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

K. G. Maier, L. Henderson, J. Narayanan, M. Alonso-Galicia, J R Falck, R. J. Roman

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66 Scopus citations

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 languageEnglish (US)
Pages (from-to)H863-H871
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume279
Issue number2 48-2
StatePublished - Sep 14 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)

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    Maier, K. G., Henderson, L., Narayanan, J., Alonso-Galicia, M., Falck, J. R., & Roman, R. J. (2000). Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid. American Journal of Physiology - Heart and Circulatory Physiology, 279(2 48-2), H863-H871.