Minor lipids profiling in subcutaneous and epicardial fat tissue using LC/MS with an optimized preanalytical phase

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.