BIOCHEMISTRY.1. INTRODUCTIION.UNDOUBTEDLY the most important biochemical event occurring during 1949was the holding of the First International Congress of Biochemistry inCambridge under the Presidency of Prof. A. C. Chibnall, F.R.S. TheCongress was attended by more than 1700 members representing 42 differentcountries and resulted in the setting up of an International Committee forBiochemistry under the Chairmanship of Sir Charles Harington, F.R.S.,with Prof. K. Linderstram-Lang as Secretary. An approach is being madeto the International Council of Scientific Unions with a request for therecognition of this Conunittee as the international body representative ofbiochemistry and with a view to the formal constitution of an InternationalUnion of Biochemistry, as soon as possible.The subject of Biochemistry has therefore reached another and Significantstage in its development as an independent scientific discipline.The work of the Congress was spread over 12 sections and a volume ofabstracts of communications was issued to each participating member.In selecting topics for this year's Report, attention has been directedto fields in which notable advances have been recorded during the year.These must necessarily be reviewed against the background of previousdiscovery and interpretation, but it is felt that, in the fields both of thecarotenoid pigments and of the hx?mopoietic factors, very significant advanceshave been achieved during 1949.c. R.2. HBMOPOIIETIC FACTORS.(FOLIC ACID AND VITAMIN 1312.)In this short review i t will be possible to mention only a small selectionof the many important papers on haemopoietic factors published within thelast four years.Folic Acid.-This subject-was last reviewed in Annual Reports for 1946,since when a very great deal of work has been produced.The literaturehas been reviewed ls2 up till 1949. Much of the difliculty met in earlywork on this subject was due to the fact that a number of closelyrelated compounds had folic acid activity for different organisms. Thetable gives the main factors shown to be members of the folic acidgroup.1 T. H. Jukes and E. L. R. Stokstad, Physiol. Reviews, 1948, 28, 51.E. L. R. Stokstad and T. H. Jukes, Ann. Reviews Biochem., L949, 18, 435230 BIOCHEMISTRY.(Adapted from T.H. Jukes and E. L. R. Stokstad.l)Factor. Source and effects. -Vitamin M.Factor U.Vitamin B,.Norite eluate factors.Folic acid.L. casei factor.Vitamin B,, and B,,,Factors R and S.Yeast extract, effective in tropical sprue.3Yeast and liver extract effective in nutritional cytopenia of theYeast extract, promoted growth in chicks.6Adsorbed on fullers' earth, prevented nutritional anaemia in theFrom yeast and liver, promoted growth of L. m ~ e i . ~Active for S. fmcaZis R, prepared from spinach.8Prepared from liver and yeast.'Promoted growth and feathering of chicks.'*Essential in chick nutrition.llmonkey.'chick.8The chemistry of these compounds has been investigated 12. l3 and toconform with these findings they are considered as derivatives of pteroicacid (I), folic acid (liver L.cusei factor) being pteroylglutamic acid (PGA)(11). I n pteroyltriglutamic acid two further mglutamic acid molecules(1.1 OHare attached by y-peptide linkages to the glutamic acid residue of pteroyl-glutamic acid, and in pteroylheptaglutamic acid (Vitamin B, conjugate)six ngfutamic acid molecules are attached by y-peptide linkages to theglutamic acid residue of PGA.Deficiency states which may be controlled by the administration of PGAhave been produced in a number of animals and insects, e.g., rat,l* guinea-s L. Wills, Brit. Med. J., 1931, I, 1059.4 P. L. Day, W. C. Langston, and W. J. Darby, Proc. SOC. Exp. BioZ. Med., 1935,ii E. L. R. Stoketad and P. D. V. Manning, J.BioZ. Chem., 1938,125,687.(I A. H. Gogan and E. M. Parrott, ibid., 1939,128, xlvi.38, 860.E. E. Snell and W. H. Peterson, J. Bact., 1940,39, 273.H. K. Mitchell, E. E. Snell, and R. J. Williams, J. Amer. Chem. Soc., 1941, 83,2284.* E. L. R. Stokstad, J. Bid. Chem., 1943,149, 573.lo G. M. Briggs, T. D. Luckey, C. A. Elvehjem, and E. B. Hart, ibid., 1943,148, 163.l1 A. E. Schumacher, G. F. Heuser, and L. C. Norris, ibid., 1940,135, 313.l2 5. H. Mowat, B. L. Hutchings, R. B. Angier, E. L. R. Stokstad, J. H. Boothe,C. W. Waller, J. Scmb, and Y. J. SubbaRow, Amer. Chem. SOC., 1948, 70, 1096.J. H. Boothe, J. H. Mowatt, B. L. Hutchings, R. B. Angier, C. W. Waller,E. L. R. Stokstad, J. Semb, A. L. Gazzola, and Y. J. SubbaRow, ibid., p. 1099.14 S.Black, J. M. McKibbin, and C. A. Elvehjem, Proc. Soc. Exp. BWE. Med., 1941,47, 308CUTHBERTSON : HBEMOPOIETIC FACTORS. 231pig,15 monkey,ls chick,f7 dog,lS mink,ls Aedes aegypti,m Triboliumand Tenebrio moZitor.22 The typical condition in mammals is a decreasedgrowth-rate together with leucopenia and ultimately an anamia of themacrocytic type. In this review, folic acid deficiency will be used for adeficiency condition alleviated by one or other of the pteroylglutamates,and the term folic acid will be used to describe compounds promoting thegrowth of L. cccsei or 8. famlis R on folic-acid-deficient media.In man, the pteroylglutamates have been found effective in the treat-ment of a variety of macrocytic ansmias and in tropical s p r ~ e .~ ~ Folicacid will cause reticulocytosis and the return of an apparently normalblood and bone-marrow appearance in patients with pernicious anaemia,but will not prevent or relieve the symptoms of sub-acute combined de-generation of the cord found in the later stages of this disease.2P VitaminB,, will both cause complete remission of the blood symptoms and preventor alleviate the nerve changes.Compounds with Anti-folic Acid Activity.-Numerous compounds areknown to antagonize the action of folic acid in bacteria. Most of these arederived from PGA by substitution in the pteridine nucleus, the most im-portant being the 4-amino-, the N10-methyl-, and the 4-amin0-N~~-methyl-derivative.2 I n animals (rat, mouse, chick, pig, guinea-pig), administrationof these substances leads to the appearance of symptoms associated withPGA deficiency, but other toxic symptoms not reversed by PGA are pro-duced, and in the guinea-pig 2s the changes in the haemopoietic system arenot completely reversed by folic acid.The predominant effect of thesecompounds is the production of a leucopenia, followed by anaemia. Theyhave been tried clinically in human cases of leucsmia. Although 4-amino-pteroyglutamic acid appears to be the most satisfactory, i t will cause onlytemporary remissions, and difficulties are encountered because of toxicity.26* 27Apart from their use in partial control of human leucsmias, anti-folicacids have been found to inhibit the growth of the Rous sarcoma in chicks.28D. W. Woolley and H.Sprince, J . Biol. Chem., 1945,157, 447.l6 P. L. Day, W. C. Langston, and W. J. Darby, Proc. Soc. Exp. Biol. Med., 1938,l7 E. L. R. Stokstad and P. D. V. Manning, J . Bwl. Glwm., 1938, 125, 687.l8 W. A. Krehl, N. Torbet, J. de la Huerga, and C. A. Elvehjem, Arch. Biochem.,A. E. Schaeffer, C. K. Whitehair, and C. A. Elvehjem, Proc. Soc. Exp. Biol. Med.,38, 860.1946, 11, 363.1946, 62, 169.2o L. Golberg, B. de Meillon, and M. Lavoipierre, J . Exp. Biol., 1945, 21, 90.21 G. Fraenkel and M. Bfewett, Nature, 1946,158, 697.22 G. Fraenkel, M. Blewett, and M. Coles, ibid., 1948, 161, 981.23 T. D. Spies, " Experiences with folic acid," " The Year Book " Publishers Inc.,24 S . 0. Schwartz and B. E. Armstrong, J . Lab. Clin. Med., 1947,32, 1427.25 J.Innes, E. M. Innes, and C. V. Moore, ibid., 1949, 34, 883.26 S. Farber, Blood, 1949, 4, 160.27 L. 11. Rleyer, Trans. N . Y . Acad. Sci., 1948, 10, 99.28 P. A. Little, A. Sampafh, and Y. J. SubbaRow, J . Lab. Clilz. Ned., 1948, 33,1144.Chicago232 BIOCHEMISTRY.Leuchtenberger et aE. reported that pteroyltriglutamic acid inhibited thegrowbh of breast cancer in mice.29Metabolism of PGA and Conjugates.-In human cases of perniciousanaemia, sprue, and nutritional macrocytic anaemia, administration ofpteroyl-di-, -tri-, or -hepta-glutamate leads to remission of symptoms andthe di- and the tri-glutamate cause urinary elimination of folic a~id.~O> 31Conflicting results have been obtained with the use of the heptaglutamate inthe treatment of pernicious anaemia in relapse.These may be associatedwith the action of substances inhibiting the release of PGA from itsconjugates.32y 33In normal persons 30450% of administered PGA is eliminated in urinewhen given at the level of 3-10 mg. per day.34 After injection of pteroyl-di- and -tri-glutamic acid T. H. Jukes et aE.35 noted folic acid excretion inthe urine. M. E. Swenseid et aZ.36 have shown that free folic acid is excretedin the urine after administration of pteroylheptaglutamate, but this effectmay be absent if materials inhibiting the release of folic acid by deconjugasepreparations are present in the extracts used. This work could thereforebe interpreted to show that the heptaglutamates must be broken down inthe gut before absorption or utilisation.Although the evidence is veryconflicting, it appears that the heptaglutarnate is often not well utilised bypernicious-anemia patients in relapse, and it has been suggested 32y 37 thatliver extract improves the utilisation of the conjugates but the resultsobtained are probably best explained by the low doses used and the presenceof conjugase inhibitors. J. F. Wilkinson and MI. C. G. Israels 38 have shownthat pteroyltriglutamic acid and " diopterin " (a synthetic a-pteroyl-diglutamic acid not known to occur in Nature) are effective in the relief ofthe haematological effects of pernicious anaemia when given orally or byinjection. Their observations, along with those of Jukes et aE.,35 show thatthe conjugates may be broken down in the body to free folic acid.After administration of PGA (or its conjugates) only about 30% of thefolic acid may be accounted for in the urine in man : the fate of the remainderis not yet known.H.E. Sauberlich and W. I). Salmon 39 noted that administration of fofic29 R. Leuchtenberger, C. Leuchtenberger, D. Laszlo, and R. Lewisohn, Science,30 T. D. Spies, 8th. Med. J., 1946, 39, 634.31 T. D. Spies, G. Garcia Lopez, R. E. Stone, F. Milanes, R. 0. Brandenberg, andT. Aramburu, Internat. Rev. Vit. Res., 1947, 19, 1.32 F. H. Bethell, M. C. Meyers, G. A. Andrews, M. E. Swendseid, 0. D. Bird, andR. A. Brown, J. Lab. Clin. Med., 1947,32, 3.33 A. E. Sharp and E. C. Vonder Heide, Amer. J. Clin. Path., 1947, 17, 761.34 R. Steinkamp, C. F. Shukers, J.R. Totter, and P. L. Day, Proc. Soc. Ezp. Biol.Med., 1946, 63, 556.3 5 J . Lab. Clin. Med., 1947,32, 1350.S6 Ibid., p. 23.37 R. M. Suarez, A. D. Welch, R. W. Heinle, R. M. Suarez, jun., and E. M. Nelson,ibid., 1946, 31, 1294.36 Lancet, 1949, 257, 689.1945, 101, 46.3e Fed. Proc., 1949, 8, 247CUTHBERTSON : HBMOPOIETIC FACTORS. 233acid to rats led to an increased elimination of the L. citrovorurn factor inthe urine. This substance is not PGA but may be replaced in the nutritionof L. citrovorurn by PGA in the presence of t h ~ r n i d i n e . ~ ~ The importanceof this substance in mammalian metabolism is not known.H. G. Buyze and C . Engel,41,42 state that pteroylheptaglutamate istransformed by normal gastric juice into a product from which folic acidis liberated by liver homogenate but not by conjugase preparations.In the chick and the rat PGA conjugates are effective in controlling folicacid 44Thymine in large amounts can replace folic acid for the growth of 8.fcecalis R,45 and in the presence of a purine base thymine will replace folicacid in the nutrition of L.casei,46 though in the latter instance only half themaximal growth is attained. These observations lead to the hypothesisthat folic acid acts as a coenzyme in the synthesis of thymine and relatedcompounds. The relations between thymine, PGA, p-aminobenzoic acid,and the mode of synthesis of folic acid have been discussed by J. 0. Lampenand M. J. Jones.47 A possible role of histidine in the formation of folic acidhas been reported by D.A. Hall,48 who worked with 8. fcecaEis R. A relationwith tyrosine metabolism in the guinea-pig is suggested by C. W. Woodruffet aZ.,49 who showed that PGA prevented the elimination of tyrosinedcrivatives in the urine of scorbutic guinea-pigs.H. M. Kalckaret showed that 6-formylpteridine (;‘ 6-pteridylaldehyde ”) (an anti-folicacid) inhibited the activity of xanthine oxidase. “ Dopa ’’ decarboxylaseactivity was inhibited by two folic acid displacers and this inhibition wasprevented by PGA.51 The observation that the anemia produced in ratson a low-protein diet 52 is cured by the administration of folic acid suggestsa relation between folic acid and protein metabolism.Assay of Folic Acid.-It was early shown that microbiological assay offolic acid activity with L.casei and S . fcecalis often gave discordant resultswhich did not agree with each other or with the results of animal tests onrats, chickens, or monkeys.These results are now known to be due to the multiple nature of folicacid as shown in the following table.1Folic acid may be involved in certain enzyme systems.40 H. E. Sauberlich, Arch. Biochem., 1949, 24, 224.4z Nature, 1949, 163, 135.43 M. E. Swendseid, R. A. Brown, 0. D. Bird, and R. A. Heinrich, Arch. Biochem.,44 T. H. Jukes and E. L. R. Stokstad, J . Biol. Chem., 1947, 1S8, 563.45 E. E. Snell and H. K. Mitchell, Proc. Nut. Acad. Sci. Wash., 1941, 27, 1.46 E. L. R. Stokstad, J . Biol. Chem., 1941, 139, 475.47 Ibid., 1947,170, 133,4e J .Biol. Chem., 1949, 178, 861.6o Ibid., 1948,174, 771.s1 G. J. Martin and J. M. Beiler, Arch. Biochem., 1947, 15, 201.62 0. Shehata and B. C. Johnson, Proc. Xoc. Exp. Biol. Med. N.Y., 1948,%7, 332..Biochim. Biophys. Actu, 1948, 2, 217.1948, 16, 367.48 Biochem. J., 1946,40, lv234 BIOCHEMISTRY.Biological activity of PCA and related substam.Substance.Relative activity. Activity inS. fatcalis R. L. cmei. chick. rat. - - Pteroic acid .................................... 60 0.01Pteroylglutamic acid 100 100 + +Pteroyltriglutamic acid 7.5 80 4- +Pteroylheptaglutamic acid 0.3 0.2 + +........................ .......................................Crude vitamin B, conjugase from hog kidney or chicken pancreas wasshown by 0. D. Bird et to convert the heptaglutamate into a micro-biologically active form.A. Kazenko and M. Laskowski45 showed thatthe y-peptides were attacked with loss of the terminal glutamic acid, thetriglutamate thus yielding the diglutamate. Whether the conjugases candegrade the heptaglutamate to a substance with full microbiological activityis not yet known.The problem of microbiological assay of the total folic acid content ofnatural materials is further complicated by the occurrence of conjugaseinhibitors in natural sources. Glutamic protein,55 thymus nucleicand glutamates 67 have all been shown to inhibit the action of theseenzymes. There is also the possibility that the conjugases are incapable ofliberation of folic acid from all the complexes in which it may O C C U ~ .~ ~Anti-pernicious Ansmia Factor-Vitamin B,,.-Twenty-two yearsafter G. R. Minot and W. P. Murphy 58 noted the efficacy of liver in thetreatment of pernicious anzemia, the active principle was isolated in theform of dark red crystals by two independent groups of workers, E. L.Rickes et ~ 1 . ~ ~ in America and E. Lester Smith and L. F. J. Parker inEngland. The English workers used clinical tests as a guide to isolationprocedures from liver ; their last stages of purification depended on adsorp-tion 62This anti-pernicious anzemia factor has been called vitamin B,, andthis name is now in general use. Though the vitamin was originally isolatedfrom liver, it has now been produced by fermentation, using XtreptomycesAnother red crystalline substance with anti-pernicious anaemia activityand having closely related properties has been produced in fermentation8griseus.6353 J .Biol. Chem., 1945, 157, 413.5 5 A. Z. Hodson, Arch. Biochem., 1948, 16,309; J. A. Bain and H. F. Deutsch, ibid.,54 Ibid., 1948, 173, 217.p. 221.V. Mims, M. E. Swendseid, and 0. D. Bird, J . Biol. Chem., 1947,170,367.5 7 E. L. R. Stokstad, J. Pierce, T. H. Jukes, and A. L. Franklin, Fed. Proc., 1948,58 J . Amer. Med. ABBOC., 1926, 87, 470.5s Science, 1948, 107, 396.60 Biochem. J., 1948, 43, viii.7, 193.E. Lester Smith, Proc. Int. Congr. Biochem., Cambridge, 1949.E. Lester Smith, Brit. Med. J., 1949, 11, 1367.63 E. L. Rickes, N. G. Brink, F. R. Koniuszy, T. R. Wood, and K. Folkers, Science,1948, 108, 634CUTHBERTSON HBMOPOIETIC FACTORS.235with Streptomyces aureofuciens. * This substance has been called vitaminB,, (Blm has been used to describe a hydrogenation product of Bx2).Vitamin B,, isolated in the form of dark red crystals has been shown tocontain cobalt, phosphorus, carbon, nitrogen, oxygen, and hydrogen 65, 66and to -have a molecular weight of about 1500, the molecule containing oneatom each of cobalt and phosphor~s.~~, 68Vitamin B,, is most stable at pH 5 and is inactivated by acid or alkali,even in the cold.68, e6The cobalt is held in very firm combination.6* Prolonged acid hydrolysisappears to liberate phosphoric acid, ammonia, 5 : 6-dimethylbenziminazole,two 1 -substituted 5 : 6-dimethylbenziminazoles, and 2-aminopropanol, themajor fragment being an acidic red cobalt complex which has not beencharacterised.'o On fusion with alkali, pyrrole derivatives are liberated.67Infra-red spectroscopy has shown that the molecule contains bonded O-Hor N-H groups and that there are aromatic or heterocyclic nuclei andprobably few aliphatic groups.71B,, in Animal Nutrition.-The use of more refined nutritional techniqueshas shown that a number of animals require growth factors other than folicacid and the known vitamins.Three main methods have been employedto demonstrate these requirements : (1) Use of diets based on crude vegetablematerials or on casein exhaustively extracted with alcohol. (2) Use ofyoung animals derived from parents maintained on vegetable rations.(3) Use of animals treated with thyroxine to increase their apparent vitaminrequirements.J. C.Hammond and 11. W. Titus 72 and M. Rubin and H. R. Bird 73reported that the addition of fish meal or cow manure to the diet greatlyimproved the growth of chicks on an all-vegetable ration. The factor(s)responsible for the growth effects is associated with animal protein and hasbeen known by the term " Animal Protein Factor."The cow-manure factor was concentrated 74 and shown to promote the8 4 5. V. Pierce, A. C. Page, jun., E. L. R. Stokstad, and T. H. Jukes, J . Amer. Chem.65 E. Lester Smith, Nature, 1948, 162, 144.66 E. L. Rickes, N. G. Brink, F. R. Koniuszy, T. R. Wood, and K. Folkers, Science,1948,108, 134.6 7 N. G. Brink, D. E. Wolf, E.Kaczka, E. L. Rickes, F. R. Koniuszy, T. R. Wood,and K. Folkers, J . Amer. Chem. SOC., 1949, '71, 1854.6 8 K. H. Fantes, J. E. Page, L. F. J. Parker, and E. L. Smith, appendix byD. Hodgkin, M. W. Porter, and R. C. Stiller, Proc. Roy. SOC., B, 1949, 136, 592.'O B. Ellis, V. Petrow, and 0. F, Snook, J . Pharm., Pharmacol., 1949,1, 735; E. R.Holliday and V. Petrow, ibid., p. 734 ; B. Ellis, V. Petrow, and G. F. Snook, ibid., p. 60 ;J . Pharm., Pharmacol., 1949, 1, 287, 950, 957; N. G. Brink, D. E. Wolf, E. Kaczka,E. L. Rickes, F. R. Koniuszy, T. R. Wood, and K. Folkers, J . Amer. Chem. SOC., 1949,'41, 1854; E. Lester Smith, J . Pharm., Pharmacol., 1949, 1, 500.Soc., 1949, '71, 2952.71 R. Barer, A. R. H. Cole, and H. W. Thompson, Nature, 1949, 183, 198,72 Poultry Sci., 1944, 23, 49, 471.7s J .Biol. Chem., 1946, 163, 387.HI. R. Bird, M. Rubin, and A. C . Groschke, ibid., 1948,174,611236 BIOCHEMISTRY.growth of the chick and the hatchability of eggs and to be stored for 10-15weeks in the hen.75 Sardine meal 7, produces effects very similar to thoseobtained with cow manure, and the properties of the cow-manure factorand animal-protein factor of fish solubles are very similar. 76Vitamin B,, has been shown to promote chick growth under con-ditions similar to those used in investiga.tion of the animal-proteinfactor.77R. J. Lillie, C. A. Denton, and H. R. Bird78 have shown that the effectsassociated with the cow-manure factor may be obtained by the administrationof Vitamin B,, orally or by injection.The identity of the animal-protein factor and vitamin B,, is renderedprobable because, in a fermentation product, microbiological activitytowards Lb.Zeichmannii 313 and chick-growth activity both increasedduring purification, and a highly concentrated extract controlled perniciousanaemia.79 This material was shown to have B,, activity, the active fractionbehaving on paper chromatograms in the same way as that from liverextracts. 8oA nutritional deficiency in rats on a vegetable protein diet has beendescribed by L. M. Zucker and T. F. Zucker.81 The condition is preventedby animal protein, and this growth factor has been called zoopherin. C. A.Cary et u Z . ~ ~ have described a deficiency in rats fed on extracted caseinand ascribe this to absence of “nutritional factor X.” The deficiencyproduced and the properties of the factors make it appear that zoopherinand factor X are identical.Here again the deficiency may be overcomeby use of vitamin BI2. Further support to the view that B,, factor Xand the animal-protein factor are the same substance, or closely relatedsubstances, is given by the observation that the amounts required rise withincreasing protein 82bB. H. Ershoff 84 has shown that the administration of thyroxine increasesvitamin requirements : in thyroid-treated rats normal growth and survivalmay be obtained by administering whole liver. Workers using vegetablediets with added thyroid or iodinated casein have demonstrated B,,75 M. Rubin, A. C. Groschke, and H.R. Bird, Proc. Soc. Exp. BWZ. Med., 1947,66, 36.76 A. R. Robblee, C. A. Nichol, W. W. Cravens, C. A. Elvehjem, and J. C. Halpin,J . Biol. Chem., 1948, 173, 117.7 7 W. H. Ott, E. L. Rickes, and T. R. Wood, ibid., 1948,174, 1047.78 Ibid., 1948, 176, 1477.7* E. L. R. Stokstad, A. Page, J. Pierce, A. L. Franklin, T, H. Jukes, R. W. Heinle,80 W. F. J. Cuthbertson and E. Lester Smith, Biochem. J., 1949, 44, v.81 L. M. Zucker and T. F. Zucker, Arch. Biochem., 1948, 16, 115; Proc. SOC. Exp.B2 Fed. Proc., 1946, 5, (a) 128, (b) 137.e3 A. M. Hartman, L. P. Dryden, and C. A. Caw, A&. Biochem., 1949, 23, 165;H. R. Bird, M. Rubin, and A. C. Groschke, Off. Rep. of 8th World Poultry Congr., 1948,No. 23, p. 187.M. Epstein, and A. D. Walsh, J.Lab. CZim Ned,, 1948.33, 860.Biol. Med., 1948, 68, 432.Arch. Biochem., 1947, 15, 365CUTHBERTSON : HBMOPOIETIC FACTORS. 237deficiency in the rat,S5 in the chick,86 and in the mouse,87 and B,, assaymethods have been developed from these findings. P. H. Erschoff 88failed to demonstrate B,, deficiency in thyroid-treated rats. In theReporter’s laboratory, rats on vegetable diets given L-thyroxine developeda deficiency, which was not completely relieved by B,, but prevented byadministration of B,, and B13, prepared by the method of A. F. Novakand S. M. H a ~ g e . ~ ~ Using rats and chicks on diets closely similar to thoseused in B,, investigations, Novak and Hauge obtained a. deficiency attributedto the absence of B13; this has not yet been isolated in the pure state butfrom its chemical properties is clearly different from B12.Stokstad et a1.W have shown that vegetable diets may lead to deficienciesnot corrected by B,, or other known factors.Vitamin B,, is probablyessential in the nutrition of the dog 91 and the ~ i g . ~ l aReviewing the evidence as a whole it can be stated that vitamin B,,can replace factor X, zoopherin, and the cow-manure factor, which areprobably identical with, or closely related to, B,,, but this has not yet beenconfirmed by isolation of these factors in the pure state. Animal-proteinfactor activity can usually be completely replaced by B,,, but here againidentity has not been established. In some instances animal-protein factoractivity may be due, not to B,,, but to B13, to the substance responsiblefor Erschoff’s results, or to some other unknown factor(s).Microbiological Assay of Vitamin BI2,--A hitherto undescribed growth-factor for LactobaciEZus lactis Dorner ATCC 8000 was shown by IM.s. Shorb 92to be present in liver extracts. In 1948, crystalline B,, was shown to havevery high activity in the promotion of growth of Lb. Z ~ c t i s . ~ ~ A number ofother organisms has been shown to require B,, under certain conditions,and assay methods have been published involving the use of Lb. lactisATCC 8000 and ATCC 1097,9* Lb. Zuctis 1175,95 Lb. Zeichmnnii 313 ATCC86 U. D. Register, W. R. Ruegamer, and C. A. Elvehjem, J . Biol. Chem., 1949,177,129.8 6 C. A. Nichol, L. S. Dietrich, W. W. Cravens, and C.A. Elvehjem, Proc. SOC. Exp.BioE. Ned., 1949, 70, 40; A. R. Robblee, C. A. Nichol, W. W. Cravens, C. A. Elvehjem,and J. G. Halpin, ibid., 1948, 67, 400.87 D. K. Bosshardt, W. J. Paul, K. O’Doherty, J. W. Huff, and R. H. Barnes,J . Nutrit., 1949, 37, 21.88 J . Exp. Med., Surgery, 1948, 6, 438.O0 Ibid., 1949, 180, 647.O1 W. R. Ruegamer, W. L. Brickson, N. J. Torbet, and C. A. Elvehjern, J . Nutrit.1948, 36, 425.R. W. Heinle, A. D. WeIch, and J. A. Pritchard, J . Lab. Clin. Med., 1948, 33,1647; A. L. Neumann, M. F. James, J. L. Krider, and B. C. Johnson, Fed. Proc.,1949, 8, 391; A. L. Neumann, J. L. Krider, and B. C. Johnson, Proc. SOC. Exp.Biol. Ned., 1948, 69, 513; A. G. Hogan and G. C. Anderson, Fed. Proc., 1949, 8, 385;R. Braude, Brit.J . Nutrit., 1949, in the press.O2 J . BioE. Chem., 1947,169,455.O8 M. S. Shorb, Science, 1948,107, 397.94 M. C. Caswell, L. K. Koditschek, and D. Hendlin, J . Biol. Chem., 1949,180, 125.O5 Von K. Kocher and 0. Schindler, Internat. Review Vit. Res., 1949,20,369.J . Biol. Chem., 1948,174, 647238 (BIOUHEMISTRY.7830,96 EugZem gracilis var. bacillu~is,~~ Lb. luctis ATCC 8000,98 and Lb.Zeichmannii ATCC 4797.99A cup-pla te method loo developed from penicillin-assay technique over-comes many of the difficulties encountered in the tube method, though itis not as sensitive (lower limits about 0.05 vg./mf. compared with 0.001pg./ml. with tube methods) and is more subject to interference.Great difficulty has been experienced with B,, assays by normal micro-biological procedures, owing to the effects of oxygen and reducing agentson the growth of Lb.Zuctis and Lb. leichrn~nnii.~5~ lo1 Further difficultieswere encountered in the development of assay methods with Lb. luctisATCC 8000 in that this strain, unlike the variant used by Sh0rb,~3 requiresan oleic acid source '' Tween 80." g9, 100, l01a* lo2Thymidine lo3 and other deoxyribosides have been shown to replace B,,for the growth of Lb. Eactis and Lb. leichmannii.lo3* lU43 lo5The activity of the deoxyribosides is of the order of 1/1000-1/5000ththat of B1,, but interference from these compounds is readily detected inthe plate test lo6 and may be inhibited by high salt concentrations,lW or paperchromatography may be used to remove these corn pound^.^^^In tube assays, alkali treatment which destroys B,, may be employedto differentiate between B,, and deoxyriboside activity.lo7Paper psrtition-chromatographic methods have been used to identifyand investigate the number of factors responsible for the B,, activity ofliver extracts.About 0.003-043 vg. of total activity is applied to thepaper which is then developed (usually with n-butanol). The developedstrip is then applied to the surface of a B,,-deficient agar medium seededwith Lb. Eactis 80* lo5 or Lb. leichmannii.lOs On development overnight, zonesof growth appear at sites corresponding to the B,,-active fractions. WithLb. lactis the clinically active factors B,, and B,, give dense growth-zones,but the deoxyribosides produce areas of faint growth.In our hands, the96 C. E. Hoffrnann, E. L. R. Stokstad, A. L. Franklin, and T. H. Jukes, J . Biol.Chem., 1948,176, 1465.97 S. H. Hutner, L. Provasoli, E. L. R. Stokstad, C. E. Hoffmann, M. Belt, A. L.Franklin, and T. H. Jukes, Proc. SOC. Exp. Biol. Med., 1949, 70, 118.98 G. E. Shaw, Nature, 1949, 164, 187.99 H. R. Skeggs, J. W. Huff, L. D. Wright, and D. K. Bosshardt, J . Biol. Chem.,1948,176, 1459.100 W. F. J. Cuthbertson, Biochem. J., 1949, 44, v ; J. C. Foster, J. A. Lally, andH. B. Woodruff, Science, 1949, 110, 507.101 (a) W. Shive, J. M. Ravel, and R. E. Eakin, J . Amer. Chem. Soc., 1948, 70, 2614;( b ) R. D. Greene, A. J. Brook, and R. B. McCorrnack, J . Bid. Chem., 1949, 178, 999;(c) L. K. Koditschek, D.Hendlin, and H. B. Woodruff, ibid., 1949, 179, 1093.102 L. D. Wright, H. R. Skeggs, and J. W. Huff, ibid., 1948, 175, 475.103 E. E. Snell, E. Kitay, and W. S. McNutt, ibid., p. 473.104 V. Kocher, Internat. Review Vit. Res., 1949, 20, 441.105 E. Lester Smith and W. F. J. Cuthbertson, Biochem. J., 1949, 45, xii.106 W. F. J. Cuthbertson, Internat. Congr. Biochem., Cambridge, 3949.107 C. E. Hoffmann, E. L. R. Stokstad, B. L. Hutchings, A. C. Dornbush, and T. H.108 W. A. Winsten and E. Eigen, ibid., 1949,177, 989.Jukee, J . BioZ. Chem., 1949,181,635CUTHBERTSON : HBMOPOIETIC FACTORS. 239zones produced on Lb. Zeichmannii plates with B,, and the deoxyribosidesare closely similar. Various factors have been found in liver extracts andtentatively identified in this way.lo4Microbiological assay methods may be applied to refined liver extractsand fermentation concentrates with success, but little is known of theapplication of the techniques to crude materials, e.g., foodstuffs, and suitableextraction procedures have not yet been described.Role of B,, in Intemediaxy Metabolism.-Vitamin B,, is thought toplay a part in vital metabolic processes. Work on bacteria has shown thatit may be particularly involved in the biochemistry of the deoxyribosidesthat are of great importance in nucleic acid metabolism. More recent workwith vertebrates indicates that B,, may play some role in the metabolismof the methyl group.Thymidine and the other deoxyribosides have been shown to replaceB,, (at least in part) in the nutrition of Lb.Zactis,101c*104,105 and Lb.leichmannii.99~ 109Folic acid, thymine or the other pyrimidines or purines, or deoxyribosewill not replace B,, in the nutrition of these bacteria; consequently.it issupposed that B,, plays a role in an enzyme system that brings about thecombination between the purine or pyrimidine and deoxyribose.lo2M. Friedkin et al.l10 have shown that deoxyribose phosphate under theinfluence of liver nucleoside phosphorylase may combine with hypoxanthineto form hypoxanthine deoxyriboside.The observation that the other deoxyribosides may be used equally aswell as thymidine while deoxyribose itself is ineffective for growth mayperhaps be explicable on the assumptions that combined deoxyribose is theessential requirement and that the deoxyribose in this state may be freelytransferred from one purine or pyrimidine base to another by an enzymesystem in these bacteria.The discovery by Friedkin et al. that deoxyribosephosphate may react with hypoxanthine to form the deoxyriboside opensthe attractive speculation that B,, is essential for an enzyme system whichphosphorylates deoxyribose, the sugar phosphate then reacting with thepurine and pyrimidine bases to form the deoxyribosides.Vitamin B,, has been shown by I. 2. Roberts et ~ 1 . 1 ~ ~ to increase the rateof synthesis of deoxyribonucleic acid in Lb. Zeichmnnii and increase therate of phage (T4J formation in E . coli ; 112 this again emphasises the possiblerole of the vitamin in nucleic acid metabolism.Vitamin B,, is required by Lb.Zuctis under aerobic conditions but maybe dispensed with under anaerobic condition^.^^^ loo$ lola It may therefore beconcerned with biological oxidation reactions. Prom the evidence availableit appears to be essential for the combination of deoxyribose with purineor pyrimidine bases under aerobic conditions.Vitamin B,, may have some part to play in the metabolism of methionine,los E. Kitay, W. S. McNutt, and E. E. Snell, J . Biol. Chem., 1949, 177, 993.110 Ibid., 1949,178,527.lf2 R. B. Roberts and M. Sands, ibid., p. 710.ll1 J . Bact., 1949, 58, 709240 BIOCHEMISTRY.for in E . coZi it may be replaced by methionine.l13 A study of B,, deficiencyin the rat and the chick has also indicated a relation between B,,, methionine,and choline.From the literature it is not possible to decide whether theseeffects are best interpreted as showing that B,, enters into methyl-groupmetabolism directly in the animal body or indirectly via the intestinal floraof the gut.A. E. Schaefer et aZ.l14 showed that, in the rat and chick, B,, in thediet markedly reduced symptoms of choline deficiency. M. B. Gillis andL. C . Norris 115 showed that a liver paste rich in B,, and of low choline contentwas more effective than choline itself in preventing deficiency symptomsin chicks on low-methyl diets. C. A. Hall and W. A. Drill 116 showed thatliver extracts prevented hepatic fibrosis and fatty livers normally encounteredin rats on the Himsworth-Glynn diet.A.E. Schaefer et ~ 1 . ~ 1 ~ claim that, in chicks, choline spares B,, and thatB,, lowers the need for choline. It is of interest to note that the chickdiets used by Stockstad et aLgO for B1,-deficiency studies are supplemented withmethionine and choline. At the moment the conclusion may be drawn thatthe addition of B,, to the diet reduces the methyl requirement in the ratand chick.Treatment of Pernicious Ansmh-Crystalline B,, has been shown bya number of workers 11* to control the haematological condition in perniciousanaemia. Unlike folic acid, B,, also controls the neurological lesi0ns.1~~C. C. Ungley 12* has given a very full account of the amounts of B,, requiredin the control of pernicious anaemia. In most instances 10 pg. per fortnightby injection suffice, but doses of 20 pg.per week are recommended for generaluse although initial treatment and control of nervous lesions may need muchlarger amounts.Vitamins B12b (probsbly identical with Smith's second red factor) andB,, are also effective in the treatment of pernicious anzemia. From thesmall amount of work published on BlZn, i t seems to be rather less activethan B,,.Vitamin B,, has been shown to be effective in the control of nutritionalmacrocytic anaemia and sprue,121+ but ineffective in some cases of macrocyticanzemia of pregnancy and infancy.113 W. Shive, Amer. Acad. Sci., 1950, in the press.11* Ped. PTOC., 1949, 8, 395.11' Ibid., 1949,71, 202.11s R. MTest, Science, 1948, 107, 398; C. C. Ungley, Lancet, 1948, I, 771; Brit.Med.J., 1948, 11, 154; T. D. Spies, R. M. Suarez, G. Garcia Lopez, G. Fernando Milanes,R. E. Stone, Lopez R. Toea, T. Aramburu, and S. Kartus, J . Amer. Ned. ASSOC., 1949,139, 521 ; IF. H. Bethell, M. C. Meyers, and R. B. Neligh, J . Lab. Clin. Med., 1948, 33,1477.J . Biol. Chem., 1949, 178, 487.PTOC. SOC. Exp. Biol. Med., 1949, 69, 3.11s M. Finland and W. B. Castle, New Erzgland J . Med., 1948, 239, 328.120 Brit. Med. J., 1949, 11, 1370.121 T. D. Spies, R. E. Stone, G. Garcia Lopez, G. Fernando Milanes, Toca R. Lopez,182 J. C. Patel, Brit. Med. J., 1948,II, 934.and T. Aramburu, Lancet, 1948, 255,519CUTHBERTSON : HLEMOPOIETI(3 FACTORS. 241Relation between Folic Acid and Vitamin B,, in Pernicious Anaemia.--Inanimal nutrition, both vitamins have independent effects, i.e., all resultsin animal feeding may be explained by the independent action of these twosubstances ; thus the administration of folic acid to B,,-deficient animalshas no effect, and conversely B,, will not relieve folic acid deficiency.123 I nhuman nutritional macrocytic anamias, evidence is now accumulatingthat these diseases may be due to dietary insufficiency (or failure of absorp-tion) of one or both of these factors.Cases have been reported that respondto only one or the other type of vitamin. In bacterial nutrition the samething is met, folic acid will not replace B,,, nor will B,, replace folic acidfor growth of the lactobacilli under conditions studied hitherto. Investig-ations with bacteria have so far shown that these vitamins are concernedwith different stages of pyrimidine and purine metabolism.Folic acidenters into the reactions essential for the synthesis of thymine, but B,, isrequired (under aerobic conditions at least) for the formation of the nucleo-sides, i.e., for the formation of the purine and pyrimidine deoxyribosides.In human Addisonian pernicious anaemia the position is rather different.In these patients there is not only the macrocytic anaemia and megaloblasticbone marrow typical of the nutritional macrocytic anamias, but there isalso an alteration in the stomach which fails to secrete acid and changesin appearance. The disease is not nutritional in origin, but appears to bedue to an error in metabolism which leads to a condition very like thenutritional macrocytic anamias.Patients with Addisonian perniciousanamia often show other symptoms, such as glossitis, and sooner or laterif the anamia is not adequately controlled the condition of sub-acutecombined degeneration of the spinal cord with attendant nervous symptomsmay arise.Either folic acid (by mouth or by injection) or B,, (by injection) willcontrol the hamatological condition. If folic acid alone is used then thecondition of subacute combined degeneration of the cord will appear intime, and this condition is not improved even with large doses of folicacid.12* I n the control of the hamatological signs, pteroylglutamic acid isrequired in doses of 2-10 mg. per day, but B1, is effective a t the rate of1-2 pg.per day. F. H. Bethell et ~ 1 . ~ ~ suggested that liver extracts actedby liberating free folic acid from the bound forms existing in natural foods.In experiments to test this hypothesis the findings were conflicting. Theresults have been reviewed and reported by Wilkinson and I ~ r a e l s , ~ ~ whohold that the hypothesis can no longer be maintained, but L. S. P. Davidson 125thinks that the supposition is correct. Even in serious cases of perniciousanamia, the response to B,, treatment is usually rapid. Not much is knownof the amount of unavailable (bound) folic acid in the patient’s diets, butit is unlikely that these diets contain sufficient to account for the rapidresponses to BI2. If, therefore, this hypothesis is t o be tenable, the boundlZs E.Koditmhek and K. J. Carpenter, Biochem. J., 1948, 43, i.lap M. C. G. Israels and J. F. Wilkinson, B&. Med. J., 1949,11, 1072.la6 Lancet, 1949, 257, 814242 BIOCHEMISTRY.folk acid must be stored in the patient's body and utilised only when B,,is administered-this bound folic acid may not be a conjugate but mightbe the compound investigated by Buyze and Engel *l* *3 which is formed onincubation of folic acid with gastric juice.B$,-deficiency in the rat and the chick leads to a decreased growth rate,but anaemia is not a predominant symptom, although in folic acid deficiencyleucopenia and anaemia are readily obtained. An anmmia in dogs has beendescribed as not responding to folic acid but responding to liver extract,and in chicks 126 the administration of B,, together with folic acid increasedthe rate of haemoglobin regeneration after the induction of a severe anaemiawith phenylhydrazine.Observations on folic acid antagonists may be used to throw light on therelation between folic acid and B12.L. M. Meyer et aZ.12' treated pernicious-ansmia patients with a folic acid antagonist (" Met-Fol-B," methylpteroicacid) which they found prevented the action of vitamin B,, in promoting therelief of hzematological symptoms. These experiments tend to show thatvitamin B,, does act, at least in part, through the mediation of folic acidwhich is essential for its activity. Unfortunately, in these experiments i tis not certain that the " Met-Fol-B " and " Amethopterin " acted only byantagonising folic acid, for these workers did not show that the action ofthese substances could be overcome by the administration of pteroyl-glutamic acid.This criticism is offered because J. Innes et have shownthat, although aminopterin will produce, in the guinea-pig, a hamatologicalcondition resembling that expected in folic acid deficiency, the effects arenot reversed by folic acid. The anti-folic acids are highly toxic and i t maywell be that they act on the hzemopoietic system not only by blocking theeffects of folic acid but also in other ways. Thymidine in large doses hasbeen shown to cause a haematological response in pernicious anaemia.128 Inbacteria this compound may replace B,, or enable organisms to grow whenfolic acid metabolism is blocked.lo1C* lo** lo5~ lo% 129 The mode of action in thepernicious-anamia patient is not known.An assay method for vitamin B,, under consideration by a U.S.P.Committee depends on the observation that in the presence of sulphanilamide(which prevents the metabolism of p-aminobenzoic acid) B,, is apparentlyrequired for growth of E .coli (private communication). These observationsagain indicate a relation between B,, and folic acid metabolism.Sufficient facts are not available to interpret the finding that either folicacid or B,, will control the hzematological condition in pernicious anzemia ;at the moment the speculation which seems most worthy of investigation isthat B,, is needed to make available folic acid from some bound form (orprecursor) present in the diet or stored in the body.12* C.A. Nichol. A. E. Harper, L. S. Dietrich and C. A. Elvehjem, Fed. Proc., 1949,l27 Amer. J . Med. SGi., 1949, 818, 197.12* W. Shive, R. E. Eakin, W. M. Harding, J. M. Ravel, and J. E. Sutherland,8, 233.lSa K. Hausmann, Lancet, 1949,157,962.J . Amer. Chem. Soc., 1948, 70, 2299CUTHBERTSON : HBMOPOIETIC FACTORS. 243Egtrinsic and Intrinsic Faetor.-The classical experiments of Castle(reviewed by Ungley 13*) showed that for oral treatment of pernicious anmmiacertain food materials, though ineffective by themselves, did producehmmatological responses if they were digested with normal human gastricjuice before administration. On the basis of this work, Castle postulatedthat in these foods (beef muscle) there was present a dietary extrinsic factorand that in normal gastric juice there was an intrinsic factor.On incubationthese substances were thought to interact to produce the material thatbrought about the hmnatological response. The fact that refined liverextracts have only slight activity when given orally, although they may bevery active when injected, has been recognised for a long while and it hasbeen shown 131 that the oral activity of such extracts may be much increasedby incubation with normal gastric juice. In spite of these observations,the activity of liver extracts has been thought to be due in the main to theinteraction product of Castle’s extrinsic and intrinsic factors. RecentlyL. Berk et ~ 1 .~ 3 , have shown that 5 pg. of B,,, given orally, per day had noeffect on pernicious-anemia patients, but that if this amount were ad-ministered with neutralised human gastric juice a hematological responsewas obtained. These observations show that B,, can act as the extrinsicfactor. Although satisfactory B,, assays are not available for beef muscle(the most commonly used source of extrinsic ‘factor), the B,, content isprobably about 0.05 pg./g. (rat tests 133 and unpublished preliminary testswith Lb. Zuctis in the Reporter’s laboratory). This concentration of B,, issufficient to explain the effects of beef muscle without postulation of afurther type of B,,.J. L. Ternberg and R. E, Eakin134 state that gastric juice contains aheat-labile substance that combines with B,, to form a microbiologicallyinactive complex from which the B,, may be released by heating.Thisheat-labile substance, which they term “ apoerythein,” may be obtained inrelatively large amounts from normal gastric juice and hog gastric mucosa(both of which are good sources of Castle’s intrinsic factor), but only smallamounts are present in the gastric juice from pernicious-anemia patients.It is too early to identify this substance with Castle’s intrinsic factor, butthe possibility would appear to be worth investigation.Berck et ~ 1 . l ~ ~ and F. H. Bethel1 et aZ.118 have shown that pernicious-anemia patients in relapse eliminate large amounts of B,, in the feces.From the foregoing evidence i t is probable that normal gastric juice(the intrinsic factor) potentiates the absorption of B,, or prevents its lossin the alimentary tract.At present no firm conclusions may be drawn about the relation between130 C.C. Ungley, Nature, 1936, 137, 210.131 F. Reimann and F. Fritsch, 2. klin. Med., 1934, 126, 469.New England Med. J., 1948,239, 911.188 U. J. Lewis, U. D. Register, H. T. Thompson, and C. A. Elvehjem, Proc. SOC.134 J . Amer. Chem. SOC., 1949,71, 3868.Exp. Biol. Ned., 1949, 72, 479244 BIOCHEMISTRY.folic acid and vitamin B,, in metabolism or about the fundamental bio-chemical defects in pernicious anaemia. Much of the evidence is conflictingand further facts are urgently required, although the main problems maybe very near to solution. Pernicious anaemia may, however, now be definedas caused by B,, deficiency which is brought about by a failure in absorptionowing to lack of the intrinsic factor.W. F. J. C.8. CARO!L’ENOIDS, VITAMIN A, AND VISUAL PIGmNTS.Carotenoids.-A new book gives a comprehensive account of thechemistry of the carotenoids and provides a firm foundation for morebiochemical studies. The work of Karrer’s school on the 5 : 6-epoxidesof carotenoids, which undergo isomerization to furanoid structures is fullydescribed, and among the other interesting results are the findings thatviolaxanthin is the diepoxide of zeaxanthin, and flavoxanthin the furanoidisomer of lutein epoxide.The biogenesis of carotenoids is a difficult problem. In the chromato-graphy of many plant extracts a colourless zone, adsorbed below the colouredbands, is indicated by its fluorescence when exposed to ultra-violet rays ina dark room.2p3 The effect is usually due to phytofluene * (C,oHs4), acolourless carotenoid containing 7 double bonds (of which 5 are conjugated)and showing sharp maxima at 332, 348, and 367 mp.in h e ~ a n e . ~ , ~ Arelated compound, phyt~fluenol,~ has been obtained from tomatoes.Phytofluene also occurs in the yeast Rhodotorula rubra, the pigment fractionof which contains torulene (purple red; 13 conjugated double bonds),y-carotene, 8-carotene, two yellow pigments (principal maxima at 440 and400 mp.), and phytofluene. Ultra-violet irradiation of the yeast producesmutants (orange, yellow, or white) : the white mutant produces no phyto-fluene.8 It is suggested9 that the stages in pigment biosynthesis in thered yeast are as follows :Block in Block inalbino mutant orange mutantUnknown -:+ phytofiuene _3 yellow and orange -!+ red pigments.precursors pigmentsGenetical blocking is a very promising approach to biogenesis in yeastsKarrer and Jucker, “ Carotenoide,” 1948, Verlag Birkhausen, Basel.H.H. Strain, Nature, 1936, 137, 946.L. Zechmeister and A. Polgar, Science, 1944, 100, 317.L. Zechmeister and A. Sandoval, Arch. Biochem., 1945, 8, 425.0 L. Zechmeister and A. Sandoval, J . Amer. Chem. SOC., 1946, 68, 197.7 L. Zechmeister and J. H. Pinckard, Experientia, 1948, 4, 474.* H. H. Strain, “ Leaf Xanthophylls,” 1938, Carnegie Inst.J. Bonner, A. Sandoval, V.W. Tang, and L. Zechmeister, Arch. Riochem., 1946.L. Zechmeister, American Scientist, 1948, 56, 605.10, 113MORTON : CAROTENOIDS, VITAMXN A, AND VISUAL PIGMENTS. 245although it does not necessarily follow that the stages will be the same inleaves. In plant organs which produce considerable quantities of carote-noids in the absence of chlorophyll, a substance showing a deep-bluefluorescence and one broad absorption band with Amax. 343-349 mp. (inhexane) is found. Another compound with hma,. 284 mp. does not fluoresce.Galloxanthin,lo a carotenoid from chicken retinas, shows maxima at 380,401, and 422 mp. (antimony trichloride colour test, Amax. ca. 790 mp.), andmust fall between phytofluene and @-carotene in respect of conjugateddouble bonds.Provitamins A.-The symmetrical molecule of all-trans-(3-carotene remainsthe most important as well as the most potent provitamin A foiind.inNature. An intact half of the (3-carotene molecule is found in a considerablenumber of carotenoids each of which yields vitamin A in vivo. Certainepoxides act as provitamins presumably because the oxygen can he: removedin vivo.The work begun by Gillam 11 and greatly extended by Zechmeister andhis colleagues displays the importance of cis-tvuns-isomerism in the caro-tenoids. Of the 11 double bonds in @-carotene, 2 are fixed in the ringsystems and, according to Pauling, 4 others are spatially hindered fromrearrangement (but see L. Pauling ll~). The remaining 5 double bondspermit the existence of 20 isomers, 13 of which have been observedThe chromatographically. Several of them have been prepared pure.12, l3all-truns-form of a carotenoid is the deepest in colour; all the isomersshow spectra more or less displaced in the direction of shorter wave-lengths.The relationship between stereochemical configuration and provitamin Aactivity has been carefully studied.The isomers of all-trans- p-caroteneare labelled neo-@-carotene A, B . . . if adsorbed less strongly and neo-(3-carotene T, U . . . if adsorbed more strongly, A and T being nearest to theall-trans-form on a column. On a scale in which the provitamin A activityof all-trans-@-carotene is 100, the activities by the growth test of othercarotenoids are as follows : =-carotene, all-trans- 53,14 neo-U (9 mono-cis ? ) 13,14 neo-B 16; l5 p-carotene, all-trans- 100, neo-B 53,15 neo-U (3mono-cis) 38 ; l7 y-carotene, all-trans- 43,15 pro-y-carotene (a poly-cis-10 G.Wald, J . Qen. PhysioZ., 1948, 31, 377.11 A. E. Gillam, M. S. el Ridi, and S. K. Kon, Biochem. J., 193?,31, 1605.11a L. Pauling, Helv. Chim. Acta, 1949, 32, 2241.l2 E. M. Bickoff, L. M. White, A. Bevenue, and K. T. Williams, J . Ass. 03. Agric.13 F. T. Jones and E. M. Bickoff, ibid., p. 776.l4 H. J. Deuel, jun., E. Sumner, C. Johnston, A. Polgar, and L. Zechmeister, Arch.15 L. Zechmeister, Bull. SOC. Chim. biol., 1949, 31, 961.l6 H. J. Deuel, S. M. Greenberg, E. Straub, T. Fakin, A. Chatterjee, and L. Zech-1 7 H. J. Deuel, jun., C. Johnston, E. Sumner, A. Polgar, and L.Zechmeister, ibid.,Chem., 1948, 31, 633.Biochem., 1945, 6, 157.rneister, Arch. Biochem., 1949, 23, 239.1944, 5, 107246 BXOUHEMISTRY .form) 44 21 (41),Z2 neo-P 21,15 mixture of neo-forms 10; l5 cryptoxanthin,all-trans- 57,185Judged by the amount of storage of vitamin A in the liver and kidneysF3the relative activities are : all-trans-p-carotene 100, neo-p-carotene-B 48,neo-p-carotene-U 33, all-tram-a-carotene 25. The last produced smallerstores of vitamin A than its growth-promoting power would lead one toexpect.Some of the isomerides occur in Nature; thus one analysis of the p-carotene of grass shows all-trans, 77.7y0 ; neo-U, 12.9% ; neo-B, 9.4%.Bending of a-, p-, or y-carotene and cryptoxanthin molecules reducesthe biological activity by half or two-thirds.The results make a tidypattern in spite of the fact that accurate comparisons by biological methodsare not easy in this field. Whether or not, as a result of feeding thesestereoisomers, cis-trans-isomers of vitamin A are formed, is not known.So far no direct carotenoid precursor of vitamin A, is known and itseems likely that vitamin A, is always formed by dehydrogenation of vitaminA or its precursors in vivo.Biochemical Roles of 0arotenoids.-The part played by carotenoids inthe leaf is still far from being ~nderstood.~~ In diatoms (Nitzschia dissipatu,Nitxschia spec. cf. ovalis) there is ifi fucoxanthin-chlorophyll-protein com-plex ; the living cells show selective absorption a t 500-560 mp. attributableto the fucoxanthin-protein.Light so absorbed may cause chlorophyll tofluoresce and also may participate in photosynthesis, with chlorophyll asa necessary mediator. The mode of transfer of energy in the complex isnot known.25 The phenomenon is unusual since the light absorbed bycaroterroids in green alga or the higher plants does not contribute tophotosynthesis .24aCarotenoids act as photo-receptor substances in the phototropic bendingof etiolated oat seedlings and in the phototropic responses of unicellularspore bearers of the mould Phywrnyces. There is fairly good correspondence(a) between the absorption spectra of the carotenoids concerned and thephototropic sensitivity curves and (b) between the sites of carotenoiddeposition and the photosensitive zones.26 The phototropic responses ofgreen flagellates seem to be due to light absorbed by astaxanthin.72e0-A,~~* 2o 42, neo-U 27.15I8 H.J. Deuel, j u n , E. B. Meserve, C. H. Johnston, A. Polgar, and L. Zeichmeister,1s G. S. Fraps and A. R. Kemmerer, Ind. Eng. Chem, Anal., 1941, 13, 806.20 H. J. Deuel, jun., E. B. Meserve, A. Sandoval, and L. Zechmeister, Arch. Bwchent.,21 L. Zechmeister, Abstrtccts 1st International Congress of Biochemistry, 1949, 244.z2 L. Zechmeister, J. H. Pinckard, S. M. Greenberg, E. Straub, J. Fukui, and H. J.23 R. M. Johnson and C . A. Baumann, ibid., 1947, 14, 361.24 G. Wald, Vit. and Hor., 1943, 1, 195.24a J, H. C . Smith, J . Chem. Educ., 1949, 28, 631.s5 E. C. Wassink and J. A. H. Kersten, Enzymologia, 1946, la, 3.26 G.Wald, ‘‘ Harvey Lectures Series,” 1946, 41, 117.Arch. Biochem., 1945, 7, 447.1946, 10, 491.Deuel, Arch. Biochem., 1949, 23, 242MORTON : CAROTENOIDS, VITAMIN A, AND vIsufi PIGMENTS. 247SpiriIlo~anthin,~7 the major pigment obtained from the bacteriumRhodospirilZum rubrum, is probably rhodoviobscin.as It apparently is notconcerned in phototactic responses which are attributed to residual caro-tenoids and bacteriochlorophyll.29Several carotenoids take part in reproductive cycles and a very interest-ing but difficult aspect of comparative biochemistry is being opened up.In some algae the pigment of the male gametes is mainly @-carotene whilstthe female gametes contain fucoxanthin and chl~rophyll.~~ Recent workon rainbow trout (Sulmo irideus Gibb) implies the existence of " fertilizationhormones "-androgamones AI and AII and gynogamones GI and GII.The trout eggs contain astaxanthin, @-carotene, and lutein ; astaxanthincauses the GI biological response, it activates the spermatozoa and antago-nises AI.31 Sea urchins (Arbaciu pustulosa), however, contain echinochrome 32(3 : 5 : 6 : 7 : 8-pentahydroxy-2-ethyl-1 : 4-naphthaquinone) as the GI sub-stance-a very surprising structural variation compared with astaxanthin.The carotenoids of the brown trout (Sulmo truttu Linn.) have been studiedthroughout the life-cycle.33 The adult trout tissues contain @-carotene,lutein, and astaxanthin ; muscle contains free hydroxy-carotenoids butlittle or no carotene, the liver contains carotene and lutein but no astaxanthin,whilst the red and the yellow chromatophores of the skin contain luteinand astaxanthin, both esterified.The ova of the spawning female containall three pigments, a large part of the total having been transferred from themuscle tissue, but the chromatophores of the adult do not suffer depletion.Lutein and astaxanthin are free in the egg yolk but esterified in the embryo ;p-carotene, although present in freshly spawned ova, is not detectable inthe embryo a t any stage, presumably because it is used to form vitamin A.Lutein and astaxanthin are transferred without loss from the yolk to thebody of the embryo rather late in the larval period and are responsible forthe development of xanthophores and erythrophores.In the larva, theyolk-sac carotenoids are concentrated in a lipoid droplet which rises to thetop when the urethane-narcotized larva is held head-downwards in a glasstube. The portion of the sac containing the pigmented droplet of fat maybe tied off and cut away. This removal of about 90% of the yolk caro-tenoids results later in larvae slightly below normal in size and with very fewchromatophores and those pale, but the larvae show no other obvious defect.The carotenoid distribution suggests that the maintenance of normal2 7 P. B. van Niel and J. H. C. Smith, Arch. mikrobiol., 1935, 6, 219.28 A. Polgar, P. B. van Niel, and L. Zechmeistsr, Arch. Biochem., 1944, 5 , 343.29 A. Manten, " Phototaxis, Phototropism and Photosynthesis," Dissertation,30 P.W. Carter, L. C. Cross, I. M. Heilbron, and E. R. H. Jones, Biochem. J., 1948,31 M. Hartmann, F. G. Medem, R. Kuhn, and H-J. Bielig, 2. Naturforsch., 1947.32 R. Kuhn and K. Wallenfals, Ber., 1939, 72, 1497; 1940, 73, 458; 1942, 75,33 D. M. Steven, J . exp. Biol., 1948, 25, 369; 1949, 28, 295.Univ. Utrecht, 1948.43, 349.24,330.407248 BIOCHEMISTRY.colour pattern is important, but whether the carotenoids are essential forother more physiological processes during the larval period is doubtful.Some insects do not appear to need carotenoids or vitamin A,34935 butothers contain various carotenoids so distributed about the body as tosuggest that they may be functional. The integument of locusts 36 (Locustamigratoria migratorioides R and E' and Schistocerca gregaria Forsk) contains@-carotene and free astaxanthin.Both pigments also occur in the eyes andthe wings, astaxanthin in the wings as a protein complex. Only @-caroteneis found in the fatty tissues, blood, and eggs 37 of locusts and the grasshopperblelanoplus bivattus Solitary and gregarious locusts show nocharacteristic differences in carotenoid content or distribution. Newlylaid eggs contain @-carotene which disappears as astaxanthin appears duringincubation. In the early hopper stages astaxanthin accounts for 70% ofthe total carotenoids but in the mature insect less than 30% is accountedfor in this way.38 The blood is bright green and carotene (present in highconcentration) is the only pigment; it must occur as a protein complex.Locusts contain no vitamin A and, as in other organisms lacking the vitamin,astaxanthin seems to be the key substance in visual processes.36Esterified astaxanthin occurs in the hypodermis of the lobster Hlomarusvulgaris Edw.and the prawn Nephrops norvegicus L. The carapace seemslike the eggs to contain unesterified astaxanthin; the ova contain mainlythe greenish astaxanthin-protein, ovoverdin. The lobster hepatopancreascontains P-carotene as the only carotenoid and that in very small amount:lbut vitamin A is present.42Northern krill (Meganictiphunes norvegica and Thysanoessa inerrnis)contain astaxanthin and very little p-carotene, but pre-formed vitamin Ais present in appreciable amount.43 The common shrimp Crangon vuEgarisand other crustacea contain small amounts of vitamin A.The whaleswhich feed on krill ingest large amounts of vitamin A and relatively little@-carotene.A study of carotenoids and vitamin A in frogs (Ram temporaria) through-out the life-cycle reveals a complicated picture.44 Young tadpoles containchlorophyll (from undigested food) and carotenoids. At first, xanthophyllsaccumulate in the body more quickly than carotene (which undergoes someconversion into vitamin A) but just before metamorphosis the rates ofstorage are more nearly equal. During metamorphosis the weight decreases84 C. M. McCay, Physiol. Zool., 1938, 11, 89.35 R. E. Bowers and C. M. MCay, Science, 1940, 92, 291.36 T. W. Goodwin and S. Srisukh, Biochem. J., 1949,45,263.37 R. Chauvin, Ann.SOC. ent. Fr., 1941, 110, 133.3s T. W. Goodwin, Biochem. J., 1949, 45,472.39 J. 3%. Grayson, Iowa State CoEE. J . Sc., 1942, 17, 69.J. M. Grayson and 0. E. Tauber, ibid., p. 191.41 T. W. Goodwin and S. Srisukh, Biochem. J., 1949, 45, 268.*% T. B. Nielands, Arch. Biochem., 1947, 13, 415.48 S. K. Kon and S . Y. Thompson, Biochem. J . , 1949, 45, xxxi.44 R. A. Morton and G. D. Rosen, ibid., p. 612MORTON: CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 249and the carotenoids disappear unselectively. Ram tempmaria formvitamin A, 45 but some other frog species appear to form vitamin A,; 46indeed, Wald47 has recorded a sharp transition a t metamorphosis fromporphyropsin (vitamin A,) eyes to rhodopsin (vitamin A,) eyes in thebullfrog Rana cutesbiana.This does not apply to all frog species.48 Adultfrogs store " xanthophyll," carotene, and vitamin A in the liver and thekidneys : the amount of vitamin A in the eyes and kidneys is relativelylarge. Carotenoids, especially xanthophylls, are freely stored in the skinand, a t certain seasons, in the fat-bodies. The deposition of carotenoids inthe ovaries is so large that marked difference results between the sexes incarotenoid storage and utilization, but reproduction also drains the carotenoidreserves of the male.Such distribution studies in animals may be no more than preliminaryto the elucidation of function. The results depend upon superimposedpatterns of intake, assimilation, storage, and utilization of precursors andmetabolites, and of enzyme capacities and " detoxication '' processes.Here, the provitamin A role is clear, as are the photo-receptor functionsalready referred to ; carotenoids play several parts in reproductive processes,and in many respects they confer biological advantages a t concentrationlevels higher than those associated with indispensability.Only a beginning has been made in these comparative studies, and withregard to modes of action and the specificity of structure in relation to functionmuch remains to be done.Some of the difficulty arises from the maskingeffect of ecological variables ; carotenoid accumulation indeed often appearsto be physiologically fortuitous, or a t least not fundamentally significant.Vitamin-A Activity.-The definition of the unit of vitamin A (see p.250)as 0.3 pg. (potency of vitamin-A, alcohol, 3.33 x lo6 i.u./g.) makes p-carotene half as potent, weight for weight (potency of all-trans-@-carotene1.66 x lo6 i.u./g. from the definition 0.6 pg. for the unit of provitamin Aactivity).The liver reserves are retained durifig hibernation.This implies under appropriate conditionsin vccoC40H56 - c20H28x C20H28yif fission occurs a t the central double bond, only half of the p-carotenemolecule finally yielding the vitamin. The aldehyde Cl9H2,*C€€O is readilyreduced to vitamin A, in vivo and could well be an intermediate.These considerations point to the formation of vitamin A, as the generalmethod by which the carotenoids exert their growth-promoting action.The fact that the acid C,,H,,*CO,H 50 and the ether C,,H2,*CH,*OMe 51, 5245 A.E. Gillam, Biochem. J., 1938, 82, 1496.4~ E. Lederer and F. H. Rathmann, ibid., p. 1252. *' G. Wald, The Harvey Lectures, 1946, 41, 117.48 M. Lovs and R. A. Morton, unpublished observation.4s J. F. Arens and D. A. van Dorp, Nature, 1946, 151, 190.50 I. M. Sharman, Brit. J. Nutrit., 1949, 3, viii.ti1 A. R. Hanze, T. W. Conger, E. C. Wise, and D. I. Weisblat, J . Amer. Chem. Soc.,5a 0. Isler, M. Kofler, W. Euber, and A. Ronco, Experientia, 1946, 2, 31.1948, 'SO, 1253250 BIOCHEMISTRY.both exhibit very high growth-promoting power for rats deficient in vitaminA calls for caution, particularly as no sign of any substance akin to vitaminA, can be found in animals treated with the acid.53 The possibility cannotbe ignored that vitamin A and some of its derivatives may themselves beprecursors of an unknown degradation product responsible for some of thesystematic effects attributed t o the vitamin.Standards.-The International Conference held in London in 1949 underthe auspices of the World Health Organization recommended the adoptionof vitamin A acetate as the reference substance and defined the unit ofvitamin A activity as that shown by 0.344 pg.of the pure acetate. Stoicheio-metrically, this corresponds to 0.3 pg. of vitamin A, (C2,H,,*OH) the potencyof which is thus 3-33 x lo6 i.u./g. Since the intensity of absorption of theacetate at 328 mp. (in certain solvents) is Ei&. 1525, and 1750 for the alcoholat 326 my.the conversion factor is necessarily 1900: To use this factorfor converting observed E,l:m. values for oils and concentrates into i.u./g.is, however, legitimate only when the absorption spectrum of the sample isdemonstrably free from any irrelevant contribution at 326-328 mp.The p-carotene standard, in use since 1934, is to be retained as a yardstickfor provitamin A activity only; its main use will be in the determinationof p-carotene but i t will also be of service in relating provitamin A activityto stereochemical configuration. The preparation of the purest @-caroteneand of stable solutions in oil is difficult and the continued availability 54of the standard preparation will be welcomed.The problem of vitamin-A standardization has now probably been solvedand a brief review of its history may be opportune.The need for a referencestandard was evident in 1931; at that time carotene and vitamin A wereknown to be distinct substances each capable of curing or preventingavitaminosis A, but only carotene had been obtained in crystalline form.The “ unit ” was therefore defined as 1 pg. of carotene. By 1934 a-caroteneand &carotene had been differentiated, and the original specimen of caroteneshown to be heterogeneous. The unit was then re-defined as 0.6 Vg. ofpure p-carotene so as to preserve continuity, and a fresh reference materialwas brought into use.A considerable number of vitamin-A-containing oils had been assayedby the growth test on rats with parallel experiments using the growthresponse to P-carotene in known amounts.The intensity of absorption inthe ultra-violet (Ei?m. a t 328 mp,) had been determined for each fish liveroil tested biologically and it was found that by multiplying by 1600 theEi& value (observed on rich oils or on the-unsaponifiable fraction of pooroils) results agreeing closely with biological assays were obtained. In theU.S.A., however, a conversion factor of 2000 was preferred to 1600. Theuse of alternative conversion factors led t o much difficulty, mitigated bythe tacit acceptance of the E value as the measure of potency.The situation was clarified by concerted biological assays on crystalline63 Unpublished observations, Reporter’s laboratory.64 From the Director of Biological Standards, National Institute of Medical ResearchMORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS.251vitamin A and the acetate, and by the use of photo-electric spectrophoto-meters. The potency of vitamin A in terms of the @-carotene unit was foundto correspond with a conversion factor between the limits 1800 (Britishworkers) and 1900 (American workers).It had been shown 55 that the original conversion factor of 1600 wasempirically satisfactory for gross i3:& values on oils of moderate to highpotency uncorrected for irrelevant absorption. A simple geometricalprocedure for obtaining corrected E values is widely agreed to be ~erviceable.~~The U.S. Pharmacopmia adopted in 1948 a new reference standardpreparation (labelled 10,000 i.u./g.) consisting of vitamin A acetate in oil,and showing E:Fm.a t 328 mp., 5.23. The 1949 International Conference 57recommended the general use of such a preparation for which a conversionfactor of 1900 is necessary.Provitamin-Apotency and vitamin-A potency cannot be regarded as necessarily inter-changeable or additive. Biological assays represent the combination of(a) provitamin-A or vitamin-A content and (6) the availability to theanimal of that content as compared with the availability of the standardpreparation.In determining the vitamin-A content of fish-liver oils and concentrates,the E value (at 328 mp. where the vitamin absorption is maximal) cancorrectly be multiplied by 1900 (to give i.u./g.) when absorption a t thatwave-length due to substances other than vitamin A has been eliminatedor allowed for.The standard preparation can be used to fix the correctabsorption curue by plotting E values between 250 mp. and 380 mp. on a,scale where Em,,. = 1-00. If the curve for an oil under study is plotted inthe same manner irrelevant absorption will bring about obvious distortionas compared with the standard curve. The curve may then be correctedgeometrically and the reduced E,,,.multiplied by 1900, or the sample maybe purified by chromatographic or other methods. A new departure is thusto be noted in the use of a standard preparation; in addition to its directuse as a biological standard it provides a test for absorption curves distortedby irrelevant absorption and applicable to whatever type of spectrophoto-meter is in use.This is, of course, not the same as using it to test the correctbehaviour of the instrument; that is best ascertained by using a standardglass or a simple solution such as potassium chromate.Biological Standardization.-The usual procedure using growth responseshas been supplemented by a histological method of assessing the degree ofmyelin degeneration shown in vitamin-A deficien~y.~~ Microscopic lesionsAttention needs to be drawn, however, to certain pitfalls.5 5 R. A. Morton and A. L. Stubbs, Biochem. J., 1948, 42, 195.5 6 Idem, Analyst, 1946, 71, 356.5 7 Expert Committee on Biological Standardization, Bulletin World HealthOrganization 1950. (Recommendations accepted August, 1949, and now official.)In this country the standard preparations are distributed by the Director of BiologicalStandards, National Institute of Medical Research ; next issue April lst, 1960.ii8 H.K. Coetzee, Biochem. J., 1949, 45, 628252 BIOCHEMISTRY.in the brains of chicks deficient in vitamin A have also been noted.60Coetzee’s method when applied to a range of fish-liver oils led to estimatesof potency which agreed very closely indeed with those based on the Evalues at 328 mp. corrected by the procedure of Morton and Stubbs.56Considerable work has been devoted to an attempt to use the liver storageof vitamin A as a biological-assay method.59 There is no doubt that themethod has possibilities particularly as the animal rejects to a large extentmaterials giving rise to great irrelevant ultra-violet absorption (e.9.thosepresent in some whale-liver oil preparations). The animal thus acts as a“ filter,” but the final assessment rests on spectrophotometric readings onthe liver unsaponifiable fraction from the experimental animal. Much willdepend on the anti-oxidants present in the oil under study.61-68 Thewhole problem of storage, in relation to dose levels of vitamin, propertiesof the “ carrier ” oil, distribution of storage between Kupffer cells and trueliver cells, and permanence or otherwise of the vitamin store, is very com-plicated and bio-assay methods based on this approach need to be usedwith circumspe~tion.~~Special Analytical Prob1ems.-Vitamin A in many samples of whale-liver oil is difficult to determine because of the presence of other absorbingsubstances.These include kitol,?O a divitamin A [C,,€€,,(OH),] showingLax. at 286 mp. but lacking vitamin-A potency. This substance occurs insome whale-liver oils as a diester which has been obtained fairly p ~ r e . ~ 1A good method of obtaining a vitamin-A fraction by Chromatography onalumina of the unsaponifiable matter of whale-liver oil has been worked0 ~ t , 7 ~ and an alternative method making use of chromatography of the wholeoil on weakened alumina gives similar results more r e a d i l ~ . ~ lThe existence of neo-vitamin A may give rise to further complication^.^^neo-Vitamin A, is said to be a stereoisomer of the all-trans-vitamin A,. Itmelts a t 58-60’ as compared with 62-64’ for the ordinary form, and itsabsorption maximum (E;Trn* at 328 mp., 1645) ’* is slightly different from5s K.Guggenheirn and W. Koch, Biochem. J . , 1944, 38, 261.So F. B. Adamstone, Arch. Path., 1947, 43, 301.6 1 F. Week and F. J. Sevigne, J . Nutrition, 1949, 39, 233.62 Idem, ibid, p. 251.63 T. Moore, A. J. P. Martin, and K. R. Rajogopal, “Vitamin E Symposium,”64 A. W. Davies and T. Moore, Nature, 1941, 147, 794.65 T. Moore, Vit. and Horm., 1945, 3, 12.6 8 HI. C. D. Hickman, P. L. Harris, and M. R. Woodside, Nature, 1942, 150, 91.87 P. L. Harris, M. W. Kaley, and K. C. D. Hickman, J . BioZ. Chem., 1944,152,313.68 K. C. D. Hickman, M. W. Kaley, and P. L. Harris, ibid., pp. 303, 321.69 Unpublished work by A. D. MacQueen and J.Glover, Reporter’s laboratory.70 J. G. Baxter, F. B. Clough, H. 1%. Kascher, and C. D. Robeson, Science, 1947,7 1 R. K. Barua and R. A. Morton, Biochem. J., 1949, 45, 308.72 N. T. Gridgeman, J. P. Savage, and G. P. Gibson, Analyst, 1948, 73, 662.7 3 J. G. Baxter and C. D. Robeson, J . Amer. Chem. SOC., 1947, 69, 136.74 J. D. Cawley, C. D. Robeson, L. Weisler, E. M. Shantz, N. D. Embree, and J. G.Heffer, Cambridge, 1939.105, 436.Baxter, Science, 1948, 107, 346MORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 253that of the all-trans-form (E::&. a t 326 mp., 1750). Its biological activitymay be slightly lower than that of all-trans-vitamin A,. Synthetic products 74and many fish-liver oils 75 contain considerable proportions of neo-vitaminA, as judged by an analytical procedure making use of the different ratesat which neo-vitamin A and all-trans-vitamin A combine with maleicanhydride.It is perhaps early to say whether the existence of neo-vitamin A requiresreconsideration of analytical methods, but it is very unlikely that muchwill be gained by routine examination for isomers.Vitamin-A Requirements-of Adult Humans.---The full report 76 appearedin 1949 of an experimental study of vitamin A deprivation in man, carriedout during the War by a British team of workers.Conscientious objectorswho volunteered to act as subjects were kept on a diet very low in caroteneand vitamin A but otherwise adequate, until (in some of them) unmistakablesigns of vitamin-A deficiency appeared.The amount of (3-carotene orvitamin A needed to remove the symptoms was then determined.The Report is long and technical and the Reporter, as one who participatedin the work, is conscious that any short summary is inadequate. Thevolunteers (20 young men and 3 young women) were given a diet whichprovided a t most 42 pg. of ( ( carotene” per day and was shown byfeeding tests on rats to possess negligible vitamin-A activity. The wholeexperiment lasted over two years (1942-1944).Five subjects were given throughout the experiment about 5000 i.u.of @-carotene daily in various forms, and two others were given 2500 i.u.of vitamin A per day in the form of a diluted concentrate. Sixteen subjectsreceived the unsupplemented diet until they showed a serious drop (orderof 50%) in plasma vitamin A and a considerably delayed dark-adaptation,or in a few cases until they dropped out of the experiment.The manifestlydepleted subjects were given either p-carotene or vitamin A.The best techniques available under war-time conditions were used fordetermining capacity for darlr-adaptation and also total carotenoids,(‘carotene,” and vitamin A in food, blood, and faxes. Clinical tests,including blood counts, skin biopsies, audiometric tests, and slit-lampexaminations were made regularly together with psychological appraisals.Although some of the work could today be improved upon as a result ofpost-war re-equipment, the investigation as a whole was a most successfuleffort in co-operative research which it would be difficult to repeat.The first change caused by the deficient diet was a decrease in plasmacarotenoids from about 0.9 pg.to about 0.24 pg. per ml., little of the persistingpigment being “ carotene.” A steady state was reached after 3 monthsand no marked change was noticeable during the next 5 months. Then the75 P. Meunier and J. Jouannateu, BUZZ. SOC. Chim. biol., 1948, 30, 260.7 6 A Report of the Vitamin A Sub-Committee of the Accessory Food FactorsCommittee of the Medical Research Council (compiled by E. M. Hums and H. A. Krebs),M.R.C. Special Report, Series No. 264, H.M.S. Office, 1949. The report gives the namesof sl1 the workers who participated in the experiment264 BIOCHEMISTRY.plasma-vitamin-A levels (normally of the order 1.2 i.u./ml.) began to fallin 10 out of 16 subjects and later reached 0.5 i.u./ml.in 4 men.Dark-adaptation tests showed that the normal cone-rod transitiontime varied between 5 and 12 minutes and the normal final cone-rodthreshold between 1-37 and 2.3 log mp. lamberts. Three subjects showedincreased transition times (up to 33i minutes) and had low plasma-vitamin-A levels at the critical time. One of them received 1300 i.u. ofvitamin A in oil daily and his capacity for dark-adaptation was graduallyrestored to a level which could not be improved by larger doses of vitamin A.With a daily dose of 750 pg. (1250 i.u.) of @-carotene in oil, one depletedsubject showed an increase in plasma-vitamin-A but a worsened cone-rodthreshold. Another depleted subject given 2500 i.u.per day of p-carotenein oil showed improvement by both criteria. After 5$ months of supple-mentation the first ( ( carotene ’’ subject (1250 i.u. per day) improved markedlyon transfer to an unrestricted diet, whereas the other subject (admittedlynot quite so markedly depleted) recovered completely in 3 weeks with 2500i.u. per day of @-carotene.Two subjects given for 14 and 17 months, respectively, a prophylacticdose of 2300 i.u. per day of vitamin A in oil were maintained in vitamin-Abalance although the plasma levels subsequently rose on an unrestricteddiet.The great majority of the clinical examinations showed no significantdifferences between the deprived and non-deprived groups.In all cases a variable proportion of the carotene administered appearedin the fzeces, and the part not excreted is called the maximum effectivedose.The average amount of carotene excreted, expressed as a percentageof the measured intake, was about 75 for carrots, 59 and 73 for cabbage a ttwo different dosages, 57 for homogenised spinach, 29 for carotene inmargarine, and 26 for carotene in arachis oil.In the prophylactic tests with carotene given in different ways the effectivedose varied from 1250 to 3700 i.u. daily and except for a doubtful period atthe lower level no significant signs of depletion appeared. This was notsurprising as 71 civilians accidentally killed in 1941-44 possessed liverstores of the order of 500,000 i.u. This reserve “ buffers ” the plasma-vitamin-A level making it unresponsive to short-term marginal intake ofvitamin A or provitamin A.The minimum requirement provided wholly as preformed vitamin Ais near 1260 i.u.daily with 2500 i.u. providing a margin of safety. Similarlythe requirements of p-carotene are 1500 i.u. daily with 3000 i.u. for safety,but if the recommended intake is corrected for incomplete absorption, thegross figures would be : for carotene contained in cooked carrots 12,000,cabbage or spinach 7500, p-carotene in oil 4000 i.u. per day (perhaps 7500i.u. daily for mixed foods).The Report stresses that the results apply mainly to healthy youngadult males, and it is impossible to say what modifications should be madefor children, pregnant and lactating women, and diseased persons.ThMORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 255results, moreover, do not provide an infallible guide for assessing the adequacyof a diet in nutritional surveys.In some ways the most important finding is that the lengthened cone-rodtransition time emerges as the least equivocal and most reproduciblymeasurable test of vitamin-A deficiency, and the view that rhodopsinregeneration is conditioned by vitamin-A supply is fully borne out.Conversion of Provitamins into Vitamins A.-An increase in the liverstore of vitamin A, as a result of ingesting carotene, was established in1929.77 The idea that the conversion takes place in the liver was widelyentertained but the evidence was unsatisfactory. None of the experimentswith liver tissue and colloidal solutions of carotene in vitro was unequivocallypositive and many were negative.Drummond and his colleagues carriedout experiments in vivo all of which were negative in the sense that conversionin the liver could not be d e m o n ~ t r a t e d . 7 ~ ~ ~ Vitamin A cannot be detectedby fluorescence microscopy 81 in the liver of depleted rats after parenteraladministration of carotene although there is no doubt that the hydrocarbonreaches the liver.82The first pointer to a site other than the liver was the discovery of largea-nnounts of vitamin A on the lining of the intestines of many kinds offishes; 8 3 ~ ~ 4 this was at once followed up commercially but its theoreticalimplications were not, partly because the phenomenon does not occur inmammals or even in all fishes.85It is now clear that injected carotene may be freely stored in the Kupffercells of the liver but in the rat conversion there into vitamin A is on a scaletoo small to be demonstrable.Deuel's group 86 has shown that, irrespectiveof the route, parenterally administered carotene is ineffective and that theliver may be rich in carotene without any relief of the symptoms of avita-minosis A. Incubation of the intestines with colloidal carotene failed toshow Conversion into vitamin A, but in spits of this the intestine was latershown to be the site of the p r o ~ e s s . ~ ~ - ~ ~The Liverpool group had been impressed by the fact that some speciesdo not naturally store carotenoids; thus in sheep and goats little caroteneis found in blood plasma, milk fat, or body fat in spite of a large intake infood. This was a serious obstacle to accepting the liver as the site of con-7 7 T.Moore, Riochem. J., 1930, 24, 692.79 J. C. Drummond, H. P. Gilding, and R. J. MacWalter, J. Physiol., 1934, 82, 75.J. L. Rea and J. C. Drurnrnond, 2. Vitaminforsch., 1932, 1, 177.J. C. Drummond and R. J. MacWalter, ibid., 1935, 83, 236.H. Pepper, Arch. Path., 1941, 31, 766.82 A. Vinet, M. Plessier, and Y. Raoul, Bull. SOC. Chim. biol., 1943, 25, 87.J. A. Lovern, J. R. Edisbury, and R. A. Morton, Nature, 1937, 140, 276.** J. A. Lovern, R. A. Morton, and J. Ireland, Biochem. J., 1939, 33, 325.8 5 J. A. Lovern, T. H. Mead, and R. A. Morton, ibid., p. 338.8 6 E.L. Sexton, J. W. Mehl, and J. Deuel, jun., J. Nutrition, 1946, 31, 299.8 7 S. Ball, J. Glover, T. W. Goodwin, and R. A. Morton, Biochem. J., 1947, 41,xxix.F. H. Mattson, J. W. Mehl, and H. J. Deuel, Arch. Biochem., 1947, 14, 65.8B C. F. Wiese, J. W. Mehl, and H. J. Deuel, ibid., 1947, 17, 75256 BIOCHEMISTRY.version. No carotene could be detected in portal or systemic blood ofsheep or goats given large doses of carotene direct into the du~denum.~OThe conversion of vitamin-Al-aldehyde (p. 249) into vitamin A wasshown to occur in rats in the intestinal wall.86,g1 This was followed bydirect proof of the conversion of carotene into vitamin A in uiuo and inv i t ~ o . ~ ~ The finding was soon confirmedg3 and extended to pigs in a fulls t ~ d y .~ 4 A rise in the concentration of vitamin A in thoracic lymph in thegoat after feeding it with @-carotene was e~tablished.~~ Finally, it wasshown that carotene could be converted into vitamin A in rats in which theliver was isolated by a ligature on the portal vein.96 All this makes itcertain that the intestinal wall is the main, and possibly the sole site of theprovitamin + vitamin conversion in mammals.Thnoid Activity and Vitamin A,--Contradictions in the literatureconcerning the interrelation between the thyroid hormone and vitamin Ahave been largely explained.Thyroidectomized animals depleted of vitamin A may be cured ofxerophthalmia by orally administered carotene 97 but not by injectedcarotene.98, g9 That is not now difficult to understand (see p.255), but theobservation, if it can be reproduced, that carotene appears in the milk ofthyroidectomized goats 1, may be more significant. When liver storageof vitamin A was used as a measure of carotene conversion, desiccatedthyroid gave an increase and thiouracil a decrease compared with controls,whereas with a mixture of the thyroactive and antithyroid substancesnormal storage o ~ c u r r e d . ~ , ~ At a much lower level of carotene intakesimilar effects might be expected when the growth test is used to assess theefficiency of conversion. Thiouracil not only retards the rate of depletionof hepatic stores of vitamin A but also has itself a growth-inhibiting actioncorrectable by adding desiccated thyroid but not by vitamin A ; the amountof carotene needed to produce half the maximal growth in both the controlsand the thiouracil-treated animals was the same.This experiment pointsaway from the idea that thiouracil inhibits the carotene _t vitaminconversion.O0 T. W. Goodwin, A. D. Dewar, and R. A. Gregory, Biochem. J., 1946, 40, x.O1 J. Glover, T. W. Goodwin, and R. A. Morton, Biochem. J., 1947, 41, xv.92 Idem, ibid., 1948, 43, 512.O3 F. H. Mattson, J . Biol. Chem., 1948, 176, 1467.O4 S. Y. Thompson, J. Ganguly, and S. K. Kon, Brit. J . Nutrition, 1947,1, v ; 1949,O 5 T. W. Goodwin and R. A. Gregory, Biochem. J., 1948, 43, 505.96 R. F. Kraus and H. B. Pierce, Arch. Biochem., 1948, 19, 145.D7 R. E. Remington, P. L. Harris, and C. L. Smith, J . Nutrition, 1942, 24, 597.Q8 V.A. Drill and A. P. Truant, EndocrinoEogy, 1947, 40, 259.Q9 J. M. Canadell and F. G. Valdescasas, Experientiu, 1947, 3, 35.T. Fellenberg and F. Greuter, Biochem. Z., 1932,253, 42.F. Fasold and E. R. Heidemann, 2. ges. ezp. Med., 1933, 92, 53.R. M. Johnson and C. A. Baumann, J . Biol. Chem., 1947,171, 513.B. Kelley and H. C. Day, ibid., 1948, 175, 163.C. E. Wiese, J. W. Mehl, and H. J. Deuel, ibid., p. 21.3, 50MORTON: CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 257Recent work casts doubt on the observation that hypothyroid goats giveyellow milk, indeed no carotene was found in the blood or the milk.'Rabbits on a carotene-rich diet and given large doses of thiouracil showedno carotene in the systemic blood. Thiouracil has no effect on the stabilityof carotene in witro, but thiouracil-treated rats excrete a larger fraction ofthe dose of (3-carotene (under different dietary conditions) than do untreatedsnimals.899 This shows that the anti-thyroid drug reduces caroteneabsorption, which accounts for the previously observed decreased storage ofvitamin A.3 The growth-test experiments were perhaps a t dose levelsbelow the threshold for interference with absorption.The claim lo thatthyroactive substances influence the enzymic conversion of carotene intovitamin is probably unjustified.8Mobilization of Liver Reserves of Vitamin A-Pfasma concentrationsof free vitamin-A alcohol rise quite slowly with large increases in the totalliver store of vitamin A, after heavy doses of vitamin A.A much closerrelationship obtains between plasma-vitamin-A concentration and the freevitamin A of the liver.ll The liver store is distributed over the Kupffercells and the true liver cells, and the former appear to contain only the esterwhereas the latter contain some alcohol. This agrees with the absence oflipases (and esterases?) from the Kupffer cells, and their presence in thetrue liver cells.13 It is not clear how vitamin-A esters are liberated fromthe Kupffer cells. It is possible that frequent small doses of vitamin Awill result in a more favourable distribution of the store than higher dosesat longer intervals apart. Much of a massive dose is probably immobilizedin the phagocytic Kupffer cells. This line of thought has importantimplications in the therapeutic use of vitamin A.Claims that ethyl alcohol ingestion mobilizes vitamin A fromliver to blood and that adrenaline affects vitamin-A levels have not beenconfirmed, 149 15Physiology of Vitamin A.-In rats, metaplasia of the epithelium of theurinary tract is an early result of vitamin-A deficiency, and i t appears thatmucosie become avitaminotic because the mitochrondia are low in vitamincontent.16 Cessation of weight increase is considered to be secondary tomucosal dysfunction.In growing chickens l7 the typical post-mortem signs-urates in the kidneys, nodules on the oesophagus, etc.--are rarely seenR. M. Johnson and C . A. Baumann, Fed. Proc., 1948, '7, 290.V. R. Smith, R. P. Niedenneiser, and L. Schultz, J .Animal Sci., 1948, 7 , 544.H. R. Cama and T. W. Goodwin, Biochem. J., 1949, 45, 248.s Idem, ibid., p. 317.lo T. A. Balaba, J. Physiol. U.S.S.R., 1940, 29, 318.l1 J. Glover, T. W. Goodwin, and R. A. Morton, Biochem. J., 1947, 41, 101;lS J. GIover and R. A. Morton, Biochem. J., 1948, 43, Proc. xii.l4 See ref. 76.l5 T. W. Goodwin and A. A. Wilson, Biochem. J., 1949,45, 370,l7 M. W. Taylor and W. C . Russell, Poultry Sci., 1947, 26, 234.1948, 43, 512.M. Bracco and H. v. Euler, Arkiv IZemi, Min., Geol., 1948, A , 26, 1.REP.-VOL. XLVI. 258 BIOCHEMISTRY.before the last stages of deficiency, but inco-ordination possibly caused bybony overgrowths with sequele on nervous tissue occurs early. Thesesigns recall Mellanby’s observations on dogs l8 and Wolbach and Bessey’son rats.lg Vitamin-A deficiency exacerbated cecal coccidiosis when itoccurred in chicks.20 The earliest sign of deficiency was a slight reddeningabout the eyes, followed rapidly by secondary infection.The minimumrequirement of laying hens for provitamins A for good egg production andhigh hatchability is rather high (ca. 3000 i.u./lb. of feed); if it is provided,the plasma-vitamin A is of the order of 1.5 i.u./ml.Liver stores of vitamin A are depleted more quickly in rats growingnormally than in those stunted by inadequacies of calories, aneurin, ortryptophan. Halving the growth rate is more important for vitamin-Aretention than a threefold increase in metabolic rate induced by desiccatedthyroid. In normally growing rats, a decrease in liver reserves is acccm-panied by a rise in kidney vitamin A, but this does not happen in rats whosegrowth was restricted during the depletion period.21Rats deficient in vitamin A show symptoms resembling those of scurvy.22Blood ascorbic acid is a t least halved23 and the adrenals show considerablehypertrophy with lowered vitamin4 content.The striking effect firstobserved by Moore 2 4 7 2 5 that vitamin-A storage is enhanced by an adequatevitamin-E intake is complemented by the observation on humans thatblood-vitamin-E levels rise significantly after prolonged high dosage withvitamin A.26 Similar rises in plasma cholesterol occurred but carotene andvitamin-C levels were unaffected.Vitamin A is much more effectively stored when given dispersed inaqueous media than in oily solution.27~ 28 Lecithin enhances absorptionof carotene and of vitamin A.29 With extremely high intake and storage,hypervitaminosis A becomes a reality.3O The characteristic lesions bearsome resemblance to those found in human and in experimental scurvy.It will be seen that the elucidation of the mode or modes of action ofvitamin A is distinctly hampered by a plethora of physiological evidenceand by vitamin interrelations.The co-enzymic role which might clarifythe situation is still to be found.E. Mellanby, J . Physiol., 1941,99, 467.l* S . B. Wolbach and 0. A. Bessey, Arch. Path., 1941, 91, 599.2o M. W. Taylor, J. R. Stern, and W.* C. Russell, Poultry Xci., 1947, 26, 243.21 R. M. Johnson and C.A. Baumann, J . Nutrition, 1948,35, 703.2z J. Mayer and W. A. Krehl, Arch. Biochem., 1948, 16, 313.23 G. Johnson, A. L. Obel, and K. Sjoberg, 2. Vitaminforsch., 1942, 12, 300.24 T. Moore, A. J. P. Martin, and K. R. Rajogopal, “Vitamin E Symposium,”25 T. Moore, Biochem. J., 1940,34, 1321.26 J. T. v. Bruggen and J. V. Straumfjord, J . Lab. Clin. Med., 1948, 33, 67.27 A. E. Sobel, M. Sherman, J. Lichblan, S. Snow, and B. Kramer, J . Nutrition,28 H. Popper and B. W. Vole, Proc. SOC. exp. biol., 1948, 68, 562.29 G. C. Esh and T. B. Sutton, J . Nutrition, 1948, 36, 391.30 T. Moore and Y . L. Wang, Biochem. J., 1945, 39,222.Heffer, Cambridge, 1939.1948, 35, 225MORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 259Biochemical Aspects of Vision.-The light receptors of the visual layerof the retina are called rods and cones but the implied difference in shapeis not clear-cut.Nocturnal animals have many more rods than cones butthe opposite is true for diurnal animals. During dark-adaptation, a highlyphotosensitive pigment accumulates in the rods but the cones are alwaysrelatively insensitive to light. With intensities of about 0.1 metre-candle,both types of receptor are in action; a t lower intensities the rods only area t work and a t higher intensities vision is mediated only by the cones. Therod pigment, visual purple or rhodopsin, reaches its maximum concentrationin man after about 45 minutes of dark-adaptation; very dim light is thenperceptible partly because of the sensitivity of the pigment and partlybecause many rods are connected via bipolar and ganglion cells to one opticnerve fibre.31The minimum perceptible intensity of light decreases as the timepreviously spent in darkness increases; the curve of intensity against timeshows a sharp break a t the cone-rod threshold and the cone-rod transitiontime.The discontinuity has been confirmed by electrophysiological methods.If very weak monochromatic radiations are used, a dark-adapted personcannot recognize colour-differences but the minimum perceptible intensitydepends upon the wave-length.For rod (or scotopic) vision and cone (or photopic) vision the luminositycurves are broad and show maxima a t 500 mp. and 560 mp., respectively.Photopic vision is not necessarily accompanied by colour vision.Rhodopsin may be extracted from retinas obtained by dissecting eyesin In solution it exhibits an absorption spectrum which agreesvery closely with the scotopic luminosity curve.The methods which yieldrhodopsin solutions (Amax. 500 mv.) from mammalian retinas result in theextraction of a related pigment porphyropsin (A,,,. 520-530 mp.) from theretinas of certain fresh-water fishes. A pigment iodopsin has been postulatedto account for the photopic luminosity curve.The Young-Helmholtz trichromatic theory of colour vision postulatedthree types of cones or three types of receptor per cone. Recent physiologicalresearches have enriched the evidence; thus Granit and his colleagueshave perfected electrophysiological methods of exploring the retina witha micro-electrode touching a single nerve-fibre from a ganglion cell on thevitreal surface, preferably using decerebrate animals with the cornea andlens excised.The second electrode is placed in contact with the sclera andthe currents obtained are amplified and recorded. The responses to mono-chromatic light are in the form of impulses, the frequencies of whichincrease with increasing intensity of monochromatic light. With eyes ofmany species a broad scotopic dominator curve ( hmax. 500 mp.) is obtained inthe dark-adapted state; but fresh-water fishes such as tench and carpshow Am,,. 530 mp. With light-adapted eyes the results vary from onenerve-fibre to another and from one species to another. Eyes of rats andred light.81 R.Granit, " Sensory mechanim of the retina," Oxford Univ. Press, 1947260 BIOCHEMISTRY.guinea-pigs show Amax. 500 mp. but those of frogs and cats exhibit a photopicdominator curve with Amax. 560 mp. Some nerve fibres produce a curvewith an inflexion near 600 mp., whilst others show modulator curves abouthalf as wide as the dominator curves. The following maxima were recorded :rat (few cones), modulators at 500 mp. and 610 mp; guinea-pig, 460, 500,530, and 600 mp.; frog, 450470, 530, 580, and 600 mp.; cat, 36% offibres, dominator 560 mp., 64% of fibres, 530 and 560 mp.; tortoise, conedominator 610 mp., modulator 540 mp.; carp and tench, cone dominator610 mp., modulators 540 and 650 mp.Granit 32 has obtained further evidence of modulator curves by selectivelight adaptation and by the use of polarizing currents.Hartridge 33 interprets a very wide range of bio-physical measurements asfollows : at least 7 types of cones can be differentiated according to theirspectral sensitivities; 3 types, responding mainly to blue, green, and redlight, respectively, are regarded as main receptors, and 3 subsidiary re-ceptors are complementary to the others; the different types are randomlydistributed except that clusters of similar cones may occur ; sufficientlynarrow pencils of white light or filtered light may stimulate a cluster ofcones or possibly single cones, and fixation points may be recorded, eachcorresponding with a rather narrow strip of the visible spectrum.Manyfindings 34, 35 are difficult to reconcile with the simple trichromatic theory,and the hypothesis of additional receptors gives more “ elbow room ”without necessarily making it easier to compel assent.Excellent reviews36 of recent advances in the physiology of vision areavailable, and the foregoing paragraphs are intended mainly to drawattention to the challenge to chemists in that the action spectra of thedominators and modulators present something very definite to be accountedfor.Rhodopsin.Visual purple is a conjugated protein, the prostheticgrouping of which is in some way related chemically to vitamin A, andporphyropsin seems to be similarly related t o vitamin A,. Rhodopsinoccurs mainly in the outer segments of the rods; these are often easilydetached by shaking the retinas in d i n e .A good method is to dissectout retinas in a weak red light and shake them in a 40% (w/v) solution ofsucrose ; 37 on centrifuging, the rods remain in suspension. Dilution withwater followed by re-centrifuging throws down the rods, which are thenhardened with alum. After the liquor has been poured off and the residue32 B. Gernandt and R. Granit, Nature, 1947, 159, 806.33 H. Hartridge, Phil. Trans., 1947, 232, 592.34 R. W. Pickford, Nature, 1948, 162, 684.35 E. N. WilImer and W. D. Wright, Nature, 1945, 156, 119.as E. N. Willmer, “ Retinal Structure and Colour Vision,” 1947, London ; W. D.Wright, ‘‘ Researches on Normal and Defective Colour Vision,” 1946, London ; “ Docu-menta Ophthalmologica, Advances in Ophthalmology,” 1949, Vol.3, Junk S-Gravenhage,Edited by F. P. Fisher, Utrecht, A. J. Schaeffer, and A. Sorsby. M. H. Pirenne,“ Vision and the Eye,” 1949, Chapman & Hall.31 Z. Saito, Tohoku J . Exp. Med., 1938,32,432MORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 261washed, the rhodopsin dissolves on treatment with a 1% solution ofd i g i t ~ n i n . ~ ~ The best preparations show it nearly symmetrical absorptioncurve with Amax. 500 my.; the intensity of absorption at 400 mp. is aboutone quarter of that at 500 mp. and there is a weak maximumat 340-350mp.393 40 The absorption rises steeply in the ultra-violet, showing “ protein ”absorption (due to tyrosine and tryptophan). The maximum (at 275 mp.) maybe as low as 2-2 times 393 40 the intensity of the 500 mp.peak, but is usuallymuch more intense because it is difficult to eliminate colourless contaminatingproteins. The molecularweight has been estimated to be 270,00041 but that figure may needThere is so far no fully satisfactory test of purity.revision.Cepholopsin. Cepholopsin is an interesting analogue of rhodopsinobtained from the eyes of squids (Loligo v u t g a r i ~ , ~ ~ Loligo pe~lii).*~ It isa light-stable red pigment (Amax. 495 mp.) which becomes light-sensitive ontreatment with formaldehyde, and retinene is released.43 However, St.George and Wald 44 applied to squid retinas the normal method for extractingrhodopsin. The solution showed maxima at 490, 365 (weak), and 279 mp.“ In the light it undergoes a photochemical change followed by a ‘ dark ’reaction, comparable with the transformation of rhodopsin to lumi- andmeta-rhodopsin.The squideye seems to contain both free and bound retinene.45Photochemical Changes. Exposure of isolated retinas to light destroysrhodopsin, and vitamin A, is set free. Photochemical bleaching of rhodopsinsolutions can take place at very low temperatures, and a labile compound,transient o r i ~ n g e , ~ ~ , ~ ~ is formed. This material is unstable a t room tem-perature, and yields indicator yellow, which, as its name implies, is pH-sensitive (Amax. 360 mp. in alkaline solutions, 440 mp. in acid solutions).By extracting freshly-bleached retinas with light petroleum, Wald 489 49obtained a new carotenoid-like material which he called retinenel. I nchloroform solution it showed Lax.385-387 mp. and gave with the antimonytrichloride reagent a blue colour having Amax. 664 mp. Wald also obtainedretinene, by extracting bleached rhodopsin solutions. Porphyropsinsolutions similarly treated gave retinene2 (Lax. 405 mp. in chloroform,705 mp. with the antimony trichloride reagent).The two retinenes were obviously key substances to which Wald hadThe material is very similar to rhodopsin ”.3* K. Tansley, J . Physiol., 1931, 71, 442.39 G. Wald, ‘‘ Documenta Ophthalmologica,” 1949, VoI. 3, p. 94.40 F. D. Collins and R. A. Morton, Biochern. J., 1950, in the press.41 S. Hecht and E. G. Pickels, Proc. Nut. Acad. Sci. Wash., 1938, 24, 172.42 J. E.-Desrivihres, E.Lederer, and M.-L. Verrier, Compt. rend., 1938, 207, 1447.43 A. F. Bliss, J . Biol. Chern., 1948, 178, 563.4p R. C. C. St. George and G. Wald, Biol. Bull., 1949, 97, 248.45 G. Wald, J. Dwell, and R. C. C. St. George, Science, in the press.46 R. J. Lythgoe and J. P. Quilliam, J . Physiol., 1938, 94, 339.47 E. E. Broda and C. F. Goodeve, Proc. Roy. SOC., 1941, B, 130,217.48 G. Wald, J . Gen. Physiol., 1935-6, 19, 351.p9 Idem, ibid., p. 781262 BIOCHEMISTRY.attached valuable labels although neither could be obtained pure or inadequate quantity for characterization. The only plausible explanationof their spectra and colour tests was the hypothesis 50 that they wererespectively the aldehydes corresponding with vit.amins A, and A,.Thisidea was tested and ~onfirmed.~~Retinene, may conveniently be prepared by leaving vitamin-A alcoholin light petroleum over solid manganese dioxide at room temperat~re,~~Vitamin A,, free from vitamin A,, is not a t all readily accessible, but amixture of vitamins A, and A, may be oxidised similarly, and the tworetinenes separated by chr~matography.~~ Both substances have beenobtained crystalline and fully characterized.Spectroscopic Data for Vitamin A and Some Related Compounds.OscillatorCompound. Amax. (ml.1. Emax.. strength, f.* Solvent.Vitamin-A aIcohoI ............ 326 48,300 0-97 cyclohexaneVitamin-A acetate ............ 328 48,500 0-92 cyclohexaneVitamin A28 .................. 35 1 41,600 0.99 ethanolRetinene, ( C ~ O H ~ ~ O ) .........385 39,800 0.84 ethanolRetinene, (C,,H,,O) ......... 386 41,200 0.90 light petroleum* f = 4.31 x 1W8 ycdv = ca. 1.0. s[v is in wave numbers (crn.-l), s is the deoadic molecular extinction coefficient]The availability of retinene, (vitamin-A-aldehyde) in reasonable amountsmade possible experiments in which it was administered orally and parenter-ally to rats. Retinene, was found to be readily converted into vitamin A,by an enzymic reduction. The orally administered aldehyde is convertednearly quantitatively into vitamin A in the gut waIl.54 Retinene, is similarlyconverted into vitamin A,. Both aldehydes undergo Ponndorf reductionto give the vitamins.The retina contains an enzyme system which readily reduces retinene, ;the change, to vitamin A can be effected in vitro using coenzyme I andfructose 1 : 6-dipho~phate.~~ The change is reversible since a rabbit-liverDPN-specific alcohol dehydrogenase preparation in the presence of coenzymeI and pure vitamin A (dispersed by a detergent), together with bisulphiteor cyanide to “trap ” aldehyde, results in a 40% c0nversion.5~ Isolatedrods appear to contain the reductase.6o R.A. Morton, Nature, 1944,153, 69.61 R. A. Morton and T. W. Goodwin, ibid., p. 405.S. Ball, T. W. Goodwin, and R. A. Morton, Biochem. J., 1948, 42, 516.R. A. Morton, M. K. Salah, and A. L. Stubbs, Nature, 1947,159, 744.J. Glover, T. W. Goodwin, and R. A. Morton, Biochem. J., 1948,43,10, 109.6 5 G. Wald and R. Hubbard, J . Gen. Physiol., 1949, 32, 367.ti6 A.IF. Bliss, BioE. Bull., 1949, 97, 221MORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 263In essentials the sequence is as follows :light 1 rhodopsin? transient orangeI IYindicator yellowI I retinene reductase \ $vitamin A + protein I__ retinene, + protein \ (DPN-PH + fructose 1 : 6-diphosphafx + dehydro-genase system)Retinenel + DPN4H + vitamin A + DPN(normally DPN, etc., is washed out of the rods when they are separated).The rhodopsins obtained from different species are not necessarilyidentical, the prosthetic group may well be the same in all cases but theprotein need not. There is in fact some evidence of species differences inthe precise position of Amax., e.g. 503 mp. for frog rhodopsin and 498 mp. forrat rhodopsin.57 The E,lTm.is of the order of 6.6 58 although this needsconfirmat ion.The conversion of rhodopsin into indicator yellow has been studied insome detail. The first clue to the nature of the pH-sensitive product wasthe preparation of compounds closely analogous to it by the interaction ofretinene and many amines and proteins. 59? 6oA typical experiment made use of a protein solution from sheep retinas.When retinene was added, Amax. was a t 387 mp., displaced to 365 mp. whenthe solution was made alkaline ; subsequent acidification resulted in ashife of h,,,. to 440 mp. Em,,. values at 387 and 440 mp. were practicallythe same.The change in intensity of absorption ( ~ E ~ O O ~ ~ . ) which occurs on com-plete bleaching of rhodopsin solution measures the photochemical destruc-tion, and the increase in absorption a t 370 mp. (hE37omP.) measures theformation of alka.line indicator yellow.AE370mp. fAE500mp. is found to be0.75 at pH 9.2, and a t pH 1-76 eE440mp./AE500mfi. = 0.37.61 Ereshly bleached,neutral solutions of rhodopsin contain not only the indicator-yellow systembut also some retinene. It appears that retinene is formed from indicatorWith aliphatic amines such as methylamine (but not dimethylamine)pure retinene forms pH-sensitive derivatives, having hmaX. 440 mp. in acidyellow.67 F. D. Collins and R. A. Morton, Biochem. J., 1950, in the press.58 E. E. Broda, C. F. Goodeve, and R. J. Lythgoe, J. Physiot., 1940, 98, 297.6s S. Ball, F. D. Collins, R. A. Morton, and A. L.Stubbs, Nature, 1948, 161, 424.61 F. D. Collins and R. A. Morton, ibid., 1950, in the press.S. Ball, F. D. Collins, P. D. Dalvi, and R. A. Morton, Bioehem. J., 1949, 45, 304264 BIOCHEMISTRY.and 365 mp. in alkali. Two molecules of retinene appear to react with oneof methylamine :. .(full conjugation restored, Amax. 440mp.)(I) corresponds to alkaline and (11) to acid indicator yellow. If thesemethylamine derivatives are true analogues of indicator yellow its structuremust be similar, with an amino-group of a protein replacing that ofmet hylamine.Detailed study of the spectra 62 has led to the following figures : 63cr = 39,000, E , = 40,00On, = 49,00On, and E , = 47,200m;where c7, E,, q,, and €6 are the maximal molecular extinction coefficientof retinenel, indicator yellow, rhodopsin, and alkaline indicator yellow,respectively, and p = the number of C,, (retinene or vitamin A) residuesin the rhodopsin chromophore, and n and rn the number of such residues inacid and alkaline indicator yellow, respectively.Both n and m probablyequal 2, hence E, = 80,000 and cb = 98,000.Transient Orange, This is conveniently prepared by cooling a thinlayer of rhodopsin solution to -70" and illuminating the solid from allsides.63 The freshly frozen solution is pink but it becomes orange onirradiation. At low temperatures transient orange is quite stable to light.After irradiation the material is allowed to reach room temperature incomplete darkness. The absorption spectrum is then determined and anapparent 50% regeneration of rhodopsin is recorded.The whole operationcan be repeated and there is then 25% regeneration expressed in terms ofthe original rhodopsin absorption. The formation of transient orange andregeneration can be observed several times until the E value a t ca. 500 mp.becomes very low. The maximum appears to be displaced some 8 mp. inthe direction of shorter wave-lengths after the first regeneration but remainsunchanged in subsequent regenerations. The regenerated product iscalled '' isorhodopsin " because it is not identical with rhodopsin. Theabsorption curves show that indicator yellow is formed in amounts corre-sponding with the rhodopsin which has disappeared. The quantitativedata are consistent with the following scheme := 49,3OOp,rhodopsin + nhv ---+ transient orangeisorhodopsin + nhv ___p transient orange*I F.D. Collins and R. A. Morton, Nature, 1949,164,528.2 transient orange --+ indicator yellow + isorhodopsin2 transient orange --+ etc.d8 Idem, ibid., in the pressMORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 265Freezing to -70" stops the sequence at the transient orange stage andwarming to room temperature in the dark isolates the thermal change.If the rhodopsin chromophoric group is(with many resonance forms)transient orange will be a free radical :rhodopsin + nhv 4 transient orange +and dismutation will result in half the transient orange molecules losinganother electron and forming indicator yellow. Ordinary rhodopsin isassumed to consist of two chromophoric groups attached to the same proteinmolecule and manifesting some mutual interaction so that Amax.is shiftedslightly. After the absorption of light and the subsequent dismutationof transient orange, one chromophoric group will have been converted intoindicator yellow leaving one isolated isorhodopsin chromophoric group.Subsequent dismutation of transient orange will be intermolecular and willresult in the continued formation of isorhodopsin.If, as has been suggested, the emax. value for rhodopsin is 48,OOOp andp = 2 the molecular extinction coeft3cient will be 96,000. It has previouslybeen shown that E,,,. x y = 24,000, where y is the quantum effi~iency.~~Hence the overall value of y will be 0.25. As the dismutation of transientorange reduced the quantum yield by SO%, y for the primary process willbe 0.5.Given that Emax. is 96,000 and the intensity of absorption forrhodopsin is about E',&. = 6.6 the upper limit for the carrier weight isabout 145,000. This is roughly half the molecular weight of 270,000 observedby Hecht and Pickels in 1938 and it supports the idea of two prostheticgroups in rhodopsin itself.Wald has founds5 that the photochemistry of rhodopsin is possiblymore complicated than is suggested above, and the detailed account of hisresults is awaited with interest. The interpretation suggested by Collinsand Morton should be regarded as EL first attempt a t a consistent scheme,it may need modification in detail, but there is no real conflict with Wald'sresults.Cone Pigments. The attempts to isolate cone pigments have not beenvery successful and some of the published evidence is technically questionable,but sinte rhodopsin accounts for the scotopic luminosity curve it is natural64 E. E. Schneider, C. F. Goodeve, and R. J. Lythgoe, R o c . Roy. Soc., 1939, A ,170, 102.Lecture at First International Congress of Biochemistry, 1949266 BIOCHE&5XSTRY.to postulate the existence of iodopsin to account for the photopic luminositycurve. Wald,66 using the weakest red light in which he could work, obtainedan aqueous digitonin extract of chicken retinas and measured the absorptionspectrum before and after exposure to light of wave-length 650 mp. Thedifference spectrum with Amax. at 575 mp. was attributed to iodopsin. Bliss 67carried out similar experiments and found A,,,, a t 560 mp. The destructionof iodopsin resulted in the liberation of retinene.The photopic luminosity curve is a reality-the problem is whether itis due to a single pigment, iodopsin, or to a summation of three modulatorcurves. Difference curves throw no light on this and the occurrence ofretinene as a “ bleaching ” product would fit either view. Wright 68determined the luminosity curve with a small foveal patch of low brightnessand found Amax. 560 mp. with an inflexion near 600 mp. confirmedthis by a different approach on the cat-dominator curve. A good case canbe made out that the photopic dominator is a synthesis of three modulatorcurves-recalling in some ways the Young-Helmholtz trichromatic theory.The experimental evidence leads to the view that the status of “ iodopsin ”is rather doubtful.One striking fact, however, remains, namely that as the key substancefor cone vision as well as rod vision, no alternative to vitamin A has emerged,and the narrow modulator curves are a persistent challenge. As is well-known, vitamin A and the retinenes give rise to deeply coloured blue orblue-green solutions with antimony trichloride in chloroform. The sharp-ness of the absorption bands (coupled with the transient nature of the bluecolours) suggests that under suitable conditions vitamin A or retinene mightgive rise to ionized or halochromic molecules 70 resembling Granit’smodulators.I n fact when vitamin A or retinene is dissolved in concentrated sulphuricacid or syrupy phosphoric acid a t temperatures near 0” coloured solutionsare formed which exhibit well-defined selective absorption with maximacorresponding closely with those of Granit’s modulators : 71Absorption Maxima in Strongly Ionizing Media.GranitConc. H,SO,. Conc. H,P04.Vitamin A, ......... 465, 520-530, 590-620 mp. 440-480, 520, 620Vitamin A, .... .....Retinene . . . . . . . . .Re tinene, . . . . . . . . .540, 560, 590, 660, 680, (720)450, 525, 570, 664470, 525, 570590 persistent, (695, transient)470, 500, 550, 590, 664470, 505, 570, 600 (at first)470, 505*, 560, 590 (after 2 hrs.) * Enhanced on storage.Granit’s modulators 450-465, 500, 520-530, 580-610.O6 G. Wald, Nature, 1937, 140, 546.67 A. F. Bliss, J . Gen. Phyxiol., 1946, 29, 277, 299.W. D. Wright, Nature, 1943, 161,6@ R. Granit, Proc. Physical SOC., 1945, 57, 447.‘O P. Meunier and A. Vinet, ‘‘ Chromatographie et MbomBrie,” 1947, Masson71 8. Ball and R. A. Morton, Biochem. J., 1949, 45, 298.et lie, ParisMORTON : CAROTENOIDS, VITAMIN A, AND VISUAL PIGMENTS. 267Although the conditions are not physiological, the agreement indicatesthat vitamin A and the retinenes are sufficiently versatile to permit theappearance of substances spectroscopically analogous to the modulators.The outstanding problems are (a) to produce a rhodopsin-like pigmentfrom the indicator-yellow analogues and (b) to produce the modulatoranalogues under conditions similar to those obtaining in the retina. Therecan be little doubt, however, that the evidence being collected by variousgroups of workers will lead to a unified picture. This is due in no smallpart to the labours of physiologists which are inadequately reported here,During the period covered by the Report the subject of vision sustained a,great loss in the death of Selig Hecht, who had done much pioneer workof great and lasting value.72 R. A. M.W. F. J. CUTHBERTSON.C. RIMINGTON.R. A. MORTON.Described in an obituary notice by G. Wald, J . Gen. PhysioZ, 1949, 32, 1