Enzymatic and free radical formation of cis- and trans- epoxyeicosatrienoic acids in vitro and in vivo.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) oxidation that have important cardioprotective and signaling properties. AA is an ω-6 polyunsaturated fatty acid (PUFA) that is prone to autoxidation. Although hydroperoxides and isoprostanes are major autoxidation products of AA, EETs are also formed from the largely overlooked peroxyl radical addition mechanism. While autoxidation yields both cis- and trans-EETs, cytochrome P450 (CYP) epoxygenases have been shown to exclusively catalyze the formation of all regioisomer cis-EETs, on each of the double bonds. In plasma and red blood cell (RBC) membranes, cis- and trans-EETs have been observed, and both have multiple physiological functions. We developed a sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay that separates cis- and trans- isomers of EETs and applied it to determine the relative distribution of cis- vs. trans-EETs in reaction mixtures of AA subjected to free radical oxidation in benzene and liposomes in vitro. We also determined the in vivo distribution of EETs in several tissues, including human and mouse heart, and RBC membranes. We then measured EET levels in heart and RBC of young mice compared to old. Formation of EETs in free radical reactions of AA in benzene and in liposomes exhibited time- and AA concentration-dependent increase and trans-EET levels were higher than cis-EETs under both conditions. In contrast, cis-EET levels were overall higher in biological samples. In general, trans-EETs increased with mouse age more than cis-EETs. We propose a mechanism for the non-enzymatic formation of cis- and trans-EETs involving addition of the peroxyl radical to one of AA's double bonds followed by bond rotation and intramolecular homolytic substitution (SHi). Enzymatic formation of cis-EETs by cytochrome P450 most likely occurs via a one-step concerted mechanism that does not allow bond rotation. The ability to accurately measure circulating EETs resulting from autoxidation or enzymatic reactions in plasma and RBC membranes will allow for future studies investigating how these important signaling lipids correlate with heart disease outcomes.