Total glycerolipids were quantified from their fatty acids: in an aliquot fraction, 5 μg of 15:0 (internal standard) were added and the fatty acids present were converted to methyl esters (FAME) by a 1-hour incubation in 3 mL 2.5% H2SO4 in pure methanol at 100°C [29 (link)]. The reaction was stopped by addition of 3 mL water and 3 mL hexane. The hexane phase was analyzed by gas chromatography-flame ionization detector (GC-FID) (Perkin Elmer) on a BPX70 (SGE) column. FAMEs were identified by comparison of their retention times with those of standards (Sigma) and quantified using 15:0 for calibration. Extraction and quantification were performed at least 3 times. To quantify the various classes of neutral and polar glycerolipids, 300 μg of lipids were separated by thin layer chromatography (TLC) onto glass-backed silica gel plates (Merck) using two distinct resolving systems [30 (link)]. To isolate neutral lipids including DAG and TAG, lipids were resolved by TLC run in one dimension with hexane:diethylether:acetic acid (70:30:1, v/v). To isolate membrane glycerolipids, lipids were resolved by two-dimensional TLC. The first solvent was chloroform:methanol:water (65:25:4, v/v) and the second one chloroform:acetone:methanol:acetic acid:water (50:20:10:10:5, v/v). Lipids were then visualized under UV light, after spraying with 2% 8-anilino-1-naphthalenesulfonic acid in methanol, and scraped off the plate. Lipids were quantified by methanolysis and GC-FID directly from the scraped silica as described above.
Free full text: Click here
