Moran-Garrido Maria, Camunas-Alberca Sandra M, Sáiz Jorge, Gradillas Ana, Taha Ameer Y, Barbas Coral
Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte, Madrid, Spain.
Department of Food Science and Technology, College of Agriculture and Environmental Sciences, University of California, Davis, CA 95616, USA; West Coast Metabolomics Center, Genome Center, University of California, Davis, CA 95616, USA; Center for Neuroscience, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA.
J Pharm Biomed Anal. 2025 Mar 15;255:116587. doi: 10.1016/j.jpba.2024.116587. Epub 2024 Nov 29.
Oxylipins are signaling lipids derived from the oxidation of polyunsaturated fatty acids (PUFAs). In lipidomic studies, human plasma may be subjected to various storage conditions and freeze-thaw cycles, which may impact the analysis of these compounds. In this study, we used liquid chromatography coupled with mass spectrometry (LC-MS) to examine the influence of up to five freeze-thaw cycles (FTCs) on free and total (mostly esterified) oxylipins in human plasma and the influence of temperature and storage duration (4 °C for up to 120 h and -20 °C and -80 °C for 1-98 days) in the presence or absence of butylated hydroxytoluene (BHT) on extracted oxylipins stored in LC-MS amber vials. In fresh plasma subjected to several FTCs, approximately 48 % of the detected free oxylipins were significantly altered by the third cycle, with increases in cytochrome P450 (CYP450) and lipoxygenase (LOX)-derived compounds and reductions in trihydroxylated oxylipins. In contrast, multiple FTCs did not significantly alter esterified oxylipins. At 4 °C, the extracted oxylipins did not change significantly for up to 120 h (5 days). Oxylipin levels remained stable for 98 days at -80 °C but decreased by 98 days at -20 °C. The antioxidant activity of butylated hydroxytoluene (BHT) did not influence oxylipin stability at 4 °C for 120 h or at -80 °C for 98 days, but it reduced oxylipin degradation at -20 °C at 98 days. Conversely, prostaglandin F (PGF) exhibited substantial increases at -20 °C and -80 °C, independent of BHT. This study demonstrates that (i) unlike free oxylipins, the esterified oxylipin pool remains stable following repeated FTCs, (ii) extracted oxylipins are stable at 4 °C for up to 120 h and at -80 °C for up to 98 days, but not at -20 °C for 98 days, and (iii) BHT may minimize oxylipin degradation of sample extracts stored at -20 °C. This study provides a framework for measuring oxylipins under various freeze-thaw and storage conditions.
氧化脂质是由多不饱和脂肪酸（PUFA）氧化衍生而来的信号脂质。在脂质组学研究中，人体血浆可能会经历各种储存条件和冻融循环，这可能会影响这些化合物的分析。在本研究中，我们使用液相色谱-质谱联用（LC-MS）技术，研究了多达五次冻融循环（FTC）对人体血浆中游离和总（主要是酯化）氧化脂质的影响，以及在存在或不存在丁基羟基甲苯（BHT）的情况下，温度和储存时间（4℃下长达120小时，-20℃和-80℃下1-98天）对储存在LC-MS琥珀色小瓶中的提取氧化脂质的影响。在经历多次FTC的新鲜血浆中，约48%检测到的游离氧化脂质在第三次循环时发生显著变化，细胞色素P450（CYP450）和脂氧合酶（LOX）衍生化合物增加，三羟基化氧化脂质减少。相比之下，多次FTC并未显著改变酯化氧化脂质。在4℃下，提取的氧化脂质在长达120小时（5天）内没有显著变化。氧化脂质水平在-80℃下98天保持稳定，但在-20℃下98天下降。丁基羟基甲苯（BHT）的抗氧化活性在4℃下120小时或-80℃下98天时不影响氧化脂质稳定性，但在-20℃下98天时可减少氧化脂质降解。相反，前列腺素F（PGF）在-20℃和-80℃下显著增加，与BHT无关。本研究表明，（i）与游离氧化脂质不同，酯化氧化脂质库在重复FTC后保持稳定，（ii）提取的氧化脂质在4℃下长达120小时和-80℃下长达98天稳定，但在-20℃下98天不稳定，（iii）BHT可使储存在-20℃下的样品提取物的氧化脂质降解最小化。本研究为在各种冻融和储存条件下测量氧化脂质提供了一个框架。
