Omega oxidation of 3-hydroxy fatty acids by the human CYP4F subfamily enzyme CYP4F11

Madhurima Dhar, Daniel W. Sepkovic, Vandana Hirani, Ronald P. Magnusson, and Jerome M. Lasker

Institute for Biomedical Research, Hackensack University Medical Center, Hackensack, NJ 07601

Corresponding Author: jlasker{at}humed.com

Long-chain 3-hydroxydicarboxylic acids (3-OHDCA) are thought to arise via ß-oxidation of the corresponding DCA, although long-chain DCA are neither readily transported into nor ß-oxidized in mitochondria. We thus examined if w-hydroxylation of 3-hydroxy fatty acids (3-OHFA), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their w-hydroxylated metabolites 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 ± 0.5 and 1.46 ± 0.30 nmol product formed/min/mg protein, respectively (mean ± SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal w-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%) whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed w-hydroxylation of 3-hydroxystearate while CYP4F3b, CYP4F12 and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were KM = 55 µM and VMAX =8.33 min-1 while those for 3-hydroxypalmitate were KM = 56.4 µM and VMAX = 14.2 min-1. CYP4F11 kinetic values resembled those of native microsomes, with a KM = 53.5 µM and VMAX = 13.9 min-1 for 3-hydroxystearate and a KM = 105.8 µM and VMAX = 70.6 min-1 for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to -hydroxylated 3-OHDCA precursors in human liver, and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted w-hydroxylation of 3-OHFA may modulate the disposition of these compounds in pathological states where enhanced fatty acid mobilization or impairment of mitochondrial fatty acid ß-oxidation increase circulating 3-OHFA levels.

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