Robert V. Rege

Pathogenesis of calcium-containing gallstones. Canine ductular bile, but not gallbladder bile, is supersaturated with calcium carbonate.

Calcium precipitation in bile is a requisite event in the initiation and growth of all pigment gallstones. Calcium solubility in bile is thus of great importance. This is the first attempt to define the ion-product of CaCO3 in bile in any species. If the ion-product: [Ca++] X [CO = 3] exceeds solubility product (Ksp), the sample is supersaturated and CaCO3 precipitation is thermodynamically possible. We have recently determined Ksp of calcite to be 3.76 X 10(-8) mol/liter at 37 degrees C and total ionic strength = 0.16 M. Gallbladder (GB) bile was obtained from 15 anesthetized dogs after 12-24-h fasts. Duct bile was obtained from three dogs (n = 12) during variable taurocholate infusion. Samples were assayed for pH, partial pressure of carbon dioxide (PCO2), total bile salt concentration ([TBS]), total calcium concentration ([Ca]), and free calcium ion concentration ([Ca++]). With increasing [TBS] in both GB and duct bile, there was a linear decline in pH, a curvilinear decline in [HCO-3] and [CO = 3], and linear increase in [Ca++] and [Ca]. All ductular samples were supersaturated with CaCO3, with saturation indices (SI) as high as 17.5 and a mean of 8.36 +/- 1.43 (SE). In sharp contrast, none of the GB samples were supersaturated, due to the marked decline in [CO = 3] upon concentration and acidification of bile. In GB bile, the SI ranged from 0.006 to 0.126, with a mean of 0.039 +/- 0.011. The gallbladder thus produced a change in the SI from a value as high as 17.5 to a value as low as 0.006 in concentrated GB bile, which is a nearly 3,000-fold change. The average change in the SI was approximately 215-fold. Since all duct samples were supersaturated, and since the dog does not normally form gallstones, the data support our previous hypotheses that: (a) in canine bile, as in canine pancreatic juice, a nucleating factor is necessary for CaCO3 precipitation; (b) bile salts are important buffers for Ca++ in bile; and (c) normal GB mucosal function (concentration and acidification of bile) plays an important role in reducing CaCO3 lithogenicity in GB bile.

Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro.

In seeking to identify nucleating/antinucleating proteins involved in the pathogenesis of cholesterol gallstones, a major acidic protein was isolated from each of 13 samples of cholesterol gallstones. After the stones were extracted with methyl t-butyl ether to remove cholesterol, and methanol to remove bile salts and other lipids, they were demineralized with EDTA. The extracts were desalted with Sephadex-G25, and the proteins separated by PAGE. A protein was isolated, of molecular weight below 10 kD, which included firmly-bound diazo-positive yellow pigments and contained 24% acidic, but only 7% basic amino acid residues. The presence of N-acetyl glucosamine suggested that this was a glycoprotein. This protein at concentrations as low as 2 micrograms/ml, but neither human serum albumin nor its complex with bilirubin, inhibited calcium carbonate precipitation from a supersaturated solution in vitro. This protein could be precipitated from 0.15 M NaCl solution by the addition of 0.5 M calcium chloride. Considering that cholesterol gallstones contain calcium and pigment at their centers, and that small acidic proteins are important regulators in other biomineralization systems, this protein seems likely to play a role in the pathogenesis of cholesterol gallstones.

Human polymorphonuclear leukocyte phagocytosis of crystalline cholesterol, bilirubin, and calcium hydroxyapatite in vitro

We tested the hypothesis that human polymorphonuclear leukocytes (PMNs) phagocytize crystalline cholesterol, bilirubin, or calcium hydroxyapatitein vitro and in the process release oxygen metabolites and enzymes involved in the inflammatory process. Chemiluminescence (CL), elicited by the respiratory burst (release and activation of oxygen metabolites and enzymes) of PMNs during phagocytosis of a target particle, was used to quantitate PMN phagocytosis of each crystal. Significant CL (P<0.05) was observed with cholesterol concentrations of 1.3–5.3 mg/ml and the dose-response was linear (r≥0.95). With bilirubin, significant CL was observed with concentrations of 0.07–0.33 mg/ml. The response to calcium hydroxyapatite was variable. Human PMNs phagocytize cholesterol, bilirubin, and to a lesser extent, calcium hydroxyapatite. PMN chemiluminescence was associated with phagocytosis, indicating that inflammatory substances are being released in the process. These results support the concept that crystals that occur in the gallbladder may initiate gallbladder inflammation.

Secretion of biliary calcium is increased in dogs with pigment gallstones

We have previously demonstrated that gallbladder bile is supersaturated with calcium bilirubinate in a canine dietary model of pigment gallstones. Supersaturation resulted from combined increases in the concentrations of both biliary calcium and unconjugated bilirubin. The elevations in biliary calcium and unconjugated bilirubin concentrations remain unexplained but could possibly be due to increases in hepatic or ductular secretion, alterations in bile composition with respect to calcium- or bilirubin-binding affinity, decreases in absorption from the gallbladder lumen, or, in the case of unconjugated bilirubin, production within the lumen by hydrolysis of conjugated bilirubin. Here, we study a single possible cause for the observed increase in biliary calcium concentration during pigment gallstone formation in dogs. Secretion of calcium into bile in dogs with pigment gallstones before and after infusion of the bile salt, taurocholate, was compared to normal dogs. A significant increase in bile acid-independent bile flow and calcium output (CaO) was observed at any given bile acid output. Thus, plots of bile flow and CaO versus bile acid output yielded two separate functions in normal dogs and dogs with pigment gallstones. The slopes of these functions were similar, but intercepts extrapolated to zero bile acid output were markedly different, indicating that bile acid-independent, but not bile acid-dependent, bile flow and CaO was increased. The increase in CaO was not due to secretion of bile with increased concentrations of calcium but rather to the increases in the rate of bile flow. These findings might, in part, explain elevated calcium concentrations since increased amounts of calcium would be presented to the gallbladder in these animals during gallstone formation.