LC–MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22
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)
- et al.
J. Lipid Res.
(2008) - et al.
J. Biol. Chem.
(2000) - et al.
Biochem. Biophys. Res. Commun.
(2005) - et al.
Arch. Biochem. Biophys.
(2003) Biochem. Biophys. Res. Commun.
(1986)- et al.
Prostaglandins Other Lipid Mediat.
(2007) - et al.
J. Invest. Dermatol.
(2007) - et al.
J. Invest. Dermatol.
(2009) - et al.
J. Invest. Dermatol.
(2007) - et al.
Biochim. Biophys. Acta
(2005)
Biochim. Biophys. Acta
J. Lipid Res.
J. Biol. Chem.
J. Biol. Chem.
Biochem. Biophys. Res. Commun.
Arch. Biochem. Biophys.
Bioorg. Med. Chem.
J. Biol. Chem.
Anal. Biochem.
J. Chromatogr.
Cited by (16)
Evaluation of ω-alkynyl-labeled linoleic and arachidonic acids as substrates for recombinant lipoxygenase pathway enzymes
2023, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsSimultaneous determination of selected eicosanoids by reversed-phase HPLC method using fluorescence detection and application to rat and human plasma, and rat heart and kidney samples
2015, Journal of Pharmaceutical and Biomedical AnalysisEicosanoids in Metabolic Syndrome
2013, Advances in PharmacologyCitation Excerpt :Hydroxylation of AA is performed by other CYP enzymes forming a series of subterminal regioisomeric HETE, 16-,17-, 18-, and 19-HETE is catalyzed by the ethanol-inducible CYP2E1 (18-, 19-HETE), CYP1A1 and CYP1A2 (16-,17-, 18-HETE); CYP2J9 that produces exclusively 19-HETE; and CYP4F22 (18-HETE) (Nilsson, Ivanov, & Oliw, 2010). A recent study on the activity of the 19-hydroxy-PGH2 CYP4F8 and CYP4F22 P450 in the metabolism of AA and ω3 PUFA showed that CYP4F22 produces 18-HETE, and CYP4F8 metabolizes ω3-PUFA to 8,9- and 11,12-epoxyalcohols (HEETS-hydroxyepoxyeicosatrienoic acid) (Nilsson et al., 2010). The human CYP4A11 ω-hydroxylase produces 20- and 19-HETEs in a ratio of 90:10, while members of the human CYP4F subfamily CYP4F2, CYP4F3A, CYP4F3B show strict regioselectivity in the ω-hydroxylation of AA (Konkel & Schunck, 2011).
The expression of epidermal lipoxygenases and transglutaminase-1 is perturbed by NIPAL4 mutations: Indications of a common metabolic pathway essential for skin barrier homeostasis
2012, Journal of Investigative DermatologyCitation Excerpt :Recently, TGM1 was found to catalyze the covalent coupling of long-chain ω-hydroxyceramides to the cornified envelope (Nemes et al., 1999; Elias et al., 2002; Zheng et al., 2011). Several of the other genes encode proteins that are involved in either epidermal lipid transport or in the 12-lipoxygenase (LOX) (hepoxilin) pathway, which converts the essential fatty acid arachidonic acid present on all cell membranes into specific epoxyalcohols essential for epidermal barrier homeostasis (Eckl et al., 2005; Hall et al., 2005; Lefèvre et al., 2006; Milger et al., 2006; Zuo et al., 2008; Nilsson et al., 2010). It was also recently shown that linoleic acid esterified to ω-hydroxylacyl-sphingosine might be a target for epidermal enzymes 12R-LOX and eLOX-3 (Zheng et al., 2011).
CYP4F22 is highly expressed at the site and timing of onset of keratinization during skin development
2012, Journal of Dermatological ScienceConstruction of an lncRNA model for prognostic prediction of bladder cancer
2022, BMC Medical Genomics