Silva M F, Ruiter J P, IJlst L, Allers P, ten Brink H J, Jakobs C, Duran M, Tavares de Almeida I, Wanders R J
Department of Clinical Chemistry and Paediatrics, University of Amsterdam, The Netherlands.
Anal Biochem. 2001 Mar 1;290(1):60-7. doi: 10.1006/abio.2000.4947.
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.
丙戊酸盐的许多不良反应是由于其干扰了包括脂肪酸氧化在内的多种代谢途径。为了确定脂肪酸氧化的哪些线粒体酶被哪些丙戊酸（VPA）中间体所抑制，我们开发了合成其辅酶A酯形式的方法。本文描述了VPA酰基辅酶A酯代谢物的合成以及VPA在大鼠肝线粒体中的代谢数据。通过化学合成获得了丙戊酰辅酶A、Δ2-丙戊酰辅酶A和3-羟基丙戊酰辅酶A。3-酮基丙戊酰辅酶A通过一种新的酶促方法制备，随后结合固相萃取和制备型高效液相色谱（HPLC）纯化。该方法被证明在获得丙戊酰辅酶A的所有β-氧化中间体方面是有效的。合成标准品用于通过HPLC测定线粒体内丙戊酰辅酶A、Δ2-丙戊酰辅酶A、3-羟基丙戊酰辅酶A和3-酮基丙戊酰辅酶A的浓度。这些水平是在完整的大鼠肝线粒体与VPA在状态3和状态4呼吸条件下孵育后测定的。结果表明，丙戊酰辅酶A以及程度小得多的3-酮基丙戊酰辅酶A是VPA在线粒体中的主要代谢物。这些信息对于解析VPA抑制线粒体过程（如脂肪酸氧化）所涉及的机制将非常有用。
