Abstract
20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a principal arachidonic acid (AA) metabolite formed viaP450-dependent oxidation in hepatic and renal microsomes. Although 20-HETE plays an important role in the regulation of cell and/or organ physiology, the P450 enzyme(s) catalyzing its formation in humans remain undefined. In this study, we have characterized AA ω-hydroxylation to 20-HETE by human hepatic microsomes and identified the underlying P450s. Analysis of microsomal AA ω-hydroxylation revealed biphasic kinetics (KM1 andVMAX1 = 23 μM and 5.5 min−1; KM2 andVMAX2 = 144 μM and 18.8 min−1) consistent with catalysis by at least two enzymes. Of the human P450s examined, CYP4A11 and CYP4F2 were both potent AA ω-hydroxylases, exhibiting rates of 15.6 and 6.8 nmol 20-HETE formed/min/nmol P450, respectively. Kinetic parameters of 20-HETE formation by CYP4F2 (KM = 24 μM;VMAX = 7.4 min−1) and CYP4A11 (KM = 228 μM;VMAX = 49.1 min−1) resembled the low and high KM components, respectively, found in liver microsomes. Antibodies to CYP4F2 markedly inhibited (93.4 ± 6%; n = 5) formation of 20-HETE by hepatic microsomes, whereas antibodies to CYP4A11 were much less inhibitory (13.0 ± 9%; n = 5). Moreover, a strong correlation (r = 0.78; P < .02) was found between microsomal CYP4F2 content and AA ω-hydroxylation among nine subjects. The correlation (r = 0.76; P < .02) also noted between CYP4A11 content and 20-HETE formation stemmed from the relationship (r = 0.83; P < .02) between hepatic CYP4A11 and CYP4F2 levels in the subjects. Finally, immunoblot analysis revealed that in addition to liver, both P450s also were expressed in human kidney. Our results indicate that AA ω-hydroxylation in human liver is catalyzed by two enzymes of the CYP4 gene family, namely CYP4F2 and CYP4A11, and that CYP4F2 underlies most 20-HETE formation occurring at relevant AA concentrations.
Footnotes
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Send reprint requests to: Dr. Jerome M. Lasker, Department of Biochemistry/Box 1020, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029.
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↵1 This work was supported by National Institutes of Health grant AA07842, and by the Liver Transplant, Procurement and Distribution System (DK62274).
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↵2 The P450 enzymes described in this report are designated according to the nomenclature of Nelson et al(1996).
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↵3 Jin R, Koop DR, Raucey JL and Lasker JM (1998) Role of human liver CYP4F2 in leukotriene B4 catabolism, submitted for publication. As detailed therein, CYP4F2 was identified as such based on its NH2-terminal amino acid sequence of Ser-Leu-Ser-Trp-Leu-Gly-Leu-Gly-Pro-Val-Ala-Ala-Ser-Pro-Trp-Leu-Leu, which corresponds to the sequence deduced from the human liverCYP4F2 cDNA (Kikuta et al, 1994). CYP4F2 is a potent leukotriene B4 ω-hydroxylase, whereas CYP4A11 does not catalyze this activity.
- Abbreviations:
- AA
- arachidonic acid
- HETE
- hydroxyeicosatetraenoic acid
- EET
- epoxyeicosatrienoic acid, 20-HETE, 20-hydroxy-5,8,11,14-eicosatetraenoic acid
- 19-HETE
- 19-hydroxy-5,8,11,14-eicosatetraenoic acid, diHETE, dihydroxyeicosatrienoic acid
- P450
- cytochrome P450
- b5
- cytochrome b5
- P450 reductase
- NADPH:P450 oxidoreductase
- kDa
- kilodaltons
- IgG
- immunoglobulin G
- KPO4
- potassium phosphate
- DLPC
- l-α-dilauroylphosphatidylcholine
- HPLC
- high-performance liquid chromatography
- Received November 4, 1997.
- Accepted February 2, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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Metabolism of Arachidonic Acid to 20-Hydroxy-5,8,11,14-eicosatetraenoic Acid by P450 Enzymes in Human Liver: Involvement of CYP4F2 and CYP4A11
