Tr13421

Fecal samples were collected at 7:30–8:00 a.m. and stored at −80 °C. Liver tissues were snap-frozen in liquid nitrogen and stored at −80 °C until lipid extraction. Lipids were extracted from powdered liver tissue or faecal pellets using a modified version of Folch et al.^{51 (link)} method. In brief, 800 μL 2:1 chloroform-methanol (v/v) was added to ~25 mg of powdered liver or a single faecal pellet. The tissue was homogenised with a motorised pellet pestle, followed by 10 pulses with a probe sonicator, then vortex mixed for 20 s. Samples were digested for 1 h at room temperature on a rocker. Four hundred microlitres 0.6% NaCl was added to each sample and then vortex mixed. Samples were centrifuged (3000 × g, 10 min) and the bottom phase (lipid extract) was collected. Centrifugation and collection of the lipid extract was repeated once. The lipid extract was dried under a steady stream of nitrogen in a TurboVap^® Evaporator (Biotage, USA). The extract was resuspended in 0.4 mL 95% ethanol and heated to 37 °C prior to lipid assays. Colorimetric assays were used to measure triglycerides (Pointe Scientific, T7532) and cholesterol (Thermo Scientific, TR13421), and fluorometric assay used to measure NEFA (Wako Chemical, 279-75401). All assays were conducted according to the manufacturer’s protocols. Results were normalised to mass of liver tissue or faecal sample.