K. Nithipatikom et al., Determination of EETs using microbore liquid chromatography with fluorescence detection, AM J P-HEAR, 279(2), 2000, pp. H857-H862
Citations number
31
Categorie Soggetti
Cardiovascular & Hematology Research
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachi
donic acid involved in the regulation of vascular tone. The method of micro
bore column high-performance liquid chromatography with fluorescence detect
ion 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. E
ETs were reacted with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate
(NT) to form highly fluorescent derivatives. A C-18 microbore column and a
water-acetonitrile mobile phase were used for separation. Samples were exci
ted at 259 nm, and the fluorescence was detected at 395 nm. The overall rec
overies were 88% for EETs and 40% for TA. EETs were detected in concentrati
ons as low as 2 pg (signal-to-noise ratio = 3). The method was used to dete
rmine the EET production from endothelial cells (ECs). Bradykinin and metha
choline (10(-6) M) stimulated an increase in the production of EETs by ECs
two- and fivefold, respectively. This sensitive method may be used for dete
rmination of EETs at low concentrations normally detected in complex biolog
ical samples.