Stability of total oxylipins – influence of plasma generation and long-term storage

International audienc

Extraction of lipids and oxylipins from plasma for quantification of total oxylipins – Challenges and strategies

Several eicosanoids and other oxylipins are potent lipid mediators which are involved in theregulation of physiological functions such as inflammation. It is believed that they act predominantlyin their free, i.e. non-esterified, form. However, a major portion of oxylipins is found as esters, e.g. inpolar lipids.Only little information is available on the biological activity of these esterified oxylipins. An importantstep towards a better understanding of their biological role is a comprehensive comparison of thechanges in the pattern of free vs. esterified mediators induced by pharmacological intervention ordietary supplementation as well as during onset and progression of diseases.While several LC-MS based methods for the detection of free oxylipins have been developed,esterified oxylipins are commonly quantified as a sum of free and esterified oxylipins following basehydrolysis. However, different approaches have been described for preparation of samples beforehydrolysis and for the solid phase extraction. Here, we present a three-step strategy for thequantification of total oxylipins including extraction of total lipids, saponification to liberate esterifiedoxylipins and solid phase extraction of free oxylipins. We thoroughly investigated different liquidliquidextraction procedures and protein precipitation in terms of extraction efficiency for variouslipid classes from plasma. Moreover, to optimize sample throughout we compared extraction of freeoxylipins via solid phase extraction on cartridges with 96-well plates.In conclusion, our results emphasis the challenges related to the extraction procedures and providedifferent strategies for reliable oxylipin quantification with a focus on efficient and reproducibleextraction

Stability of oxylipins during plasma generation and long-term storage

International audienceOxidized unsaturated fatty acids - i.e. eicosanoids and other oxylipins - are lipid mediators involved in the regulation of numerous physiological functions such as inflammation, blood coagulation, vascular tone and endothelial permeability. They have raised strong interest in clinical lipidomics in order to understand their role in health and diseases and their use as biomarkers. However, before the clinical translation, it is crucial to validate the analytical reliability of oxylipins. This notably requires to assess the putative artificial formation or degradation of oxylipins by (unsuitable) blood handling during plasma generation, storage and sample preparation. Using a liquid chromatography-mass spectrometry method covering 133 oxylipins we comprehensively analyzed the total (free + esterified) oxylipin profile in plasma and investigated the influence of i) addition of additives during sample preparation, ii) different storage times and temperatures during the transitory stage of plasma generation and iii) long-term storage of plasma samples at -80 degrees C. Addition of radical scavenger butylated hydroxytoluene reduced the apparent concentrations of hydroxy-PUFA and thus should be added to the samples at the beginning of sample preparation. The concentrations of all oxylipin classes remained stable (within analytical variance of 20%) during the transitory stage of plasma generation up to 24 h at 4 degrees C or 4 h at 20 degrees C before centrifugation of EDTA-whole blood and up to 5 days at - 20 degrees C after plasma separation. The variations in oxylipin concentrations did not correlate with storage time, storage temperature or stage of plasma generation. A significant increase of potentially lipoxygenase derived hydroxy-PUFA compared to immediate processing was only detected when samples were stored for longer times before centrifugation, plasma separation as well as freezing of plasma revealing residual enzymatic activity. Autoxidative rather than enzymatic processes led to a slightly increased concentration of 9-HETE when plasma samples were stored at - 80 degrees C for 15 months. Overall, we demonstrate that total plasma oxylipins are robust regarding delays during plasma generation and long-term storage at -80 degrees C supporting the application of oxylipin profiling in clinical research