To clarify the relationship between intracellular concentrations of methylmalonic acid and metabolic and growth inhibition in vitamin B,,deficient rats, hepatic methylmalonic acid levels were assayed and inhibition of glucose and glutamic acid metabolism by methylmalonic acid was studied in isolated hepatocytes. Vitamin B,,deficient rats (14 weeks old) excreted more urinary methylmalonic acid and had lower body weights than the control rats. Hepatic methylmalonic acid levels (3-6 (SD 1.30)-5.3 (SD 0.51) pmol/g tissue; 7.9 (SD 290)-11-8 (SD 1-14) mM) were increased and correlated with the extent of the growth retardation during vitamin B,,deficiency. Isolated hepatocytes and mitochondria from normally fed rats were labelled with ["Cc(Ullglucose and [14C(LJ)]glutamic acid respectively, in the presence or absence of Sm-methylmalonic acid. Although methylmalonic acid did not atTect the incorporation of "C into protein and organic acid fractions in the hepatocytes, it inhibited 14C02 formation (an index of glucose oxidation by the Krebs cycle) by 25 % and incorporation of I4C into the amino acid fraction by 30%. In the mitochondria, methylmalonic acid inhibited "CO, formation (indicating glutamic acid oxidation by the Krebs cycle) by 70%, but not the incorporation of I4C into the protein fraction. The incorporation of I4C into the organic acid fraction was significantly stimulated by the addition of methylmalonic acid. These results indicate that the unusual accumulation of methylmalonic acid caused by vitamin B,,deficiency disrupts normal glucose and glutamic acid metabolism in rat liver, probably by inhibiting the Krebs cycle.
Mammalian livers have been reported to contain NADH- and NADPH-linked aquacobalamin reductases, which are distributed in both mitochondria and microsomes. The four aquacobalamin reductase isozymes have been purified and characterized from rat liver. It is unclear which aquacobalamin reductase among the four reductase isozymes participates in the synthesis of cobalamin coenzymes. To clarify the physiological roles of the aquacobalamin reductase isozymes, human mutant fibroblasts (cblC and cblA cells) with defects in cobalamin reductases involved in the coenzyme synthesis were used. In the cblC cells, the activity of the mitochondrial NADH-linked aquacobalamin reductase was reduced significantly, compared with normal human fibroblasts but the mitochondrial NADPH-linked enzyme was not. The reduced specific activity of the NADH-linked enzyme was not due to reduction in levels of the enzyme, but in its affinity for NADH. Although there was not a significant difference in the mitochondrial NADH-linked enzyme activity between normal and cblA cells, the activity of the mitochondrial NADPH-linked enzyme was not detectable in the mutant cells. These results indicate that the defects in the mitochondrial NADH- and NADPH-linked aquacobalamin reductases underlie cblC and cblA disorders, respectively.
Rat liver contains both NADH- and NADPH-linked aquacobalamin reductases, which are involved in the synthesis of the vitamin B-12 coenzymes and are distributed in both the mitochondrial and microsomal membranes. To clarify the physiological roles of these hepatic enzymes, vitamin B-12-deficient rats were used to study the effect of the deficiency on the enzyme activities. Male rats fed a vitamin B-12-deficient diet for 11 wk developed a severe vitamin B-12 deficiency with a high urinary methylmalonate excretion (214.3 +/- 115.2 mumol/d) and approximately 96% lower hepatic vitamin B-12 content. Tissues of the vitamin B-12-deficient rats were assayed for NADH- and NADPH-linked aquacobalamin reductase activities. The specific activities of both enzymes in homogenates of liver, kidney or upper intestine were shown to be three- to 20-fold greater in the vitamin-deficient rats than in the control rats. In liver, the vitamin deficiency specifically elevated the specific activities of the mitochondrial NADH-linked and microsomal NADPH-linked enzymes. These are likely the isozymes involved in vitamin B-12 coenzyme synthesis.
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