Measurement of serum adiponectin, leptin and insulin

Peripheral venous blood samples were collected from patients in the morning after an overnight fasting. Blood samples were centrifuged immediately at 4 °C and separated serum samples were stored at − 80 °C. Serum adiponectin levels were measured by enzyme linked immunoassay (ELISA) method using human adiponectin ELISA kit (Mediagnost, Reutlingen, Germany). Serum insulin levels were measured by ELISA method using human insulin kit (Monobind Inc., Lake Forest, CA (92630), USA). Serum leptin levels were measured using human leptin ELISA kit (Mediagnost, Reutlingen, Germany). Homeostatic model assessment of insulin resistance (HOMA-IR) is a method for assessment of insulin resistance and β-cell function based on the fasting plasma insulin and glucose concentrations. Insulin resistance was estimated using HOMA 2-IR software, as described before⁴⁵. Blood sampling was performed on the day of clinical visit and assessment of hepatic steatosis by ultrasound and CAP measurement.