Determination of the cytochrome P-450 IV marker, omega-hydroxylauric acid, by high-performance liquid chromatography and fluorimetric detection
Abstract
The formation of omega-hydroxylauric acid from lauric acid is an indicator of the activity of cytochrome P-450 IV family proteins. The two main metabolites of lauric acid, (omega-1)-and omega-hydroxylauric acid, have been completely separated by reversed-phase high-performance liquid chromatography. Measurement of lauric acid hydroxylase activity in microsomal liver samples, based on derivatization of the substrate and metabolites with the fluorescent agent 4-bromomethyl-6,7-dimethoxycoumarin, is a precise method (coefficient of variation = 7.6 and 10% for omega and (omega-1) metabolites, respectively) with good sensitivity (signal-to-noise ratio in microsomal samples of untreated rats greater than 20). In microsomal fractions from livers of rats treated with di-(2-ethylhexyl)phthalate the extent of omega-hydroxylation of lauric acid increased dose-dependently (ca. ten-fold). The (omega-1)-hydroxylase activity was not altered. A strong correlation between immunochemically determined cytochrome P-450 IVA1 and lauric acid omega-hydroxylase activity was found (r = 0.94, n = 30).
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