LC–MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22

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Abstract

CYP4F22 and CYP4F8 are expressed in epidermis, and mutations of CYP4F22 are associated with lamellar ichthyosis. Epoxyalcohols (HEETs) and epoxides (EETs) of 20:4n−6 appear to be important for the water permeability barrier of skin. Our aim was to study the MS/MS spectra and fragmentation of these compounds and to determine whether they were oxidized by CYP4F22 or CYP4F8 expressed in yeast. HEETs were prepared from 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 12-HPETE, and their [2H8]labeled isotopomers, and separated by normal phase-HPLC with MS/MS analysis. CYP4F22 oxygenated 20:4n−6 at C-18, whereas metabolites of HEETs could not be identified. CYP4F8 formed ω3 hydroxy metabolites of HEETs derived from 12R-HPETE with 11,12-epoxy-10-hydroxy configuration, but not HEETs derived from 15S-HPETE. 8,9-EET and 11,12-EET were also subject to ω3 hydroxylation by CYP4F8. We conclude that CYP4F8 and CYP4F22 oxidize 20:4n−6 and that CYP4F8 selectively oxidizes 8,9-EET, 11,12-EET, and 10,11R,12R-HEET at the ω3 position.

Section snippets

Materials

20:4n−6 (>99%) was from Sigma. 12(S)-Hydroperoxyeicosa-5(Z),8(Z),10(E),14(Z)-tetraenoic acid (12S-HPETE) was from Larodan Fine Chemicals (Malmö, Sweden). 14(S),15(S)-trans-epoxy-13(S)-hydroxy-5(Z),8(Z),11(Z)-eicosatrienoate (13S,14S,15S-HEET) methyl ester, 14(S),15(S)-trans-epoxy-11(S)-hydroxy-5(Z),8(Z),12(E)-eicosatrienoate (11S,14S,15S-HEET) methyl ester, and 14(S),15(S)-trans-epoxy-11(R)-hydroxy-5(Z),8(Z),12(E)-eicosatrienoate (11R,14S,15S-HEET) methyl ester were from Lipidox (Stockholm,

HEETs from 15S-HPETE

HEETs were generated by hematin treatment of 15S-HPETE. Fig. 2 shows the separation of the HEETs on NP-HPLC (3% isopropanol in hexane). Four HEETs were generated as main products: the erythro isomer 13S,14S,15S-HEET (peak I) and the threo isomer 13R,14S,15S-HEET (peak II), 11S,14S,15S-HEET (peak III), and 11R,14S,15S-HEET (peak IV). The minor peaks likely consisted of the HEETs with cis epoxy conformation as judged from their MS/MS spectra.

11,14S,15S-HEET and 13,14S,15S-HEET fragmented adjacent

Discussion

We report expression of recombinant CYP4F22 with arachidonate ω3 hydroxylase activity. This confirms that the open reading frame of CYP4F22 codes for a functional enzyme, but unfortunately the enzyme appeared to be poorly expressed. As we expected that CYP4F22 also could metabolize HEETs, we performed a systematic study of the MS/MS spectra of HEETs in order to determine metabolites formed by CYP4F22 and by CYP4F8.

18-HETE cannot be formed non-enzymatically. Biosynthesis of this metabolite by

Acknowledgments

This work was supported by Vetenskapsrådet Medicin (3X-06523), Stiftelsen Lars Hiertas Minne (KDB269/08), and The Knut and Alice Wallenberg Foundation (2004.0123).

References (40)

  • M. Fer et al.

    J. Lipid Res.

    (2008)
  • J. Bylund et al.

    J. Biol. Chem.

    (2000)
  • F.P. Guengerich et al.

    Biochem. Biophys. Res. Commun.

    (2005)
  • K. Stark et al.

    Arch. Biochem. Biophys.

    (2003)
  • P.M. Woollard

    Biochem. Biophys. Res. Commun.

    (1986)
  • G. Fürstenberger et al.

    Prostaglandins Other Lipid Mediat.

    (2007)
  • J.L. Moran et al.

    J. Invest. Dermatol.

    (2007)
  • K.M. Eckl et al.

    J. Invest. Dermatol.

    (2009)
  • F. Lesueur et al.

    J. Invest. Dermatol.

    (2007)
  • Z. Yu et al.

    Biochim. Biophys. Acta

    (2005)
  • C.R. Pace-Asciak

    Biochim. Biophys. Acta

    (1994)
  • A.A. Spector

    J. Lipid Res.

    (2009)
  • P.A. Ladd et al.

    J. Biol. Chem.

    (2003)
  • L.A. Cowart et al.

    J. Biol. Chem.

    (2002)
  • J. Bylund et al.

    Biochem. Biophys. Res. Commun.

    (1999)
  • K. Stark et al.

    Arch. Biochem. Biophys.

    (2005)
  • I.V. Ivanov et al.

    Bioorg. Med. Chem.

    (2002)
  • T.A. Dix et al.

    J. Biol. Chem.

    (1985)
  • E.H. Oliw et al.

    Anal. Biochem.

    (2006)
  • E.H. Oliw

    J. Chromatogr.

    (1983)
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