Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig University Hospital, Paul-List-Str. 13-15, 04103 Leipzig, Germany.
Nutrients. 2022 Mar 22;14(7):1319. doi: 10.3390/nu14071319.
Lipids are increasingly recognized as bioactive mediators of extracellular vesicle (EV) functions. However, while EV proteins and nucleic acids are well described, EV lipids are insufficiently understood due to lack of adequate quantitative methods. We adapted an established targeted and quantitative mass spectrometry (LC-MS/MS) method originally developed for analysis of 94 eicosanoids and seven polyunsaturated fatty acids (PUFA) in human plasma. Additionally, the influence of freeze-thaw (FT) cycles, injection volume, and extraction solvent were investigated. The modified protocol was applied to lipidomic analysis of differently polarized macrophage-derived EVs. We successfully quantified three PUFAs and eight eicosanoids within EVs. Lipid extraction showed reproducible PUFA and eicosanoid patterns. We found a particularly high impact of FT cycles on EV lipid profiles, with significant reductions of up to 70%. Thus, repeated FT will markedly influence analytical results and may alter EV functions, emphasizing the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs. EV lipid profiles differed largely depending on the polarization of the originating macrophages. Particularly, we observed major changes in the arachidonic acid pathway. We emphasize the importance of a standardized sample pretreatment protocol for the analysis of bioactive lipids in EVs.
脂质越来越被认为是细胞外囊泡 (EV) 功能的生物活性介质。然而，尽管 EV 中的蛋白质和核酸已有很好的描述，但由于缺乏适当的定量方法，EV 中的脂质仍未得到充分的了解。我们对原用于分析人血浆中 94 种花生四烯酸和 7 种多不饱和脂肪酸 (PUFA) 的靶向和定量质谱 (LC-MS/MS) 方法进行了改编。此外，还研究了冻融 (FT) 循环、进样体积和提取溶剂的影响。改良后的方案应用于不同极化巨噬细胞衍生的 EV 的脂质组学分析。我们成功地定量了 EV 内的三种 PUFAs 和八种花生四烯酸。脂质提取显示出重现性的 PUFA 和花生四烯酸模式。我们发现 FT 循环对 EV 脂质谱的影响特别大，减少高达 70%。因此，重复 FT 将显著影响分析结果，并可能改变 EV 的功能，这强调了标准化样品预处理协议对于分析 EV 中生物活性脂质的重要性。根据起源巨噬细胞的极化，EV 脂质谱有很大的差异。特别是，我们观察到花生四烯酸途径发生了重大变化。我们强调标准化样品预处理协议对于分析 EV 中生物活性脂质的重要性。
