The Influence of Vitamin B12 on the Content, Distribution and in Vivo Synthesis of Thiamine Pyrophosphate, Flavin Adenine Dinucleotide and Pyridine Nucleotides in Rat Liver

Abstract

Rats depleted of their vitamin B₁₂ reserves were used to study the effects of dietary vitamin B₁₂ on (1) the content and distribution in liver cytoplasm of thiamine pyrophosphate (TPP) and free thiamine, flavin adenine dinucleotide (FAD) and non-FAD riboflavin (FMN + free riboflavin) and total pyridine nucleotides (PN), and (2) the in vivo synthesis of these cofactors from the respective vitamins. In the normal, stock diet-fed animals, about 30 % of total liver TPP was localized in the mitochondria while the rest was found almost exclusively in the supernatant fraction (including microsomes). Free thiamine was distributed between the supernatant (65%) and the nuclear fraction (23%). About 60% of total liver FAD content was associated with mitochondria while about 55% of the total non-FAD content was contained in the supernatant fraction, the balance in either case being almost equally distributed between the remaining two fractions. A major portion (60%) of the total liver PN was localized in the supernatant; the nuclear fraction also contained appreciable amounts (25%). The liver content of the cofactors was markedly affected in vitamin B₁₂ deficiency with average reductions of 45, 48 and 64%, respectively, in TPP, FAD and PN. The effects were largely confined to the mitochondrial fraction and were not accompanied by corresponding changes in the content of the free vitamins. A decrease in oxidized pyridine nucleotides in liver homogenates with proportional lowering of the ratio of oxidized to reduced pyridine nucleotides (PN/PNH) was observed. The incorporation of injected thiamine, riboflavin and nicotinamide into the liver coenzymes was impaired in vitamin B₁₂ deficiency. The impairment in TPP and PN synthesis was reflected to a greater extent in the nuclear fraction than in other fractions, while that in FAD synthesis was seen to an almost equal extent in all fractions. In the vitamin B₁₂-deficient animal there was also appreciable incorporation of administered vitamins into their cofactors; the concentration of the free forms of the vitamins was, however, relatively greater. The results, suggesting an impairment in the biosynthesis of these cofactors in vitamin B₁₂ deficiency, are discussed. Conditions arising out of possible damage to mitochondrial integrity, as well as the effects of the vitamin in relation to the mode and site of synthesis of the cofactors, are discussed.