Valproyl-dephosphocoa: A novel metabolite of valproate formed in vitro in rat liver mitochondria
Valproyl-dephosphocoa: A novel metabolite of valproate formed in vitro in rat liver mitochondria
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DOI：
10.1124/dmd
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发表时间：
2004-11-01
影响因子：
3.9
通讯作者：
Wanders, RJA
中科院分区：
文献类型：
--
作者：
Silva, MFB;Ijlst, L;Wanders, RJA
体外研究了丙戊酸（VPA）的线粒体代谢，以阐明其β -氧化途径，因为尚未完全实现丙戊酸中间体酰基辅酶a硫酯形式（而不仅仅是游离酸形式）的表征。用[4,5- h -3(2)] VPA和[2- h -3] VPA孵育完整大鼠肝脏线粒体。采用反相高效液相色谱法（HPLC）、放射性同位素和紫外检测法对中间体丙戊酰辅酶a、δ (2(E))-丙戊酰辅酶a、3-羟丙戊酰辅酶a和3-氧丙戊酰辅酶a进行了分析。检测到来自两种标记底物的未知代谢物。通过快速原子轰击质谱法（FAB-MS）对其HPLC峰段进行分析，鉴定为丙戊酰去磷coa （valproyl- dephcoa）。真实化学合成的丙戊酰-深氢辅酶a的FAB-MS谱图与未知化合物的谱图一致。丙戊酰深层辅酶a是由线粒体中的丙戊酰辅酶a产生的，可能是通过磷酸酶催化的反应。这种转化更依赖于涉及[AXP] （[AXP] = [ATP] + [ADP] + [AMP]）和[磷酸盐]浓度的能量状态，而不是严格的线粒体[ATP] /[ADP]比率。结果表明，较高浓度的AXP和磷酸抑制丙戊酰辅酶a的去磷酸化。全面了解丙戊酰-去氢辅酶A在体内形成的毒性意义，作为脂肪酸β -氧化的潜在抑制剂，对于阐明vpa相关肝毒性的发病机制非常重要。
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.