Determination of 14,15-epoxyeicosatrienoic acid and 14,15- dihydroxyeicosatrienoic acid by fluoroimmunoassay

Kasem Nithipatikom, J R Falck, Rama K. Bhatt, Craig J. Hanke, William B. Campbell

Research output: Contribution to journalArticle

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Abstract

A fluoroimmunoassay (FIA) for 14,15-epoxyeicosatrienoic acid (14,15- EET) and 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), cytochrome P450 epoxygenase products of arachidonic acid, was developed using fluorescence polarization. 14,15-EET was hydrolyzed and analyzed as 14,15-DHET. 14,15- DHET was conjugated to thyroglobulin and a specific antibody was raised in rabbits. Both [3H8]14,15-DHET in radioimmunoassay or fluorescein-labeled 14,15-DHET (14, 15-DHET*) in FIA bound to this antibody and were competitively displaced by 14,15-DHET. The binding activity and cross- reactivity of 14,15-DHET antibody were also studied by RIA compared to FIA. The antibody cross-reacted ≤1% with 11,12-DHET and 14,15-EET and <0.1% with other regioisomeric DHETs and arachidonic acid metabolites. The detection limit of 14,15-DHET was 2 pg/0.6 ml by FIA. Using this method, we found that A23187 stimulated the production of 14,15-EET by endothelial cells and angiotensin II stimulated 14,15-EET release from zona glomerulosa cells. The production of 14,15-EET in these samples was confirmed by gas chromatography/mass spectrometry. These studies demonstrate a sensitive and specific FIA for 14,15-EET and 14,15-DHET and that agonists stimulate the release of these eicosanoids in two cell types, bovine coronary artery endothelial cells and bovine zona glomerulosa cells.

Original languageEnglish (US)
Pages (from-to)253-259
Number of pages7
JournalAnalytical Biochemistry
Volume246
Issue number2
DOIs
StatePublished - Mar 15 1997

Fingerprint

Fluoroimmunoassay
Zona Glomerulosa
Antibodies
Endothelial cells
Arachidonic Acid
Endothelial Cells
Fluorescence Polarization
Eicosanoids
14,15-dihydroxyeicosatrienoic acid
14,15-epoxy-5,8,11-eicosatrienoic acid
Thyroglobulin
Calcimycin
Metabolites
Fluorescein
Angiotensin II
Gas chromatography
Gas Chromatography-Mass Spectrometry
Cytochrome P-450 Enzyme System
Radioimmunoassay
Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Determination of 14,15-epoxyeicosatrienoic acid and 14,15- dihydroxyeicosatrienoic acid by fluoroimmunoassay. / Nithipatikom, Kasem; Falck, J R; Bhatt, Rama K.; Hanke, Craig J.; Campbell, William B.

In: Analytical Biochemistry, Vol. 246, No. 2, 15.03.1997, p. 253-259.

Research output: Contribution to journalArticle

Nithipatikom, Kasem ; Falck, J R ; Bhatt, Rama K. ; Hanke, Craig J. ; Campbell, William B. / Determination of 14,15-epoxyeicosatrienoic acid and 14,15- dihydroxyeicosatrienoic acid by fluoroimmunoassay. In: Analytical Biochemistry. 1997 ; Vol. 246, No. 2. pp. 253-259.
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