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

Research output: Contribution to journalArticle

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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)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume279
Issue number2 48-2
StatePublished - 2000

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Arachidonic Acid
High Pressure Liquid Chromatography
Urine
Hydroxyeicosatetraenoic Acids
Acids
Extracellular Fluid
Microsomes
Gas Chromatography-Mass Spectrometry
Cytochrome P-450 Enzyme System
20-hydroxy-5,8,11,14-eicosatetraenoic acid
Kidney
Lipids

Keywords

  • 20-Hydroxyeicosatetraenoic acid
  • Cytochrome P-450
  • Eicosanoids
  • Epoxyeicosatrienoic acids
  • Fluorescent
  • High-performance liquid chromatography
  • Kidney

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

Cite this

Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid. / Maier, K. G.; Henderson, L.; Narayanan, J.; Alonso-Galicia, M.; Falck, J R; Roman, R. J.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 279, No. 2 48-2, 2000.

Research output: Contribution to journalArticle

Maier, K. G. ; Henderson, L. ; Narayanan, J. ; Alonso-Galicia, M. ; Falck, J R ; Roman, R. J. / Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid. In: American Journal of Physiology - Heart and Circulatory Physiology. 2000 ; Vol. 279, No. 2 48-2.
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N2 - 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.

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