title: Kinetic study of cytochrome P450 3A4 activity on warfarin by capillary electrophoresis with fluorescence detection.
abstract: The use of capillary electrophoresis (CE) for the determination of cytochrome P450 3A4 (CYP3A4) activity with R-warfarin as a substrate was investigated. CYP3A4 activity was determined by the quantitation of the product, 10-hydroxywarfarin, based on separation by CE. The separation conditions were as follows: capillary, 80.5 cm (75 microm i.d., 60 cm effective length); 50 mM sodium phosphate buffer (pH 6.5); 23 kV (90 microA) applied voltage; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 37 degrees C. With the developed CYP3A4 activity assay and the Lineweaver-Burk equation, the Michaelis-Menten parameters Km and Vmax for formation of 10-hydroxywarfarin from R-warfarin in the presence of CYP3A4 were calculated to be 166 +/- 12 microM and 713 +/- 14 pmol/min/nmol (or 91.4 pmol/min/mg) CYP3A4, respectively.
authors: Laboratory of Pharmaceutical Chemistry and Drug Analysis, Faculty of Pharmaceutical Sciences, K. U. Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium.
authors: Jie
authors: J
authors: Zhang
authors: Pham Thi Thanh
authors: PT
authors: Ha
authors: Yijia
authors: Y
authors: Lou
authors: Jos a
authors: Ja
authors: Hoogmartens
authors: Ann
authors: A
authors: Van Schepdael
mesh_terms: D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007700:Kinetics; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014859:Warfarin / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*
chemical_list: D014859:Warfarin; C065719:10-hydroxywarfarin; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human