Liver fatty acid profile.

Liver tissues were collected and immediately placed in dry ice prior to storage at −70/−80°C prior to processing. Liver tissues (50 mg × sample) were homogenized at 4°C with an Ultra-Turrax homogenizer (IKA, Staufen, Germany) in homogenization buffer (1 μM 2,6-di-tert-butyl-4-methylphenol, 1 mM diethylenetriamine penta-acetic acid, 2 mM ethylenediamine tetra-acetic acid, 5 mM 3-(N-morpholino)propanesulfonic acid, with 180 mM potassium chloride, and adjusted to pH 7.4). Samples were normalized by protein content (Bradford assay). Tissue fatty acid profiles were based on reported methodology (64).

(i) Fatty acid methyl ester preparation. Total lipids from liver homogenates were extracted via chloroform-methanol (2:1 [vol/vol]; 3 times) with 0.01% butylated hydroxytoluene. The chloroform phase was evaporated under nitrogen, and the fatty acids were transesterified by incubation in 2.5 ml of 5% methanolic HCl at 75°C for 90 min. Following transesterification, 2.5 ml of n-pentane and 1 ml of saturated NaCl solution were added to extract fatty acid methyl esters (FAMEs). The n-pentane phase was separated, evaporated under N₂ gas, and redissolved in 80 μl of carbon disulfide. Two microliters was used for subsequent GC analysis.

(ii) Gas chromatography conditions. Gas chromatography analyses was performed on a GC system 7890A with a series injector 7683B and a flame ionization detector (Agilent Technologies, Barcelona, Spain), equipped with a DBWAX capillary column (length of 30 m, inner diameter of 0.25 mm, and film thickness of 0.20 μm). The injection port was maintained at 220°C and the detector at 250°C. Injections were performed using the splitless mode. The flow rate of carrier gas (helium 99.99%) was maintained at 1.8 ml/min. The temperature program was 5 min at 145°C, then 2°C/min to 245°C, and finally, the temperature was held at 245°C for 10 min with a postrun of 250°C for 10 min.

(iii) Data analysis. Identification of FAMEs was made by comparison with authentic standards (Larodan Fine Chemicals, Malmö, Sweden). The fatty acid profile detected plus identified plus quantified represents more than 95% of the total chromatogram. Results were expressed as percent moles. Here, we normalized against pooled CON samples.