Measurement of fatty acids by GC-FID

For fatty acid analysis by GC-FID, lipid samples were extracted from tissues by the method of Bligh and Dyer. C23:0 (Supelco n-Tricosanoic acid, Sigma–Aldrich) was added to the extracted samples as an internal standard. Then, the fatty acids were methylated with the Fatty Acid Methylation Kit (Nacalai Tesque), and purified using the Fatty Acid Methyl Ester Purification Kit (Nacalai Tesque) following the manufacturer's instructions. Fatty acid methyl ester samples were characterized with GC-2010 Plus system (Shimadzu) equipped with an FID. The flow rate of carrier gas (He) was set at 45 cm/s linear velocity. The temperature of the injection unit and the detector were 240°C and 250°C, respectively. The oven temperature was initiated at 140°C, then raised to 200°C at a rate of 11°C/min, then increased to 225°C at a rate of 3°C/min, and finally elevated to 240°C at a rate of 20°C/min and held at this temperature for 5 min. The injection volume was 2 μl in the split injection mode. For separation, a capillary column (FAMEWAX, 30 m, 0.25 mm ID, 0.25 μm; Restek Corporation, Bellefonte, PA) was used. Fatty acid methyl esters were identified and quantified using a mixture of fatty acid methyl ester standards (Supelco 37 Component FAME Mix and DPA (n-3) from Sigma–Aldrich; DPA (n-6) from Nu-Chek Prep, Inc., Elysian, MN; DTA (n-6) from Cayman) for calibration.