The Influence of Vitamin B12on the Content, Distribution and in Vivo Synthesis of Thiamine Pyrophosphate, Flavin Adenine Dinucleotide and Pyridine Nucleotides in Rat Liver1
URMILA MARFATIA, D. V. REGE, H. P. TIPNIS ANDA. SREENIVASAN Department of Chemical Technology, University of Bombay, Matunga, Bombay
Apart from the well-known interrelation (FAD) and pyridine nucleotides (PN), and ships among the B vitamins, there is rea to their in vivo synthesis from the corre
son to believe that folie acid and vitamin sponding administered vitamins, in the rat Downloaded from Bu may influence the functioning of other liver. Data on the distribution of these vitamins as cofactors. Thus, dietary folie cofactors in liver cells of the normal rat acid has been known to determine rat are available in the works of Goethart ('52) liver stores of coenzyme A (CoA) and and Dianzani and Dianzani Mor ('57) on adenotriphosphate (ATP) (Popp and Tot TPP, of Carruthers and Suntzeff ('54) and ter, '52; Totter, '53); a decrease in liver Dianzani ('55) on pyridine nucleotides DPN is also caused by aminopterin2 (PN) and of Schneider and Hogeboom jn.nutrition.org (Strength et al., '54). The in vivo incor (Schneider, '56) on FAD. poration of nitcotinamide into pyridino- nucleotides in rat liver is affected in a EXPERIMENTAL deficiency of vitamin B«(Nadkarni et al., Young, male Wistar rats weighing ap '57). Low blood level of citrovorum factor proximately 100 gm each were used. The by guest on April 19, 2011 in the hyperthyroid, vitamin Bi2-deficient animals, housed individually in raised rat is corrected by administration of vita mesh-bottom cages, were initially depleted min B«(Pfander et al., '52). In liver ho- of their vitamin B!2 reserves by mainte mogenates from vitamin Bu-deficient hens, nance on a purified, iodo-casein ration. the synthesis of citrovorum factor from This consisted of the following percentage added folie acid is less than in those from composition : hot, alcohol-extracted casein, animals injected with the vitamin (Doctor 18; iodinated casein,3 0.15; arachis oil, 6; et al., '54). The potentiating effect of vita shark liver oil, 2; sucrose, 9.85; maize min Bu in the mobilization of folie acid starch 60; and salt mixture (U.S.P. XIV), has also been reported from this laboratory 4; with vitamins to provide in milligrams (Sreenivasan, '51; Fatterpaker et al., '55a). per kilogram of diet: thiamine-HC1, 6; ribo- The general influence of vitamin Bis on flavin, 10; nicotinic acid, 30; calcium pan- carbohydrate and lipid metabolism has tothenate, 20; pyridoxine-HCl 6; biotin, been linked to a primary relation to sul- 1; folie acid, 5; p-aminobenzoic acid, 100; phydryl biosynthesis (Ling and Chow, '54; choline-Cl, 500; inositol, 500; 2-methyl-l, Register, '54; Kasbekar et al., '56, '59a). 4-naphthoquinone, 10; and a-tocopherol, Distinguished from these apparently col lateral findings is the reported elevation of Received for publication August 5, 1959. CoA in livers of vitamin Bu-deficient rats 1This work was supported by a research grant and mice (Boxer et al., '53, '55; Wong and from the Williams-Waterman Fund, Research Corporation, New York. Schweigert, '56). 1 Strength, D. R., and N. I. Mondy 1953 Che- The present work relates to a study of line dehydrogenase activity and DPN content of the influence of vitamin Biz on the intra- rat livers following aminopterin injection. Fed eration Proc., 12: 276 (abstract). cellular distribution of thiamine pyrophos- 3Protomone, obtained from Cerophyl Labora phate (TPP), flavin adenine dinucleotide tories, Kansas City, Mo.
J. NUTBITION, 70: '60 283
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53QO-SQE-W52E 284 URMILA MARFATIA AND OTHERS
50. The vitamin additions provided in this for 18 hours at 37°C,the TPP content basal diet were considered adequate for being calculated by difference. The total the hyperthyroid condition. At the end of and non-FAD (flavin mono nucleotide, 4 weeks, animals were divided into two FMN plus free riboflavin) riboflavin were groups, one of which continued to receive determined by the fluorometric procedure the basal diet modified by the omission of of Bessey et al. ('49) and the FAD content iodinated casein and p-aminobenzoic acid derived from these values with use of and substitution by 2% of succinyl sulpha- proper conversion factor, as indicated by thiazole. The latter addition was compen these authors. The determination of total sated for by adjusting the percentage of PN in homogenates and in fractions, as starch. The second, control group, received well as the differential determination of the modified diet with a supplement of their oxidized and reduced forms in whole vitamin Bu (150 ug/kg of diet). After a homogenates, was carried out fluoro- further 5-week period, the animals in both metrically by the procedure outlined by groups were divided into 4 sub-groups of Dian/ani ('55). 7 to 8 rats each, one of which was killed For determinations of blood erythrocyte immediately to establish the liver distribu count and hemoglobin content and of
tion pattern of the cof actors in the vitamin plasma vitamin Bi2 concentration, the ani Downloaded from Biz-deficient and replete states. The re mals were bled from tail veins and ade maining three sub-groups of each group quate samples collected in heparinated were used to study the synthesis of vials. The erythrocyte count was made by each of the three cof actors from the respec the standard method. Hemoglobin was de tive, administered vitamins. The rats were termined by acid-hematin method in a injected, intraperitoneally, on 5 successive Klett-Summerson photoelectric colorimeter days, with 5 mg of thiamine-HCl, 5 mg (Kolmer et al., '51). Plasma vitamin Bu jn.nutrition.org of riboflavin, or 1 mg of nicotinamide per was determined by the method of Ross rat per day. All animals were killed on the ('52) using Euglena gracilis as the test 5th day, 8 hours following the final injec organism. tion of the test-vitamin. Portions of liver homogenates were incu The normal intracellular distribution bated at 37°Cfor 12 hours under toluene by guest on April 19, 2011 pattern of the cofactors was secured on with papain (25 mg/gm of fresh liver) to liver of 10 adult male rats weighing ap liberate the bound vitamin Bu, which was proximately 200 gm, maintained on the assayed using E. gracilis according to the laboratory stock diet consisting of (gm method of Hoff-Jorgenson ('54). per 100 gm of diet): whole wheat Hour, The results were analyzed for statistical 75; whole milk powder, 2; casein, 12; dried significance by calculating the t value of yeast, 2; arachis ou, 3; shark liver on, 2; Fisher (Fisher and Yates, '53). Only the sodium chloride, 2; and calcium carbonate, differences with a t value corresponding 2. to a probability P < 0.05 were accepted as Animals were exsanguinated and livers significant. were perfused with ice-cold 0.25 M sucrose, promptly excised, blotted and madd into RESULTS 10% homogenates with 0.25 M sucrose in Content and distribution of cofactors a Potter-Elvehjem homogenizer. The ho and free vitamins in normal liver cyto mogenates were separated by the differ plasm. The data obtained with liver ho ential centrifugation procedure of Schnei mogenates of normal, stock diet-fed ani der and Hogeboom ('50) into nuclear, mito- mals are reported in table 1. About 30% chondrial and microsomal plus super of the TPP of the homogenate was found natant fractions using an International contained in mitochondria, while the rest (PR-2) refrigerated centrifuge. Total and was present almost exclusively in the super free thiamine in portions of whole homog natant. The distribution pattern compares with data reported by others (Goethart, enates and fractions were determined by '52; Dianzani and Dianzani Mor, '57) un a modification of the fluorometric method of Hennessy ('41) before and after hydrol der similar conditions. Free thiamine, also ysis of TPP complexes with taka-diastase maximally localized in the supernatant O>IO00 ini-i oÕ01à COrH +lg +1+1in rH•fiO5CO•i•fii00ood•fico SOl COM ^S -LI» 1(503*»HCO£ S*"*t
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The ing that of TPP (P < 0.05). effects of vitamin Bu deficiency are sum The distribution pattern of FAD differed marized as follows: (a) there was a from that of TPP, in its greater association marked reduction in the liver content of with the mitochondria than with the super the cofactors studied, being about 44% natant fraction. The proportion of FAD for TPP, 48% for FAD and 64% for total found in association with the mitochondria PN; (b) the mitochondria exhibited the was similar to that reported (about 65% ) largest depletion of the cytoplasm frac by Schneider and Hogeboom (Schneider, tions. The reductions were 75, 66 and '56). Although about 60% of the non-FAD 75% , respectively, for TPP, FAD and PN. riboflavin was contained in the super Significant reductions in the concentra natant, the proportion associated with tions of these cofactors were also apparent mitochondria was small as compared with in the other two fractions, especially in the FAD content of this fraction. the supernatant; (c) the content and dis A major fraction of the total liver PN tribution of free thiamine remained es was localized in the supernatant, in con sentially unaltered. With non-FAD ribo firmation of earlier observations (Car- flavin, there was small but significant ele Downloaded from ruthers and Suntzeff, '54; Dianzani, '55). vation in both nuclear (P < 0.005) and A significant proportion was, however, con supernatant (P < 0.05) fractions, the total tained in the nuclear fraction as well. being not significantly altered; and (d) the Alterations in vitamin B« deficiency. observed decrease in the proportion of The data on blood erythrocyte count and oxidized to reduced forms of pyridine nu- hemoglobin concentration and especially cleotides (PN/PNH ratio) in livers of vita jn.nutrition.org on plasma and liver content of vitamin B>2 min Bi2-deficient animals (table 6) is in (table 2), apart from observations on accordance with the findings of Nadkarni growth noted below, connote the severity of et al., '57). It is of interest to note that the deficiency attained. During the 5-week the changes in PNH content were much period for which the animals were main less pronounced in vitamin Bu deficiency. by guest on April 19, 2011 tained on the succinyl sulphathiazole- con Effect of -vitamin B¡2onincorporation of taining diets, the vitamin B^-deficient ra administered vitamins into liver cofactors. tion promoted an average gain in body Tables 3 to 5 also include the results of weight of 8 gm/week compared with an experiments on the conversion by liver average of 22 gm using the control ration enzymes in vivo of the administered vita with vitamin En (table 2). Data on blood mins into their respective cofactors. In erythrocyte count and hemoglobin concen corporation of thiamine, riboflavin and tration and especially on plasma and liver nicotinamide into their coenzyme forms is content of vitamin Bi2 (table 2) also con considerably less in the vitamin Bi2-defi- TABLE 6 Effect of vitamin Bu on oxidized and reduced PN in liver homogenstes Oxidized Reduced No. of pyridine pyridine Group rats nucleotides nucleotides PN/PNH (PN) (PNH) Normal 10 639 ±22' 206 ±9 3.10 ±0.09 Vitamin Bn-deficient 8 237 ±18 155 ±5 1.53 ±0.11 Vitamin Bi2-fed 8 476 ±23 165 ±6 2.88 ±0.13 Vitamin Bi2-deficient—• injectedVitaminnicotinamide ±26809 ±7250 ±0.143.24 Bi2-fed nicontinamide injected78343 ±19221 ±111.55 ±0.19 1Standard error of mean. VITAMIN BIÃŽ AND LIVER COFACTORS 289 cient animals, although the extent of in partial displacement from the mitochon corporation is appreciable even in this drial into the supernatant fraction, where group. A relatively greater proportion of DPNase is very active. Since both vitamin free thiamine and of non-FAD riboflavin Biz and choline could exert similar lipo- was observed in the deficient group. The tropic effects, it is probable that the same PN/PNH ratio was not significantly altered types of causes operate in either deficiency. as a result of nicotinamide administration, A rapid depletion of rat liver and its the difference between the deficient and mitochondrial vitamin Bi2 could result control groups being maintained (table 6). from hyperthyroidism (Kasbekar et al., The decreased ability in vitamin Biz de '59a) and acute carbon tetrachloride poi ficiency of the liver enzymes for conver soning (Kasbekar et al., '59b). These con sion of the administered vitamins into ditions are known to cause morphological their respective cof actors is reflected in all damage to mitochondrial structure with of the cytoplasmic fractions and is largely consequent displacement of intramitochon- seen in the nuclear fraction for TPP and drial constituents into the surrounding me PN and in the mitochondrial fraction for dium (Dianzani, '54, '55; Dianzani and FAD. The deficient animals showed higher Dianzani Mor, '57; Maley and Lardy, '55; gains of free thiamine in the nuclear and Kasbekar and Sreenivasan, '56). The pro Downloaded from mitochondrial fractions, whereas the dif tection afforded by prior administration of ferences with respect to non-FAD riboflavin vitamin B)2under these conditions of stress were confined to the supernatant fraction. (Fatterpaker et al., '55b; Kasbekar et al., '59a, '59b) could arise from its general DISCUSSION lipotropic effect and from its known in The present observations demonstrate fluence on sulphydryl conservation which, an impairment in the retention of TPP, in turn, is essential for maintenance of jn.nutrition.org FAD and PN in the vitamin Bi2-deficient mitochondrial integrity (Tapley, '56; Hun rat liver, as well as in their biosynthesis ter et al., '56). It has also been suggested from the corresponding vitamins adminis that vitamin Bi2 may be necessary for the tered. In general, the observed effects synthesis of porphyrin-containing proteins point to greater depletion of cof actors from of the cell (O'Dell et al., '55) which, apart by guest on April 19, 2011 mitochondria than from other fractions. from their function as respiratory carriers, If these changes are not always reflected in are apparently also of importance in deter liver levels of the free vitamins, it may be mining mitochondrial morphology (Gam because of some losses through excretion; ble, '57). The observed alterations in mito this may also imply a decreased use of the free vitamins for synthesis of coen- chondrial cofactors in vitamin B]2 de zymes. Dianzani ('55) and Dianzani and ficiency may thus have an important Dianzani Mor ('57) have reported similar though indirect, bearing on this function of the vitamin in the maintenance of mito modifications in the distribution of TPP chondrial organization in so far as the and PN in mitochondria from fatty livers caused by choline deficiency or CC14poison nucleotidation and phosphorylation reac tions involved in the synthesis of cofactors ing. As discussed by these authors, such are ATP-dependent and any structural changes could result from decreased rates of synthesis of the cofactors or their in damage to mitochondria renders the syn thesis of ATP inoperative (Kielley and creased degradation or from both causes. Kielley, '51; Dianzani, '54). Thus it is possible that the synthesis of TPP through phosphorylation of thiamine The effect of vitamin Bi2 also has to be and of DPN through the Kornberg reaction assessed in terms of its known relationship is diminished in vivo as a consequence of to nucleotide biosynthesis. Although the the reduced concentration of ATP in fatty nature of this relationship is obscure, evidence exists to suggest its involvement livers; increased decomposition of TPP and in the biosynthesis of both ribose4 and de- DPN may be favored by increased acid phosphatase activity and through pyrophos- 4Ling, C. T., and B. F. Chow 1954 Effect of phorolytic cleavage, respectively. Increased vitamin Biz on ribose formation in erythrocytes. degradation of PN may also occur through Federation Proc., 13: 253 (abstract). 290 URMILA MARFATIA AND OTHERS oxyribose (Downing and Schweigert, '56; SUMMARY Wong and Schweigert, '57) moieties of Rats depleted of their vitamin Bu re nucleic acids. serves were used to study the effects of It is interesting to note that the injec dietary vitamin Ea on (1) the content and tion of a vitamin could enhance the liver distribution in liver cytoplasm of thiamine content of the corresponding cofactor de pyrophosphate (TPP) and free thiamine, spite dietary adequacy of the vitamin con flavin adenine dinucleotide (FAD) and cerned. Kaplan and coworkers ('56) had non-FAD riboflavin (FMN + free ribo observed a 10-fold increase in liver PN fol flavin) and total pyridine nucleotides lowing administration of nicotinamide into (PN), and (2) the in vivo synthesis of normal mice. these cofactors from the respective vita According to Hogeboom and Schneider mins. ('52), the DPN synthesis is localized in the In the normal, stock diet-fed animals, liver cell nucleus. In the vitamin B«- about 30 % of total liver TPP was localized deficient rats, the extent of impairment in in the mitochondria while the rest was PN synthesis from administered nicontin- found almost exclusively in the super amide is more pronounced in the nuclear natant fraction (including microsomes). fraction (table 5). A similar impairment Free thiamine was distributed between the Downloaded from in TPP synthesis from administered thia- supernatant (65% ) and the nuclear frac mine is again better reflected in the nuclear tion (23% ). About 60% of total liver fraction than in the mitochondria and FAD content was associated with mito least in the supernatant fraction (table 3); chondria while about 55% of the total non- this nuclear impairment is accompanied FAD content was contained in the super by an appreciable rise in free thiamine in natant fraction, the balance in either case this fraction, suggesting that TPP, like being almost equally distributed between jn.nutrition.org DPN, may be synthesized in the liver nu the remaining two fractions. A major por cleus from thiamine, a process susceptible tion (60% ) of the total liver PN was local to vitamin Biz-deficiency. ized in the supernatant; the nuclear frac On the other hand, the impairment in tion also contained appreciable amounts FAD synthesis from riboflavin administered (25% ). by guest on April 19, 2011 to the vitamin Bu-deficient rat (table 4) is The liver content of the cofactors was better reflected in the mitochondrial frac markedly affected in vitamin En deficiency tion. The increase in the proportion of with average reductions of 45, 48 and non-FAD riboflavin in the vitamin Bis- 64%, respectively, in TPP, FAD and PN. deficient animals, with or without ribo The effects were largely confined to the flavin administration, is, however, con mitochondrial fraction and were not ac fined mainly to the supernatant fraction. companied by corresponding changes in The synthesis of FAD from FMN and ATP the content of the free vitamins. A de is also localized in the supernatant frac crease in oxidized pyridine nucleotides in tion (Schneider, '56). liver homogenates with proportional lower An increase in the proportion of re ing of the ratio of oxidized to reduced pyri duced PN in fatty livers (Dianzani, '55) dine nucleotides (PN/PNH) was observed. and in those from vitamin Bu-deficient rats The incorporation of injected thiamine, (Nadkarni et al., '57) has been reported. riboflavin and nicotinamide into the liver Such a condition may provoke predom coenzymes was impaired in vitamin Bu ination of fatty acid synthesis as compared deficiency. The impairment in TPP and with breakdown (Lynen, '54). The de PN synthesis was reflected to a greater ex creased content (table 6) of oxidized PN tent in the nuclear fraction than in other in vitamin B«deficiency (Nadkarni et al., fractions, while that in FAD synthesis was '57) may point to its selective destruction seen to an almost equal extent in all frac by the DPNase (Mcllwain and Rodnight, tions. '49; Zatman et al., '53) contained in mito In the vitamin Biz-deficient animal there chondrial and supernatant fractions, as was also appreciable incorporation of ad well as to decreased synthesis. ministered vitamins into their cofactors; VITAMIN Biz AND LIVER COFACTORS 291 the concentration of the free forms of the Analysis, vol. 1. Interscience Publishers Inc., vitamins was, however, relatively greater. New York, p. 81. Hogeboom, G. H., and W. C. Schneider 1952 The results, suggesting an impairment Cytochemical Studies. VI. The synthesis of in the biosynthesis of these cofactors in diphosphopyridine nucleotide by liver cell nu vitamin Bu deficiency, are discussed. Con clei. J. Biol. Chem., 197: 611. ditions arising out of possible damage to Hunter, Jr., F. E., J. Davis and L. Cariât 1956 The stability of oxidative and phosphorylative mitochondrial integrity, as well as the systems in mitochondria under anaerobic con effects of the vitamin in relation to the ditions. Biochim. Biophys. Acta, 20: 237. mode and site of synthesis of the cofactors, Kaplan, N. O., A. Goldin, S. R. Humphreys, M. M. are discussed. Ciotti and F. E. Stolzenbach 1956 Pyridine nucleotide synthesis in the mouse. J. Biol. LITERATURE CITED Chem., 219: 287. Bessey, O. A., O. H. Lowry and R. H. Love 1949 Kasbekar, D. K., and A. Sreenivasan 1956 Labil The fluorometric measurement of the nucleo- ity of intramitochondrial components in ex tides of riboflavin and their concentration in perimental liver injury. Nature, 178: 989. tissues. J. Biol. Chem., 380: 755. Kasbekar, D. K., D. V. Rege and A. Sreenivasan Boxer, G. E., W. H. Ott and C. E. Shonk 1953 1956 Protective action of vitamin Bi2 in ex Influence of vitamin Biz on the coenzyme A perimental liver injury. Ibid., 378: 989. content of the liver of chicks. Arch. Biochem. Kasbekar, D. K., W. V. Lavate, D. V. Rege and A. Sreenivasan 1959a A study of vitamin Biz Biophys., 47: 474. Downloaded from Boxer, G. E., C. E. Shonk, E. W. Gilfillan, G. A. protection in experimntal thyrotoxicosis in the Emerson and E. L. Oginsky 1955 Changes in rat. Biochem. J., 72: 374. coenzyme A concentration during vitamin Tin 1959b A study of vitamin Biz protec deficiency. Ibid., 59: 24. tion in experimental liver injury to the rat Carruthers, C., and V. Suntzeff 1954 The dis by carbon tetrachloride. Ibid., 72: 384. tribution of pyridine nucleotides in cellular Kielley, W. W., and R. K. Kielley 1951 Myo- fractions of some normal and malignant tissues. kinase and adenosinetriphosphatase in oxidative Cancer Res., 14: 29. phosphorylation. J. Biol. Chem., 193: 485. jn.nutrition.org Dianzani, M. U. 1954 Uncoupling of oxidative Kolmer, J. A., E. H. Spaulding and H. W. Robin phosphorylation in mitochondria from fatty son 1951 Approved Laboratory Technic, ed. livers. Biochim. Biophys. Acta, 14: 514. 5, Appleton-Century-Crofts, New York. 1955 Content and distribution of pyri Ling, C. T., and B. F. Chow 1954 The influence dine nucleotides in fatty livers. Ibid., 17: 391. of vitamin Biz on carbohydrate and lipide Dianzani, M. U., and M. A. Dianzani Mor 1957 metabolism. J. Biol. Chem., 206: 797. by guest on April 19, 2011 Displacement of thiamine pyrophosphate from Lynen, F. 1954 Participation of coenzyme A in swollen mitochondria. Ibid., 24: 564. the oxidation of fat. Nature, 374: 962. Doctor, V. M., J. F. Khun, P. Sparks, C. M. Lyman Mcnwain, H., and R. Rodnight 1949 Break and J. R. Couch 1954 Studies on the con down of oxidized forms of coenzymes I and II version of folie acid to citrovorum factor by by an enzyme from the central nervous system. avian liver homogenate. Arch. Biochem. Bio Biochem. J., 45: 337. phys., 48: 249. Maley, G. F., and H. A. Lardy 1955 Efiiciency Downing, M., and B. S. Schweigert 1956 Role of phosphorylation in selected oxidations by of vitamin Biz in nucleic acid metabolism. IV. Metabolism of CI4-labeled thymidine by Lacto- mitochondria from normal and thyrotoxic rat livers. J. Biol. Chem., 235: 377. bacillu$ leichmannii. J. Biol. Chem., 220: 521. Nadkarni, G. B., D. S. Wagle and A. Sreenivasan Fatterpaker, P., U. Marfatia and A. Sreenivasan 1957 Vitamin B«and the metabolism of pyri 1955a Role of folie acid and vitamin Biz in dine nucleotides. Nature, 380: 659. transmethylations. I. Formation of creatine O'Dell, B. L., J. S. Gordon, J. H. Bruemmer and in vitro and in vivo. Ind. J. Med. Res., 43: 43. A. G. Hogan 1955 Effect of a vitamin Bi2 1955b Protective action of vitamin Bi2 deficiency and of fasting on oxidative enzymes in the hyperthyroid rat. Nature, 176: 165. in the rat. J. Biol. Chem., 237: 1955. Fisher, R. A., and F. Yates 1953 Statistical Tables for Agricultural, Biological and Medical Pfander, W. H., L. S. Dietrich, W. J. Monson, Research. Oliver and Boyd, Edinburgh. A. E. Harper and C. A. Elvehjem 1952 Citro Gamble, Jr., J. L. 1957 Aggregation of mito vorum factor, vitamin Biz, and folie acid activ chondria, mitochondrial fragments, and micro- ity of whole blood of several species. Proc. Soc. somes by cytochrome C. Biochim. Biophys. Exp. Biol. Med., 79: 219. Acta, 23: 306. Popp, E. M., and J. R. Totter 1952 Studies on Goethart, G. 1952 Thiamine pyrophosphate in the relationship between folie acid and co fractions from rat-liver homogenates—the in enzyme A. J. Biol. Chem., 399: 547. fluence of thiamine deficient diet. Ibid., 8: 479. Register, U. D. 1954 Effect of vitamin Bn Hennessy, D. J. 1941 Chemical methods for the on liver and blood non-protein sulphydryl com determination of vitamin Bi. Ind. Eng. Chem., pounds. Ibid., 206: 705. (Anal, ed.), 13: 216. Ross, G. I. M. 1952 Vitamin B«assay in body Hoff-Jorgensen, E. 1954 Microbiological assay fluids using Euglena gracilis. J. Clin. Pathol., of vitamin Bis. In Methods of Biochemical 5: 250. 292 URMILA MARFATIA AND OTHERS Schneider, W. C. 1956 Structural factors in Tapley, D. F. 1956 The effect of thyroxine and metabolic regulations. Proc. 3rd Int. Congr. other substances on the swelling of isolated rat Biochem., Brussels, 1955. Academic Press, New liver mitochondria. J. Biol. Chem., 222: 325. York, p. 305. Totter, J. R. 1953 The biological function of Schneider, W. C., and G. H. Hogeboom 1950 pteridine derivatives. J. Cell, and Comp. Iiitr.u ellular distribution of enzymes. V. Fur Physiol., Suppl. 1, 41: 241. ther studies on the distribution of cytochrome Wong, W. T., and B. 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