Our recently completed work has established the importance of a clearly significant role for certain biliary proteins by reason of their capacity to inhibit cholesterol monohydrate nucleation, the initial step in the pathway leading to cholesterol gallstone formation. In part, this finding at last explains why many normal persons with gallbladder bile supersaturated in cholesterol do not form gallstones. Almost nothing is presently known about these nucleation inhibitory proteins, i.e., their number, relative concentrations, biochemical properties and precise mechanism of action etc. Moreover, very little is understood concerning the relative role of this newly identified second risk factor (additional to cholesterol supersaturation) in the pathogenesis of cholesterol cholelithiasis. In light of these major informational gaps, the present proposal centers entirely in logical sequence upon a series of studies concerning the nucleation inhibitory proteins. Some of the aspects of the subject we propose to pursue are: i) separation and isolation of the relevant proteins (nucleation inhibitors) using a carefully planned chromatography scheme following delipidation. Biliary proteins will be separated first on the basis of their isolelectric points (chromatofocusing); then by their hydrophobic interaction; finally by their charge (HPLC-ion exchange). As purification proceeds, the separated proteins will be analyzed electrophoretically and by functional activity. ii) qualitative characterization of their properties. The functional proteins will be analyzed electrophoretically, immunologically using rabbit antisera raised with purified proteins, and by analytical centrifugation. iii) studies of possible mechanism(s) of action whereby the nucleation inhibitory effect is mediated. These studies will deal with the question of which cholesterol-containing particle in bile preferentially associates with the purified functional proteins and the consequences of this association. iv) quantiation of the specific proteins using radioimmunoassay and related techniques with normal bile. v) application of the quantitation methodology to clinically-relevant problems for the purpose of assessing the relationship between apparent synthesis and secretory rates. For this purpose we will assess both bile and serum. vi) study of physiological control mechanisms determining the rate of biliary secretory output of these proteins with a view towards the possible future modulation of this using pharmacologic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK017562-16
Application #
3225771
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1976-12-15
Project End
1990-03-31
Budget Start
1988-12-01
Budget End
1990-03-31
Support Year
16
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Secknus, R; Darby, G H; Chernosky, A et al. (1999) Apolipoprotein A-I in bile inhibits cholesterol crystallization and modifies transcellular lipid transfer through cultured human gall-bladder epithelial cells. J Gastroenterol Hepatol 14:446-56
Ginanni Corradini, S; Yamashita, G; Nuutinen, H et al. (1998) Human gallbladder mucosal function: effects on intraluminal fluid and lipid composition in health and disease. Dig Dis Sci 43:335-43
Ginanni Corradini, S; Yamashita, G; Nuutinen, H et al. (1997) Variations in pigment and carbohydrate content of gallbladder bile affect accurate quantitation of total protein when using the fluorescamine method. Scand J Gastroenterol 32:340-9
Yamashita, G; Secknus, R; Chernosky, A et al. (1996) Comparison of haptoglobin and apolipoprotein A-I on biliary lipid particles involved in cholesterol crystallization. J Gastroenterol Hepatol 11:738-45
Secknus, R; Yamashita, G; Ginanni Corradini, S et al. (1996) Purification and characterization of a novel human 15 kd cholesterol crystallization inhibitor protein in bile. J Lab Clin Med 127:169-78
Nuutinen, H; Abei, M; Schwarzendrube, J et al. (1995) Biliary alpha 1-acid glycoprotein concentrations in gallstone-free controls and in patients with multiple or solitary cholesterol gallstones. Dig Dis Sci 40:1786-91
Nuutinen, H; Ginanni Corradini, S; Jungst, D et al. (1995) Correlation between biliary alpha 1-acid glycoprotein concentration and cholesterol crystal nucleation time in gallstone disease. Dig Dis Sci 40:1174-8
Yamashita, G; Ginanni Corradini, S; Secknus, R et al. (1995) Biliary haptoglobin, a potent promoter of cholesterol crystallization at physiological concentrations. J Lipid Res 36:1325-33
Abei, M; Nuutinen, H; Kawczak, P et al. (1994) Identification of human biliary alpha 1-acid glycoprotein as a cholesterol crystallization promoter. Gastroenterology 106:231-8
Abei, M; Kawczak, P; Nuutinen, H et al. (1993) Isolation and characterization of a cholesterol crystallization promoter from human bile. Gastroenterology 104:539-48

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