Triglyceride standard

One microlitre of a solution of 4% (w/w) fat in hexane was injected into the gas chromatograph. The TAG solution was analysed on a GC Dani 1000 gas chromatograph equipped with a flame ionization detector (FID) held at 360 °C and a temperature-programmed vaporizer (PTV) injector. An Rtx 65TG (mod. 17008) capillary column (30 m × 0.25 mm i.d.; 0.10 μm film thickness) from Restek (Bellefonte, PA, USA) was used with high-purity helium as a gas carrier at 1.5 mL/min. The injection was performed with a split ratio of 1/30 (v/v) and a heating programme of 50 °C for 0.3 min, followed by a 500 °C/min increase to 300 °C with a 7-min hold. The column oven temperature programme was 250 °C for 2 min, followed by an increase of 6 °C/min to 360 °C for 10 min, as described in Romano et al. [25 (link)]. The flow rate of the gas carrier, He, was 1.2 mL/min. TAGs were identified by comparing their retention times with those of a triglyceride standard (Sigma, Saint louis, MO, USA). These samples allowed response factors (Rfs) to be calculated, which converted peak areas into weight percentages. The reference standard for cholesterol was purchased from Sigma Chemical Co. (Saint louis, MO, USA).

Triglyceride in the liver was measured as previously described (Le Marchand et al., 1973 (link); Fujii et al., 2008 (link); Komazaki et al., 2017 (link); Sasaki et al., 2018 (link)). Briefly, the aliquots of liver lysates were added to a microcentrifuge tubes containing 37.5% KOH and heated at 70°C for 30 min. The tubes were placed in 55°C water bath overnight (n = 4). Subsequently, 50% ethanol was added, and the tubes were centrifuged. The supernatants were separated, treated with MgCl₂, left on ice for 10 min, and then centrifuged again. The supernatants and a triglyceride standard (Sigma Aldrich [St. Louis, MO, USA]) were placed in a 96 well black plate with a clear flat bottom, and triglyceride levels were measured using a commercially available kit (Triglyceride quantification kit, Sigma Aldrich [St. Louis, MO, USA]). Preparation was performed by following the manufacturer’s instruction. Fluorescence intensity (λex = 540 nm/λem = 590 nm) was measured with a plate reader (BMG Labtech FLUOstar OPTIMA-6).