Determination of 14,15-epoxyeicosatrienoic acid and 14,15-dihydroxyeicosatrienoic acid by fluoroimmunoassay.
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.
Nithipatikom
K
K
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA.
Falck
J R
JR
Bhatt
R K
RK
Hanke
C J
CJ
Campbell
W B
WB
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D000906:Antibodies; D002417:Cattle; D002478:Cells, Cultured; D003429:Cross Reactions; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology; D015200:Fluoroimmunoassay / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis*; D011817:Rabbits; D011863:Radioimmunoassay; D015384:Zona Glomerulosa / Q000737:chemistry / Q000166:cytology
C107016:14,15-dihydroxyeicosatrienoic acid; D000906:Antibodies; D006893:Hydroxyeicosatetraenoic Acids; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D015126:8,11,14-Eicosatrienoic Acid