Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195, USA.
Department of Pharmaceutical Sciences, Campbell University, PO Box 1090, Buies Creek, NC 27506, USA.
Free Radic Biol Med. 2017 Nov;112:131-140. doi: 10.1016/j.freeradbiomed.2017.07.015. Epub 2017 Jul 19.
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 (Si). 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.
环氧二十碳三烯酸（EETs）是花生四烯酸（AA）氧化的代谢产物，具有重要的心脏保护和信号作用。AA 是一种 ω-6 多不饱和脂肪酸（PUFA），容易自动氧化。虽然氢过氧化物和异前列烷是 AA 自动氧化的主要产物，但 EETs 也是由很大程度上被忽视的过氧自由基加成机制形成的。虽然自动氧化生成顺式和反式 EETs，但细胞色素 P450（CYP）环氧合酶已被证明仅能专一地催化每个双键上所有区域异构体顺式 EETs 的形成。在血浆和红细胞（RBC）膜中，已经观察到顺式和反式 EETs，并且两者都具有多种生理功能。我们开发了一种灵敏的超高效液相色谱串联质谱（UPLC-MS/MS）测定法，可分离 EETs 的顺式和反式异构体，并将其应用于测定 AA 在苯和体外脂质体中自由基氧化反应混合物中顺式与反式 EETs 的相对分布。我们还测定了 EET 在包括人心和鼠心以及 RBC 膜在内的几种组织中的体内分布。然后，我们比较了年轻和老年小鼠心脏和 RBC 中的 EET 水平。AA 在苯自由基反应和脂质体中的 EET 形成随时间和 AA 浓度的增加而增加，并且在这两种条件下，反式 EETs 的水平均高于顺式 EETs。相比之下，生物样品中的顺式 EETs 水平总体较高。通常，随着小鼠年龄的增长，反式 EETs 的增加比顺式 EETs 更多。我们提出了一种涉及过氧自由基加成到 AA 双键之一的非酶形成顺式和反式 EETs 的机制，随后是键旋转和分子内均裂取代（Si）。细胞色素 P450 酶促形成顺式 EETs 很可能通过不允许键旋转的一步协同机制发生。准确测量血浆和 RBC 膜中自动氧化或酶促反应产生的循环 EETs 的能力将为未来研究这些重要信号脂质与心脏病结果的相关性奠定基础。
