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

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. 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. 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 β-oxidation that takes place in mitochondria and peroxisomes. Other mechanisms for the oxidation of fatty acids involve α-oxidation in peroxisomes or ω-oxidation in the smooth endoplasmic reticulum. It is generally accepted that under normal physiological conditions, fatty acid ω-oxidation is a minor pathway that accounts for ∼5–10% of total fatty acid oxidation in the liver (1Bjorkhem I. On the quantitative importance of omega-oxidation of fatty acids.J. Lipid Res. 1978; 19: 585-590Abstract Full Text PDF PubMed Google Scholar). During periods of fasting or starvation, intracellular levels of free fatty acids increase and subsequently become available as substrates for ω-oxidation (2Preiss B. Bloch K. Omega-oxidation of long chain fatty acids in rat liver.J. Biol. Chem. 1964; 239: 85-88Abstract Full Text PDF PubMed Google Scholar, 3Bjorkhem I. On the mechanism of regulation of omega oxidation of fatty acids.J. Biol. Chem. 1976; 251: 5259-5266Abstract Full Text PDF PubMed Google Scholar, 4Mortensen P.B. Gregersen N. The biological origin of ketotic dicarboxylic aciduria. In vivo and in vitro investigations of the omega-oxidation of C6-C16-monocarboxylic acids in unstarved, starved and diabetic rats.Biochim. Biophys. Acta. 1981; 666: 394-404Crossref PubMed Scopus (72) Google Scholar). The ω-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 (5Wakabayashi K. Shimazono N. Studies on omega-oxidation of fatty acids in vitro. I. Overall reaction and intermediate.Biochim. Biophys. Acta. 1963; 70: 132-142Crossref PubMed Scopus (33) Google Scholar, 6Lu A.Y. Coon M.J. Role of hemoprotein P-450 in fatty acid omega-hydroxylation in a soluble enzyme system from liver microsomes.J. Biol. Chem. 1968; 243: 1331-1332Abstract Full Text PDF PubMed Google Scholar, 7Okita R.T. Okita J.R. Cytochrome P450 4A fatty acid omega hydroxylases.Curr. Drug Metab. 2001; 2: 265-281Crossref PubMed Scopus (88) Google Scholar). The ω-hydroxy fatty acid that is produced can be oxidized further by an alcohol dehydrogenase into an ω-oxo-fatty acid (8Mitz M.A. Heinrikson R.L. Omega hydroxy fatty acid dehydrogenase.Biochim. Biophys. Acta. 1961; 46: 45-50Crossref PubMed Scopus (33) Google Scholar). Finally, this product can be converted into a dicarboxylic acid by an aldehyde dehydrogenase. Dehydrogenation of ω-hydroxy- and ω-oxo-fatty acids requires NAD+ and has been identified in the cytosol (5Wakabayashi K. Shimazono N. Studies on omega-oxidation of fatty acids in vitro. I. Overall reaction and intermediate.Biochim. Biophys. Acta. 1963; 70: 132-142Crossref PubMed Scopus (33) Google Scholar, 9Robbins K.C. In vitro enzymic omega oxidation of medium-chain fatty acids in mammalian tissue.Arch. Biochem. Biophys. 1968; 123: 531-538Crossref PubMed Scopus (37) Google Scholar, 10Kundu R.K. Tonsgard J.H. Getz G.S. Induction of omega-oxidation of monocarboxylic acids in rats by acetylsalicylic acid.J. Clin. Invest. 1991; 88: 1865-1872Crossref PubMed Scopus (23) Google Scholar). The dicarboxylic acids that are produced via the ω-oxidation pathway can be either excreted into the urine or β-oxidized in mitochondria or peroxisomes (11Kolvraa S. Gregersen N. In vitro studies on the oxidation of medium-chain dicarboxylic acids in rat liver.Biochim. Biophys. Acta. 1986; 876: 515-525Crossref PubMed Scopus (75) Google Scholar, 12Bergseth S. Hokland B.M. Bremer J. Metabolism of dicarboxylic acids in vivo and in the perfused kidney of the rat.Biochim. Biophys. Acta. 1988; 961: 103-109Crossref PubMed Scopus (27) Google Scholar, 13Vamecq J. Draye J.P. Brison J. Rat liver metabolism of dicarboxylic acids.Am. J. Physiol. 1989; 256: G680-G688PubMed Google Scholar). Several rat cytochrome P450 4A isoforms (CYP4A1, CYP4A2, CYP4A3, and CYP4A8) and two human isoforms (CYP4A11 and CYP4A22) have been characterized (7Okita R.T. Okita J.R. Cytochrome P450 4A fatty acid omega hydroxylases.Curr. Drug Metab. 2001; 2: 265-281Crossref PubMed Scopus (88) Google Scholar, 14Bellamine A. Wang Y. Waterman M.R. Gainer III, J.V. Dawson E.P. Brown N.J. Capdevila J.H.H. Characterization of the CYP4A11 gene, a second CYP4A gene in humans.Arch. Biochem. Biophys. 2003; 409: 221-227Crossref PubMed Scopus (46) Google Scholar, 15Kawashima H. Naganuma T. Kusunose E. Kono T. Yasumoto R. Sugimura K. Kishimoto T.T. Human fatty acid omega-hydroxylase, CYP4A11: determination of complete genomic sequence and characterization of purified recombinant protein.Arch. Biochem. Biophys. 2000; 378: 333-339Crossref PubMed Scopus (38) Google Scholar). 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 ω-oxidation of long-chain fatty acids in brain suggest the existence of additional hydroxylation enzymes with different chain length specificities (16Alexander J.J. Snyder A. Tonsgard J.H. Omega-oxidation of monocarboxylic acids in rat brain.Neurochem. Res. 1998; 23: 227-233Crossref PubMed Scopus (25) Google Scholar, 17Chuang S.S. Helvig C. Taimi M. Ramshaw H.A. Collop A.H. Amad M. White J.A. Petkovich M. Jones G. Korczak B.B. CYP2U1, a novel human thymus- and brain-specific cytochrome P450, catalyzes omega- and (omega-1)-hydroxylation of fatty acids.J. Biol. Chem. 2004; 279: 6305-6314Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). At present, little is known about the ω-oxidation of fatty acids with a chain length of more than 20 carbons. Very long-chain fatty acids (VLCFAs; >22 carbons) are exclusively β-oxidized in peroxisomes. In several inherited peroxisomal diseases, including X-linked adrenoleukodystrophy, peroxisomal biogenesis disorders, and two single peroxisomal enzyme deficiencies, acyl-CoA oxidase deficiency and peroxisomal bifunctional protein deficiency, VLCFAs are increased in plasma as a result of reduced β-oxidation of these fatty acids. In contrast, plasma levels of docosanoic acid (C22:0) are decreased in these patients (18Kemp S. Valianpour F. Denis S. Ofman R. Sanders R.J. Mooyer P. Barth P.G. Wanders R.J.J. Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy.Mol. Genet. Metab. 2005; 84: 144-151Crossref PubMed Scopus (73) Google Scholar). An excess of long-chain hydroxylated fatty acids and dicarboxylic acids was found in urine of patients with peroxisomal biogenesis disorders (19Rocchiccioli F. Aubourg P. Bougneres P.F. Medium- and long-chain dicarboxylic aciduria in patients with Zellweger syndrome and neonatal adrenoleukodystrophy.Pediatr. Res. 1986; 20: 62-66Crossref PubMed Scopus (57) Google Scholar). Based on these data, we postulate that ω-oxidation of (very) long-chain fatty acids may provide an alternative route for fatty acid metabolism. The aim of this study was to investigate whether C22:0 is a substrate for the microsomal ω-oxidation system. NAD+, NADPH, and glucose-6-phosphate dehydrogenase were obtained from Roche Applied Science. 22-Hydroxy-docosanoic acid (ω-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). Omeprazole, sulfaphenazole, quinidine, ketoconazole, furafylline, trimethoprim, diethyldithiocarbamate, 17-octadecynoic acid (17-ODYA), N-ethylmaleimide, miconazole, disulfiram, and l-lactate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO). 4-Chloromercuribenzoate was obtained from Janssen Chemica. All other chemicals used were of analytical grade. Microsomes were isolated from rat livers by differential centrifugation essentially as described by Baudhuin et al. (20Baudhuin P. Beaufay H. Rahman-Li Y. Sellinger O.Z. Wattiaux R. Jacques P. De Duve C.C. Tissue fractionation studies. 17. Intracellular distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase and catalase in rat-liver tissue.Biochem. J. 1964; 92: 179-184Crossref PubMed Scopus (485) Google Scholar). To this end, male Wistar rats (200–250 g) were fed ad libitum with standard rodent chow and fasted overnight before being killed. The livers were removed immediately and washed with homogenization buffer containing 250 mM sucrose, 5 mM MOPS, pH 7.4, and 2 mM EDTA, minced, and homogenized (five strokes at 500 rpm) using a Wheaton homogenizer with a Teflon pestle. The homogenate was centrifuged at 550 g for 10 min to remove the nuclei and cell debris. Subsequently, the supernatant was centrifuged at 3,200 g for 10 min, followed by centrifugation at 22,500 g for 10 min to remove the bulk of the mitochondria, lysosomes, and peroxisomes. To isolate enriched microsomal membranes, the supernatant was sonicated three times for 10 s with an interval of 1 min on ice and centrifuged for 1 h at 100,000 g. The complete isolation procedure was carried out at 4°C. The enriched microsomal membrane fraction was resuspended in phosphate-buffered saline, pH 7.4, and divided into small aliquots that were stored at −80°C until further use. Protein was measured according to the method described by Bradford (21Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217546) Google Scholar). Incubations were carried out in a reaction mixture containing glycine (100 mM)/Hepes (100 mM) buffer, α-cyclodextrin (1 mg/ml), and NAD+ or NADPH (1 mM) in a total volume of 200 μl for 15–30 min at 37°C. Incubations were carried out at pH 8.5 using NADPH as cofactor or at pH 9.5 using NAD+ as cofactor. After preincubation at 37°C for 10 min, the reaction was initiated by addition of the substrate at a final concentration of 120 μM and terminated by addition of 1 ml of hydrochloric acid to a final concentration of 1.7 M. C22:0 and ω-hydroxy-C22:0 were dissolved in dimethyl sulfoxide to a final concentration of 2.8 mM. Depletion of molecular oxygen was accomplished by gently flushing the reaction mixture with nitrogen for 20 min on ice. Experiments to determine the cofactor dependence were carried out in the standard reaction mixture supplemented with a NAD+ or NADPH regenerating system. With the NAD+ regenerating system, the reaction mixture contained pyruvate (1 mM) and l-lactate dehydrogenase (4 U/ml). To regenerate NADPH, MgCl2 (3 mM), glucose-6-phosphate (10 mM), and glucose-6-phosphate dehydrogenase (20 U/ml) were added to the reaction mixture. After termination of the reactions, the mixture was transferred to a 4 ml glass vial followed by addition of 100 μl of internal standard solution containing D4-C22:0 and D4-C24:0 in toluene and 2 ml of hexane. The samples were vortex mixed thoroughly for 30 s and centrifuged at 3,000 rpm for 1 min. Approximately 1.5 ml of the upper phase was transferred to a 16 × 125 mm glass tube, and hexane was evaporated at 37°C under a constant stream of nitrogen. Finally, the residue was dissolved in a 100 μl chloroform-methanol-water mixture containing and transferred to The samples were analyzed according to the method described by Valianpour et al. F. A.H. Wanders S.S. of very long-chain fatty acids using electrospray ionization mass Genet. Metab. 2003; PubMed Scopus Google Scholar). the of fatty acid a was for ω-hydroxy-C22:0 and hexacosanedioic The of hexacosanedioic acid was used to determine the concentration of docosanedioic acid the is not were and analyzed as described the concentration was the of the of the to the of internal The and the were used to the of the in the of fatty acids than 20 was in rat liver microsomes. We with determination of the using C22:0 as To this end, rat liver microsomes were incubated in a at different pH containing 1 mM NADPH and α-cyclodextrin to The in a pH and The activity was found at pH and this pH was for further the of an in which the NADPH concentration was and 1 mM. In the presence of NADPH, a product was formed with an of to at of NADPH 250 a second product was formed with an of that to The of this as C22:0-DCA was by the that was by the of a at which the of C22:0-DCA the of the ω-oxidation of C22:0 at different substrate from the the for C22:0 was 30 Formation of C22:0-DCA was at the substrate concentration used and increased substrate To whether the production of C22:0-DCA was to of NADPH with NAD+, the of NADPH was using This that NADPH was not with NAD+ not and that C22:0 can be converted to C22:0-DCA using NADPH as study of and of C22:0 in rat liver microsomes. The standard reaction mixture contained of 1 mM NADPH, and 120 μM were terminated at the The of the of The with an of and the and first The single The with of and the of and D4-C24:0 was used as an internal standard and has a with an of To and of C22:0, the substrate was by of rat liver microsomes with this substrate in the of the two hydroxylated products of the substrate at the terminal in with an of to the of one In a with an of was that to the product of The of the with was for the of the first from these we that the of to was To study the of the of ω-hydroxy acid to dicarboxylic we C22:0 by ω-hydroxy-C22:0 in In the presence of NADPH as as NAD+ as C22:0-DCA was formed whereas no product was when or was used as cofactor not of ω-hydroxy-C22:0 to C22:0-DCA was at pH 8.5 with NADPH as cofactor At this the for NADPH and ω-hydroxy-C22:0 were and from The were μM for ω-hydroxy-C22:0 and 4 μM for NADPH In the presence of NAD+, ω-oxidation of ω-hydroxy-C22:0 was at pH 9.5 The were and μM for ω-hydroxy-C22:0 and NAD+, To provide further for the existence of two pathways for ω-oxidation of C22:0, we studied the oxygen dependence of the and Depletion of molecular oxygen in a in the of C22:0-DCA in the presence of NADPH, whereas in the presence of NAD+ ω-oxidation activity was not significantly To further these two ω-oxidation several inhibitors were Formation of C22:0-DCA via either the or the system was inhibited strongly by the and an of a of cytochrome K. J. J. of miconazole, ketoconazole and with rat-liver PubMed Scopus Google strongly inhibited the pathway but not the system. were with disulfiram, which is an of the microsomal fatty aldehyde dehydrogenase M. Human liver fatty aldehyde microsomal and Biophys. Acta. PubMed Scopus Google and cytochrome P450 of on the of and in human J. Clin. PubMed Scopus Google of molecular oxygen and different inhibitors on the ω-oxidation of C22:0 and NADPH 1 10 docosanoic ω-hydroxy-C22:0, 22-hydroxy-docosanoic Rat liver microsomes were in the standard reaction mixture containing inhibitors at the for 10 min. After were initiated by the substrate and were to for min at 37°C. The are of three to and are to the activity in the of in a C22:0, docosanoic ω-hydroxy-C22:0, 22-hydroxy-docosanoic Rat liver microsomes were in the standard reaction mixture containing inhibitors at the for 10 min. After were initiated by the substrate and were to for min at 37°C. The are of three to and are to the activity in the of The of different cytochrome P450 enzymes was studied using ketoconazole and of cytochrome P450 inhibitors substrates in human and rat liver J. Clin. 1998; PubMed Scopus Google Scholar, K. K. N. K. of human cytochrome P450 inhibitors toward rat P450 study with of the Metab. 2003; PubMed Scopus Google of cytochrome P450 inhibitors substrates in human and rat liver J. Clin. 1998; PubMed Scopus Google N. P. of and as inhibitors of cytochrome P450 Metab. Google Wang J. and are inhibitors of and Metab. PubMed Scopus Google and F. J.R. activity and inhibition of rat cytochrome P450 2004; PubMed Scopus Google Scholar, Wang J.R. of the rat CYP4A acid metabolism and J. Physiol. Google Scholar). of C22:0 and was inhibited by ketoconazole and whereas the other inhibitors no on the hydroxylation of the two substrates In this we that C22:0 is a substrate for the ω-oxidation system in rat liver microsomes. The first reaction step, the hydroxylation of the terminal methyl group, is and molecular This reaction is by miconazole, a cytochrome P450 that one or more of the cytochrome P450 family are the terminal and the can be The hydroxylation of the at the or with a of for the we found that of microsomes with C22:0 in the presence of NADPH not only produced ω-hydroxy-C22:0 but also in the of of microsomes with ω-hydroxy-C22:0 demonstrated the of C22:0-DCA in the presence of either NADPH or Based on the in pH substrate and cofactor we the existence of two different ω-oxidation for ω-hydroxy-C22:0 in rat liver microsomes. In the presence of NADPH, the of C22:0-DCA from ω-hydroxy-C22:0 was decreased when molecular oxygen was whereas little was with NAD+ as cofactor. is known for inhibition of a microsomal fatty aldehyde dehydrogenase M. Human liver fatty aldehyde microsomal and Biophys. Acta. PubMed Scopus Google and of on the of and in human J. Clin. PubMed Scopus Google Scholar). is by the gene and is in patients with syndrome fatty alcohol oxidation in to fatty Clin. Invest. 1988; PubMed Scopus Google Scholar). In to with NADPH, no on the of C22:0-DCA in the presence of This suggests that is to be in the ω-oxidation of is that aldehyde is in rat liver which catalyzes the oxidation of into dicarboxylic an of cytochrome P450 only inhibited C22:0-DCA production when NADPH was used as cofactor. This result strongly suggests that cytochrome P450 hydroxylases are not only in the of C22:0 into ω-hydroxy-C22:0 but also in the oxidation of ω-hydroxy-C22:0 into A mechanism for the two pathways is in In the first the of C22:0 into ω-hydroxy-C22:0 is catalyzed by a and molecular cytochrome P450 In the ω-hydroxy-C22:0 can be converted into an aldehyde by an alcohol dehydrogenase which is oxidized further by an aldehyde in C22:0-DCA ω-hydroxy-C22:0 can also be oxidized into C22:0-DCA via a and molecular to this ω-hydroxy-C22:0 be hydroxylated to which can be catalyzed either by the cytochrome P450 that is in the initial or by other cytochrome of from which can be hydroxylated to C22:0-DCA This mechanism has also been for hydroxylation in liver mitochondria as catalyzed by I. E. I. K. in acid of oxidation of 5 into acid.J. Biol. Chem. Full Text PDF PubMed Google hydroxylation of in T. F. in of to acids catalyzed by cytochrome P450 Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and oxidation of to in liver microsomes G. C. T. M. Cytochrome P-450 and of PubMed Scopus Google Scholar). of C22:0 and was inhibited by ketoconazole and that one or more cytochrome P450 enzymes belonging to the and/or are The inhibition by in strongly suggests to the of cytochrome P450 enzymes belonging to the CYP4A and CYP4F Wang J.R. of the rat CYP4A acid metabolism and J. Physiol. Google Scholar, F. J.R. activity and inhibition of rat cytochrome P450 2004; PubMed Scopus Google Scholar, K. K. N. K. of human cytochrome P450 inhibitors toward rat P450 study with of the Metab. 2003; PubMed Scopus Google Scholar). The described in this that C22:0 is a substrate for the microsomal ω-oxidation We that this alternative fatty acid oxidation route may be in several inherited peroxisomal in which plasma levels of VLCFAs are increased but C22:0 levels are decreased (18Kemp S. Valianpour F. Denis S. Ofman R. Sanders R.J. Mooyer P. Barth P.G. Wanders R.J.J. Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy.Mol. Genet. Metab. 2005; 84: 144-151Crossref PubMed Scopus (73) Google Scholar). with a peroxisomal biogenesis have a deficiency in β-oxidation increased levels of fatty acids and dicarboxylic acids in urine (19Rocchiccioli F. Aubourg P. Bougneres P.F. Medium- and long-chain dicarboxylic aciduria in patients with Zellweger syndrome and neonatal adrenoleukodystrophy.Pediatr. Res. 1986; 20: 62-66Crossref PubMed Scopus (57) Google Scholar). This that microsomal ω-oxidation of VLCFAs is also in In that C22:0 may undergo ω-oxidation to C22:0-DCA via two distinct that the first the cytochrome of ω-hydroxy-C22:0 from ω-hydroxy-C22:0 can undergo two oxidation driven by NAD+ to ω-hydroxy-C22:0 may also undergo two cytochrome to the product, It to be whether of these cytochrome P450 steps involve one or more of this enzyme The for with the electrospray ionization mass and and and for This was by from the the for the and the

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