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

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. Supplementary key words hydroxylase • cytochrome P450 • -hydroxyfatty acids • dicarboxylic acids Fatty acid oxidation plays a major role in the production of energy, notably in the heart and skeletal muscle, and is the main energy source during periods of fasting. The primary degradation route of fatty acids is via -oxidation that takes place in mitochondria and peroxisomes. Other mechanisms for the oxidation of fatty acids involve -oxidation in peroxisomes or -oxidation in the smooth endoplasmic reticulum. It is generally accepted that under normal physiological conditions, fatty acid -oxidation is a minor pathway that accounts for 5–10% of total fatty acid oxidation in the liver (1). During periods of fasting or starvation, intracellular levels of free fatty acids increase and subsequently become available as substrates for -oxidation (2–4). The -oxidation of fatty acids consists of three sequential steps in which the terminal methyl group is converted into a carboxyl group. 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 (5–7). The -hydroxy fatty acid that is produced can be oxidized further by an alcohol dehydrogenase into an -oxo-fatty acid (8). Finally, this product can be converted into a dicarboxylic acid by an aldehyde dehydrogenase. Dehydrogenation of -hydroxyand -oxo-fatty acids requires NAD and has been identified in the cytosol (5, 9, 10). The dicarboxylic acids that are produced via the -oxidation pathway can be either excreted into the urine or -oxidized in mitochondria or peroxisomes (11–13). 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. However, studies of the -oxidation of long-chain fatty acids in brain suggest the existence of additional hydroxylation enzymes with different chain length specificities (16, 17). At present, little is known about the -oxidation of fatty acids with a chain length of more than 20 carbons. Very long-chain fatty acids (VLCFAs; 22 carbons) are exclusively -oxidized in peroxisomes. In several inherited peroxisomal diseases, including X-linked adrenoleukodystrophy, peroxisomal biogenesis disorders, and two single Abbreviations: C22:0, docosanoic acid; C22:0-DCA, docosanedioic acid; -hydroxy-C22:0, 22-hydroxy-docosanoic acid; 17-ODYA, 17-octadecynoic acid; VLCFA, very long-chain fatty acid. 1 To whom correspondence should be addressed. e-mail: [email protected] Manuscript received 22 December 2004 and in revised form 9 February 2005. Published, JLR Papers in Press, February 16, 2005. DOI 10.1194/jlr.M400510-JLR200 by gest, on O cber 1, 2017 w w w .j.org D ow nladed fom