Vol. 26, Issue 4, 305-312, April 1998

Development of a Non-High Pressure Liquid Chromatography Assay to Determine [¹⁴C]Chlorzoxazone 6-Hydroxylase (CYP2E1) Activity in Human Liver Microsomes

Alison J. Draper, Ajay Madan, Jason Latham, and Andrew Parkinson

Department of Pharmacology, Toxicology and Therapeutics, Center for Environmental and Occupational Health (A.J.D., A.M., A.P.), University of Kansas Medical Center and XenoTech L.L.C. (J.L.)

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [¹⁴C]chlorzoxazone. When [¹⁴C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[¹⁴C]hydroxychlorzoxazone. Unreacted [¹⁴C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in ~45% extraction of 6-[¹⁴C]hydroxychlorzoxazone. The amount of 6-[¹⁴C]hydroxychlorzoxazone remaining in the aqueous incubation mixture (~55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[¹⁴C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[¹⁴C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[¹⁴C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.