Determination of cytochrome P450 1A activities in mammalian liver microsomes by high-performance liquid chromatography with fluorescence detection

A sensitive method for the determination of cytochrome P450 (P450 or CYP) 1 A activities such as ethoxyresorufin O-deethylase (EROD) and methoxyresoruf in O-demethylase (MROD) in liver microsomes from human, monkey, rat and mou se by high-performance liquid chromatography with fluorescence detection is reported. The newly developed method was found to be more sensitive than p revious methods using a spectrofluorimeter and fluorescence plate reader. T he detection limit for resorufin (signal-to-noise ratio of 3) was 0.80 pmol /assay. Intra-day and inter-day precisions (expressed as relative standard deviation) were less than 6% for both enzyme activities. With this improved sensitivity, the kinetics of EROD and MROD activities in mammalian liver m icrosomes could be determined more precisely. EROD activities in human and monkey liver microsomes, and MROD activities in Liver microsomes from all a nimal species exhibited a monophasic kinetic pattern, whereas the pattern o f EROD activities in rat and mouse liver microsomes was biphasic, In additi on, the method could determine the non-inducible and 3-methylcholanthrene-i nducible activities of EROD and MROD in rat and mouse liver microsomes unde r the same assay conditions. Therefore, this method is applicable to in viv o and in vitro studies on the interaction of xenobiotic chemicals with cyto chrome CYP1A isoforms in mammals. (C) 2000 Elsevier Science B.V. All rights reserved.