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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1974|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology