Lipidomics analysis of essential fatty acids in macrophages

Abstract

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.

Figure 1

GC/MS chromatogram of a mixture of fatty acid-PBF derivative standards with negative chemical ionization and SIM detection. Shown is a composite chromatogram of all SIM groups. The fatty acids are referenced in the following order: (1), 12:0; (2), 14:0; (3), 15:0; (4), 16:1; (5), 16:0; (6), 17:1; (7), 17:0; (8), α18:3; (9), 18:4; (10), γ18:3; (11), 18:2; (12), 18:1; (13), 18:0; (14), 20:4; (15), 20:5; (16), 11,14,17-20:3; (17), bishomo-20:3; (18), 20:2; (19), 5,8,11-20:3; (20), 20:0; (21), 22:6; (22), 22:4; (23), 22:5; (24), 22:2; (25), 22:3; (26), 22:1; (27), 22:0; (28), 23:0; (29), 24:1; (30), 24:0, (31), 26:0.

Figure 2

Representative fatty acid calibration curve of saturated and unsaturated fatty acids. Fatty acid standard curves were prepared for each fatty acid by injection of 1 μl of calibration standard mixture containing 0.15 ng to 500 ng of each fatty acid (0.003 ng/μl to 10 ng/μl). For regression analysis, the ratio of unlabeled fatty acid/deuterated fatty acid (^H18:0/^D18:0, ^H20:4/^D20:4) was calculated as described under “Materials and Methods” and plotted against the absolute amount of unlabeled fatty acid (^H18:0, ^H20:4) in the standard mixture. Inset shows part of the calibration curve at low analyte concentration range. Linear regression parameters were computed and used for quantitative analyses of endogenous fatty acids.

Figure 3

GC/MS chromatogram of cellular free fatty acids extracted from RAW macrophages. The fatty acids were extracted, derivatized and analyzed as described under “Materials and Methods”. No internal standard was added to this representative sample to clearly illustrate the profile of endogenous free fatty acids. For quantification, a mixture of deuterated internal standards was added to the sample prior to the extraction step. The free fatty acid content in the biological sample was calculated from the standard curve using the ratio between analyte peak area and corresponding internal standard peak area. The fatty acids in the chromatogram are referenced as described under Figure 1.