Lipid Extraction and Fatty Acid Analysis

A constant amount of cells (counting 6×10⁶ millions of cells) in a 1.5 mL vial was added with tridistilled water (2 mL) and 2:1 chloroform/methanol (4 times × 4 mL of 2:1 chloroform/methanol mixture) to extract lipids according to the Folch method [59 (link)]. The organic layers, dried on anhydrous Na₂SO₄ evaporated to dryness, gave the total lipid extract that was checked by thin layer chromatography for lipid class separation as previously reported [21 (link)]. In some of the cell samples triglycerides and cholesteryl esters were also obtained, therefore a chromatographic separation of the lipid classes was performed and differentiated conversion of the lipid classes was performed: Fatty acid-containing phospholipids were transformed to the corresponding FAME by adding 0.5 M solution of KOH in MeOH (0.5 mL), quenching the reaction after 10 min for PL fraction and 30 min for TG fraction by brine addition (0.5 mL). The derivatization of fatty acid moieties in cholesteryl esters was carried out by adding 1 M solution of NaOH in 3:2 MeOH/benzene (0.5 mL). The reaction was stirred in the dark under argon and quenched by brine (0.5 mL) after 15 min. FAME were extracted with n-hexane (3 times × 2 mL of n-hexane), dried on anhydrous Na₂SO₄, evaporated to dryness and analysed by GC in comparison with standard references. Detailed fatty acid compositions of each lipid class identified in the experiments with 150 and 300 µM palmitic, palmitoleic and sapienic acids supplementations are listed as µg/mL in Tables S1–12 of Supplementary Information.