Liver Triglyceride Extraction and Quantification

Liver triglycerides (TG) were extracted by the method of Folch and determined by a TG GPO-PAP kit (Roche Diagnostic, Australia) as previously described [18 (link)]. Briefly, 30–40mg of each liver sample was homogenized in 4 ml of chloroform/methanol (2:1) using a glass pestle tissue grinder. After the homogenization, the samples were rotated at room temperature overnight to ensure the complete solubilisation of the liver TG. The next day, 2 ml of 0.6% NaCl was added to each sample and followed by centrifugation to separate the aqueous from the organic phases. The lower chloroform layer contained liver TG were carefully transferred into a glass vial and dried completely under the nitrogen or air at 45°C. The extract was reconstituted in absolute ethanol for the determination TG using a POLARstar microplate reader (BMG Labtech, Germany).