High-performance liquid chromatographic method for measurement of cytochrome P450-mediated metabolism of 7-ethoxy-4-trifluoromethylcoumarin.
An HPLC method for analysis of deethylation of 7-ethoxy-4-trifluoromethylcoumarin (ETFMC), a substrate of various enzymes of the cytochrome P450 superfamily, was developed. ETFMC was incubated at 37 degrees C with human hepatic microsomes or microsomes prepared from a lymphoblastoid cell line that expresses human CYP2B6. Under these conditions, the highly fluorescent metabolite 7-hydroxy-4-trifluoromethylcoumarin (HTFMC) is formed. The metabolite was analyzed by reversed-phase HPLC with fluorescence detection. The limits of detection of the metabolite were 5.0 fmol per injection, a sensitivity at least one order of magnitude greater than the standard method, which does not involve HPLC. This method will be of great utility when quantities of microsomal protein from cell lines expressing human CYP enzymes are limited.
Morse
M A
MA
Division of Environmental Health Sciences, The Ohio State University School of Public Health, and The Ohio State University Comprehensive Cancer Center, CHRI, Columbus 43210, USA.
Lu
J
J
D000328:Adult; D000368:Aged; D001189:Aryl Hydrocarbon Hydroxylases; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000378:metabolism*; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000378:metabolism; D008875:Middle Aged; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D003374:Coumarins; C056033:7-ethoxy-4-trifluoromethylcoumarin; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating