Abstract
We studied the omega-oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxy-docosanoic acid (omega-hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, omega-oxidation of C22:0 produced not only the hydroxylated product, omega-hydroxy-C22:0, but also the dicarboxylic acid of C22:0, docosanedioic acid (C22:0-DCA). When rat liver microsomes were incubated with omega-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 omega-hydroxy-C22:0 with NADPH as cofactor was inhibited strongly by miconazole and disulfiram, whereas no inhibition was found with NAD+ as cofactor. Furthermore, omega-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 omega-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 omega-oxidation system in rat liver microsomes and that the product of the first hydroxylation step, omega-hydroxy-C22:0, may undergo further oxidation via two distinct pathways driven by NAD+ or NADPH.
Highlights
We studied the -oxidation of docosanoic acid (C22:0) in rat liver microsomes
The initial step, hydroxylation of the methyl group, requires NADPH and molecular oxygen and is catalyzed by microsomal enzymes belonging to the cytochrome P450 4A (CYP4A) family [5,6,7]
NADϩ, NADPH, and glucose-6-phosphate dehydrogenase were obtained from Roche Applied Science. 22-Hydroxy-docosanoic acid (-hydroxy-C22:0) and hexacosanedioic acid were purchased from Larodan Fine Chemicals (Malmö, Sweden). 22,22,22-D3-Docosanoic acid was obtained from CDN Isotopes (Québec, Canada)
Summary
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 the hydroxylated product, -hydroxy-C22:0, and the dicarboxylic acid of C22:0, docosanedioic acid (C22:0-DCA). 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. Several rat cytochrome P450 4A isoforms (CYP4A1, CYP4A2, CYP4A3, and CYP4A8) and two human isoforms (CYP4A11 and CYP4A22) have been characterized [7, 14, 15] All of these enzymes were demonstrated to have highest activity for medium-chain fatty acids, and the hydroxylation rate was found to decline with increasing chain length. In several inherited peroxisomal diseases, including X-linked adrenoleukodystrophy, peroxisomal biogenesis disorders, and two single
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