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

Abstract

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.

The RXR agonist LG100268 causes hepatomegaly, improves glycaemic control and decreases cardiovascular risk and cachexia in diabetic mice suffering from pancreatic beta-cell dysfunction

“…Biochemistry. Lauric acid hydroxylase activity was determined by using published procedures [18]. Protein measurements were determined using the BCA-Protein Assay according to the manufacturer's specifications (Pierce, Rockford, Ill., USA).…”

AZ 242, a novel PPARα/γ agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats

“…The CYP4A-dependent lauryl -hydroxylase activity was measured in liver microsomes using 14 C-labeled lauric acid. Separation and detection of the 11-and 12-hydroxy metabolites were performed using reverse-phase HPLC coupled to a radioactivity detector (33).…”

In Vivo and in Vitro Induction of Cytochrome P450 Enzymes in Beagle Dogs

“…The O-dealkylation of 7-ethoxyresorufin and 7-benzyloxyresorufin, the 6␤-, 16␣-, and 16␤-hydroxylation of testosterone, the 4-hydroxylation of nitrophenol, and the 12-hydroxylation of lauric acid were determined by methods described previously (Burke and Mayer, 1974;Wood et al, 1983;Koop, 1986;Sonderfan et al, 1987;Romano et al, 1988;Sonderfan and Parkinson, 1988;Giera and Van Lier, 1991;Tierney et al, 1992;Burke et al, 1994;Pearce et al, 1996). The incubation conditions for each of the assays are given in Table 1.…”