Quantification of Endogenous Lipids in Mouse Tissues

Endogenous lipids from mouse liver and heart were detected and quantified using several techniques. FC was quantified using straight-phase HPLC and ELS detection as previously described10 (link). Quantification was made against an external calibration curve. This chromatographic set-up was also used to fractionate DG. Quantification of CE, TG, SM, and phospholipids (all from the total extract) and DG (fractionated from the HPLC) was made by direct infusion (shotgun) on a QTRAP 5500 mass spectrometer (Sciex, Concord, Canada) equipped with a robotic nanoflow ion source, TriVersa NanoMate (Advion BioSciences, Ithaca, NJ)11 (link). For this analysis, total lipid extracts, stored in chloroform:methanol (2:1), were diluted with internal standard-containing chloroform/methanol (1:2) with 5mM ammonium acetate and then infused directly into the mass spectrometer. The characteristic dehydrocholesterol fragment m/z 369.3 was selected for precursor ion scanning of CE in positive ion mode12 (link). The analysis of TG and DG was performed in positive ion mode by neutral loss detection of 10 common acyl fragments formed during collision induced dissociation13 (link). The PC, LPC and SM were detected using precursor ion scanning of m/z 184.114 (link), while the PE, phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI) lipid classes were detected using neutral loss of m/z 141.0, m/z 185.0, m/z 189.0 and m/z 277.0 respectively15 (link)16 (link). For quantification, lipid class-specific internal standards were used. The internal standards were either deuterated or contained diheptadecanoyl (C17:0) fatty acids.
Ceramides (CER), dihydroceramides (DiCER), glucosylceramides (GlcCER) and lactosylceramides (LacCER) were quantified using a QTRAP 5500 mass spectrometer equipped with a Rheos Allegro quaternary ultra-performance pump (Flux Instruments, Basel, Switzerland). Before analysis the total extract was exposed to alkaline hydrolysis (0.1M potassium hydroxide in methanol) to remove phospholipids that could potentially cause ion suppression effects. After hydrolysis the samples were reconstituted in chloroform:methanol:water [3:6:2] and analyzed as previously described17 (link).
For the recovery experiments the tissue samples were spiked with non-endogenously present lipids (or endogenous lipids spiked at relatively high levels) and could therefore all be detected by lipid class specific scans using the shotgun approach. In the recovery experiment we therefore also included the PA and phosphatidylcholine plasmalogen (PC P) lipid class, which we could not measure endogenously using our current analytical platform. Due to poor ionization efficiency, FC was derivatized and analyzed as picolinyl esters according to previous publication18 (link). See Table 1 for details. With some exceptions, lipids are annotated according to Liebisch et al.19 (link).