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 B,t 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(Znitrobenzoic acid) measurement of the liberated coenzyme A. Methylmalonyl-CoA was measured by coupling the succinyl-CoA assay with methylmalonyl-CoA mutase. ‘Ihe 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 B1*-deprived animals than in the controls. As anticipated by the site of the known metabolic action of coenzyme Blz, methylmalonyl-CoA levels were also increased in the Blz-deprived animals, but were only 12fold greater in the Bls-deprived than in the controls. Unexpectedly, succinyl-CoA levels were found to be on an average 4-fold greater in the livers of the Blz-deprived animals than in the controls. In individual liver samples the propionyl-CoA to methyhnalonyl-CoA ratio was approximately 2:l. Acetyl-CoA levels were not reduced by the presence of increased endogenous propionyl-CoA, but were actually increased in livers from the Blz-deprived group. Study of selected Krebs’ tricarboxylic acid cycle intermediates showed increased malate and normal citraie levels in the livers of the Blz-deprived group, yielding a Z-fold increase in the malate to citrate ratio. ‘Ihus, the present study provides a method of measurement of the CoA intermediates in the B12-dependent pathway in the intact liver and demonstrates that Blz deficiency results in an increase in propionylCoA and methylmalonyl-Cob as well as in succinyl-CoA, an intermediate beyond the site of action of coenzyme B~z.