2.4. Liver Lipid Extraction and Fatty Acid Quantification

Liver tissue was collected from adult offspring (n = 10/group) at 17 weeks post-weaning and total lipids were extracted according to the Folch et al. method [13]. Briefly, 100 mg of liver tissue was treated with 2:1 chloroform: methanol and triheptadecanoic acid (17:0) was added as an internal standard to quantify fatty acids. The tissue was then homogenized and left to incubate at 4 °C overnight. Samples were then treated with 0.88% KCl and phase separation was achieved by a 10-min centrifugation at 500× g. The lipid-containing organic phase was collected and dried down under nitrogen and reconstituted in chloroform. Fatty acid methyl esters were generated and quantified as previously described [14], by which boron trifluoride/methanol (14%; Sigma-Aldrich, St. Louis, MO, USA) was added to the extracted lipids and heated at 100 °C for 60 min. Fatty acid methyl esters were analyzed by a gas chromatograph (model 430; Varian) equipped with a 30-m DB-FFAP column. The injector and flame-ionization detector were set at 250 °C and 300 °C, respectively, and the oven-temperature program was 50 °C for 1 min, increased to 130 °C at 30 °C/min, then increased to 175 °C at 10 °C/min, then increased to 230 °C at 5 °C/min and held for 9.5 min, and finally increased to 240 °C at 50 °C/min and held for 11.1 min. Helium was the carrier gas and was set to a constant flow rate of 1.0 mL/min. Fatty acids were identified using the Compass CDS chromatography software, and concentrations were calculated as ug fatty acid per gram of liver tissue as previously described [14].