DETERMINATION OF CYTOCHROME-P450 2E1 ACTIVITY IN MICROSOMES BY THIN-LAYER CHROMATOGRAPHY USING [2-C-14]CHLORZOXAZONE

A thin-layer chromatographic assay was developed as an alternative met hod for the determination of cytochrome P450 2E1 (CYP2E1) in microsome s using [2-C-14]chlorzoxazone. After incubation of microsomes with 0.1 25 mu Ci/mmol chlorzoxazone, chlorzoxazone and its single metabolite, 6-hydroxychlorzoxazone, were extracted using chloroform-2-propanol (85 :15, v/v) and chromatographed on silica gel 60 F254 plates with aceton e-hexane (45:55, v/v) as solvent. The plates were then exposed to X-ra y film for 2 days to localize the radiolabelled chlorzoxazone and 6-hy droxychlorzoxazone. The metabolite and substrate regions were scraped and counted in a liquid scintillation analyzer. This method is sensiti ve enough to determine constitutive and induced CYP2E1 activities in l iver or kidney microsomes. The precision of the method was similar to that of the HPLC method. The correlation coefficient between both meth ods was found to be 0.97 (n=21). Therefore, the TLC method constitutes a valuable tool for the determination of chlorzoxazone metabolism in microsomes.