Kronbach T, Mathys D, Gut J, Catin T, Meyer U A
Anal Biochem. 1987 Apr;162(1):24-32. doi: 10.1016/0003-2697(87)90006-6.
Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency of oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for the in vitro metabolism of (+)- and (-)-bufuralol, debrisoquine, and dextromethorphan in human liver microsomes and reconstituted purified cytochrome P-450 isozymes. These assays combine nonextractive sample preparation by precipitation of protein with perchloric acid with reversed-phase inorganic ion-pair HPLC and fluorescence detection. The minimal detectable levels of the major metabolites formed are 1'-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 ng/ml; and dextrorphan, 0.1 ng/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 micrograms of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)- and (-)-bufuralol 1'-hydroxylation and dextromethorphan O-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (less than 1 mM) studied. These data, in combination with those obtained by purified human cytochrome P-450 isozymes indicate the involvement of the same enzyme in the metabolism of all three substrates investigated. However, additional and distinct activities contribute to the metabolism of (+)- and (-)-bufuralol and dextromethorphan.
布呋洛尔、异喹胍和右美沙芬是人类中常见的氧化药物代谢遗传缺陷的三种原型底物，这种遗传缺陷被称为异喹胍/鹰爪豆碱型多态性。我们描述了在人肝微粒体和重组纯化细胞色素P-450同工酶中对(+)-和(-)-布呋洛尔、异喹胍及右美沙芬进行体外代谢的检测方法。这些检测方法将用高氯酸沉淀蛋白质的非萃取性样品制备方法与反相无机离子对高效液相色谱法及荧光检测相结合。所形成的主要代谢物的最低可检测水平分别为：1'-羟基布呋洛尔，0.1 ng/ml；4-羟基异喹胍，0.8 ng/ml；及右啡烷，0.1 ng/ml。这些代谢物的形成在至少45分钟内以及1至100微克微粒体蛋白之间呈线性。对人肝微粒体中三种单加氧酶反应的比较动力学分析显示，(+)-和(-)-布呋洛尔1'-羟基化及右美沙芬O-去甲基化呈现明显的双相性，但在所研究的底物浓度范围（小于1 mM）内，4-羟基异喹胍的形成呈单相性。这些数据与通过纯化的人细胞色素P-450同工酶获得的数据相结合，表明所研究的所有三种底物的代谢均涉及同一种酶。然而，额外且不同的活性对(+)-和(-)-布呋洛尔及右美沙芬的代谢也有贡献。
