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Synthesis and characterization of cytochrome P-450 epoxygenase metabolites of eicosapentaenoic acid

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Lipids

Abstract

Eicosapentaenoic acid, a major component of fish oil extracts, was recently shown to be metabolized to vicinal diol regioisomers by renal and hepatic cytochrome P-450 epoxygenases. The diol products were also found in the urine of people ingesting fish oil. In the present report, we developed a chemical method of making milligram amounts of the epoxide intermediates and their diol products. Eicosapentaenoic acid was reacted with 0.1 eq.m-chloroperoxybenzoic acid for 15 min. After normal- and reverse-phase high performance liquid chromatography plus capillary gas chromatography and electron impact mass spectrometry, the products were identified as 17,18-cis-epoxyeicosa-5,8,11,14-tetraenoic, 14,15-cis-epoxy-eicosa-5,8,11,17-tetraenoic, 11,12-cis-epoxy-eicosa-5,8,14,17-tetraenoic, 8,9-cis-epoxy-eicosa-5,11,14,17-tetraenoic and 5,6-cis-epoxy-eicosa-8,11,14,17-tetraenoic acids. The total epoxide yield from eicosapentaenoate was 10% per incubation. By reincubating (cycling) the unused substrate 10–20 times, the total epoxide yield could be increased to 66–88%. Epoxide regioisomers were not produced in equal amounts; epoxygenation was facilitated as the distance between the target double bond and carbomethoxy group increased. Upon hydrolyzing the epoxides, the gas-chromatographic retention times and mass spectra of the diol products were found to match those of biological metabolites. In addition, each of these standards was rapidly and quantitatively synthesized in an amount (milligram) adequate for biological tests.

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Abbreviations

c:dn-a:

“c” and “d” signify the number of carbon atoms and double bonds, respectively; the location of the last double bond is “a” carbons from the terminal methyl

DHEQ:

dihydroxyeicosaquatraenoic acid

DHET:

dihydroxyeicosatrienoic acid

ECL:

equivalent chain length

EEQ:

epoxyeicosaquatraenoic acid

EET:

epoxyeicosatrienoic acid

GC-MS:

gas chromatography-mass spectrometry

HPLC:

high performance liquid chromatography

m-CIPBA:

m-chloroperoxybenzoic acid

ODS:

octadecasilyl

RP- and NP-HPLC:

reverse-phase and normal-phase high performance liquid chromatography

TLC:

thin-layer chromatography

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Authors and Affiliations

  1. Hypertension Research Division, Henry Ford Hospital, 2799 West Grand Boulevard, 48202, Detroit, Michigan

    Mike VanRollins

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  1. Mike VanRollins
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VanRollins, M. Synthesis and characterization of cytochrome P-450 epoxygenase metabolites of eicosapentaenoic acid. Lipids 25, 481–490 (1990). https://doi.org/10.1007/BF02538092

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  • DOI: https://doi.org/10.1007/BF02538092

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