The overall objective of this project is to determine the pathogenesis of pigment gallstones using the laboratory mouse (genotype nb/nb) with hereditary hemolytic anemia as an animal model of the human disease. Although hemolysis and cirrhosis are closely associated with pigment gallstones in humans, patients without those underlying conditions have pigment stone disease. Approximately, 20-25% of the 500,000 cholecystectomies performed yearly are due to pigment gallstones. Because the etiology of pigment gallstone disease is unknown, the mouse model is an excellent opportunity to evaluate mechanisms of hemolysis-induced calcium bilirubinate (black) pigment stone formation. Specifically, we will (1) assess whether hydrolysis or isomerization of bilirubin conjugates is a mechanism for the formation of unconjugated bilirubin in bile; (2) determine the calcium binding properties of unconjugated, monoglucuronidated, and diglucuronidated bilirubin and the influence of pH, bile salts, and glycoproteins on those interactions; and (3) determine the concentrations of glycoproteins in hepatic and gallbladder bile of control and nb/nb mice with or without luminal gallstones. Whole bile or purified bilirubin conjugates will be incubated for 1 hr at 37 degrees C in the presence of ascorbate (1 mM), saccharolactone (5mM), or both and then analyzed by high performance liquid chromatography. Isomerization of bile pigments is inhibited by ascorbate and enzymatic hydrolysis by saccharolactone. Calcium binding of purified bilirubin conjugates will be monitored by a calcium ion selective membrane electrode and the absorbance 450 of the test solutions. The influence of pH, bile salts and glycoproteins on those interactions can be assessed. The concentrations of glycoprotein in bile will be measured by HPLC using a hydroxylapatite column after separation of glycoprotein from other bile components by sedimentation. These proposed studies should provide insight into the pathogenesis of hemolysis-induced gallstones in mice and inferentially in humans.
| Trotman, B W; Nair, C R; Bernstein, S E (1988) Monoconjugated bilirubin is a major component of hemolysis-induced gallstones in mice. Hepatology 8:919-24 |
