Lipid Extraction and Fatty Acid Analysis

Lipids were extracted using hexane:isopropanol [39 (link)]. A powdered sample of tissue (∼50 mg) was extracted twice into 3.6 mL of hexane/2-propanol (3:2 v/v) with BHT (50 μM; Sigma-Aldrich, St. Louis, MO, USA) added to limit lipid peroxidation. Samples were homogenized using a PRO200 Bio-Gen Series homogenizer (PRO Scientific Inc., Oxford, CT, USA) and then centrifuged at 2000× g for 10 min. The organic phase was removed, dried under nitrogen, and dissolved in 1 mL of hexane/2-propanol (3:2 v/v) containing 5% water and 50 μM BHT and stored at −80 °C under nitrogen. The FA content of the organic extract was determined by fatty acid methyl ester (FAME) analysis using a Thermo Trace-1310 equipped with a TriPlus RSH Autosampler, (Thermo Fisher Scientific, Waltham, MA, USA) and a Supelco SP-2560 capillary column (75 m, 0.18 mm ID, 0.14 µm film thickness) as previously described [38 (link)]. Fatty acid methyl esters were prepared with the use of acetyl chloride [38 (link)].