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

This study describes a fluorescent HPLC assay for measuring 20-hydroxyeicos atetraenoic acid (20-HETE) and other cytochrome P-450 metabolites of arachi donic acid in urine, tissue, and interstitial fluid. An internal standard, 20-hydroxyeicosa- 6(Z), 15(Z)-dienoic acid, was added to samples, and the l ipids were extracted and labeled with 2-(2,3-naphthalimino) ethyl trifluoro methanesulfonate. P-450 metabolites were separated on a C18 reverse-phase H PLC column. Coelution and gas chromatography-mass spectrometry studies conf irmed the identity of the 20-HETE peak. The 20-HETE peak can be separated f rom those for dihydroxyeicosatrienoic acids, other HETEs, and epoxyeicosatr ienoic acids. Known amounts of 20-HETE were used to generate a standard cur ve (range 1-10 ng, r(2) = 0.98). Recovery of 20-HETE from urine averaged 95 %, and the intra-assay variation was <5%. Levels of 20-HETE were measured i n 100 mu 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 (A BT) on the excretion of 20-HETE in rats. ABT reduced excretion of 20-HETE b y >65% and inhibited the formation of 20-HETE by renal microsomes. The avai lability of this assay should facilitate work in this field.