Targeted Lipidomics for Characterization of PUFAs and Eicosanoids in Extracellular Vesicles

Abstract

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.

Extracellular vesicles from patients with coronary artery disease (CAD) demonstrate enhanced pro-coagulatory activity

“…Comparative proteomics revealed some differences in the protein content of EVs from arterial and venous blood, but these were quite limited and, as an exploratory study, should be considered preliminary data only. It is also worth noting that focusing only on the protein profile of EVs provides a limited view of their potential bioactivity; recent advances in targeted lipidomics are revealing lipids as key bioactive mediators 36 , which should be explored in the context of the role of EVs in CAD which is beyond the scope of this manuscript. The results of our study would therefore support the further use of arterial blood EVs when investigating their role in the pathogenesis of CAD and also their use as a biomarker or target for intervention.…”