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Journal of Lipid Research, Vol. 49, 612-624, March 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

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

Madhurima Dhar^*, Daniel W. Sepkovic^*, Vandana Hirani^*, Ronald P. Magnusson and Jerome M. Lasker^1,*

^* Jurist Institute for Research, Hackensack University Medical Center, Hackensack, NJ 07601
Kinnakeet Biotechnology LLC, Midlothian, VA 23112

Published, JLR Papers in Press, December 7, 2007.

² Hirani V., A. Kozeska, and J. M. Lasker, unpublished observations.

³ We used 16-hydroxypalmitate for standard curves rather than 3,16-dihydroxypalmitate or 3,18-dihydroxystearate because the latter compounds were not commercially available and because the PDAM derivatives of medium- and long-chain fatty acids, such as laurate, oleate, and palmitate, exhibit nearly identical extinction coefficients at 275 nm (19).

⁴ When this antibody was first produced, only two human CYP4F P450s had been identified, CYP4F2 and CYP4F3a, the latter of which is found exclusively in myeloid cells. Later studies revealed the expression in human liver and kidney of CYP4F3b (the alternatively spliced form of CYP4F3a) (10), CYP4F11, and CYP4F12 (11, 40–42). Because of the extensive (75%) sequence homology among the CYP4F P450s, it is now known that our original polyclonal CYP4F2 antibody cross-reacts with all of these enzymes.

⁵ Hirani V., A. Yarovoy, A. Kozeska, and J. M. Lasker. Expression of CYP4F2 in human liver and kidney: assessment using specific peptide antibodies, submitted, 2007.

¹ To whom correspondence should be addressed. e-mail: jlasker{at}humed.com

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via β-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor β-oxidized in mitochondria. We thus examined whether -hydroxylation of 3-hydroxy fatty acids (3-OHFAs), 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 -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 -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 -hydroxylation of 3-hydroxystearate, whereas 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 K_m = 55 µM and V_max = 8.33 min^–1, whereas those for 3-hydroxypalmitate were K_m = 56.4 µM and V_max = 14.2 min^–1. CYP4F11 kinetic values resembled those of native microsomes, with K_m = 53.5 µM and V_max = 13.9 min^–1 for 3-hydroxystearate and K_m = 105.8 µM and V_max = 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 -hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid β-oxidation increases circulating 3-OHFA levels.

Supplementary key words dicarboxylic acids • -hydroxylation • dihydroxy fatty acids • cytochrome 450 enzymes

Abbreviations: b₅, cytochrome b₅; BSTFA, N,O-bis-(trimethylsilyl) trifluoroacetamide; DCA, dicarboxylic acid; LCHAD, long-chain 3-hydroxyacyl-coenzyme A dehydrogenase; MTP, mitochondrial trifunctional protein; 3-OHDCA, 3-hydroxy dicarboxylic acid; 3-OHFA, 3-hydroxy fatty acid; P450 reductase, PDAM, 1-pyrenyldiazomethane; NADPH:P450 oxidoreductase; R_T, retention time on HPLC or GC

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