Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid

Kristopher G. Maier, Lisa Henderson, Jayashree Narayanan, Magdalena Alonso-Galicia, John R. Falck, Richard J. Roman
2000 American Journal of Physiology. Heart and Circulatory Physiology  
Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid. Am J Physiol Heart Circ Physiol 279: H863-H871, 2000.-This study describes a fluorescent HPLC assay for measuring 20-hydroxyeicosatetraenoic acid (20-HETE) and other cytochrome P-450 metabolites of arachidonic acid in urine, tissue, and interstitial fluid. An internal standard, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, was added to samples, and the lipids were extracted and labeled with
more » ... trifluoromethanesulfonate. P-450 metabolites were separated on a C18 reverse-phase HPLC column. Coelution and gas chromatography-mass spectrometry studies confirmed the identity of the 20-HETE peak. The 20-HETE peak can be separated from those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatrienoic acids. Known amounts of 20-HETE were used to generate a standard curve (range 1-10 ng, r 2 ϭ 0.98). Recovery of 20-HETE from urine averaged 95%, and the intra-assay variation was Ͻ5%. Levels of 20-HETE were measured in 100 l of urine and renal interstitial fluid or 0.1 mg of renal tissue. The assay was evaluated by studying the effects of 1-aminobenzotriazole (ABT) on the excretion of 20-HETE in rats. ABT reduced excretion of 20-HETE by Ͼ65% and inhibited the formation of 20-HETE by renal microsomes. The availability of this assay should facilitate work in this field. 20-hydroxyeicosatetraenoic acid; cytochrome P-450; highperformance liquid chromatography; fluorescent; epoxyeicosatrienoic acids; eicosanoids; kidney RECENT STUDIES have indicated that arachidonic acid (AA) is primarily metabolized in the brain, kidney, lung, and vasculature by cytochrome P-450 enzymes to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (diHETEs), and
doi:10.1152/ajpheart.2000.279.2.h863 pmid:10924088 fatcat:2de2yujytba3rip67hxois7qiq