2.4.3. Biochemical Assessments
TNF-α
All participants were asked to fast for at least nine hours prior to venepuncture. Monash Health Pathology, using assays accredited by the National Association of Testing Authorities, processed blood samples for ALT, insulin, glucose, total cholesterol, triglyceride and stored serum at −80 °C. The stored serum was used for TNF-α, adiponectin, and leptin analysis via ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA & Cusabio, Wuhan, China). Analysis was performed using a microplate reader at 490 nm for TNF-α and 450 nm for adiponectin and leptin (Magellan, Phoenix, AZ, USA). The TNF-α, adiponectin, and leptin concentrations were calculated from the standard curve generated in GraphPad Prism 5.0 d for Mac OS X (GraphPad Software, La Jolla, CA, USA). To measure faecal calprotectin, faecal material was collected by individuals at home and frozen at −20 °C before being delivered to the investigators. Samples were then stored at −80 °C until the end of the study. Faecal samples were processed using Buhlmann Smart-Prep Extraction kits, followed by Buhlmann Calprotectin ELISA, following the manufacturer’s instructions (Taylor Scientific, St Louis, MO, USA). Analysis was performed using a microplate reader at 450 nm (Magellan). Faecal calprotectin concentration was calculated from the standard curve generated in GraphPad Prism 5.0 d for Mac OS X (GraphPad Software, La Jolla, CA, USA).