Abstract
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a useful tool to characterize the behavior of natural lipids within biological matrices. We report a LC-MS/MS method developed specifically to analyze CYP products of the arachidonoyl ethanolamide (anandamide, AEA), the epoxyeicosatrienoic acid ethanolamides (EET-EAs) and their hydrolyzed metabolites, and the dihydroxyeicosatrienoic acid ethanolamides (DHET-EAs). This method was used to measure EET-EA biotransformation to DHET-EAs by two human epoxide hydrolases: the soluble EH (sEH) and the microsomal EH (mEH). In general, sEH and mEH substrate preference was similar, based on kcat/KM. The 14,15-EET-EA and 11,12-EET-EA were the most efficiently hydrolyzed, followed by 8,9-EET-EA and 5,6-EET-EA. The method was also used to detect endogenous levels of these lipids in mouse tissues, although levels were below the instrumental detection limit (0.1-3.4nM). Because both AEA and EETs are biologically active, the method described herein will be invaluable in revealing the role(s) of EET-EAs in vivo.
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