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Determination of EETs using microbore liquid chromatography with fluorescence detection.
K Nithipatikom1, P F Pratt, W B Campbell
1Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Summary
A new high-performance liquid chromatography method accurately measures epoxyeicosatrienoic acids (EETs), crucial for vascular tone. This sensitive technique allows detection of EETs in endothelial cells, revealing their role in cellular responses.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Physiology
Background:
- Epoxyeicosatrienoic acids (EETs) are vital metabolites of arachidonic acid.
- EETs play a key role in regulating vascular tone.
- Accurate quantification of EETs is essential for understanding their physiological functions.
Purpose of the Study:
- To develop a sensitive method for quantifying specific EETs.
- To determine EET production in endothelial cells (ECs).
- To investigate the impact of bradykinin and methacholine on EET production.
Main Methods:
- Microbore column high-performance liquid chromatography (HPLC) with fluorescence detection.
- Derivatization of EETs with 2-(2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NT).
- Use of tridecanoic acid (TA) as an internal standard.
Main Results:
- The method achieved high recovery rates for EETs (88%) and TA (40%).
- Detection limits for EETs were as low as 2 pg (S/N=3).
- Bradykinin and methacholine significantly increased EET production in ECs (two- and fivefold, respectively).
Conclusions:
- A sensitive and reliable HPLC method for EET quantification was established.
- The method enables the study of EET production in complex biological samples like ECs.
- EETs are involved in cellular signaling pathways modulated by bradykinin and methacholine.