Dynamic biliary lipid collection and analysis
Mice were anaesthetized by intraperitoneal injection of 1 mL/kg fentanyl/fluanisone and 10 mg/kg Diazepam. The abdomen was opened and, after distal ligation of the bile duct, the gallbladder was cannulated. Bile sampling started directly after cannulation and was collected for 10 min. Bile flow was determined gravimetrically. Biliary bile salt, sterol and phospholipid concentrations were determined as described previously [32]. To determine maximal secretion rates of biliary lipid secretion, bile was diverted for 90 min to deplete the endogenous bile salt pool. Subsequently, tauroursodeoxycholate (Sigma Chemical Co., MO, USA) dissolved in PBS was infused into the jugular vein in stepwise increasing rates from 600–1,800 nmol/min/100 g body weight. Bile was collected in 10 min fractions and analyzed for bile salt and lipid content. In separate experiments, following distal ligation of the bile duct and cannulation of gallbladder, bile was diverted for 30 minutes while mice were infused with sterile PBS. Subsequently, the mice were infused continuously with 1,200 nmol/min/100 g body weight of tauroursodeoxycholate and two 30-minute fractions were collected. Sterol analyses of bile was performed by GC analysis as described above.