Chair of Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Wuppertal, Germany.
Université Clermont Auvergne, INRAe, UNH, Clermont-Ferrand, France.
Talanta. 2020 Sep 1;217:121074. doi: 10.1016/j.talanta.2020.121074. Epub 2020 Apr 25.
Oxidized 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 °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 °C or 4 h at 20 °C before centrifugation of EDTA-whole blood and up to 5 days at -20 °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 °C for 15 months. Overall, we demonstrate that total plasma oxylipins are robust regarding delays during plasma generation and long-term storage at -80 °C supporting the application of oxylipin profiling in clinical research.
氧化不饱和脂肪酸 - 即类二十烷酸和其他氧化脂类 - 是参与调节许多生理功能的脂质介质，如炎症、血液凝固、血管张力和内皮通透性。它们在临床脂质组学中引起了强烈的兴趣，以了解它们在健康和疾病中的作用及其作为生物标志物的用途。然而，在临床转化之前，验证氧化脂类的分析可靠性至关重要。这尤其需要评估在血浆生成、储存和样品制备过程中（不合适的）血液处理导致的氧化脂类的潜在人工形成或降解。我们使用一种涵盖 133 种氧化脂类的液相色谱-质谱法，全面分析了血浆中的总（游离+酯化）氧化脂类谱，并研究了以下因素的影响：i）在样品制备过程中添加添加剂，ii）在血浆生成的过渡阶段期间不同的储存时间和温度，以及 iii）-80°C 下长期储存血浆样品。添加自由基清除剂丁基羟基甲苯可降低羟基-PUFA 的表观浓度，因此应在样品制备开始时添加到样品中。在血浆生成的过渡阶段，所有氧化脂类的浓度在 4°C 下 24 小时或 20°C 下 4 小时内（在 20%的分析方差内）保持稳定，然后离心 EDTA-全血，在 -20°C 下分离血浆后，在 5 天内保持稳定。氧化脂类浓度的变化与储存时间、储存温度或血浆生成阶段无关。仅当在离心、血浆分离以及冷冻血浆之前将样品储存更长时间时，与立即处理相比，潜在的脂氧合酶衍生的羟基-PUFA 才会显著增加，这表明仍存在残留的酶活性。当在 -80°C 下储存 15 个月时，自动氧化而不是酶促过程导致 9-HETE 的浓度略有增加。总体而言，我们证明了总血浆氧化脂类在血浆生成过程中的延迟和-80°C 下的长期储存方面具有稳健性，这支持了氧化脂类谱分析在临床研究中的应用。
