Journal of Lipid Research, Vol 30, 731-738, Copyright © 1989 by Lipid Research, Inc.
Identification of the major metabolite of 12-HETE produced by renal tubular epithelial cells
JA Gordon, PH Figard and AA Spector
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
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.
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