title: NADPH-dependent formation of 15- and 12-hydroxyeicosatrienoic acid from arachidonic acid by rat epidermal microsomes.
abstract: Rat epidermal microsomes were incubated with [1-14C]-arachidonic acid for 30 min at 37 degrees C in the absence and presence of NADPH. The arachidonate metabolites that eluted in the "monohydroxy acid fraction" on reverse-phase high performance liquid chromatography (HPLC) were methylated, purified by straight-phase HPLC and analyzed by chromatography with standard compounds, UV spectroscopy and/or gas chromatography-mass spectrometry (GC-MS). In the absence of NADPH, epidermal microsomes converted arachidonic acid to two major products identified as 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE). In the presence of NADPH, the microsomal reaction produced, besides 15(S)- and 12(S)-HETE, two less polar metabolites which were characterized as 15-hydroxy-5,8,11,-eicosatrienoic acid (15-HETrE) and 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). Stereochemical analysis by chiral-phase HPLC showed that the biosynthesized 12-HETrE consisted of a mixture of optical isomers in a S/R ratio of 65:35. Formation of 15- and 12-HETrE was blocked by the mixed cyclooxygenase-lipoxygenase inhibitors quercetin and phenidone but was not affected by the cyclooxygenase inhibitor indomethacin or the cytochrome P-450 monooxygenase inhibitor metyrapone. These data indicate that rat epidermal microsomes, supplemented with NADPH, are capable of metabolizing arachidonic acid to 15- and 12-HETrE. The production of these compounds may be initiated by lipoxygenase-mediated hydroperoxidation of arachidonic acid.
authors.affiliation: Janssen Research Foundation, Beerse, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Van Wauwe
authors.forename: M C
authors.initials: MC
authors.lastname: Coene
authors.forename: G
authors.initials: G
authors.lastname: Van Nyen
authors.forename: W
authors.initials: W
authors.lastname: Cools
authors.forename: J
authors.initials: J
authors.lastname: Goossens
authors.forename: L
authors.initials: L
authors.lastname: Le Jeune
authors.forename: W
authors.initials: W
authors.lastname: Lauwers
authors.forename: P A
authors.initials: PA
authors.lastname: Janssen
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D000831:Animals, Newborn; D001095:Arachidonic Acids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004817:Epidermis / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D013058:Mass Spectrometry; D008861:Microsomes / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013237:Stereoisomerism
chemical_list: D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D009249:NADP; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid