The rate of cholesterol nucleation and crystal growth from metastably supersaturate model and native human gallbladder biles has been shown to importantly affected by factors present in biliary proteins. These kinetic factors are both inhibiting and promoting in activity. Our recent studies show that they are both glycoproteins, a well-defined 63 kDa inhibitor and most likely @ 140 kDa promotor. These glycoproteins have been found in different lectin chromatography-bound fractions with differing sugar specificities. We have developed a sensitive and reproducible new spectrophotometric assay system that facilitates not only their detection but permits estimates of their relative potency. Using this assay, we have found that both glycoproteins are present in normal biles. This suggests that in Health a balance of the kinetic factors is present and that an undefined imbalance may play a significant role in cholesterol gallstone pathogenesis. Given our present findings, with progress in coming months we expect to complete the Isolation and partial characterization of the two glycoproteins with optimal """"""""scaling-up"""""""" of production. The following logical objectives have therefore been established. Firstly, we wish to accomplish quantitation of the two effector glycoproteins. Radioimmunoassay (RIA) (and related immunoblotting for final purity assessment) seems the only reasonable methodologic approach. Secondly, a biophysicochemical characterization will be undertaken. This will consist of an assessment of the magnitude and functional importance of the sugar- moieties, followed by amino acid analysis of both effector glycoproteins. Thirdly,, their individual mechanism(s) of action will be examined. This will be done by Quasi-elastic light-scattering (QELS) analysis of the crystal growth process to assess the normal pattern as altered by the glycoproteins, especially the inhibitor. Analysis of the spectrophotometric crystal growth curve will also be undertaken to define quantifiable parameters. For study of mechanistic aspects of the promotor glycoprotein,k we propose to use Differential Scanning Calorimetry, QELS and related techniques to assess fusogenic vesicle protein-lipid interactions. Comparative surface activity measurements will also be made. Lastly, we propose to use the previously-developed quantitation methods (RIA) to determine the source(s) and natural distribution of the effector glycoprotein.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK017562-19
Application #
3225773
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1976-12-15
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
19
Fiscal Year
1992
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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