Tang W, Abbott F S
Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
J Mass Spectrom. 1996 Aug;31(8):926-36. doi: 10.1002/(SICI)1096-9888(199608)31:8<926::AID-JMS383>3.0.CO;2-P.
The hepatotoxicity of the anticonvulsant drug valproic acid (VPA) is most likely associated with the bioactivation of its metabolite 2-propylpent-4-enoic acid (4-ene VPA), which is known to induce hepatic microvesicular steatosis in rats. This paper presents an on-line liquid chromatographic/tandem mass spectrometric (LC/MS/MS) identification of new glutathione (GSH)-related conjugates of the reactive metabolites of 4-ene VPA. Bile samples collected from male Sprague-Dawley rats dosed intraperitoneally with 4-ene VPA or its [2H7]-analogue (100 mg kg-1) were injected on to an ODS column interfaced to a LC/MS/MS instrument using electrospray ionization. LC was developed such that no overlapping of peaks occurred among those metabolites which may potentially produce common fragment ions of interest. Subsequent comparison of LC retention times and MS/MS full fragment ion spectra generated for putative metabolites with that of authentic reference compounds made available by chemical synthesis confirmed the presence of the GSH, cysteinylglycine, cysteine and N-acetylcysteine (NAC) conjugates of 2-(2'-carboxypentanyl)oxirane (4,5-epoxy VPA) and (E)-2-propylpenta-2,4-dienoic acid ((E)-2,4-diene VPA), respectively. Quantitatively, the biliary thiol conjugates accounted for 5% of the dose. This observation is novel for 4-ene VPA metabolism in terms of the degradation of GSH conjugates to the corresponding mercapturic acids possibly occurring within the liver as opposed to an inter-organ process which involves the kidney. In addition, the GSH- and NAC-glucuronide di-conjugates of (E)-2,4-diene VPA were also identified as the biliary metabolites with the GSH-glucuronide di-conjugate being 10% of the corresponding mono-GSH conjugate. Taken together, these data clearly indicate that reactive metabolites of VPA can react with hepatic GSH via several different metabolic pathways and may subsequently produce depletion of GSH that leads to toxic consequences.
抗惊厥药物丙戊酸（VPA）的肝毒性很可能与其代谢产物2-丙基戊-4-烯酸（4-烯VPA）的生物活化有关，已知该代谢产物会在大鼠中诱导肝微泡性脂肪变性。本文介绍了一种在线液相色谱/串联质谱（LC/MS/MS）方法，用于鉴定4-烯VPA反应性代谢产物新的谷胱甘肽（GSH）相关共轭物。从腹腔注射4-烯VPA或其[2H7]类似物（100 mg kg-1）的雄性Sprague-Dawley大鼠收集胆汁样本，使用电喷雾电离将其注入与LC/MS/MS仪器相连的ODS柱。液相色谱的展开方式使得可能产生共同感兴趣碎片离子的那些代谢产物之间不会出现峰重叠。随后，将推测代谢产物的液相色谱保留时间和MS/MS全碎片离子光谱与化学合成提供的真实参考化合物的进行比较，证实分别存在2-(2'-羧基戊基)环氧乙烷（4,5-环氧VPA）和（E）-2-丙基戊-2,4-二烯酸（（E）-2,4-二烯VPA）的GSH、半胱氨酰甘氨酸、半胱氨酸和N-乙酰半胱氨酸（NAC）共轭物。定量分析表明，胆汁硫醇共轭物占给药剂量的5%。就GSH共轭物可能在肝脏内降解为相应的巯基尿酸而言，这一观察结果对于4-烯VPA代谢来说是新颖的，而不是涉及肾脏的器官间过程。此外，（E）-2,4-二烯VPA的GSH-和NAC-葡萄糖醛酸二共轭物也被鉴定为胆汁代谢产物，其中GSH-葡萄糖醛酸二共轭物占相应单GSH共轭物的10%。综上所述，这些数据清楚地表明，VPA的反应性代谢产物可通过几种不同的代谢途径与肝脏GSH反应，并可能随后导致GSH耗竭，从而产生毒性后果。
