Ultracentrifugal isolation of vesicular carriers of biliary cholesterol in native human and rat bile.
We have utilized ultracentrifugation of native bile-Metrizamide density gradients to isolate a vesicular transport system of biliary lipids in both man and rat. We identified vesicular structures by electron microscopy. Fresh bile specimens were obtained from bile fistula rats (unsaturated bile) and from patients 1 week after bile duct surgery (supersaturated bile). Metrizamide was dissolved in bile (33% w/v), and continuous density gradients were performed with undiluted bile (density limits = 1.020 to 1.300 gm per ml). The relative distribution of biliary cholesterol, phospholipid and bile salt was studied as a function of the density of the fractions. Approximately 50% of total rat biliary cholesterol and between 61 and 90% of human biliary cholesterol was concentrated in the lightest fractions of the gradients (density less than 1.060 gm per ml). In contrast, less than 20% of bile salts was present in fractions with densities lower than 1.060 gm per ml. The highest amounts of bile salts and phospholipids of the bile-Metrizamide density gradients were found in the density range of 1.075 to 1.100 gm per ml in both human and rat bile. More than 80% of biliary proteins was found in fractions with densities greater than 1.075 gm per ml, and only 2% was found in the cholesterol-rich fraction with density less than 1.060 gm per ml in both species. When bile salt concentration was raised in rat bile from 38 to 97 mM by adding taurocholate, the low density cholesterol-rich fraction almost disappeared. Electron microscopy of negatively stained preparations of the fractions with density less than 1.060 gm per ml showed 40 to 120 nm vesicles, which were not apparent in the other fractions. Similar vesicles were demonstrated also in fresh rat bile and within the canaliculi after acute depletion of the bile salt pool (biliary bile salt concentration of 3.45 mM; total biliary lipid concentration of 0.25 gm%). The structure of these vesicles was shown in thin sections of liver specimens. They appeared as internal cavities surrounded by a single, continuous 6-nm-thick bilayer. These studies demonstrate that a high proportion of biliary cholesterol is transported in vesicles in human supersaturated native bile and that vesicular carriers are also responsible for the transport of a significant amount of biliary cholesterol in unsaturated rat bile. The presence of vesicles in unsaturated hepatic bile strongly supports the thesis that biliary lipids may be secreted as vesicles from the hepatocyte into the canaliculi.
D000818:Animals; D001646:Bile / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000378:metabolism; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes / Q000302:isolation & purification* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D014461:Ultracentrifugation
D001647:Bile Acids and Salts; D008081:Liposomes; D010743:Phospholipids; D002784:Cholesterol