ω-Oxidation of Very Long-chain Fatty Acids in Human Liver Microsomes

Abstract

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder biochemically characterized by elevated levels of very long-chain fatty acids (VLCFA). Excess levels of VLCFAs are thought to play an important role in the pathogenesis of X-ALD. Therefore, therapeutic approaches for X-ALD are focused on the reduction or normalization of VLCFAs. In this study, we investigated an alternative oxidation route for VLCFAs, namely -oxidation. The results described in this study show that VLCFAs are substrates for the -oxidation system in human liver microsomes. Moreover, VLCFAs were not only converted into -hydroxy fatty acids, but they were also further oxidized to dicarboxylic acids via cytochrome P450-mediated reactions. High sensitivity toward the specific P450 inhibitor 17-octadecynoic acid suggested that -hydroxylation of VLCFAs is catalyzed by P450 enzymes belonging to the CYP4A/F subfamilies. Studies with individually expressed human recombinant P450 enzymes revealed that two P450 enzymes, i.e. CYP4F2 and CYP4F3B, participate in the -hydroxylation of VLCFAs. Both enzymes belong to the cytochrome P450 4F subfamily and have a high affinity for VLCFAs. In summary, this study demonstrates that VLCFAs are substrates for the human -oxidation system, and for this reason, stimulation of the in vivo VLCFA -oxidation pathway may provide an alternative mode of treatment to reduce the levels of VLCFAs in patients with X-ALD.In mammalian cells, fatty acid oxidation plays a major role in the production of energy, particularly in the heart and skeletal muscle, and is the main energy source during periods of fasting. Both mitochondria and peroxisomes are capable of degrading saturated fatty acids via ␤-oxidation. Short-, medium-, and long-chain saturated fatty acids are degraded predominantly by mitochondria, whereas very long-chain fatty acids (VLCFA, 2 Ͼ22 carbons) are ␤-oxidized exclusively in peroxisomes (1, 2). Moreover, peroxisomes also metabolize certain branched chain fatty acids, bile acid precursors, eicosanoids, and dicarboxylic acids (3).X-linked adrenoleukodystrophy (X-ALD: MIM 300100), the most common peroxisomal disorder, is a progressive neurodegenerative disease that affects the cerebral white matter, spinal cord, peripheral nerves, adrenal cortex, and testis (4). X-ALD is caused by mutations in the ABCD1 gene that encodes ALDP, an ATP-binding cassette transporter located in the peroxisomal membrane with an unknown function (5). Biochemically, X-ALD is characterized by elevated levels of saturated and monounsaturated VLCFAs in plasma and tissues due to the impaired ␤-oxidation of VLCFAs in peroxisomes (6 -8). Since the pathogenesis of X-ALD is probably due to the increased levels of VLCFAs, correction of VLCFA levels is one of the primary objectives in therapeutic approaches. These include gene replacement therapy, bone marrow transplantation, lovastatin treatment, inhibition of VLCFA biosynthesis by mono-unsaturated fatty acids, notably oleic acid (C18:19) and erucic acid (C22:19) (Lorenzo's oil), a...

Regulation of Human Cytochrome P450 4F2 Expression by Sterol Regulatory Element-binding Protein and Lovastatin

“…C22:0, C24:0, and C26:0). This suggested that the induction of these enzymes could represent a potential therapeutic target for treatment of X-linked adenoleukodystrophy (X-ALD) disease (15). X-ALD is a recessive genetic disease and is characterized by a defect in peroxisomal ␤-oxidation of very long chain fatty acids with secondary neuroinflammatory damage.…”

“…Antibody inhibition experiments (13,14) suggest that P450 4F2/4F3B in human liver and kidney microsomes contribute ϳ66% to the formation of 20-HETE. Recently, P450s 4F2 and 4F3B were found to -hydroxylate very long chain saturated fatty acids (15) as well as phytanic acid (16). Our preliminary experiments using com-mercially available Supersomes TM (BD Gentest) indicate that P450 4F2 also -hydroxylates oleic acid, stearic acid, and palmitic acid, but not lauric acid.…”

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

“…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.…”

“…Recently, we demonstrated that VLCFAs can also be oxidized via another mechanism, i.e., -oxidation (17). The first step in this pathway involves the hydroxylation of the -methyl group of the VLCFA, generating a -hydroxy-VLCFA.…”

“…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).…”