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.

Omega-3（ω-3）多不饱和脂肪酸（polyunsaturated fatty acids，PUFAs）在诸多病理过程中均具有有益作用，尤以心血管疾病为著，其具有保护活性的类二十烷酸（eicosanoid）代谢物被认为发挥了关键调控作用。然而，目前尚未全面阐明ω-3 PUFAs如何调控类二十烷酸谱。本研究针对ω-3多不饱和脂肪酸补充剂诱导产生的类二十烷酸代谢物开展了系统性分析。
我们开发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）代谢物以及37种ω-3多不饱和脂肪酸衍生产物。该方法对类二十烷酸的检出限介于0.0625至1皮克（pg）之间，且检测特异性得到优化。随后，我们对ω-3多不饱和脂肪酸补充干预后的小鼠血浆、人血浆以及小鼠主动脉组织样本中的类二十烷酸进行了定量分析。小鼠样本中，二十碳五烯酸（eicosapentaenoic acid，EPA）羟基产物、4-HDoHE、17,18-EEQ、17,18-DiHETE、血栓烷B2（thromboxane B2，TXB2）以及脂氧素A4（lipoxin A4，LXA4）的水平均发生显著变化；而主动脉样本中，2系列前列腺素（prostaglandin，PG）、EDPs以及二十二碳六烯酸（docosahexaenoic acid，DHA）羟基产物的水平亦出现显著改变。对小鼠血浆数据的关联网络分析显示，经ω-3多不饱和脂肪酸补充干预后，部分类二十烷酸的连接度或介数中心性得分高于其他代谢物。
我们对ω-3多不饱和脂肪酸补充干预后五个时间点的人血浆类二十烷酸进行了谱图分析。模糊C均值聚类（Fuzzy c-mean clustering）算法分析表明，类二十烷酸活性的时间曲线可被归纳为三种动力学模式：持续上调、短期上调以及下调。本研究首次针对ω-3多不饱和脂肪酸补充干预开展了系统性的类二十烷酸谱图分析。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，将为类二十烷酸的系统性研究提供强有力的工具。