Identification of the major metabolite of 12-HETE produced by renal tubular epithelial cells.
The identification and polarity of release of the major metabolite of 12-HETE produced by cultured canine renal tubular epithelial cells was determined. When incubated with 1.0 microM [3H]12-HETE for 1 h, cultured Madin Darby Canine Kidney (MDCK) cells converted 35% of the radiolabeled 12-HETE to a more polar metabolite. Following high performance liquid chromatography isolation and chemical derivatization, gas-liquid chromatography combined with mass spectrometry was used to identify the compound as 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. The electron impact mass spectrum of the hydrogenated derivative contained major ions at m/z = 215 and 245, corresponding to cleavage on either side of the trimethylsilyl group, and chemical ionization with NH3 yielded a major ion at m/z = 359, corresponding to the protonated molecular weight of the methyl ester. Incubation with 25 mM alpha-naphthoflavone, 20 microM nordihydroguaiaretic acid, and 0.1 mM 4-pentenoic acid failed to inhibit the formation 16:3 (8-OH), suggesting that the formation of 16:3 (8-OH) is not mediated by the cytochrome P-450, lipoxygenase, or mitochondrial beta-oxidation pathways. When grown on fibronectin-treated polycarbonate filters, MDCK cells released the 16:3 (8-OH) in both the apical and basolateral directions, irrespective of which side the 12-HETE was encountered. These results demonstrate the conversion of 12-HETE to a 16-carbon monohydroxy derivative by renal tubular epithelium and suggest that this product can be released to either the potential urinary space or the kidney parenchyma and renal microcirculation.
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
J A
JA
Gordon
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Figard
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Spector
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D002478:Cells, Cultured; D004848:Epithelium / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007684:Kidney Tubules / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction
D000963:Antimetabolites; D005229:Fatty Acids, Monounsaturated; D006893:Hydroxyeicosatetraenoic Acids; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C009785:4-pentenoic acid