Roy D, Snodgrass W R
Department of Pharmacology-Toxicology, University of Texas Medical Branch, Galveston.
J Pharmacol Exp Ther. 1990 Mar;252(3):895-900.
The role of thiols (nonprotein and protein) in the metabolic activation of phenytoin was examined. In vitro phenytoin covalent binding and metabolite formation were determined in hepatic microsomes from A/J mice. Covalent binding of a phenytoin-reactive intermediate to microsomal protein was linear with respect to time, protein concentration and phenytoin concentration. Covalent binding was inhibited by inhibitors of cytochrome P-450. Inducers of cytochrome P-450 enhanced phenytoin covalent binding as follows: phenobarbital greater than 3-methylcholanthrene greater than saline-treated controls. Low molecular weight thiols (GSH, cysteine and cysteamine), a thiol generator (methylthiazolidine carboxylate), and thiol modifying agents (N-ethylmaleimide, mercuric chloride and diamide) significantly inhibited covalent binding. Amino acids other than cysteine did not decrease the covalent binding. Formation of the metabolites, para-hydroxyphenytoin and phenytoin dihydrodiol, was greater following preincubation with GSH or cysteine. In summary, protein thiol groups appear to be important sites for in vitro covalent binding of a reactive intermediate of phenytoin. These data suggest glutathione may protect membrane-bound enzymes responsible for phenytoin metabolism from attack by an electrophilic or free radical reactive intermediate of phenytoin and GSH may inactivate a phenytoin-reactive metabolite by formation of a putative glutathione conjugate.
研究了硫醇（非蛋白质和蛋白质）在苯妥英代谢活化中的作用。在来自A/J小鼠的肝微粒体中测定了体外苯妥英共价结合和代谢物形成。苯妥英反应性中间体与微粒体蛋白的共价结合在时间、蛋白浓度和苯妥英浓度方面呈线性关系。共价结合受到细胞色素P-450抑制剂的抑制。细胞色素P-450诱导剂增强苯妥英共价结合的顺序如下：苯巴比妥＞3-甲基胆蒽＞盐水处理对照组。低分子量硫醇（谷胱甘肽、半胱氨酸和半胱胺）、硫醇生成剂（甲基噻唑烷羧酸盐）和硫醇修饰剂（N-乙基马来酰亚胺、氯化汞和二酰胺）显著抑制共价结合。除半胱氨酸外的氨基酸不会降低共价结合。在与谷胱甘肽或半胱氨酸预孵育后，代谢物对羟基苯妥英和苯妥英二氢二醇的形成更多。总之，蛋白质硫醇基团似乎是苯妥英反应性中间体体外共价结合的重要位点。这些数据表明谷胱甘肽可能保护负责苯妥英代谢的膜结合酶免受苯妥英亲电或自由基反应性中间体的攻击，并且谷胱甘肽可能通过形成假定的谷胱甘肽共轭物使苯妥英反应性代谢物失活。
