Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech Republic; 4th Medical Department, First Faculty of Medicine, Charles University and General Faculty Hospital in Prague, U Nemocnice 2, 128 08 Praha 2, Czech Republic.
Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech Republic; Faculty of Chemical Technology, University of Chemistry and Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 15;1113:50-59. doi: 10.1016/j.jchromb.2019.03.006. Epub 2019 Mar 12.
Analysis of bioactive lipids in adipose tissue could lead to better understanding of the pathogenesis of obesity and its complications. However, current MS methods are limited by a high content of triacylglycerols (TAGs), which markedly surpasses the amount of other lipids and suppresses their ionization. The aim of our study was thus to optimize the preanalytical phase of lipid analysis in adipose tissue, focusing in particular on less-abundant lipids. Next, the optimized method was used to describe the differences between epicardial and subcutaneous adipose tissues obtained from patients undergoing cardiac surgery. Lipids were extracted using a modified Folch method with subsequent detachment of TAGs by thin layer chromatography (TLC). The extracts with/without TAGs were analyzed by tandem LC/MS. The repeatability of the presented method expressed by the median of the coefficients of variation was 12/5% for analysis with/without TAGs separation, respectively. The difference in the relative abundance of TAGs gained with/without TLC was, on average, 19% and did not reach significance (p value > 0.05) for any identified TAG. The novel preanalytical step allowed us to detect 37 lipids, which could not have been detected without TAG separation, because their signal to noise ratio is <5 in current methods of untargeted lipidomics. These lipids belong predominately to ceramides, glycerophosphatidylserines, glycerophosphatidylinsitols, sphingomyelins, glycerophosphatidylcholines, glycerophosphatidylethanolamines, diacylglycerols. The two adipose tissue depots differed mainly in the following lipid classes: glycerophosphatidylcholines, glycerophosphatidylinositols, glycerophosphatidylethanolamine, and sphingomyelins. Moreover, other major lipids showed distinctly different distributions between the two adipose tissues. Among these, the changes in TAGs were the most striking, which correspond to previously published data describing the differences between omental and subcutaneous adipose tissue. Implementation of the TLC step for the elimination of TAGs was crucial for enhancing the MS detection limit of minor lipids in adipose tissue. The differences between the overall lipid profiles of subcutaneous and epicardial tissue reflect their different functions arising from their location.
分析脂肪组织中的生物活性脂质可以帮助我们更好地理解肥胖及其并发症的发病机制。然而，目前的 MS 方法受到三酰基甘油（TAGs）含量高的限制，这大大超过了其他脂质的含量，并抑制了它们的离子化。因此，我们的研究旨在优化脂肪组织中脂质分析的预分析阶段，特别是针对丰度较低的脂质。接下来，我们使用优化后的方法来描述心脏手术患者的心外膜和皮下脂肪组织之间的差异。使用改良的 Folch 法提取脂质，随后通过薄层色谱（TLC）分离 TAGs。用串联 LC/MS 分析带/不带 TAGs 的提取物。通过变异系数中位数表示的该方法的重复性为 12/5%，分别用于带/不带 TAGs 分离的分析。TLC 带/不带 TLC 获得的 TAG 相对丰度差异平均为 19%，但无统计学意义（p 值>0.05）。新的预分析步骤使我们能够检测到 37 种脂质，如果没有 TAG 分离，这些脂质是无法检测到的，因为它们在当前非靶向脂质组学方法中的信噪比<5。这些脂质主要属于神经酰胺、甘油磷脂酰丝氨酸、甘油磷脂酰肌醇、鞘磷脂、甘油磷脂酰胆碱、甘油磷脂酰乙醇胺、二酰基甘油。两种脂肪组织库主要在以下脂质类别上存在差异：甘油磷脂酰胆碱、甘油磷脂酰肌醇、甘油磷脂酰乙醇胺和鞘磷脂。此外，其他主要脂质在两种脂肪组织之间表现出明显不同的分布。其中，TAG 的变化最为显著，这与之前描述大网膜和皮下脂肪组织之间差异的已发表数据一致。实施 TLC 步骤消除 TAGs 对于提高脂肪组织中小脂质的 MS 检测限至关重要。皮下和心外膜组织的整体脂质谱差异反映了它们由于位置不同而产生的不同功能。
