Cytochrome P450/NADPH-dependent biosynthesis of 5,6-trans-epoxyeicosatrienoic acid from 5,6-trans-arachidonic acid.
Cytochrome P450/NADPH-dependent biosynthesis of 5,6-trans-epoxyeicosatrienoic acid from 5,6-trans-arachidonic acid.
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DOI：
10.1042/bj20050681
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发表时间：
2005-09
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影响因子：
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通讯作者：
U. Roy;R. Joshua;R. Stark;M. Balazy
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作者：
U. Roy;R. Joshua;R. Stark;M. Balazy
5,6-反式AA （5,6-TAA，其中TAA代表反式花生四烯酸）是最近发现的一种反式脂肪酸，起源于NO2自由基引发的AA顺-反异构化。这种反式脂肪酸已经在血液循环中被检测到，我们认为它是NO2毒性作用的脂质介质。为了了解其作为脂质介质的作用，我们研究了受NADPH刺激的肝微粒体对5,6- taa的代谢。通过液相色谱/质谱分析代谢物发现了一个复杂的氧化产物混合物，其中有四种环氧化合物，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(omega-1)/(omega-2)羟基化产生的几种HETEs（羟基二十碳四烯酸）。我们发现C5-C6反式键与三个顺式键在CYP（细胞色素P450）环氧化酶和羟化酶介导的氧化代谢中竞争。通过检测5,6-反式EET（其中EET代表环氧二碳三烯酸），5,6-红二羟基二碳三烯酸和5- hete的异构体可以证明这一点。通过碘碘化5,6- taa得到的5,6-反式eet标准用于明确鉴定独特的微粒体环氧化物，其中氧环为反式构型。其他脂质产物来源于涉及顺式键的代谢，因此这些代谢物具有反式C5-C6键。18- hete和19-HETE的5,6-反式异构体可能是CYP2E1的产物，因为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了。特别重要的是检测到AA的反式环氧化物，它可能参与了这类反式脂肪酸的代谢激活，并可能参与了它们的生物活性。与它的顺式异构体不同，5,6-反式eet明显更稳定，并且抵抗肝谷胱甘肽s-转移酶催化的微粒体水解和谷胱甘肽结合。
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.