Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE₃-ethanolamide (PGE₃-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13- and 16-hydroxy-DHEA (13- and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.

中文翻译：

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在RAW264.7巨噬细胞中鉴定的n-3多不饱和脂肪酸-乙醇胺-共轭物的新型COX-2产物。

环氧合酶2（COX-2）通过催化PUFA氧化成前列腺素（PGs）和氢过氧化物而在炎症调节中起关键作用。紧接着，COX-2可以代谢中性脂质，包括类内源性大麻素酯和酰胺。我们开发了一种基于LC-HRMS的人类重组（h）COX-2筛选试验，以检查其还能够转化n -3 PUFA衍生的N-酰基乙醇胺的能力。我们的测定从建立的PUFA和anandamide中产生了已知的hCOX-2衍生产品。随后，我们证明了EPA的N-酰基乙醇胺衍生物二十碳五烯酰基乙醇酰胺（EPEA）被转化为PGE _3-乙醇酰胺（PGE ₃-EA），并分为11-，14-和18-羟基二十二碳五烯酰基-EA（分别为11-，14-和18-HEPE-EA）。有趣的是，我们证明了二十二碳六烯基乙醇酰胺（DHEA）通过hCOX-2转化为以前未知的代谢物13-和16-羟基-DHEA（分别为13-和16-HDHEA）。这些产品也是由与DHEA孵育的脂多糖刺激的RAW267.4巨噬细胞产生的。当将选择性COX-2抑制剂塞来昔布添加到细胞中时，未检测到氧化的DHEA代谢产物，进一步突显了COX-2在新型羟基化产物形成中的作用。这项工作首次证明了DHEA和EPEA被COX-2转化为以前未知的羟基化代谢物，并鼓励对这些代谢物的生物学效应进行进一步研究。