Simultaneous radiometric and fluorimetric detection of lauric acid metabolites using high-performance liquid chromatography following esterification with 4-bromomethyl-6,7-dimethoxycoumarin in human and rat liver microsomes

Abstract

The formation of (ω-1)-hydroxylauric acid from lauric acid (LA) can be used as an indicator of the activity of cytochrome P450 2E1 (CYP2E1) in rat and human liver microsomes. A high-performance liquid chromatographic (HPLC) method that is capable of identifying and measuring the two main metabolites of lauric acid, (ω-1)- and ω-OH-LA, has been developed and used in the study of rat and human liver microsomes. Measurement of the enzymatic activities, based on the esterification of the metabolites and substrate with the fluorescent agent, 4-bromomethyl-6,7-dimethoxycoumarin, is described using both radiometric and fluorimetric detection methods. Extraction efficiencies of metabolites and residual substrate were calculated using radioactivity and were greater than 85%. The assay is accurate and reproducible and has a detection limit of 75 pg (0.37 pmol). Additionally, a strong correlation between the two techniques was found in both human ( r = 0.945, n = 15, p < 0.01) and rat ( r = 0.949, n = 18, p < 0.01) livers, for the (ω-1)-hydroxylauric acid.