Abstract
The mitochondrial metabolism of valproic acid (VPA) was investigated in vitro to elucidate its β-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 [4,5-3H2]VPA and [2-3H]VPA. The respective intermediates, valproyl-CoA, Δ2(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-dephCoA 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 β-oxidation is important to clarify the pathogenesis of VPA-associated hepatotoxicity.
Footnotes
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This work was financially supported by “Fundação para a Ciência e a Tecnologia” (Lisboa, Portugal) (POCTI/FCB/48800/2002 with partial funding of Fundo Europeu de Desenvolvimento Regional).
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ABBREVIATIONS: VPA, 2-n-propylpentanoic acid or valproic acid; Δ2(E)-VPA, 2-n-propyl-trans 2-pentenoic acid; CoA, coenzyme A; dephCoA, dephosphocoenzyme A; Pi, orthophosphate; AMP, adenosine-5′-monophosphate; ADP, adenosine-5′-diphosphate; ATP, adenosine-5′-triphosphate; ATP-γ-S, adenosine-5′-O-(3-thiotriphosphate); AXP, total adenine nucleotides (AMP + ADP + ATP); MOPS, 3-[N-morpholino]propane-sulfonic acid; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; SEM buffer, sucrose/EGTA/MOPS buffer; KPi buffer, potassium dihydrogen phosphate buffer; FAB-MS, fast atom bombardment mass spectrometry; HPLC, high performance liquid chromatography; radio-HPLC, high performance liquid chromatography with radiochemical detection; RLM, rat liver mitochondria.
- Received October 10, 2003.
- Accepted July 22, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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