Regio- and stereoselective epoxidation of arachidonic acid by human cytochromes P450 2C8 and 2C9

B. E. Daikh, J. M. Lasker, J. L. Raucy, Dennis Koop

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Abstract

In the present study, the regio- and stereoselective epoxidation of arachidonic acid by cytochromes P450 2C8 and 2C9, two members of the CYP2C gene subfamily expressed in human liver, was determined. Purified P450 isozymes, reconstituted with NADPH:P450 oxidoreductase, cytochrome b5 and lipid, or microsomes isolated from human liver, were incubated with [1- 14C]-arachidonic acid. For regioselective analysis, the epoxide metabolites formed, 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (EETs), were resolved by reverse-phase high-performance liquid chromatography. P450 2C8 produces only the 14,15- and 11,12-EETs in a 1.25:1.00 ratio. The two epoxides represent 68% of the total metabolites. P450 2C9 produces 14,15-, 11,12- and 8,9-EETs in a 2.3:1.0:0.5 ratio. The three epoxides represent 69% of the total metabolites. Neither P450 isoform catalyzes the formation of 5,6-EET. For chiral analysis, the two major epoxide metabolites, 14,15- and 11,12-EETs, were derivatized to methyl and pentafluorobenzyl esters, respectively. Enantiomers of 14,15- and 11,12-EET esters were subsequently resolved on Chiralcel OB and OD columns (J. T. Baker, Phillipsburg, PA), respectively. Both P450 2C8 and 2C9 are stereoselective at the 14,15- position, preferentially producing 14(R),15(S)-EET with 86.2% and 62.5% selectivity, respectively. Both enzymes are also stereoselective at the 11,12-position but have the opposite selectivity. P450 2C8 is 81.1% selective for 11(R),12(S)-EET; P450 2C9 is 69.4% selective for the 11(S),12(R)-EET. Immunoinhibition studies performed with anti-2C9 immunoglobulin G (which also reacts with P450 2C8) and hepatic microsomes indicate that these two P450s are important arachidonic acid epoxygenases in human liver.

Original languageEnglish (US)
Pages (from-to)1427-1433
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume271
Issue number3
StatePublished - 1994

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Arachidonic Acid
Cytochrome P-450 Enzyme System
Epoxy Compounds
Liver
Microsomes
Esters
Cytochromes b5
Reverse-Phase Chromatography
NADP
Isoenzymes
Oxidoreductases
Protein Isoforms
Immunoglobulin G
High Pressure Liquid Chromatography
11,12-epoxy-5,8,14-eicosatrienoic acid
Lipids
Enzymes
Genes
8,9-epoxyeicosatrienoic acid

ASJC Scopus subject areas

  • Pharmacology

Cite this

Regio- and stereoselective epoxidation of arachidonic acid by human cytochromes P450 2C8 and 2C9. / Daikh, B. E.; Lasker, J. M.; Raucy, J. L.; Koop, Dennis.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 271, No. 3, 1994, p. 1427-1433.

Research output: Contribution to journalArticle

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title = "Regio- and stereoselective epoxidation of arachidonic acid by human cytochromes P450 2C8 and 2C9",
abstract = "In the present study, the regio- and stereoselective epoxidation of arachidonic acid by cytochromes P450 2C8 and 2C9, two members of the CYP2C gene subfamily expressed in human liver, was determined. Purified P450 isozymes, reconstituted with NADPH:P450 oxidoreductase, cytochrome b5 and lipid, or microsomes isolated from human liver, were incubated with [1- 14C]-arachidonic acid. For regioselective analysis, the epoxide metabolites formed, 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (EETs), were resolved by reverse-phase high-performance liquid chromatography. P450 2C8 produces only the 14,15- and 11,12-EETs in a 1.25:1.00 ratio. The two epoxides represent 68{\%} of the total metabolites. P450 2C9 produces 14,15-, 11,12- and 8,9-EETs in a 2.3:1.0:0.5 ratio. The three epoxides represent 69{\%} of the total metabolites. Neither P450 isoform catalyzes the formation of 5,6-EET. For chiral analysis, the two major epoxide metabolites, 14,15- and 11,12-EETs, were derivatized to methyl and pentafluorobenzyl esters, respectively. Enantiomers of 14,15- and 11,12-EET esters were subsequently resolved on Chiralcel OB and OD columns (J. T. Baker, Phillipsburg, PA), respectively. Both P450 2C8 and 2C9 are stereoselective at the 14,15- position, preferentially producing 14(R),15(S)-EET with 86.2{\%} and 62.5{\%} selectivity, respectively. Both enzymes are also stereoselective at the 11,12-position but have the opposite selectivity. P450 2C8 is 81.1{\%} selective for 11(R),12(S)-EET; P450 2C9 is 69.4{\%} selective for the 11(S),12(R)-EET. Immunoinhibition studies performed with anti-2C9 immunoglobulin G (which also reacts with P450 2C8) and hepatic microsomes indicate that these two P450s are important arachidonic acid epoxygenases in human liver.",
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T1 - Regio- and stereoselective epoxidation of arachidonic acid by human cytochromes P450 2C8 and 2C9

AU - Daikh, B. E.

AU - Lasker, J. M.

AU - Raucy, J. L.

AU - Koop, Dennis

PY - 1994

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N2 - In the present study, the regio- and stereoselective epoxidation of arachidonic acid by cytochromes P450 2C8 and 2C9, two members of the CYP2C gene subfamily expressed in human liver, was determined. Purified P450 isozymes, reconstituted with NADPH:P450 oxidoreductase, cytochrome b5 and lipid, or microsomes isolated from human liver, were incubated with [1- 14C]-arachidonic acid. For regioselective analysis, the epoxide metabolites formed, 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (EETs), were resolved by reverse-phase high-performance liquid chromatography. P450 2C8 produces only the 14,15- and 11,12-EETs in a 1.25:1.00 ratio. The two epoxides represent 68% of the total metabolites. P450 2C9 produces 14,15-, 11,12- and 8,9-EETs in a 2.3:1.0:0.5 ratio. The three epoxides represent 69% of the total metabolites. Neither P450 isoform catalyzes the formation of 5,6-EET. For chiral analysis, the two major epoxide metabolites, 14,15- and 11,12-EETs, were derivatized to methyl and pentafluorobenzyl esters, respectively. Enantiomers of 14,15- and 11,12-EET esters were subsequently resolved on Chiralcel OB and OD columns (J. T. Baker, Phillipsburg, PA), respectively. Both P450 2C8 and 2C9 are stereoselective at the 14,15- position, preferentially producing 14(R),15(S)-EET with 86.2% and 62.5% selectivity, respectively. Both enzymes are also stereoselective at the 11,12-position but have the opposite selectivity. P450 2C8 is 81.1% selective for 11(R),12(S)-EET; P450 2C9 is 69.4% selective for the 11(S),12(R)-EET. Immunoinhibition studies performed with anti-2C9 immunoglobulin G (which also reacts with P450 2C8) and hepatic microsomes indicate that these two P450s are important arachidonic acid epoxygenases in human liver.

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