Abstract
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 2-(2,3-naphthalimino)-ethyl 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, r2 = 0.98), Recovery of 20-HETE from urine averaged 95%, and the intra-assay variation was <5%. Levels of 20-HETE were measured in 100 μl 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 >65% and inhibited the formation of 20-HETE by renal microsomes. The availability of this assay should facilitate work in this field.
Original language | English (US) |
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Pages (from-to) | H863-H871 |
Journal | American Journal of Physiology - Heart and Circulatory Physiology |
Volume | 279 |
Issue number | 2 48-2 |
DOIs | |
State | Published - 2000 |
Keywords
- 20-Hydroxyeicosatetraenoic acid
- Cytochrome P-450
- Eicosanoids
- Epoxyeicosatrienoic acids
- Fluorescent
- High-performance liquid chromatography
- Kidney
ASJC Scopus subject areas
- Physiology
- Cardiology and Cardiovascular Medicine
- Physiology (medical)