Valproic acid utilizes the isoleucine breakdown pathway for its complete β-oxidation

Valproic acid (VPA) is a simple branched medium-chain fatty acid with expanding therapeutic applications beyond its prime anticonvulsant properties. (1) To resolve the underlying basis for the interference of valproate with the isoleucine degradative pathway and (2) to shed new light on the enzymology of the β-oxidation pathway of valproate. Urine organic acids were analyzed by gas chromatography/mass spectrometry. In vitro studies were performed with heterologously expressed human 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) and fibroblasts from controls and a patient with MHBD deficiency using 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. The respective enzymatic activities were measured using optimized HPLC procedures. Short-chain enoyl-CoA hydratase (ECHS1) immunoprecipitation in a human liver homogenate was performed and hydratase activity was measured in the supernatants by HPLC, using crotonyl-CoA and Δ(2(E))-valproyl-CoA as substrates. Patients on valproate therapy had a moderately increased urinary excretion of the isoleucine metabolite 2-methyl-3-hydroxybutyric acid. MHBD was found to convert 3-hydroxyvalproyl-CoA into 3-ketovalproyl-CoA. MHBD activity in control fibroblasts was comparable using both 2-methyl-3-hydroxybutyryl-CoA and 3-hydroxyvalproyl-CoA as substrates. In fibroblasts of a patient with MHBD deficiency, there was no detectable MHBD activity when 3-hydroxyvalproyl-CoA was used as substrate. Samples with immunoprecipitated crotonase had no detectable hydratase activity using both crotonyl-CoA and Δ(2(E))-valproyl-CoA as substrates. This work demonstrates for the first time, that MHBD is the unique enzyme responsible for the dehydrogenation of 3-hydroxyvalproyl-CoA. Furthermore, we show that crotonase is the major, if not the single hydratase involved in VPA β-oxidation, next to its role in isoleucine catabolism