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
✍ Scribed by Thomas Kronbach; Daniel Mathys; Josef Gut; Therese Catin; Urs A. Meyer
- Publisher
- Elsevier Science
- Year
- 1987
- Tongue
- English
- Weight
- 669 KB
- Volume
- 162
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 I'-hydroxybufuralol, 0.1 rig/ml; 4-hydroxydebrisoquine, 0.8 rig/ml; and dextrorphan, 0.1 rig/ml. Formation of these metabolites is linear for at least 45 min and between 1 and 100 pg of microsomal protein. Comparative kinetic analysis of the three monooxygenase reactions in human liver microsomes revealed an apparent biphasicity of (+)-and (-)-bufuralol l'-hydroxylation and dextromethorphan 0-demethylation but monophasic formation of 4-hydroxydebrisoquine in the substrate concentration range (< 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.