Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation

• Published in: Analytical biochemistry Vol. 170; no. 1; p. 83
• Main Authors: Romano, M C, Straub, K M, Yodis, L A, Eckardt, R D, Newton, J F
• Format: Journal Article
• Language: English
• Published: United States 01-04-1988
• Subjects: Animals; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP4A; Fatty Acids, Unsaturated - pharmacology; Gas Chromatography-Mass Spectrometry; Hydroxylation; Male; Metyrapone - pharmacology; Microsomes, Liver - enzymology; Mixed Function Oxygenases - analysis; Rats; Rats, Inbred F344
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(88)90093-0

An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, alpha-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation [omega-, (omega-1)-, (omega-2)-] of LA may be catalyzed by different isozymes of cytochrome P-450.