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.
Gordon
J A
JA
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Figard
P H
PH
Spector
A A
AA
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