Fatty Acid Extraction and Analysis

Extraction of fatty acids was performed as previously described [30 (link)]. Firstly, 2 g of frozen tissue powder was mixed with 15 ml of n-hexane:isopropanol (3:2, v/v) and 7.5 ml of 6.7% Na₂SO₄, followed by centrifugation for 10 minutes. The supernatant was evaporated to dryness with nitrogen. Methanol:toluene:H₂SO₄ (88:10:2, v/v/v) was added to produce fatty acid methyl esters (FAMEs). After cooling, 1 ml of heptane with 0.5 g anhydrous Na₂SO₄ was added for FAME extraction. To detect fatty acids, an Agilent 6890 N gas chromatograph equipped with a flame ionization detector and a DB-WAX column (0.25 mm, 30 m, 0.25  m; J & W Scientific) was used. The injector and detector temperatures were 230°C. The initial oven temperature was 50°C, increased to 200°C at 25°C min⁻¹, then increased to 230°C at 3°C min⁻¹. Nitrogen was used as the carrier gas at 1 ml min⁻¹. Exogenous heptadecanoic acid was added as internal standard.