Effect of Vitamin B12 Deprivation on the in Vivo Levels of Coenzyme A Intermediates Associated with Propionate Metabolism

The in vivo coenzyme A intermediates involved in the metabolic pathway in which vitamin B12 serves as a coenzyme (conversion of methylmalonyl-CoA to succinyl-CoA) were measured in the livers of control and B12-deprived animals. Succinyl-CoA was assayed by the succinate thiokinase arsenolysis of succinyl-CoA coupled with 5',5'-dithiobis(2-nitrobenzoic acid) measurement of the liberated coenzyme A. Methylmalonyl-CoA was measured by coupling the succinyl-CoA assay with methylmalonyl-CoA mutase. The assays were shown to be specific and reproducible and provided excellent recovery of added exogenous coenzyme A derivatives. Utilizing the in situ freeze-clamp technique, the mean liver propionyl coenzyme A levels were approximately 17-fold greater in the B12-deprived animals than in the controls. As anticipated by the site of the known metabolic action of coenzyme B12, methylmalonyl-CoA levels were also increased in the B12-deprived animals, but were only 12-fold greater in the B12-deprived than in the controls. Unexpectedly, succinyl-CoA levels were found to be on an average 4-fold greater in the livers of the B12-deprived animals than in the controls. In individual liver samples the propionyl-CoA to methylmalonyl-CoA ratio was approximately 2:1. Acetyl-CoA levels were not reduced by the presence of increased endogenous propionyl-CoA, but were actually increased in livers from the B12-deprived group. Study of selected Krebs' tricarboxylic acid cycle intermediates showed increased malate and normal citrate levels in the livers of the B12-deprived group, yielding a 2-fold increase in the malate to citrate ratio. Thus, the present study provides a method of measurement of the CoA intermediates in the B12-dependent pathway in the intact liver and demonstrates that B12 deficiency results in an increase in propionyl-CoA and methylmalonyl-CoA as well as in succinyl-CoA, an intermediate beyond the site of action of coenzyme B12.