Evidence for two enzymatic pathways for ω-oxidation of docosanoic acid in rat liver microsomes

Abstract

We studied the -oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxydocosanoic acid ( -hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, -oxidation of C22:0 produced not only the hydroxylated product, -hydroxy-C22:0, but also the dicarboxylic acid of C22:0, docosanedioic acid (C22:0-DCA). When rat liver microsomes were incubated with -hydroxy-C22:0 in the presence of either NAD ؉ or NADPH, C22:0-DCA was formed readily. Formation of C22:0-DCA from either C22:0 or -hydroxy-C22:0 with NADPH as cofactor was inhibited strongly by miconazole and disulfiram, whereas no inhibition was found with NAD ؉ as cofactor. Furthermore, -oxidation of C22:0 was reduced significantly when molecular oxygen was depleted. The high sensitivity toward the more specific cytochrome P450 inhibitors ketoconazole and 17-octadecynoic acid suggests that hydroxylation of C22:0 and -hydroxy-C22:0 may be catalyzed by one or more cytochrome P450 hydroxylases belonging to the CYP4A and/or CYP4F subfamily. This study demonstrates that C22:0 is a substrate for the -oxidation system in rat liver microsomes and that the product of the first hydroxylation step, -hydroxy-C22:0, may undergo further oxidation via two distinct pathways driven by NAD ؉ or NADPH. -Sanders, R-J., R. Ofman, F. Valianpour, S. Kemp, and R. J. A. Wanders. Evidence for two enzymatic pathways for -oxidation of docosanoic acid in rat liver microsomes. J. Lipid Res. 2005. 46: 1001-1008.

Characterization of the human ω‐oxidation pathway for ω‐hydroxy‐very‐long‐chain fatty acids

“…1F) was used to calculate the kinetic constants K m and V max ( Table 1). The optimal pH for the NAD ϩ -and NADPH-dependent -oxidation routes were similar to the previously observed pH optima in rat liver microsomes (20). These optimal conditions were used to determine the enzyme kinetic parameters for the two pathways with both -hydroxy-C22:0 and -hydroxy-C26:0 using a postnuclear supernatant fraction as the source of enzyme (Table 1).…”

“…Because -hydroxy-VLCFAs can also be metabolized by cytochrome P450 enzymes in the NADPH-driven -oxidation pathway, we investigated the effect of 17-ODYA on enzyme activity (20). Previously, we demonstrated that the NADPH and molecular oxygen-dependent conversion of -hydroxy-C22:0 was inhibited markedly by 17-ODYA, at least in rat liver microsomes (20).…”

“…The experimental conditions to study the -oxidation of -hydroxy fatty acids were the same as those described previously (17,20) with minor modifications, as discussed below.…”

“…Previously, we demonstrated that the NADPH and molecular oxygen-dependent conversion of -hydroxy-C22:0 was inhibited markedly by 17-ODYA, at least in rat liver microsomes (20). 17-ODYA is a specific inhibitor of CYP450 enzymes belonging to the CYP4 family (29,30).…”

“…This reaction requires NADPH and molecular oxygen as cofactors and is catalyzed by two cytochrome P450 (CYP450) enzymes belonging to the CYP4F subfamily, namely CYP4F2 and CYP4F3B (17). Subsequently, the -hydroxy-VLCFA is oxidized further into the corresponding dicarboxylic acid either via a CYP450-mediated route or via an as yet unidentified NAD ϩ -dependent pathway (17)(18)(19)(20)(21). Long-chain dicarboxylic acids are ␤-oxidized in peroxisomes to shorter-chain dicarboxylic acids followed by excretion into the urine (22).…”