously held in association withnegatively charged protein ions 83 and the excretion of the K+ by the kidneymight be expected to bring about the retention of Na+ in order to maintainosmotic pressure. Nevertheless, as this author himself points out, deoxy-77 G. W. Thorn and M. Clinton, J . Clin. Endocrinol., 1943,3, 335.78 Q. W. Thorn, G. F. Koepf, R. A. Lewis, andE. F. Olsen, J . Clin. Invest., 1940,19,70 J. A. Russell, Amer. J. Physiol., 1943, 140, 98.8o E. G. Fry and C. N. H. Long, Ann.N . Y . A d . Sci., 1943,43, 383.82 F. G. Young, Biochem. J., 1945, 39, 515.83 P. J. Boyle and E. J. Conway, J . Physwl., 1941,100, 1.813.B. B. Wells and E. C. Kendall, Proc. StaJ Meet. Mayo Clin., 1940, 15, 324228 BIOCHEMISTRY.corticosterone, which has little influence on the metabolism of carbohydrateand protein, is particularly active with respect to electrolyte balance andthere can be little doubt that this adrenal steroid exerts a direct action on thekidney. We must therefore assume that the main action of the deoxy-corticosterone series of steroids is complementary to that of the corticosteroneseries, the action of the latter on tissue proteins resulting in the liberationinto the intercellular fluids of K+, the excretion of which by the kidney isfacilitated by the steroids of the deoxycorticosterone series.It should benoted that steroids of the corticosterone series strikingly increase the activityof the liver with respect to the action of the enzyme arginase 84 which isintimately associated with the disposal of the products of the deaminationof amino-acids. Whether this is a direct action of the steroids, or an indirectone, mediated by the products of proteolyeis in the peripheral tissues, is notcertain. There can be little doubt, however, that the substances of thecorticosterone series exert more than one type of activity in the body, sinceas well as stimulating the catabolism of protein, with consequent accumu-lation of glycogen in the liver, they inhibit the action of insulin and theoxidation of carbohydrates in the muscular tissues.(c) Action on Lymphoid Tissue and Antibody Titre.-It has long beenrecognized that the thymus fails to degenerate to the normal extent in animalssuffering from adrenal deficiency, and it has now been shown that the adminis-tration of adrenal steroids of the corticosterone series causes rapid regressionof the thymus in rats,85 together with an absolute lymphopenia.86 Since anincreased production of antibodies has been demonstrated in animals treatedwith adrenal extract, but not with deoxycorticosterone acetate, it seemsprobable that it is adrenal substances of the corticosterone series that controlthe release of antibody from the lymphocytes.86> 87(d) Influence on Resistance to Stress.-The results of the majority ofinvestigations designed to reveal any activity of adrenal preparations ininducing increased resistance to stress in normal animals have been negative.Nevertheless the resistance of normal rats to peptone shock,80 to reducedatmospheric and to high environmental temperat~re,~~ can beraised by treatment under some conditions with adrenal preparations, butonly in the experiments concerning the effects of high environmental tempera-ture did deoxycorticosterone acetate exert any significant action.s0Control of the Secretory Activity of the Adrenal Cortex.The activity of the adrenal cortex is under the control of the anterior lobeof the pituitary gland through one of its secretions, adrenocorticotropin.slH. Fraenkel-Conrat, M.E. Simpson, and H. M. Evans, J . Biol. Chem., 1943,147,99.D. J. Ingle, Proc. SOC. Exp. Biol. Med., 1940, 44, 174.T. F. Dougherty and A, White, Endocrinology, 1944, 36, 1.(a) T . F. Dougherty, A. White, and J. H. Chase, Proc. Soc. Exp. Biol. Med., 1944,D. J. Ingle, Amer. J . Physiol., 1944, 142, 191.G. W. Thorn, 35. Clinton, B. M. Davis, and R. A. Lewis, Endocrinology, 1945,36,381.V. Hermanson and F. A. Hartman, Amer. J . Physiol., 1946,144, 108.H. G. Swam, Physiol. Rev., 1940, 20, 493.56, 28; ( b ) T. F. Dougherty, J. H. Chase, and A. White, ibid., 1945, 58, 135YOUNG : BIOCHEMISTRY OF THE ADRENAL CORTEX. 229Most of the effects of the administration of adrenal extracts can be reproducedin animals with intact adrenal glands by the administration of pituitaryadrenocorticotropin.Atrophy of the adrenal glands occurs in normal rats(but not in hypophysectomised animals) as the result of the administration oflarge doses of adrenal cortical extract,92 and it seems probable that thesecretions of the adrenal cortex act on the anterior pituitary gland to depressthe release of adrenocorticotropin. In this way a simple automatic controlof adrenal activity is achieved.There is no evidence that the secretion of adrenal steroids is under directnervous control,93, 94 but the intravenous infusion of physiological amounts ofadrenaline induces an immediate, lasting, and substantial increase in the basaloutput of adrenal cortical substances in the eviscerated dog or cat.94 Thusnervous influences may directly affect the secretion of adrenal corticalsteroids via the secretion of adrenaline by the adrenal medulla.The Biological Precursors of Adrenal Xteroids.For some years it has been assumed, without direct evidence, thatadrenal steroids, together with other physiologically active substancesderived from a perhydrocyclopentenophenanthrene nucleus, are formed inthe body from cholesterol.Since, however, i t has been known for evenlonger that the animal body can synthesize cholesterolg5 there seems noobvious reason why it should not also synthesise steroid hormones directly.Recently the biological conversion of cholesterol to pregnanediol has beenclearly demonstrated in a pregnant woman,96 so it is clear that shorteningof the hydrocarbon side-chain a t C17 of the cholesterol molecule can takeplace in vivo.C. N. H. Long and his colleagues 97 have shown that in therat a single dose of pure adrenocorticotropin reduces the adrenal cholesterolcontent to *-$ of its original value within 3 hours. Later (24 hours after theadministration) the adrenal cholesterol content is normal or high.97, 98Adrenaline also induces a rapid fall of adrenal cholesterol 99 and a fall alsooccurs under the influence of many different types of stress.lo0¶ lol Since theadministration of adrenocorticotropin and adrenaline, and the imposition ofconditions of stress, are all circumstances in which the rate of secretion ofadrenal steroids is greatly enhanced there can be little doubt that in such92 D.J. Ingle, Arner. J. Physiol., 1938, 124, 369.O3 W. E. MacFarland, J . Exp. Zool., 1944, 95, 345.R4 ( a ) M. Vogt, J . Physiol., 1944, 103, 317; ( b ) idem, ibid., 1945, 104, 60.s5 (a) H. J . Channon, Biochem. J., 1925, 19, 424; ( b ) K. Bloch and D. Rittenberg,O6 K. Bloch, ibid., 1945, 157, 661.O 7 G. Sayers, M. A. Sayers, E. G. Fry, A. White, and C. N. H. Long, Y a l e J . of Biol.O 8 R. A. Carreyett, Y. M. L. Golla, and M. Reiss, J . Physiol., 1945, 104, 210.99 C. N. H. Long and E. G. Fry, Proc. SOC. Exp. BioZ. Med., 1945, 59, 67.loo (a) N. V. Bekauri, A. A. Danilov, and E. A. Moisseev, Compt. rend. Acad. Sci.U.R.S.S., 1944, 43, 238; ( b ) L. Levin, Endocrinology, 1945,37, 34; (c) G. Sayers, M. A.Sayers, Tsan-Ying Liang, and C.N. H. Long, ibid., 1945, 37, 96.J . Biol. Chern., 1942, 143, 297.and Med., 1944, 16, 361.lol G. Popjak, J . Path. Bact., 1944, 56, 485230 BIOUHEMISTRY .instances the rapidly disappearing cholesterol is converted, at least in part,to adrenal steroids. It is of particular interest that under some conditionsthe disappearance of adrenal cholesterol is accompanied by the appearanceof substances in the adrenal tissue which react with phenylhydrazine.101The possibility that aldehydic or ketonic substances are formed intermediarilyin the conversion of adrenal cholesterol to adrenal steroids must be thusconsidered.Another interesting possibility is that the androgens secreted normallyby the adrenal cortex are by-products in the conversion of cholesterol tocorticosterone and similar substances.16 The increased production ofandrogens in the adrenal-genital syndrome l6 might then be considered toresult from a pathological derangement of the normal process.On the otherhand the isolation from urine of androstane-3 : ll-diol-17-one (XIII) (orpossibly the corresponding 1 l-keto-compound) lo2 suggests the possibilitythat urinary androgens may arise as degradation products of adrenal steroidsof the corticosterone series. In any case the estimation of urinary androgens(neutral 17-keto-steroids) is of particular value clinically as an indication ofnormal or abnormal functioning of the adrenal cortical tissues.l4, l5, lo3Me(XII.) (XIII.)Pregnanediol Androstane-3(a) : 1 l(fi)-diol-l7-one(Pregnane-3(a) : 20(a)-diol)The Fate of Secreted Adrenal Androgens.Secreted adrenal steroids are partly destroyed in the tissues,104 partlyexcreted in the urine in a physiologically active form,lo5 and partly excretedin the urine as inactive catabolic products.ls9 lo6 The urine from patientsloa H.L. Mason, J. Biol. Chem., 1945, 158, 719.LOa (a) N. H. Callow, R. K. Callow, and C. W. Emmens, J. Endocrinol., 1945, 2, 88;(b) N. B. Talbot and A. M. Butler, J. Clin. Endocrinol., 1942, 2, 724; ( c ) G. P ~ C U E ,ibid., 1943, 3, 301 ; ( d ) M. M. Hoffman, McGiZZ Med. J., 1944, 13, 177; (e) N. H. Callowand A. C. Crooke, Lancet, 1944, 248,464.l o 5 (a) E. H. Venning, M. M. Hoffman, and J. S. L. Browne, J. BWZ. Chem., 1943,148, 455; (b) R. A. Shipley, R.I. Dorfman, and B. N. Horwitt, Amer. J. Physiol., 1943,139, 742; (c) E. H. Venning, M. M. Hoffman, and J. S. L. Browne, Endocrinology, 1944,35, 49; ( d ) R. I. Dorfman, B. N. Horwitt, R. A. Shipley, and W. E. Abbott, ibid., 1944,35, 15; ( e ) R. I. Dorfman, B. N. Horwitt, and R. A. Shipley, ibid., 1944,35, 121.lo6 (a) W. K. Cuyler, C. Ashley, and E. C. Hamblen, ibid., 1940, 2'9, 177; (b) U.Westphal, 2. physiol. Chem., 1942, 273, 13; (c) M. M. Hoffman, V. E. Kazmin, andJ. S. L. Browne, J. Bwl. Chern., 1943, 147, 259; (d) W. R. Fish, B. N. Horwitt, andR. I. Dorfman, Science, 1943, 97, 227 ; ( e ) B. N. Horwitt, R. I. Dorfman, R. A. Shipley,and W. R. Fish, J. Biol. Chm., 1944,155,213.lo* M. Vogt, J. Physwl., 1943, 102, 341MORGAN THE SPECIFIC ELOOD-GROUP SUBSTANCES.231who have had surgical operations and in whom the secretion of adrenalsteroids is much enhanced is surprisingly rich in substances possessingadrenal steroid activity. 106The administration of deoxycorticosterone is followed by the excretion ofpregnane-3 : 20-diol (XII) in the urine in many species.lo6 If the reductionof the primary alcoholic group a t C,, in deosycorticosterone precedes thechanges in ring A of this compound, progesterone will be formed inter-mediarily in the transformation to pregnanediol. Such a process mightaccount for the slight progestational activity of deoxycorticosteroneacetate.106a, lo7F. G. Y.3. THE SPECIFIC BLOOD-GROUP SUBSTANCES.At the beginning of this century Landsteiner and his pupils discoveredthat human bloods can be divided into four main serological groups, A, B,AB, and 0, based on the presence or absence of the agglutinable substancesA and B within the erythrocyte.The occurrence or absence of these specificsubstances in the red-cells was determined by means of agglutination testsemploying the agglutinins anti-A and anti-B which occur naturally in theserum of group B and A persons respectively. For many years group 0erythrocytes were considered to be cells devoid of the agglutinable sub-stances A and B and were recognised by the absence of these biologicallyimportant factors and not by the possession of a specific and characteristicagglutinogen of their own. Heredity studies of the blood groups demon-strated that the A- and B-factors were each inherited as simple Mendeliandominants and, according to Bernstein's theory, which postulates theexistence of three allelic genes, A, B, and 0, and is now generally accepted,erythrocytes of the genotype 00 could possess st specific O-factor corres-ponding to the A and B agglutinogens.The recognition of the O-factorhad to await the discovery of a reliable and specific anti-0 serum, for thisagglutinin occurs only rarely in man. The modern technique of bloodtransfusion that has evolved from Landsteiner's original discovery is nowregarded as an established and safe medical procedure, but until quiterecently practically nothing was known as to the nature of the substancespresent in the erythrocytes belonging to the different blood groups which wereresponsible for the characteristic and specific immunological behaviour of theerythrocytes.The reason is in no small measure due to the difficulty ofobtaining, in quantities suitable for chemical investigation, the specific A-, B-,and O-substances. An observation by F. Schiff,l that commercial peptonecontained a blood group A-factor and could serve as a readily availablesource of this blood group substance, enabled a chemical investigation to belo' (a) J. van Heuverswyn, V. J. Collins, W. L. Williams, and W. U. Gardner, Proc.SOC. E x p BioZ. Med., 1939, 41 552; ( b ) J. M. Robson, J . Physiol., 1939, 96, 21P; (c)J. H. Leatham and R. C. Crafts, Endocrinology, 1940,27 283; ( d ) R. D. Lawrence, Brit.Med. J., 1943, i, 12; (e) G.W. Raleigh and H. F. Philipsborn, jun., Arch. Path., 1944,87, 213.Zentr. Bakt. Par., 1930 98, 94232 BIOCHEMISTRY.undertakem2 B. Brahn, F. Schiff,and F. Weinman ; and F. Schiff showed that commercial pepsin was alsoa good source of the blood group A-substance. A particularly rich supply ofthis material was discovered in hog gastric mucin 6s during a study of theglycoproteins occurring in gastric mucosa, and a polysaccharide that possessedintense A-activity and contained N-acetylglucosamine and galactose inequimolecular quantities was isolated but was considered to be only about75% pure because of its positive Ehrlich diazo-reaction. K. Landsteinerand R. A. Harte extended the examination of the active polysaccharideand showed that it contained a component rich in amino-acids.With aview to obtaining results which could be employed subsequently as a basisfor the elaboration of a method for the isolation of the blood group sub-stances from human erythrocytes, tissues, and fluids, W. T. J. Morgan andH. K. King9 devised two methods by which the A-substance could be re-covered from hog gastric mucin without employing conditions of acidity andalkalinity too far removed from neutrality and which could be carried outa t normal temperatures. The undegraded A-substance obtained by thesemethods shows a high viscosity (yj 2.8 a t a concentration of 0-57< in saline).A typical analysis of the substance gave C, 45; H, 6.0; N, 6.0; Ac, 10%.Examination of the material at pH 4.0 and 8.0 in the Tiselius electrophoresisapparatus showed the preparation to be essentially homogeneous.Hydr-olysis of the A-substance with mineral acid gives about 50% of reducing sugars,30-33% of glucosamine, 5.0% of a-amino-N (van Slyke), and 2.5% ofm-amino-acid-N.8,In most of the earlier work the activity of the A-substance isolated fromdifferent sources was determined by the hsmolytic inhibition test, with theresult that any destruction of another important serological property of thenative A-substance, its power to inhibit iso-agglutination, was overlooked,It was observed,s however, that treatment of the crude A-substance withformamide a t 150" for 1 hour gave a purified material which showed only afraction of its original power to inhibit iso-agglutination, whereas its capacityto inhibit the hzemolysis of sheep cells by an anti-A rabbit serum was actuallyincreased beyond the original value.These and other observations indicatedthat if the specific blood group substances are to be obtained in their " native "state a carefully controlled isolation procedure, such as one of those describedby W. T. J. Morgan and H. K. King,g must be employed. The A-substanceprepared by these methods is similar in composition to that described byK. Landsteiner and R. A. Harte but differs from it in a number of importantphysical and immunological properties. The material has high serologicalactivity as determined by the inhibition of iso-agglutination, shows the highviscosity that is so characteristic of native gastric mucin, and forms anSubsequently, F.Schiff and G. Weiler ;W. F. Goebel, J. Exp. Med., 1938, 68, 221.Klin. Woch., 1932, 11, 1592.K. Landsteiner and H. W. Chase, J . Exp. &led., 1936, 63, 813.K. Meyer, E. Smyth, and J. Palmer, J . Biol. Chem., 1937,119, 73.J . Exp. Med., 1940, 71, 651.Biochem. Z . , 1931, 235, 454.6 Deut. med. Woch., 59, 199.9 Biochem. J., 1943, 37, 640MORGAN : THE SPECIFIC BLOOD-GROUP SUBSTANCES. 233elastic gel on the addition of borate buffer at pH 8.5. These properties arerapidly lost on heating in neutral, acid, or alkaline solution a t 100". Treat-ment of the active substance with O.O~N-N~,CO, a t 100" for a few minutescauses the complex to break up in such a manner that at least two-thirds of i tpasses through a cellophane membrane.The amino-acid components arelargely retained by the membrane and are still associated with a carbohydratestructure.lo The indiffusible material is hvorotatory -20°), electro-phoretically homogeneous, and practically non-reducing, and shows only asmall fraction of the original serological activity. The diffusate, on the otherhand, shows strongly reducing properties without further acid hydrolysis andgives an immediate colour with Ehrlich's reagent. The development of animmediate colour with p-dimethylaminobenzaldehyde under these conditionssuggests that an oxazole ring structure is formed in a similar manner to thechange which is known to occur when iV-acetylglucosamine and other N -derivatives of glucosamine are similarly treated with dilute alkali.11-14 Itseems probable that the alkali-labile linkages in the A-substance are thoseglycoside linkages which join C atom 1 of the N-acetylglucosamine to othercomponents of the A-complex.The extreme alkali lability of the substanceis a characteristic property and was not encountered during the examinationof several complex polysaccharide substances which are known to containhexosamine molecule^.^ M. Stacey l5 has stated that the A-substanceisolated from commercial pepsin contains d-mannose and Z-fucose in additionto d-galactose and AT-acetylglucosamine, but details of this work are not yetpublished.A preliminary qualitative examination l6 of the amino-acids present inthe A-substance has been made by the chromatographic method describedby R.Consden, A. H. Gordon, and A. J. P. Martin.17 At least 15 amino-acids are present as components of the complex and it seems probable thatthreonine and hydroxyproline are present in higher concentrations than arenormally found in proteins. The isolation ofthreonine from A-substance has been described by K. Freudenberg, H. Walsh,and H. Molter.lsSeveral workers 19-25 have attempted to isolate the blood group10 H. K. King and W. T. J. Morgan, Biochenz. J., 1944,38, X.l 1 W. T. J. Morgan and L. A. Elson, ibid., 1034, 28, 988.l2 Idem, ibid., 1936, 30, 909.13 Idem, Chem. and Ind., 1938, 1191.l 4 T. White, J., 1940, 428.l6 i V . T. J. Morgan, Brit. Med. Bull., 1944, 2, 165.1 7 Biochent.J., 1944, 38, 224.19 F. Schiff and L. Adelsbergor, 2. Immun. Forsch., 1924, 40, 335.20 B. Brahn and F. Schiff, Klin. Woch., 1926, 1455.21 K. Landsteiner and J. van der Scheer, J . Exp. Med., 1925, 42, 123.23 H. Dold and R. Rosenburg, K l i n . Woch., 1928, 394.23 C. Hallauer, Schweiz. med. Woch,., 1929, 121 ; 2. Immun. Forsch., 1929, 63, 287;i&id., 1932, 76, 119; ibid., 1934, 83, 114.24 F. Ottensooser, ibid., 1932, 77, 140.Cystine appears to be absent.l5 Chem. cincl l l i d . , 1943, 110.Is Naturwiss., 1942, 30, 87.Biochimia, 1940, 5, 547.H 234 BIOCHEMISTRY.agglutinogens from human erythrocytes by extracting them with simpleaqueous reagents or with organic solvents, but without success. Substanceshave been frequently obtained, however, which though devoid of antigenicpower show intense blood group specificity.C. Hallauer 23 described theisolation of specific non-antigenic substances from all three (A, B, and 0)blood groups. Apart from the conclusion based on a few qualitative teststhat the specific substances are largely carbohydrate, the chemical nature ofthe preparations was not determined. The composition of the materialisolated, which is very similar for each of the blood group substances, was C,43-46; H, 7-1-85; N, 6.8-7-9; P, 15-21 %. The high phosphorus content,if present in organic combination, is of considerable interest, but no furtherdetails have yet been given. More recently A. V. Stepanov, A. Kusin, Z.Makajeva, and P. Kosjakov 25 have obtained similar materials fromerythrocytes.The possibility of obtaining the specific blood group substances from othersources has been investigated.E. Witebsky and N. C. Klendshoj 26 haveisolated a material showing group B specificity from gastric juice. Thesubstance contained 1.5% of N and gave 75% of reducing sugars after acidhydrolysis. A similar polysaccharide substance was also obtained 27 bythese workers from the gastric juice of secretors (persons who secrete theirblood group substance in a water-soluble form) belonging to group 0. Theserologically active material contained 2.8% of N and gave 40% of reducingsugars after acid hydrolysis. Owing to lack of material a more detailedexamination of the specific substances was not possible. As a result of thedetection of the specific blood group substances in human urine,28 attemptshave been made 29 to isolate the blood group substances from this source.Apolysaccharide material which contained galactose, aminohexose, and 10%N-acetyl was isolated and shown to possess intense blood group A-specificity.Treatment of the polysaccharide with alkali to remove some of the acetylgroups resulted in the loss of serological activity whereas re-acetylation bymeans of keten restored the A-specificity.Specific blood group substances showing A-, B-, and O-specificity have beenobtained from human saliva.30 The material obtained from secretors be-longing to groups A, B, and 0 showed little difference chemically and con-tained about 5.5% of N, 26y0 of a-amino-acid N, and 23% of glucosamine;it gave 4 5 4 8 % of reducing sugars after acid hydrolysis.An A-specificsubstance obtained from horse saliva appeared to be similar to the humanA-su bstance .The concentration of the blood group substances in saliva, and gastricjuice is high when compared31 with that of many other tissue fluids andsecretions, but even here the active substance represents only a small part of2 6 J. Exp. Med., 1940, 72, 663.2 8 K. Yosida, 2. ges. exp. Med., 1928,63, 331.29 K. Freudenberg and H. Eichel, Annalen, 1934, 510, 240; ibid., 1935, 518, 97.30 K. Landsteiner and R. A. Harte, J. Biol. Chern., 1941,140, 673.31 A. S. Weiner " Blood groups and blood transfusion " (1943).27 Ibid., 1941, 73, 655MORGAN : THE SPECIFIC BLOOD-GROUP SUBSTANOES. 235the total solid matter of the secretions which are, moreover, diiZcult toobtain in useful quantities.The examination 32 of pseudo-mucinousovarian cyst fluids obtained from secretors revealed that these fluids are aconvenient and potent source of the group specific substances A, B, and 0.The A- and O-substances were found 1% 33 to be closely similar in chemicalcomposition in spite of the different serological specificity. Analysis showedthem to contain C, -5; H, 6.6-6.8; N (Dumas), 5.9-6.2%. Bothsubstances behaved as did the hog mucin A-substance on treatment withEhrlich’s reagent after they had been heated with dilute alkali for a fewminutes. The production of a reddish-purple coloration from A-, B-, and 0-substances under these conditions appears to be a characteristic property ofthis biologically important group.The removal of the amino-acids from the specific blood group substanceswith retention of full specificity has not yet been accomplished, and it seems,probable that the con6guration of the amino-acid-containing componentcontributes to, or is entirely responsible for, the serological specificity ofthe blood group substances.The products of acid hydrolysis were verysimilar for A- and O-substances lo, 33 and about 46% of the total N is presentin a-amino-acids and at least 81 yo in a-amino-groups. Both substances gaveabout 33% of glucosamine and 48% of reducing substances after hydrolysiswith dilute acid.The immunological properties of the A-, B-, and O-substances have beenstudied in some detail,349 353 363 379 39 and their conversion to active antigeniccomplexes is reported.36, 379 38 A method €or the quantitative determinationof the amount of blood group agglutinin in normal and immune sera has beendescribed .40It has been recorded 273 329 419 42 that saliva and gastric juice obtained fromsecretors belonging to groups A and B inhibit the action of anti-0 agglutininon human 0 cells.The purified A-substance from hog gastric mucin is soactive in this respect that the O-substance itself is not noticeably more activein inhibiting the action of anti-0 serum on 0 cells.33 The results of theseexperiments indicate that the homogeneous A-substance possesses both A-and O-specificity. The A-substance isolated from ovarian cyst fluid, on theother hand, fails to inhibit the agglutination of group 0 cells by anti-0 serumand therefore possesses A-specificity only.No significant differences in the,3a W. T. J. Morgan and R. van Heyningen, Brit. J. Exp. Path., 1944,25, 6.33 Idem, andM. B. R. Waddell, ibid., 1945,26, 387.34 E. Witebsky, N. C. Klendshoj, and C. McNeil, Proc. Sac. Exp. B i d . Med., 1944,55,35 A. S. Weiner, R. Soble, and H. Polivka, ibid., 1945, 58, 311.38 W. T. J. Morgan and W. M. Watkins, Bvit. J . Exp. Path., 1944, 25, 221.37 S. G. Rainsford and W. T. J. Morgan, Lancet, 1946, 154.38 W. T. J. Morgan, Brit. J . Exp. Path., 1943, 24, 41.*9 Idem and W. M . Watkins, ibid., 1945, 26, 247.40 E. A. Kabat and A. E. Bezer, J . Exp.Med., 1945,82, 207.4 1 H. Sasaki, 2. Immun. Forsch., 1932, 77, 101.4 1 F. Schiffand H. Saeaki, Klin. Woch., 1932,11, 1426.167236 BIOCHEMISTRY.chemical composition of the A-substances isolated from hog mucin orovarian cyst fluids has yet been reported. It will be of considerable interestto know whether, in the A-substance of animal origin, it is the amino-acid-containing component or the polysaccharide that is responsible for the0 -specificity.During the last few years there has been a very rapid increase in know-ledge of the chemical and immunological properties of the specific bloodgroup substances. As yet, however, we know nothing of the chemical natureof the M and N agglutinogens and of the recently discovered Rhesus group ofblood group factors.An almost inexhaustible field of immunochemicalinvestigation on the different blood group and tissue antigens awaitsexploration.W. T. J. M.4. HYALURONIC ACID AND HYALURONIDASE.K. Meyer and J. W. Palmer reported the isolation from vitreous humorof a sulphur-free polysaccharide which contained a uronic acid, an amino-sugar, and possibly a pentose. Somewhat later 2, the same polysaccharidewas obtained from umbilical cord. This material contained 3.2% N, 11.5%Ac, and 45% hexuronic acid; it gave viscous aqueous solutions and yieldedreducing sugar after acid hydrolysis equivalent to 62.2% glucose and 40.3%hexosamine. The equivalent weight was 441. The amino-sugar wasisolated and identified as glucosamine hydrochloride. Oxidation of thepolysaccharide gave saccharic acid, which was isolated as acid potassiumsalt and identified by its crystal habit and by formation of the typicalthallium salt.Mucic acid was not found. The sugar acid is, therefore,glucuronic acid and not galacturonic acid. The analytical figures for thepolysaccharide acid, which was later called hyaluronic acid, agree closelywith those calculated for an anhydride of acetylhexosamine and hexuronicacid containing 2 or 3 molecules of water. In the presence of variousproteins the hyaluronic acid is precipitated from solution by acetic acid as a“mucoid,” similar in many ways to the mucoids obtained from naturalsource^.^ Hyaluronic acid has also been isolated from Group A and Chzemolytic streptoc~cci,~ from synovial fluid,6 from fowl sarcoma,’ and fromSkin.81An electrophoretic examination lo$ l1 of synovial fluids and vitreous bodyhas revealed that the hyaluronic acid present is not combined with protein1 J .BWZ. Chem., 1934,107, 629.‘I C. T. Morner, 2. physiol. Chem., 1894,18, 233.Ibid., 1936, 114, 689.K. Meyer, Symposia on Quantitative Biology, 1938, VI, 91.P. E. Kendall, J. W. Palmer, and.M. Heidelberger, J . BWZ. Chem., 1937, 118, 61.K. Meyer, E. M. Smyth, and M. H. Dawson, ibid., 1939,128, 319.E. A. Kabat, ibid., 1939, 130, 143.A. Claude, PTOC. SOC. Exp. Biol. Med., 1940, 43, 684.K. Meyer, J. Biol. Chem., 1940, 138, 491.lo H. Hesselvik, 2. physiol. Chem., 1938, 254, 144.l 1 G. Blix, Actaphyswl. Scad., 1940, 1, 29MORGAN : HYALURONIC ACID AND HYALURONIDASE.237but most probably exists as a salt with inorganic bases. The results of aninvestigation on the molecular shape and size of native hyaluronic acid havebeen published by G. Blix and 0. Snellman,12 who considered carefully theobservations of earlier workers on the preparation of undegraded hyaluronicacid and, in view of the degradation brought about by mild oxidants,13carried out the isolation procedure in an atmosphere of nitrogen. The Nvalues of 12 preparations of sodium hyaluronate from vitreous humor variedbetween 3-01 and 3.47 yo. The uronic acid content showed wider variation ;from 42.7 to 49.7%. The S content of the different preparations was usuallyless than 0.1 yo. Optical examinations for investigating the streaming doublerefraction and viscosity of the material were carried out in a Kundt’s 14, l5rotation apparatus with an inner rotating cylinder. It has been shown thatfor particles of high polymers, whose length is very much greater than theirthickness, the change in the angle of extinction with the velocity gradientenables values for the length of the particles to be obtained which agreewith those obtained from ultracentrifugation data.The relative viscosityof sodium hyaluronate is markedly influenced by electrolytes,16, 175 l* a valueof 9-45 for a 0.15% solution in water falling to 2.69 in O-lON-NaC1.ll Thehyaluronate solutions show a positive double refraction of flow, are poly-disperse, and possess an average particle length of about 4800 A.for materialobtained from vitreous humor and synovial fluid. Lower values, from 1000-2000 A., were obtained for preparations made without the exclusion of air.The greatest particle lengths, about 7000 A,, were recorded for hyaluronateobtained from umbilical cord. Values of this order are almost beyond theupper limits that can be determined by the apparatus employed; neverthe-less it seems probable that minimum molecular weights of the native hyal-uronates are of the order 200,000--500,000. The viscosity and birefringenceof hyaluronic acid dissolved in 0-1N-NaOH decrease a t room temperature.The particle length of the. alkali-degraded material was estimated a t about1300 A., a value, assuming an unbranched chain, corresponding to a mole-cular weight of 50,000.The same preparation was examined at a concen-tration of 0.25% for sedimentation and diffusion constants, and from thedata obtained (S = 1-78 x and D = 3.75 x lo-’) the average mole-cular weight was estimated as about 37,000. Blix and Snellman concludethat hyaluronic acid in its native state has a long chain structure and a mole-cular weight of 200,000--500,000 and that the difference in chain lengthfound in material obtained from different tissues indicates that the hyaluronicacid exists in its native state in different degrees of polymerisation. Althoughthe results are not incompatible with the presence of short side chaiiis, thesedimentation and diffusion constants do not support the idea of a branched12 Arkiv Kemi, Min.GeoE., 1945, 19, 1.13 B. Skanse and L. Sundblad, Acta physiol. Scand., 1943, 6, 3.14 0. Snellman and Y. Bjornstahl, Kolloid Beih., 1941, 52, 403.15 A. L. von Muralt and J. T. Edsall, J. Biol. Chem., 1930, 89, 315, 351.16 J. Madinaveitia and T. H. H. Quibell, Biochem. J., 1940, 34, 625.17 W. van B. Robertson, M. W. Ropes, and W. Bauer, J. Biol. Chem., 1940,133, 261,18 D. McClean, Biochem. J., 1941, 35, 159238 BIOCHEMISTRY.chain structure for hyaluronic acid. It has been shown that the action ofascorbic acid on hyaluronic acid is catalysed by copper and that this actioncan be inbibited by sodium diethyldithiocarbamate. The action of reducingagents 2o on hyaluronic acid and the influence of some environmental condi-tions 21 on the activity of hyaluronidase are recorded.K.Meyer, R. Dubos, and E. M. Smyth22 first reported the hydrolyticaction of enzymes obtained from pneumococcus on hyaluronic acid isolatedfrom vitreous humor, umbilical cord, and streptococcus. Enzymes were alsoobtained from culture filtrates of CZ. wekhii 1 7 9 23 and from group A haemo-lytic streptococci which hydrolysed hyaluronic acid with the formation ofabout 70% of the theoretical reducing power calculated from the N-acetyl-glucosamine and glucuronic acid content of the hyaluronic acid preparationand expressed as equivalents of glucose. Similar observations were madeusing partially purified enzymes.18924Some years before hyaluronic acid was discovered a number of investi-gations concerned with the capacity of a substance in testis extract, and incertain bacterial filtrates, to increase tissue permeability 259 269 27 were made,the results of which have contributed considerably to later studies whichhave dealt with the action of various enzyme preparations on hyalur*onicacid.E. Chain and L. A. Duthie 28 were the first to demonstrate that testisextracts and other preparations which contain “ spreading or diffusing factor ”decrease the viscosity of synovial fluid and vitreous humor and liberatereducing substances, and as a result of these observations suggested that“ spreading or diffusing factor’’ is probably identical with the mucinase(hyaluronidase) which hydrolyses the viscous polysaccharides in these fluids.Quantitatively good agreement was found when spreading activity andhyaluronidase activity of testis and leech extracts 29 were compared.Bacterial filtrates, especially those from CZ.wekhii and the pneumococcusand certain snake venoms,30 showed a somewhat. bigger spread in the skinthan would have been expected from the hyaluronidase activity, butChain and Duthie consider that this inconsistency is accounted for bythe fact that the injection of toxic bacterial filtrates and snake venomsis always followed by considerable oedema, due to capillary damage.Furthermore, since the spread in the skin caused by hyaluronidase can19 A. Pirie, Brit. J . Exp. Path., 1942, 23, 277.20 C. W. Hale, Biochern. J., 1944, 38, 362.21 Idem, ibid., 1944, 38, 368.23 K. Meyer, G. L. Hobby, E. Chaffee, and M.H. Dawson, J . Exp. Med., 1940, 71,24 K. Meyer, E. Chaffee, G. L. Hobby, andM. H. Dawson, ibid., 1941,73,309.25 F. Duran-Reynals, Compt. rend. SOC. Biol., 1929, 99, 6 ; J . Exp. Med., 1929,50, 327; D. C. Hoffman and F. Duran-Reynals, ibid., 1931,53,387; F. Duran-Reynals,ibid., 1933, 58, 161 ; ibid., 1939,69, 69.22 J . Biol. Chem., 1937, 118, 71.137.z 6 D. McClean, J . Path. Bact., 1930,33, 1045; 1931,34,459; 1936,42, 477.27 J. Madinaveitia, Bwchem. J., 1938, 32, 1806; 1939, 33, 347, 1470.2 8 Nature, 1939, 144, 977 ; Brit. J . Exp. Path., 1940, 21, 324.2o A. Claude, J . Exp. Med., 1937,66, 363.30 G. Favilli, Nature, 1940, 145, 866MORGAN : HYALURONIC AUID AND HYALURONIDASE. 239be influenced by non-specific irritants, it need not be represented correctlyby the viscosimetric determination of hyaluronidase.The degree of hydro-lysis of hyeluronic acid was determined by measuring the liberation of reducingsubstances and of N-acetylglucosamine and by the fall in viscosity. Otherworkers,31 however, consider it is not justifiable to assay skin-diffusing factorsby merely measuring their hyaluronidase activity. Another method ofassaying hyaluronidase, which depends on the destruction by the enzyme ofthe power of a substrate-protein complex to form a typical mucin clot on theaddition of acetic acid, has been developed. The conclusion is reached thatall three methods of assay, (a) diffusing activity, ( 6 ) viscosimetry, and ( c )mucin clot prevention, measure the activity of the same agent.l7? 327 33Guinea pigs can be used in place of rabbits for assaying hyaluronidaseactivity.34 The reduction of the viscosity of hyaluronic acid and the liber-ation of reducing substances and N-acetylglucosamine by bacterial enzymescan be completely neutralized by appropriate antisera which also inhibitdiffusion in the skin.18The results of a more detailed study of the action of partially purifiedhyaluronidase preparations obtained from leeches, bull testes, and culturefiltrates of CZ.welchii have recently been published.35 The leech hyaluroni-dase, acting on hyaluronic acid obtained from vitreous humor at 37" and pH4.6, gave 26% reduction, calculated as glucose by the method of Hagedornand Jensen. The same enzyme acting on hyaluronic acid derived from thecapsule of group A hzmolytic streptococci 36 (a substance considered to beidentical with the material obtained from vitreous humor) gave 50% reduction,corresponding to the amount expected on the assumption that the hyaluronicwas broken down to disaccharide units. On the other hand, hyaluronic acidderived from both these sources gave 70% reduction after hydrolysis withhyaluronidase derived from pneumococci.The purified hyaluronidaseobtained from CZ. welchii gave 60% of reducing substances when acting onsynovial fluid hyaluronic acid, and yielded hydrolysis products which gave areddish-purple colour with p-dimethylaminobenzaldehyde reagent after theyhad been kept at room temperature with dilute alkali. The reactive substancesgave negative reactions for glycuronic acid and were shown to be mono-saccharides, one of which behaved as if it were N-acetylglucosamine while theother was absorbed much more strongly on charcoal than N-acetylglucos-amine and contained 3.4% of N.The hydrolysis products arising throughthe action of leech and partially purified testicle hyaluronidase on theother hand gave a positive test for glucuronic acid and consisted of oneor more oligo-saccharides which show a strong absorption on charcoal andare not eluted by ephedrine. By the action of another enzyme in testiculars1 J. Madinaveitia, A. R. Todd, A. L. Bacharach, and M. R. Chance, Nature, 1940,3a C. V. Seastone, J . Exp. Med., 1939,70, 361.33 D. McClean, Biochern. J., 1943, 37, 169.34 J.H. Humphrey, ibid., p. 177.35 L. Hahn, Arkiv Kemi, Min. Geol., 1945, 19A, 1 ; 21A, 1.36 G. K. Hirst, J . Exp. Med., 1941, 73, 493.146, 197240 BIOCHEMISTRY.preparations the oligosaccharide could be hydrolysed to monosacchar-ides. The products obtained from hyaluronic acid by means of testishyaluronidase, but not those derived by means of leech hyaluronidase,behaved on treatment with dilute alkali a t room temperature like the materialderived by the action of Cl. welchii hyaluronidase, and gave with p-dimethyl-aminobenzaldehyde reagent a strong reddish-purple colour. It appears,therefore, that leech, testis, and CZ. welchii hyaluronidases give differenthydrolysis products when they act on the same hyaluronic acid. Additionalevidence 3’ of similar differences has also been obtained.Testicularhyaluronidase, unlike the streptococcal enzyme, leaves material, not diffus-able through cellophane, which will still adaptively enhance the production ofhyaluronidase by streptococci if added to growth media for the organisms.Reviews covering the subject have been p~blished.~8# 391 40W. T. J. M.5 . CYTOCHEMISTRY.been the subject of several reviews.l> 2, 3, *3 5, 6,will be discussed here.The chemical components of the cell nucleus and cytoplasm have recentlyOnly the salient pointsThe Cytoplasm.Considerable interest has centred recently round the pentose poly-nucleotides of the cell cytoplasm. The pentose polynucleotide of yeast has,of course, been known for a long time but similar material has also beenisolated from beef pancreas and sheep liver,g and from dog liver, intestine,and kidney.1° The pentose of the polynucleotide from yeast l1 and fromsheep liver has been proved conclusively to be ribose, but the general andrather unsatisfactory term ribonucleic acid is usually applied indiscriminatelyto pentose polynucleotides from all sources.Ribonucleic acid in the cell cytoplasm gives a negative Feulgen test(p.244) and, in the caae of rapidly proliferating cells, shows a high absorptionin the ultra-violet a t a wave-length of 260 mp which is characteristic of the37 H. J. Rogers, Biochem. J . , 1945, 39, 435.38 K. Wallenfels, Angew. Chem., 1941, 234.40 L. Hahn, Fermentfwsch.., 1944,17, 417.F. Duran-Reynals, Bact.Rev., 1942, 6, 197.A. E. Mirslry, Advances in Enzymology, 1943, 3, 1.J. N. Davidson and C . Waymouth, Nut. Abs. Rev., 1944, 14, 1.J. P. Greenstein, Advances in Protein Chemistry, 1944, 1, 209.J. Brachet, “ Embryologie Chimique,” Liege, 1944.E. Stedman, Edin. Med. J., 1944, 51, 353; Bwchem. J., Proc. Biocliem. SOC., 10thJ. N. Davidson, Edin. Med. J . , 1945, 52, 344; Biochem. J . , Proc. Biochem. SOC.,j H. S. Loring, Ann. Rev. Biockem., 1944, 13, 296.Nov., 1945.10th Nov., 1945.8 E. Jorpes, Acta Med. Xcand., 1928,68, 253; Biochem. J . , 1934, 28,2102.9 J. N. Davidson and C . Waymouth, ibid., 1944, 38, 375, 379.lo A. M. Brues, M. M. Tracey, and W. E. Cohn, J . Biol. Chem., 1944,155, 619.l 1 J. M. Gulland and G. R. Barker, J . , 1943, 625DAVIDSON : CYTOCHEMISTRY. 241conjugated double bonds of pyrimidines and purines.12 Histochemically itis revealed in the cell cytoplasm as material which is deeply basophilic, i.e.,which readily takes up such stains as pyronine or toluidine blue. It can bedissolved out of the cytoplasm, which then loses its basophilic properties, bytreatment with the enzyme ribonuclease which breaks down ribonucleic acidbut not deoxyribonucleic acid.l3 Nuclear staining is unaffected by suchtreatment.This is the basis of a useful histochemical test (the Rrachet test)which has been extensively employed for the detection of ribonucleicacid,l4, 1 5 3 1% 1 7 3 18, 2% 21 and may even be used to assess roughly theribonucleic acid content of different tissues.Results obtained by thismeans 14, 21 agree well with direct pentose estimations on fresh tissue,22 esti-mations made on the extracted nucleic acids,23 and ultra-violet absorptionmeasurement^.^^ The concentration of ribonucleic acid is low in brain,.muscle, and heart, and in endocrine glands such as the thyroid or the isletcells of the pancreas. It is high in all tissues in which protein synthesisis vigorous for purposes either of secretion or of cell multiplication, e.g., thepancreas, the salivary glands, the gastric and intestinal mucosa, the Mal-pighian layer of the skin, oocytes in course of vitellogenesis, the imaginaldiscs of insects, and embryonic tissues. It is high in the Nissl's granules ofnerve cells, in the young cells (e.g., lymphoblasts) of the hzemopoieticsystern,14, 25 and in simple organisms capable of rapid proliferation, e.g.,yeasts, bacteria, and plant viruses.The basophily of such cells, and therefore their ribonucleic acid content,may depend on the physiological state of the organ concerned.Thus theribonucleic acid content of glandular tissues diminishes after prolongedstimulation by electrical means or by pilocarpine,26, 27 whereas in thegranules of the anterior pituitary i t increases during pregnancy or after12 T. Caspersson, Skand. Arch. Physiol., 73 Suppl. No. 8 ; J . Roy. Microscop. SOC.,1940, 60, 8 ; T. Caspersson and J. Schultz, Nature, 1938,142, 294; ibid., 1939,143, 602.l 3 J. Brachet, ConLpt. rend. SOC. Biol., 1940, 133, 88, 90; Arch. Biol., 1940, 51, 151,167.l * 5.Brachet, ibid., 1941, 53, 207.l5 J. Desclin, Compt. rend. SOC. Bwl., 1940, 133, 457.l6 T. S. Painter and A. N. Taylor, Proc. Nat. A d . Sci., 1942, 28, 311.l i J. Gersh and D. Bodian, Biol. Symposia, 1943, 10, 163.l 8 5. N. Davidson and C. Waymouth, Proc. Roy. SOC. Edin., 1944, 62, 96.l9 J. S. Mitchell, 21st Ann. Rep. Brit. Emp. Cancer Campaign, 1944, p. 62,2O G. J. Roskin and A. S. Ginsburg, Compt. rend. Acad. Sci. U.R.S.S., 1944, 43, 122;21 J. J. Biesele, Cancer Research, 1944,4, 529, 737 ; J. J. Biesele, H. Poyner, and T. S.22 J. Brachet, Enzymologia, 1941, 10, 87.23 J. N. Davidson and C. Waymouth, Biochem. J., 1944, 38, 39.24 T. Caspersson, Naturwiss., 1941, 29, 33.25 T. Caspersson, H. Landstrom-Hydh, and L. Aquilonius, Chromosoma, 1941, 2,111; H.Landstrom-Hydh, Acta physiol. Scand., 1943, 6, Suppl. 17; H'. Landstrom-HydBn, T. Caspersson, and G. Wohlfart, 2. mi1cr.-anat. Porsch., 1941, 49, 534.G. J. Roskin and G. Kharlova, ibid., 1944,44, 389.Painter, Univemity of Texas Publications, 1942, No. 4243.26 J. Verne, Bull. Hist. appl., 1927, 55, 569.27 E. Riea, 2. Zellforsch,., 1935, 22, 523242 BIOOHEMISTRY .administration of o e ~ t r o n e . ~ ~ In nerve cells the ribonucleic acid content ofthe cytoplasm varies with the degree of excitation.25Pentose polynucleotide exists in the cell cytoplasm as phospholipin-ribonucleoprotein complexes of two different sizes.28 The " large particles "(0-5-2*0p diameter) can easily be seen under the microscope. They includethe mitochondria and the secretory or zymogen granules of liver and pancreas.The " small particles " or " microsomes " (50-200 mp diameter) are sub-microscopic but can be shown in the dark field microscope as highly re-fringent small bodies in continuous Brownian movement.They form thechromophilic ground substance of the cell, constituting up to 25% of its totaldry substance.I n the liver cell, glycogen is also present in particulate form.29The particulate components of the cytoplasm can be isolated from cellularextracts by a process of differential centrifugation employing high and lowspeeds alternately.28, 30 The particles from liver, pancreas, and leukamiccells have been most extensively investigated. They contain protein richin -SH groupings,31 pentose polynucleotide,28 lipoid material (some two-thirdsof which is phospholipin including acetal phospholipin 28y 30), inositol,28sterols, and vitamin A.30 Analytical figures from different laboratories vary,but it is generally agreed that the total fat content is higher in the smallparticles (40--45%, Claude ; 28 42-51 %, Bensley 30) than in the largeparticles (20-27%, Claude ; 28 32-38y0, Bensley 30), The phosphoruscontent is 1.1-1-7% in the large particles and about 1.5% in the smallparticles except in particles from pancreas, embryo, and tumour cells whichhave a higher phosphorus content (2.1 yo).2*Nearly all the phospholipin of the cytoplasm,30 and, in the adult cell,nearly all the ribonucleic acid, 31 is present in these particles.In certainrapidly proliferating cells or tissues such as amphibian ,eggs in course ofdevelopment, chick embryos, young oocytes, and yeast, the particles accountfor only 2 0 4 0 % of the total ribonucleic acid content. The remainderoccurs as " free " ribonucleic acid which is not brought down by tbe ultra-centrif~ge.3~Succinic dehydro-genase and cytochrome oxidase are found in granules from liver 31* 32 (butnot in the interparticulate fluid 30), y e a ~ t , ~ and heart muscle.33 Phosphataseis found in particles from kidney 34 and liver.4 Liver particles also containThe enzyme content of the particles is important.28 A. Claude, Science, 1943, 97, 451; Biol. Symposia, 1943, 10, 111; J . Exp. Med.,aQ A. Lazarow, Science, 1942, 95, 49; Biol.Symposia, 1943, 10, 1; Arch. Bwchem.,1944, 80, 19.1945, '7, 337.R. R. Bensley, Science, 1942, 96, 389.31 5. Brachet and H. Chantrenne, Acta Bwl. Belg., 1942, 4,451; R. Jeener andJ. Brachet, ibid., 1941,1, 476; 1942,2, 273.3a A. Lazarow and E. S. G. Barron, Anat. Rec., 1941,79, Suppl. 41 ; E. S. G. Barron,Bwl. Symposia., 1943, 10, 27.33 K. G. Stern, Cold S p r i n g Harbor Symp. Qwtnt. Biol., 1939, 7, 312.34 E. A. Kabat, Science, 1941, 93, 43DAVIDSON : CYTOCHEMISTRY. 243amylase, cathepsin, dipeptidase, ribonuclease, and adenyl deaminase, butthese enzymes are also present in the interparticulate fluid.4* 31 Both largeand small particles contain flavoproteins (as does the interparticulate fluid),SObut one flavoprotein, the d-amino-acid oxidase, is found in the liver in thelarge particles only.28Brachet has pointed out that the cytoplasmic ribonucleoprotein particlesconstitute excellent organs for protein synthe~is.~~ 35 They contain enzymesof the protease type which play a part in protein .synthesis together withoxidation-reduction systems which may be able to provide the energynecessary for such an endothermic reaction.Moreover the particles areabundant and the concentration of cytoplasmic ribonucleic acid is par-ticularly high in cells in which protein synthesis is vigorous.Claude28 has put forward the suggestion that the cytoplasmic particlesare endowed with the power of self-duplication, a property which appears tobe a feature of systems composed of polynucleotide associated with protein.It is found for example in the genes and in the filterable viruses. While plantviruses appear t o consist of simple ribonucleoproteins, the animal virusesare phospholipin-nucleoprotein complexes containing either ribonucleic acid(Rous sarcoma virusF6 equine encephalomyelitis virus 37), or deoxyribonucleicacid (rabbit papilloma virus,38 vaccinia elementary bodies,38* 39 influenzavirus40).The similarity between the cytoplasmic particles and the animalviruses, and possible relationships, have been commented on by severalauthors.41Phospholipin-ribonucleoprotein complexes with thromboplastic activityhave been isolated from lung tissue.42 In liver tissue the liponucleoproteinsconstitute storage material. Fasting is accompanied by a fall in liver weightrelative to total body weightys a loss in granular material from the cytoplasm,43a decrease in cytoplasmic volume, a fall in the total ribonucleic acid (but notin the deoxyribonucleic acid) content of the livery7, and a fall in phospholipinand protein.44 A similar loss of cytoplasmic particles follows administrationof a protein-poor diet.4435 J.Brachet, Ann. Soc. Roy. Zool. Belg., 1942, 73, 93.36 A. Claude, Science, 1938, 87, 467; 1939, 90, 213; 1940, 91, .77.37 A. R. Taylor, D. G. Sharp, D. Beard, and J. W. Beard, J . Infect. Dis., 1943,38 A. R. Taylor, D. Beard, D. G. Sharp, and J. W. Beard, ibid., 1942, 71, 110.S9 C. L. Hoagland, G. L. Lavin, J. E. Smadel, andT. M. Rivers, J . Ezp. Med., 1940,40 A. R. Taylor, J .Bwl. Chem., 1944,153, 675.4 1 H. G. Du Buy and M. W. Woods, Phytopathol., 1943, 33, 766; C. D. Darlington,Nature, 1944, 154, 164; A. Haddow, ibid., 1944, 154, 194; P. Koller, ibid., 1943, 151,244; J. W. Beard, Proc. Inst. Med. Chicago, 1945, 15, No. 13; V. R. Potter, Science,1945,101,609.72, 31.72, 139.48 E. Chargaff, A. Bendich, and S. S. Cohen, J . Biol. Chem., 1944,156, 161.p3 W. Berg, 2. mikr.-anut. Forsch., 1927, 12, 1.44 H. W. Kosterlitz, Nature, 1944, 154, 207; H. W. Kosterlitz and R. M. Campbell,Nut. Abs. Rev., 1946, 15, 1244 BIOCHEMISTRY.The Nucleus.Several methods of obtaining cell nuclei have been described. With theexception of that of BehrensF5 in which powdered freeze-dried tissue isallowed to sediment out in columns of organic solvents of graded density,most methods involve treatment of the finely divided tissue with a weak acid,e.g., citric acid, and isolation of the nuclei by differential centrifugation.46-50From avian erythrocytes, nuclei have been obtained by laking with water,51by freezing and thawing,52 -or by treatment with lysolecithin 53 or saponin.54Nuclei have also been obtained from cells which have been disrupted by sonicvibrations.55The cell nucleus contains deoxyribonucleic acid and basic proteins of thehistone type. Lipin is present in amounts similar to those in whole tissue,49but the phospholipin-cholesterol ratio is unusually low.*’ Glycogen isabsent from the nuclei of liver cells.49 Pigments may be present, e.g.,xanthophyll in chicken erythrocyte nuclei.The enzymes which have been reported in isolated liver cell nuclei includeargina~e,4~, 49 cytochrome oxidase, esterase, alkaline phosphatase (in par-ticularly high concentration), and acid phosphatase, but little or no catalase orsuceinic dehydrogena~e.~~ Esterase and peptidase 56 have been found int henuclei of oocytes, and acid phosphatase in those of chicken erythrocyte^.^^Phosphatase has been demonstrated histochemically in the chromosome~.58~ 59E.Stedman and (Mrs.) E. Stedman 6o have reported the discovery in thenucleus of an acidic protein, “ chromosomin,” which may comprise some30-70% of the total nuclear material. They suggest that the chromosomesconsist of compressed cylindrical spirals of chromosomin with a central coreof nucleic acid which also fills the interstices between the coils and con-stitutes the nuclear sap.6, 6oThe discovery of chromosomin may necessitate a revision of the usualinterpretation of the Feulgen nuclear reaction.61 This test depends upon thefact that the hydrolysis products of deoxyribonucleic acid will restore thecolour to a solution of basic fuchsin which has been decolourised with sulphur45 M. Behrens, 2.physwl. Chem. 1938, 253, 185; 1939, 258, 27.46 G. Crossman, Science, 1937, 85, 250.4 7 C. A. Stoneburg, J. Bwl. Chem., 1939, 129, 189.4 5 A. Marshak, J . Qen. Physiol., 1941, 25, 275.A. L. Dounce, J . Biol. Chem., 1943,147, 685; 1943,151, 221.50 D. M. Ziegler, Anat. Rec., 1945, 91, 169.5 1 D. Ackermann, 2. physiol.Chem., 1904-5, 43, 299.52 0. Warburg, ibid., 1910, 70, 413.53 M. Laskowski, Proc. SOC. Exp. Biol. N.Y., 1942, 49, 354.54 A. L. Dounce and T. H. Lan, Science, 1943,97,584.55 C. A. Zittle and R. A. O’Dell, J. Biol. Chem., 1941, 140, 899.J. Brachet, Compt. rend. SOC. Biol., 1938,127, 1455.5 7 A. L. Dounce and D. Seibel, Proc. SOC. Exp. Biol. N . Y., 1943, 54, 22.6 8 E. N. Wilber, J. Exp. Biol., 1942, 19, 11.59 J. F. Danielli and D. G. Catcheside, Nature, 1945,156,294.60 Ibid., 1943,152, 267, 503, 556; 1944,153, 500.61 R. Feulgen and H. Rossenbeck, 2. physiol. Chem., 1924,135, 203DAVIDSON : CYTOCHEMISTRY. 245dioxide. Stedman maintains that when this test is applied to tissue sectionsthe fully coloured stain is developed in the nucleus and is then taken up bychromosomin, so that, although deoxyribonucleic acid can be detected in thenucleus, it cannot of necessity be located in the chromosomes. Chromosomescan in fact be stained by " developed nucleal stain " prepared by interactionof Feulgen's reagent with hydrolysed deoxyribonucleic acid.62 While thisview has been vigorously contested,63 it would appear that the original simpleinterpretation of the Feulgen technique may be inadequate-aDeoxyribonucleic acid exists in the nucleus in combination with basicproteins of the histone type as material which is usually referred to loosely as" chromatin." Such deoxyribonucleoprotein can be extracted from cellnuclei by concentrated sodium chloride solutions and precipitated by dilutionwith water,65 or it can be isolated in threads by a process of differentialcentrifugation.66 Chromatin prepared in the latter way from liver cells isreported to stimulate cellular proliferation and to accelerate the healing ofexperimental wounds.67On the basis of histochemical tests and pentose estimations on isolatednuclei Brachet has concluded that 10% of the nucleic acid of the nucleus isribonucleic acid.l*~ 22 Some of this is present in the nucleolus (vide infra),but a small amount is reported in the genetically inert chromatin fractiontermed the heterochromatin, which consists therefore of histone, deoxyri-bonucleic acid, and small amounts of ribonucleic acid. The remainder of thechromatin is the gene-bearing euchromatin, which is composed of deoxyri-bonucleic acid together with higher proteins of the globulin G8 Therelationship, if any, of these proteins to chromosomin is not yet clear.The Nucleolus.Evidence that the nucleolus contains ribonucleic a,cid and not deoxyri-bonucleic acid is given by ( a ) a negative Feulgen test,69 ( b ) strong absorptiona t 260mp 69 which is diminished by treatment with ribonuclease,70 (c) apositive Brachet test,14 and (d) positive histochemical tests for pent~ses.'~The ribonucleic acid appears to be combined with histone and to bear arelationship to the cytoplasmic ribonucleoproteins. The nucleolus is large inthose cells where a strongly basophilic cytoplasm, indicating a high ribo-nucleic acid content, is associated with active protein synthesis, and is small62 H. C. Choudhuri, Nature, 1943, 153, 475.63 H. G. Callan, ibid., 1943, 152, 503; H. N. Barber and H. G. Callan, ibid., 1944,153, 109; T. Caspersson, ibid., 1944, 153, 499; R. E. Stowell, Stain Technology, 1945,20, 45.64 J. G. Cam, Xature, 1945, 156, 143.65 A. E. Mirsky and A. W. Pollister, Proc. Nut. Acud. Sci., 1942, 28, 344.6(i A. Claude and J. S. Potter, J. Exp. Med., 1943, 77, 345.6 i A. Marshak and A. C. Walker, Proc. SOC. Exp. Biol. N . Y., 1944, 58, 62.6 8 T. Caspersson and L. Santesson, Acta Radiol., Suppl., No. 46.61 T. Caspersson and 5. Schultz, Nature, 1938,142, 294; ibid., 1939, 143, 602; Proc.70 J. Gersh, quoted by Mirsky (1).71 Brit. J . Exp. Path., 1942, 23, 285, 296, 309, Brit. J . Radiol., 1943, 16, 339.Nat. Acad. Sci., 1940, 26, 507246 BIOOHEMISTRY.in, or apparently absent from, cells where cytoplasmic growth does notO C C U ~ . ~ ~ ~ G6Growth and Cell Division.In primitive organisms, such as the sea urchin egg, nuclear deoxyri-bonucleic acid is apparently synthesised entirely a t the expense of an initiallyabundant store of cytoplasmic ribonucleic acid.4 In higher organisms bothtypes of nucleic acid are synthesised on a large scale, but a balance appearsto be preserved between the two types. Thus in the sheep embryo, althoughribonucleic acid is present in high concentration in most tissues, the ratio ofribonucleic acid to deoxyribonucleic acid for any one tissue is of the same orderin the embryo as in the corresponding adult tissue.23 At the same time it ispossible that some, at least, of the nuclear deoxyribonucleic acid is syn-thesised by way of cytoplasmic ribonucleic acid perhaps even in the chromo-s0mes.3~ On the basis of ultra-violet absorption measurements on the cyto-plasm of the cells of human tumours exposed to X - or gamma-rays, J. S.Mitchell has suggested that ribonucleotides are formed in the cytoplasm fromunknown precursors and are reduced in the nucleus to deoxyribonucleotideswhich are finally polymerized to deoxyribonucleic acid.71The nature of the chemical changes occurring during the process ofmitotic division have been followed both by ultra-violet absorption methodsin conjunction with the quartz microscope 25 and by histochemical tech-nique.12 During prophase deoxyribonucleic acid accumulates in the chromo-8omes reaching a peak concentration at metaphase. At the same time ribonu-cleic acid decreases in the cytoplasm and the nucleolus and, according toBrachet, becomes concentrated in the chromosomes and the spindle.12 Attelophase the deoxyribonucleic acid concentration of the chromosomesdecreases while ribonucleic acid reappears in the cytoplasm and the nucleolusis reformed. Caspersson holds that the heterochromatin controls thesynthesis of histones which together with ribonucleic acid form the nucleolus,while the euchromatin is responsible for the production of higher proteins ofthe globulin type. Some of the histones of the nucleolus diffuse to thenuclear membrane where they stimulate the formation of cytoplasmicribonucleic acid and 68On the other hand Stedman regards the histones as regulators of mitosis.In resting nuclei sufficient histone is present to combine with all the deoxyri-bonucleic acid. When the histone content is low, as in the rapidly pro-liferating cells of embryonic or tumour tissue, nucleic acid is available tocombine with chromosomin forming a self-reproducing 72J. N. D.J. N. DAVIDSON.F. DICKENS.W. T. J. MORGAN.F. G. YOTTNC,.72 E. Stedman and (Mrs.) E. Stedman, Nuture, 1943, 152, 666