Faculty Collaboration Database

Determination of 14,15-epoxyeicosatrienoic acid and 14,15-dihydroxyeicosatrienoic acid by fluoroimmunoassay. Anal Biochem 1997 Mar 15;246(2):253-9

Date

03/15/1997

Pubmed ID

9073364

DOI

10.1006/abio.1997.2014

Scopus ID

2-s2.0-19244371743 (requires institutional sign-in at Scopus site)   11 Citations

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 < or = 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 by 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.

Author List

Nithipatikom K, Falck JR, Bhatt RK, Hanke CJ, Campbell WB

Authors

William B. Campbell PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Craig J. Hanke PhD Assistant Dean, Associate Professor in the Medical School Regional Campuses department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

8,11,14-Eicosatrienoic Acid
Animals
Antibodies
Cattle
Cells, Cultured
Cross Reactions
Endothelium, Vascular
Fluoroimmunoassay
Gas Chromatography-Mass Spectrometry
Hydroxyeicosatetraenoic Acids
Rabbits
Radioimmunoassay
Zona Glomerulosa