HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR 4-NITROPHENOL HYDROXYLATION, A PUTATIVE CYTOCHROME-P-4502E1 ACTIVITY, IN HUMAN LIVER-MICROSOMES

A high-performance liquid chromatographic method which measures formation of product 4-nitrocatechol (4NC) has been developed and applied to the study of human liver microsomal 4-nitrophenol (4NP) hydroxylation. Following diethyl ether extraction, 4NC and the assay internal standard (salicylamide) were separated by reversed-phase (C18) liquid chromatography. Extraction efficiencies of 4NC and internal standard were both >90%. The assay, which has a limit of detection of 15 pmol injected (or an incubation 4NC concentration of 0.25 muM), is accurate, reproducible and straightforward. With a chromatographic time of 12 min, 40-50 samples may be analyzed per day. Rates of 4NC formation were linear with time and protein concentration to 50 min and 0.5 mg/ml, respectively. Preliminary studies of 4NP hydroxylation showed that this reaction followed single enzyme Michaelis-Menten kinetics in human liver microsomes.