Systematic Metabolomic Analysis of Eicosanoids after Omega‑3 Polyunsaturated Fatty Acid Supplementation by a Highly Specific Liquid Chromatography–Tandem Mass Spectrometry-Based Method

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) have beneficial effects in many pathological processes, especially cardiovascular disease, and their protective eicosanoid metabolites are thought to play important roles. However, how ω-3 PUFAs affect the eicosanoid profile has not been elucidated comprehensively. Here, we systematically analyzed the eicosanoid metabolites induced by ω-3 PUFA supplementation. We developed an LC–MS/MS-based method covering 32 arachidonic acid (ARA) metabolites and 37 ω-3 PUFA-derived products. The limits of detection for eicosanoids were between 0.0625 and 1 pg and the detection specificity was optimized. We then quantified eicosanoids in mouse and human plasma and mouse aorta samples after ω-3 PUFA supplementation. Levels of EPA hydroxyl products, 4-HDoHE, 17,18-EEQ, 17,18-DiHETE, TXB2, and LXA4 were significantly changed in both mouse samples, and those of 2-series PGs, EDPs and DHA hydroxyl products were changed in aorta samples. Correlation network analysis of mouse plasma data revealed that some eicosanoids had higher connection degree or betweenness centrality score than others after ω-3 PUFA supplementation. Eicosanoids in human plasma were profiled across five time points after ω-3 PUFA supplementation. Fuzzy c-mean clustering algorithm suggested that the time curves of eicosanoid activity could be described with three kinetic patterns: sustained upregulation, short-term upregulation, and downregulation. This is the first systematic profiling of eicosanoids with ω-3 PUFA supplementation. The highly specific eicosanoid metabolomic and related data analysis methods would be powerful tools for comprehensive eicosanoid study.

Ω-3（ω-3）多不饱和脂肪酸（polyunsaturated fatty acids, PUFAs）在多种病理过程中展现出有益效应，尤其在心血管疾病领域，其具有保护作用的类二十烷酸（eicosanoid）代谢产物被认为发挥着关键功能。然而，ω-3 PUFAs如何调控类二十烷酸谱的分子机制尚未得到全面阐明。本研究系统分析了ω-3 PUFAs补充后诱导产生的类二十烷酸代谢产物。我们建立了一种基于液相色谱-串联质谱（LC–MS/MS）的检测方法，可覆盖32种花生四烯酸（arachidonic acid, ARA）代谢产物以及37种ω-3 PUFAs衍生代谢产物。该方法对类二十烷酸的检测限介于0.0625至1 pg之间，且检测特异性得到了优化。随后，我们对ω-3 PUFAs补充后的小鼠血浆、人血浆及小鼠主动脉样本中的类二十烷酸进行了定量分析。在小鼠样本中，二十碳五烯酸（EPA）羟基代谢产物、4-HDoHE、17,18-EEQ、17,18-DiHETE、血栓素B2（TXB2）以及脂氧素A4（LXA4）的水平发生了显著变化；而在主动脉样本中，2系列前列腺素（PGs）、环氧二十二碳五烯酸衍生物（EDPs）以及二十二碳六烯酸（DHA）羟基代谢产物的水平出现改变。对小鼠血浆数据的相关网络分析显示，经ω-3 PUFAs补充后，部分类二十烷酸的连接度或介中心性得分高于其他类二十烷酸。我们还对ω-3 PUFAs补充后五个时间节点的人血浆类二十烷酸进行了谱图分析。模糊C均值聚类算法表明，类二十烷酸活性的时间曲线可划分为三种动力学模式：持续上调、短期上调以及下调。本研究首次对ω-3 PUFAs补充后的类二十烷酸进行了系统性谱图分析。这种高特异性的类二十烷酸代谢组学及相关数据分析方法，将为全面的类二十烷酸研究提供有力工具。