Total bile acids assay kit

Fecal cholesterol was determined by same procedure employed for hepatic cholesterol determination, using total cholesterol assay kit (Cellbiolabs). Fecal bile acids were extracted using the slightly modified method of de Wael et al. [18] . Dried fecal specimen was weighed, powdered and 100 mg transferred to a glass tube. After adding 1 mL of 4% KOH in ethylene glycol, the samples were heated at 200°C for 15 min with an air reflux condenser on the test tube. Meanwhile, the contents were mixed three or four times. After cooling, 1 mL of 20% NaCl solution was added. After mixing, 0.2 mL of concentrated HCl was added and the contents mixed. Following this, 6 mL of diethyl ether was added to the acidified solution. The tube was shaken for 1 min, followed by centrifugation at 2,000 × g for 3 min. The upper layer was collected in a new glass tube. This procedure was repeated three times. From the combined extracts, the diethyl ether was completely evaporated at 40°C and the residue dissolved in 1 mL of methanol. This was used for enzymatic determination of bile acids using total bile acids assay kit (Crystal Chem, Downers Grove, IL, USA).