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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid
  • Cytochrome P-450 CYP2D6
  • Cytochrome P-450 Enzyme System / metabolism*
  • Debrisoquin / metabolism
  • Dextromethorphan / metabolism
  • Ethanolamines / metabolism
  • Humans
  • Isoenzymes / metabolism*
  • Kinetics
  • Microsomes, Liver / enzymology*
  • Microsomes, Liver / metabolism
  • Mixed Function Oxygenases / metabolism*
  • Oxidoreductases / metabolism*
  • Oxidoreductases, O-Demethylating / metabolism*
  • Polymorphism, Genetic


  • Ethanolamines
  • Isoenzymes
  • Dextromethorphan
  • bufuralol
  • Cytochrome P-450 Enzyme System
  • Mixed Function Oxygenases
  • Oxidoreductases
  • Oxidoreductases, O-Demethylating
  • dextromethorphan O-demethylase
  • Cytochrome P-450 CYP2D6
  • bufuralol 1'-hydroxylase
  • Debrisoquin