Synthesis and intramitochondrial levels of valproyl-coenzyme A metabolites.

A number of valproate adverse reactions are due to its interference with several metabolic pathways, including that of fatty acid oxidation. In order to resolve which mitochondrial enzymes of fatty acid oxidation are inhibited by which VPA intermediates we have developed methods to synthesize their CoA ester forms. This paper describes the synthesis of VPA acyl-CoA ester metabolites as well as data on the fate of VPA in rat liver mitochondria. Valproyl-CoA, Delta2-valproyl-CoA, and 3-OH-valproyl-CoA were obtained through chemical synthesis. 3-Keto-valproyl-CoA was prepared by a novel enzymatic procedure followed by a combination of solid-phase extraction and preparative HPLC purification. This approach proved to be efficient in obtaining all the beta-oxidation intermediates of valproyl-CoA. The synthetic standards were used for the determination of intramitochondrial concentrations of valproyl-CoA, Delta2-valproyl-CoA, 3-OH-valproyl-CoA, and 3-keto-valproyl-CoA by HPLC. These levels were determined after incubation of intact rat liver mitochondria with VPA under conditions of state 3 and state 4 respiration. The results show that valproyl-CoA and to a much lesser extent 3-keto-valproyl-CoA are the main metabolites of VPA in mitochondria. This information will be of great use in resolving the mechanisms involved in the inhibition of mitochondrial processes like fatty acid oxidation by VPA.