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.
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.
D002851:Chromatography, High Pressure Liquid; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003647:Debrisoquin / Q000378:metabolism; D003915:Dextromethorphan / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism*; D011110:Polymorphism, Genetic
D004983:Ethanolamines; D007527:Isoenzymes; D003915:Dextromethorphan; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D010090:Oxidoreductases, O-Demethylating; C052861:dextromethorphan O-demethylase; D019389:Cytochrome P-450 CYP2D6; C043872:bufuralol 1'-hydroxylase; D003647:Debrisoquin