High-performance liquid chromatographic assays for bufuralol 1′-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver

摘要: Abstract Bufuralol, debrisoquine, and dextromethorphan are three prototype substrates of the common genetic deficiency oxidative drug metabolism in man known as debrisoquine/sparteine-type polymorphism. We describe assays for vitro (+)- (−)-bufuralol, human liver microsomes reconstituted purified cytochrome P -450 isozymes. These combine nonextractive sample preparation by precipitation protein with perchloric acid reversed-phase inorganic ion-pair HPLC fluorescence detection. The minimal detectable levels major metabolites formed 1′-hydroxybufuralol, 0.1 ng/ml; 4-hydroxydebrisoquine, 0.8 dextrorphan, ng/ml. Formation these is linear at least 45 min between 1 100 μg microsomal protein. Comparative kinetic analysis monooxygenase reactions revealed an apparent biphasicity (−)-bufuralol 1′-hydroxylation O-demethylation but monophasic formation 4-hydroxydebrisoquine substrate concentration range ( m ) studied. data, combination those obtained isozymes indicate involvement same enzyme all investigated. However, additional distinct activities contribute to dextromethorphan.