NADPH-dependent formation of 15- and 12-hydroxyeicosatrienoic acid from arachidonic acid by rat epidermal microsomes

• Published in: Eicosanoids Vol. 4; no. 3; p. 155
• Main Authors: Van Wauwe, J, Coene, M C, Van Nyen, G, Cools, W, Goossens, J, Le Jeune, L, Lauwers, W, Janssen, P A
• Format: Journal Article
• Language: English
• Published: Germany 1991
• Subjects: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Animals, Newborn; Arachidonic Acids - metabolism; Chromatography, High Pressure Liquid; Epidermis - metabolism; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids - metabolism; Mass Spectrometry; Microsomes - metabolism; NADP - metabolism; Rats; Rats, Inbred Strains; Stereoisomerism
• ISSN: 0934-9820

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.