Valproyl-dephosphocoa: A novel metabolite of valproate formed in vitro in rat liver mitochondria

The mitochondrial metabolism of valproic acid (VPA) was investigated in vitro to elucidate its beta-oxidation pathway since the characterization of VPA intermediates in the acyl-CoA thioester form, and not just in their free acid form, has not been fully achieved. Intact rat liver mitochondria were incubated with [4,5-H-3(2)] VPA and [2-H-3] VPA. The respective intermediates, valproyl- CoA, Delta(2(E))-valproyl-CoA, 3-hydroxyvalproyl-CoA, and 3-oxovalproyl-CoA were analyzed by reverse phase high performance liquid chromatography ( HPLC) with radioisotope and UV detection. An unknown metabolite, originating from both labeled substrates, was detected. It was identified as valproyl- dephosphoCoA (valproyl-dephCoA) by fast atom bombardment mass spectrometry (FAB-MS) analysis of the corresponding HPLC peak fraction. The FAB-MS spectrum of the authentic chemically synthesized valproyl-dephCoA proved to be consistent with that of the unknown compound. Valproyl-deph-CoA is produced from valproyl-CoA in mitochondria, probably via a phosphatase-catalyzed reaction. This conversion was shown to be more dependent on the energy state involving [AXP] ([ AXP] = [ATP] + [ ADP] + [ AMP]) and [ phosphate] concentrations rather than the strict mitochondrial [ ATP]/[ ADP] ratio. The results indicate that higher concentrations of AXP and phosphate inhibit the dephosphorylation of valproyl-CoA. A complete understanding of the toxic significance of valproyl-dephCoA formation in vivo as a potential inhibitor of fatty acid beta-oxidation is important to clarify the pathogenesis of VPA-associated hepatotoxicity.