2.7. Measurement of bile acid pool size

The total bile acid pool size was determined as bile acid contents in the small intestine, gallbladder and liver. In brief, liver tissues (100–150 mg) were weighed, homogenized in 0.5 mL PBS, and mixed with 1 mL ethanol. The mixture was rotate mixed for 1 h and then centrifuged at 3500 rpm for 5 min. The supernatant was transferred to a new tube; the pellet was extracted for bile acid with 1.5 mL ethanol (rotating for 1 h) again. The supernatant was combined and stored at −20℃. The concentrations of bile acids were determined using a Total Bile Acid Assay Kit (Diazyme Laboratories, Poway, CA). The entire small intestine sample and gallbladder sample were each homogenized with 3 mL PBS and extracted with 3 mL ethanol for bile acid as described above. The pool size was expressed as micromoles of bile acid/100 g of body weight.