LC-MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22.
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 [(2)H(8)]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 omega3 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 omega3 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 omega3 position.
Nilsson
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Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Biomedicum, Uppsala University, Uppsala, Sweden.
Ivanov
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IV
Oliw
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EH
D000438:Alcohols / Q000032:analysis* / Q000378:metabolism; D016718:Arachidonic Acid / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003315:Cornea / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004852:Epoxy Compounds / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D010100:Oxygen / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000438:Alcohols; D004852:Epoxy Compounds; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D003577:Cytochrome P-450 Enzyme System; C559989:CYP4F22 protein, human; D010100:Oxygen