Highly sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes

J Chromatogr B Biomed Sci Appl. 1999 Jan 8;721(1):13-9. doi: 10.1016/s0378-4347(98)00472-1.

Abstract

A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • Coumarins / metabolism*
  • Cytochrome P-450 Enzyme System / metabolism*
  • Humans
  • Isoenzymes / metabolism*
  • Kinetics
  • Microsomes, Liver / enzymology*
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence

Substances

  • Coumarins
  • Isoenzymes
  • 7-ethoxycoumarin
  • Cytochrome P-450 Enzyme System
  • coumarin