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 T H le One 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. 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