TY - JOUR
T1 - Determination of 14,15-epoxyeicosatrienoic acid and 14,15- dihydroxyeicosatrienoic acid by fluoroimmunoassay
AU - Nithipatikom, Kasem
AU - Falck, J R
AU - Bhatt, Rama K.
AU - Hanke, Craig J.
AU - Campbell, William B.
N1 - Funding Information:
These studies were supported by the grants from the National Institutes of Health (HL-51055, HL-52159, GM-31278, and DK-38226).
PY - 1997/3/15
Y1 - 1997/3/15
N2 - 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.
AB - 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.
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U2 - 10.1006/abio.1997.2014
DO - 10.1006/abio.1997.2014
M3 - Article
C2 - 9073364
AN - SCOPUS:19244371743
SN - 0003-2697
VL - 246
SP - 253
EP - 259
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -