Unconjugated bililurbin (UCB) and/or derivatives of UCB (calcium bilirubinate or bilirubin polymers) make up part of the nidus of most cholesterol gallstones and are essential parts of the matrix of pigment gallstones, biliary sludge and the insoluble bile pigment precipitates which clog the bile ductules and canaliculi of patients with cholestatic liver disease. We recently demonstrated that bilirubin monoglucuronide (BMG), the major bile pigment found in animal models of gallstone formation and premature neonates who are particularly prone to cholestasis, undergoes non-enzymatic alkaline aqueous hydrolysis to insoluble UCB in model bile systems (and in guinea pig bile) much more rapidly than bilirubin diglucuronide (BDG). We prostulated that continuous non-enzymatic alkaline hydrolysis of BMG to UCB within the gallbladder (or within the bile ductules in cholestasis) may be the major source of precipitated UCB and its derivatives, and thus, ultimately the nidus for gallstone formation. We will use preparative and analytical HPLC to further study the hydrolysis of bilirubin conjugates using pure BDG and BMG in a variety of concentrations of biliary lipids and calcium in model bile systems and we will also use human and animal biles to determine in more detail what factors affect bile pigment hydrolysis and precipitation. We will continue our previous viscometric studies to determine the effect of changes in bile pigment and calcium concentration on the viscosity of bile. Increases in viscosity that occur with the addition of bilirubin to bile may contribute to the development of cholestasis in premature infants where the driving force for bile secretin may not be sufficient to oppose high intracanalicular viscosities. We will continue using derivative spectroscopy to study bile salt-bilirubin interactions. Fourier Transform Infrared Spectroscopy (FTIR) will be used to study intra-and inter- molecular interactions of biliary lipids and bile pigments both in the solution and in the solid phase. FTIR will be useful in determining hydrogen bonding o bilirubin in solution, the formation of bilirubin polymers and hydorphobic interaction of bilirubin with the non-polar portion of bile salts and phospholipids. Analytical ultracentrifugation will be useful in determining aggregation and self-association phenomena. Mass spectrometry and FTIR will be used to determine the structure of bilirubin covalent dimer, a possible precursor to polymer pigment stones formed during BMG hydrolysis. Such physical-chemical studies are essential in understanding bile pigment solubility, aggregation, and hydrolysis that lead to bile pigment precipitation.
