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

Abstract

We studied the ω-oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxy-docosanoic acid (ω-hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, ω-oxidation of C22:0 produced not only the hydroxylated product, ω-hydroxy-C22:0, but also the dicarboxylic acid of C22:0, docosanedioic acid (C22:0-DCA). When rat liver microsomes were incubated with ω-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 ω-hydroxy-C22:0 with NADPH as cofactor was inhibited strongly by miconazole and disulfiram, whereas no inhibition was found with NAD+ as cofactor. Furthermore, ω-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 ω-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 ω-oxidation system in rat liver microsomes and that the product of the first hydroxylation step, ω-hydroxy-C22:0, may undergo further oxidation via two distinct pathways driven by NAD+ or NADPH.

Keywords

• hydroxylase
• cytochrome P450
• ω-hydroxy-fatty acids
• dicarboxylic acids