Quantifying Liver Biomarkers and Oxidative Stress

Serum glucose, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were analyzed by enzymatic enzyme methods (Sigma-Aldrich, St. Louis, MO, USA). For hepatic triglyceride (TG) quantification, lipid was extracted as described in a previous study [28 (link)]. Briefly, liver tissue was homogenized in 1 mL solution containing 5% Nonidet P-40 (NP-40) substitute (Amresco, Solon, OH, USA). The homogenates were slowly heated to 80–100°C in a water bath for 2–5 min until the NP-40 became cloudy, then cooled down to room temperature. Samples were then centrifuged for 2 min to remove any insoluble materials. Hepatic TG levels were measured by enzymatic assay (Sigma-Aldrich) and normalized to their respective protein concentrations. Lipid peroxidation (MDA) levels in the liver tissue were measured by a OxiSelect^TM TBARS assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s protocol, normalized to their respective protein concentrations, and expressed as fold change compared to LETO control group.