Fatty Acid Extraction and Derivatization

Fatty acid extraction and derivatization was performed according to a previously described method [17 (link)]. Briefly, pieces of liquid nitrogen-frozen tissue were coarsely fragmented and homogenized for about 1 min in a 20-mL glass vial containing 2 mL of 12% w/v, 1.5 M boron trifluoride-methanol (BF3-MeOH, purchased from Acros Organics, Geel, Belgium). Then, vials were sealed and kept at 100 °C for 1 h, to allow FA transesterification. After cooling down the vials, 2 mL of n-Hexane (Carlo Erba Reagents Srl., Val de Reuil, France) were added to the mixture and vortexed, resulting in the formation of an upper n-Hexane transparent layer containing fatty acid methyl esters (FAMEs). FAMEs were then extracted, put into a 2 mL glass GC-vial and air-dried. Later, FAMEs were resuspended in 400 μL of n-Hexane and injected in the GC-FID for analysis.