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.
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