Division of Human Nutrition and Health, Wageningen University, Wageningen, The Netherlands.
Laboratory of Organic Chemistry, Wageningen University, Wageningen, The Netherlands.
Methods Mol Biol. 2023;2576:49-66. doi: 10.1007/978-1-0716-2728-0_5.
The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.
内源性大麻素和相关 N-酰基乙醇胺的定量分析可能很复杂，因为它们的内源性水平低、结构多样性和代谢复杂。因此，需要使用 LC-MS 等先进的分析方法来定量分析这些分子在血浆、组织和其他基质中的含量。已经表明，由 n-3 多不饱和脂肪酸 (n-3 PUFAs) 合成的内源性大麻素类似物，如二十二碳六烯酰乙醇胺 (DHEA) 和二十碳五烯酰乙醇胺 (EPEA)，具有有趣的免疫调节和肿瘤抑制特性。最近的研究表明，DHEA 和 EPEA 可以进一步被环加氧酶 2 (COX-2) 酶促代谢，形成氧化代谢物。本文描述了一种基于 LC-MS 的方法，用于定量分析 n-3 PUFA 衍生的内源性大麻素类似物 DHEA 和 EPEA，该方法也适合测量更广泛的内源性大麻素。该章节包含了一个用于分析血浆中（n-3）内源性大麻素的分步方案，包括样品采集和固相萃取、LC-MS 分析和数据处理。此外，还提供了方案修改，以允许定量分析组织和细胞培养介质中的 n-3 PUFA 衍生的内源性大麻素及其 COX-2 代谢物。最后，简要讨论了改变内源性大麻素浓度的条件。
