Roy Uzzal, Joshua Robert, Stark Russell L, Balazy Michael
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Biochem J. 2005 Sep 15;390(Pt 3):719-27. doi: 10.1042/BJ20050681.
5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis-trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (omega-1)/(omega-2) hydroxylations. We found that the C5-C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5-C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.
5,6-反式花生四烯酸（5,6-TAA，其中TAA代表反式花生四烯酸）是一种最近发现的反式脂肪酸，它源自由二氧化氮自由基引发的花生四烯酸（AA）的顺反异构化。这种反式脂肪酸已在血液循环中被检测到，我们认为它作为二氧化氮毒性作用的脂质介质发挥作用。为了解其作为脂质介质的作用，我们研究了用烟酰胺腺嘌呤二核苷酸磷酸（NADPH）刺激的肝微粒体对5,6-TAA的代谢。通过液相色谱/质谱对代谢产物进行分析，发现了氧化产物的复杂混合物，其中包括四种环氧化物、它们各自的水解产物（二羟基二十碳三烯酸）以及由烯丙基、双烯丙基和（ω-1）/（ω-2）羟基化产生的几种羟基二十碳四烯酸（HETEs）。我们发现C5-C6反式键与三个顺式键竞争由细胞色素P450（CYP）环氧化酶和羟化酶介导的氧化代谢。这通过检测5,6-反式环氧二十碳三烯酸（5,6-trans-EET，其中EET代表环氧二十碳三烯酸）、5,6-赤藓糖型二羟基二十碳三烯酸和5-羟基二十碳四烯酸（5-HETE）的一种异构体得到证明。通过5,6-TAA的碘内酯化获得的5,6-反式-EET标准品用于明确鉴定环氧乙烷环为反式构型的独特微粒体环氧化物。其他脂质产物源自涉及顺式键的代谢，因此这些代谢产物具有反式C5-C6键。18-羟基二十碳四烯酸（18-HETE）和19-羟基二十碳四烯酸（19-HETE）的5,6-反式异构体可能是CYP2E1的产物，因为一种中和抗体部分抑制了它们的形成，而对环氧化物的形成没有影响。我们的研究揭示了一种反式脂肪酸微粒体氧化代谢的新途径，其中顺式和反式键都参与其中。特别重要的是检测到了花生四烯酸的反式环氧化物，它可能参与此类反式脂肪酸的代谢活化，并可能有助于它们的生物活性。与它的顺式异构体不同，5,6-反式-EET明显更稳定，并抵抗微粒体水解以及肝脏谷胱甘肽S-转移酶催化的与谷胱甘肽的结合。
