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Am J Physiol Heart Circ Physiol 279: H857-H862, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 2, H857-H862, August 2000

SPECIAL COMMUNICATION
Determination of EETs using microbore liquid chromatography with fluorescence detection

Kasem Nithipatikom, Phillip F. Pratt, and William B. Campbell

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11,12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C18 microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10-6 M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.

cytochrome P-450; endothelial cells; epoxyeicosatrienoic acids; high-performance liquid chromatography


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