Pre-analytical monitoring and protection of oxidizable lipids in human plasma (vitamin E and ω-3 and ω-6 fatty acids): An update for redox-lipidomics methods

: Sample manipulation for storage and storage itself, interfere with the stability of labile lipids in human plasma, including vitamin E (α-tocopherol), polyunsaturated fatty acids (PUFAs), and their enzymatic and free radical-derived oxidation metabolites. This remains a main limit of lipidomics studies that often lack of sufficient standardization and validation at the pre-analytical level. In order to characterize the stability of these lipids in human plasma and to develop a standardized pre-analytical protocol for lipidomics methods, the oxidation metabolites of α-tocopherol, the free form of ω3 and ω6 PUFAs, and some arachidonic acid (AA)-derived eicosanoids were investigated in human plasma during storage at different freezing temperatures. The effect of a protection/defense cocktail of antioxidants and lipoxygenase inhibitors (PD solution) on these lipid parameters was also evaluated. The temperature of storage markedly affected the formation of α-tocopheryl quinone (α-TQ), the main lipoperoxyl radical-derived oxidation metabolite of vitamin E, with the lowest production rate observed in samples stored at -80 °C or in liquid nitrogen. A similar effect of the storage temperature was observed for the free form of the ω-3 species eicosapentaenoic and docosahexaenoic acid, and for the ω-6 AA. Freezing samples at -20 °C resulted in a time-dependent formation of the pro-inflammatory eicosanoid LTB4. The PD solution prevents non-specific alterations of these lipid parameters in samples that are processed for direct analysis and protects from the temperature-dependent modifications of free PUFAs. Combining PD solution and preservation at -80 °C or in liquid nitrogen, resulted in levels of α-TQ and PUFAs that remained stable over 1 month and up to 8 months of storage, respectively. This method paper provides indications for the optimal processing and storage of human plasma utilized in lipidomics studies.