Institute of Analytical Chemistry, Johannes Kepler University, Altenberger Strasse 69, 4040 Linz, Austria.
Institute of Analytical Chemistry, Johannes Kepler University, Altenberger Strasse 69, 4040 Linz, Austria.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Sep 15;1153:122271. doi: 10.1016/j.jchromb.2020.122271. Epub 2020 Jul 16.
A typical lipidomics approach aims at the simultaneous analysis of a multitude of lipid species from different lipid classes with highest possible sensitivity for all target lipids. Efficient extraction of lipids from the biological matrix is a crucial step in the analytical workflow. Whereas numerous applications of classical and more recently published extraction methods have been reported for blood serum or plasma samples, very little is known about the applicability of these methods for cerebrospinal fluid (CSF). CSF though represents a highly interesting biofluid for the investigation of neurological disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, or brain cancer. Since CSF comprises substantially lower endogenous lipid concentrations compared to serum or plasma, the use of highly efficient extraction methods is of utmost importance. In addition, literature on lipid extraction methods is often inconsistent in terms of methodological parameters like temperature, mixing times, or the number of repeated extraction cycles. In this study, four liquid-liquid extraction methods (Folch, Bligh & Dyer, MTBE and BUME) and one protein precipitation method (MMC method) were evaluated using a pooled CSF sample, followed by the investigation of key process parameters (temperature and mixing times) and modifications of the most promising methods, in order to achieve a broad coverage of lipid classes as well as high recoveries and repeatabilities. A modified Folch method turned out as most suitable for the efficient extraction of a broad range of lipid classes from CSF including glycerophospholipids, glycerolipids and sphingolipids. In addition, using cooled solvents and equipment was shown to significantly improve lipid extraction efficiencies. Mixing times should be thoroughly optimized for the lipid classes of interest in order to achieve high recoveries without lipid degradation due to unnecessarily long mixing. Finally, acidification led to improved extraction efficiency for acidic glycerophospholipids.
一种典型的脂质组学方法旨在同时分析来自不同脂质类别的多种脂质，对所有目标脂质的灵敏度尽可能高。从生物基质中高效提取脂质是分析工作流程中的关键步骤。虽然已经有许多关于经典和最近发表的提取方法的应用报告用于血清或血浆样本，但对于这些方法在脑脊液（CSF）中的适用性知之甚少。尽管 CSF 是研究神经退行性疾病（如阿尔茨海默病、帕金森病、多发性硬化症、肌萎缩侧索硬化症或脑癌）的极具吸引力的生物流体，但 CSF 中内源性脂质浓度要低得多，因此使用高效的提取方法至关重要。此外，关于脂质提取方法的文献在方法参数（如温度、混合时间或重复提取循环次数）方面往往不一致。在这项研究中，使用 pooled CSF 样本评估了四种液-液萃取方法（Folch、Bligh & Dyer、MTBE 和 BUME）和一种蛋白质沉淀方法（MMC 方法），然后研究了关键工艺参数（温度和混合时间）和最有前途方法的改进，以实现广泛的脂质类别的覆盖范围以及高回收率和重复性。改良的 Folch 方法最适合从 CSF 中高效提取广泛的脂质类，包括甘油磷脂、甘油酯和鞘脂。此外，使用冷却的溶剂和设备可以显著提高脂质提取效率。混合时间应针对感兴趣的脂质类进行彻底优化，以在不由于不必要的长时间混合而导致脂质降解的情况下实现高回收率。最后，酸化可提高酸性甘油磷脂的提取效率。
