Search

Several proteins are known to modulate cholesterol crystallization. We recently demonstrated that haptoglobin has cholesterol crystallization promoting activity. However, this effect is still not well understood mechanistically. The current study examined the distribution of haptoglobin compared to apolipoprotein A-I (apo A-I) to micelles, vesicles and crystals as an initial step in providing a focus for further studies of the mechanism of cholesterol crystallization activity. Specific protein purification was accomplished by immunoaffinity chromatography. The crystallization-promoting activity of biliary haptoglobin, albumin and commercial apo A-I was measured by a photometric crystal growth assay. The distribution of micelles, vesicles and proteins in model bile was determined by Sepharose CL-6B column chromatography. Detection of the presence of test proteins in cholesterol crystals was determined using specific 125I-radiolabelled proteins. Haptoglobin (20 micrograms/mL) showed a significant crystallization promoting-activity, whereas apo A-I (30 micrograms/mL) only tended to show a slight inhibitory activity. The cholesterol crystal-bound protein in each case was found to be less than 1% of the total concentration of that protein that had been added to the model bile system. The elution profile of commercial apo A-I from a Sepharose CL-6B column was strikingly altered when it was added to model bile prior to elution. In contrast, the column elution profiles for both haptoglobin and albumin were unchanged when model bile was similarly added to the sample. Haptoglobin increased the amount of cholesterol found in the vesicular fraction when compared to apo A-I. Haptoglobin does not bind tightly to either biliary lipid particles or to cholesterol crystals but does increase the amount of cholesterol in vesicles by inducing a shift from micellar cholesterol (P = 0.046). This shift appears to explain in part its promoting effect on cholesterol crystallization.

Pooled, normal human gallbladder biles were initially separated on a molecular sieving chromatography column to remove soluble mucin glycoproteins as well as high molecular weight proteins (greater than 200,000). The remaining lower molecular weight proteins and other bile components were then examined by lectin affinity chromatography with four different types of lectin. The separated bound fractions were compared for inhibiting and promoting activities with a newly devised sensitive cholesterol crystal growth assay and for differences in electrophoretic patterns on SDS-gels. Protein factors (presumably glycoproteins) were found to have both inhibiting and promoting activities, even in the absence of cholesterol gallstone disease. The promoting effect was indicated by shortened crystal detection times and increases in crystal growth rate; whereas the inhibiting effect was indicated by decreases in crystal growth rate and reductions in the final crystal concentration as determined by the growth assay. Affinity chromatography mitigated the major problems of removing both lipids and pigment from the glycoproteins. In addition, partial purification of bound fractions with potent cholesterol crystal nucleation-altering activity can be obtained by this technique.

Among the factors affecting the stability of supersaturated bile that culminates in the nucleation and growth of cholesterol crystals are biliary lipids. The abundance and composition of these lipids affect the composition of biliary vesicles that, if rich in cholesterol, are themselves unstable and that constitute precursor particles from which cholesterol crystals somehow arise. Kinetic factors can affect stability in either of two ways: They can inhibit cholesterol crystallization, thus stabilizing the system, or they can promote the process of cholesterol crystal nucleation, thus stabilizing the system.

Rapid nucleation of cholesterol crystals has previously been shown to provide a sharp discrimination between abnormal (cholesterol gallstone-associated) and normal human gallbladder bile. In the present study, we sought to further clarify the crystal nucleation process by time-lapse microscopy using a novel high-resolution video-enhanced microscopy technique. Using a previously described method for removal of particles from abnormal biles, we found a strikingly rapid rate of de novo formation of unilamellar vesicles, soon followed by massive vesicular aggregation, culminating in crystal formation. In normal biles, by contrast, this rapid aggregation process was not observed and the isolated unilamellar vesicles showed prolonged stability. Morphometric analysis of interval particle counts showed statistically significant differences. The process of cholesterol monohydrate crystal nucleation in supersaturated human bile is characterized by a sequential combination of vesicle formation, vesicle aggregation, and subsequent crystal formation. The primary distinction between abnormal and normal biles resides only in the consistent rapidity of onset and completion of these events in the abnormal biles.

The onset time for cholesterol crystal nucleation of supersaturated normal human gallbladder biles is consistently prolonged when compared with biles from patients with cholesterol gallstone disease. Investigation of the factor(s) responsible for the suspended supersaturation (metastability) of normal human biles revealed that model bile solutions of cholesterol saturation index (CSI) and molar lipid composition identical to individual gallbladder bile specimens had much shorter crystal nucleation times, i.e., exhibited decreased metastability. Unsaturated normal biles, after supplementation with lecithin, cholesterol, and sodium taurocholate to a 'standard' supersaturated lipid composition, also demonstrated nucleation times three- to 15-fold longer than the comparable standard model bile. Total lipid extracts of normal biles, however, when similarly supplemented, did not differ in nucleation time from the control model solution. Gallbladder biles were fractionated by gel chromatography and the eluted fractions were pooled into two fractions. The fractions eluting in about the first 25% of the included volume when mixed with the supersaturated standard model bile induced a modest increase in nucleation time of approximately 1.5 times the control value. The fractions eluting in the second 25% of the included volume and which contained all of the bile lipids, were concentrated and supplemented with lipids to the standard composition. The nucleation times of these supplements were 3-10 times longer than the control nucleation times. Delipidated bile protein mixtures, purified by discontinuous sucrose gradient centrifugation, were recombined with purified lipids at the standard composition used previously. The nucleation times of these mixtures were significantly prolonged to the same extent as those associated with the second chromatographic fraction. These observations demonstrate that the delayed onset (inhibition) of cholesterol crystal nucleation observed in normal human gallbladder bile is produced by a factor(s) present in the biliary protein fraction.

The development and validation of a direct method for measuring maximum cholesterol solubility in bile is described. Application of this method to five large mammalian species, including man, produced a micellar zone significantly smaller than that previously reported. Further studies on in vitro model solutions patterned after bile confirmed this new micellar zone. Thus, direct evidence demonstrates that the micellar zone boundary derived in vitro from model solutions is applicable to human gallbladder bile. Using the present criteria, normal human bile, in contrast to bile from other mammalian species, is commonly supersaturated with cholesterol. A male-female difference in bile composition is not demonstrable despite the well-established female preponderance of cholelithiasis. Bile from patients with cholesterol cholelithiasis has a micellar zone similar to normals but differs compositionally in that there is a greater excess of cholesterol above saturation. We conclude that cholesterol supersaturation may be a necessary but not solely sufficient cause for gallstone formation.

6 patients with radiolucent cholelithiasis underwent randomized successive 3-week trials on each of the following medications: beta-glycerophosphate, linoleic acid, or purified soybean lecithin. Bile-rich duodenal fluid was obtained prior to the study and following each treatment period. Soybean lecithin feeding effected a qualitative change in biliary lecithin with increased fatty acid unsaturation, but no significant improvement in biliary cholesterol saturation or lipid composition changes including a proportionate increase in biliary phospholipids resulted from any treatment program. A 6-month therapeutic trial with soybean lecithin plus cholic acid failed to show a therapeutic response indicative of gallstone dissolution in the 6 patients.

The phase diagram approach to defining solute relations in solutions at equilibrium was originally conceived more than a century ago by Josiah Willard Gibbs, the great American physical chemist of the late 19th century (1). Small and co-workers in a series of papers published a decade or more ago (2–4) observed that applying such a plotting system to lipid solute containing analogs for native human bile could provide, for the first time, a means for defining in thermodynamic terms the maximum solubility limit of cholesterol in bile. Based on such a definition, it was concluded that a clear separation of normal from abnormal (choles­terol gallstone-associated) human biles was possible because the latter were supersaturated, whereas normals were not. Subsequent studies (5–7) failed to confirm the precision of the claimed sep­aration of normal from abnormal (cholesterol gallstone-associated) biles. In addition, results of studies using chenodeoxycholic acid therapy for patients with cholesterol gallstone disease demon­strated discrepancies in the saturation index definition of bile from untreated patients compared with post-treatment observations (8). The combination of these findings therefore necessitated a reassessment of this initial cholesterol saturation limit definition. An important outcome of this work (9) resulted in observations indicating the need for a significant reduction in the phase diagram boundary limit for maximum cholesterol solubility. This was based primarily on the recognized need in these later studies to take into account the factor of time-dependent equilibration. According to the revised view, the still somewhat puzzling observation that biliary cholesterol supersaturation, as defined by the new criteria, occurred frequently in healthy man also became apparent; whereas such was not the case in a variety of large mammals. Further studies have documented that cholesterol supersaturation is likely to be enhanced in the presence of physiologic factors such as obesity (10, 11) or the effects of estrogen (12, 13). Such enhance­ment has also been well documented as resulting from pathophysio­logic processes such as ileal disease or resection, wherein a broken enterohepatic circulation creates a deficiency of bile salts, important solubilizers for cholesterol in bile.

The incidence of several forms of liver disease associated with inflammatory bowel disease has been putatively ascribed to a toxic effect on the liver of portal vein bile acids abnormal in type or amount. To examine this possibility, total bile acid concentrations (sulphated and non-sulphated) were measured by gas-liquid chromatography in inferior mesentric vein serum of 19 patients undergoing colectomy for severe inflammatory bowel disease (IBD). Similar determinations were obtained on a control group of eight patients requiring colectomy for other non-inflammatory conditions. Mean values for mesenteric vein serum bile acid concentrations (muM/1) were 19.6+/-1.8 in controls and 16.3+/-2.0 in IBD. The mean sulphated bile acid fraction did not exceed 10% of total, although there was considerable variability (up to 40% of total). Lithocholic acid levels (entirely sulphated in all patients) were low. Although the IBD group showed a more than two-fold increase in mean lithocholate concentration (0.54+/-0.15 muM/1) over controls (0.21 +/- muM/1), this difference was not statistically significant. No significant intra-group difference was noted in the non-sulphated and sulphated fractions for cholic, chenodeoxycholic, and deoxycholic acid species, respectively. No unidentified or unusual bile acids were observed. There was no correlation between bile acid measurements and liver histology. These findings fail to support the hypothesis that liver disease often found in association with severe inflammatory bowel disease represents a form of bile acid toxicity. The invariable finding of total sulphation of the lithocholic acid fraction even in the presence of severe mucosal disease was unexpected.

Despite solute dilution and reduced total lipid concentrations, an unexplained increase in protein concentration has been reported to occur in the gallbladder bile of cholesterol gallstone patients.Solutes in gallbladder bile from gallstone-free controls and from four study groups were measured using standard methods. Total proteins were measured using amino acid analysis and a conventional fluorescamine method.Bile salts and pigment content were greater in gallstone-free controls than in all other study groups, including morbidly obese gallstone-free subjects. Total biliary protein concentration, as determined by amino acid analysis in the gallstone-free control group was higher than in non-obese gallstone patients with multiple stones and in morbidly obese gallstone-free subjects. Total biliary proteins as measured with fluorescamine, however, did not show intergroup differences. A major problem of the conventional fluorescamine assay is shown to be an artefact arising from the high pigment content of the more concentrated samples.Very dilute gallbladder bile samples are often found in the presence of gallstone disease. This also occurs in morbidly obese subjects, even in the absence of gallstones. Although the contribution of protein secretion/absorption by the gallbladder can also be relevant, especially in the presence of morbid obesity, the protein concentration in gallbladder bile, when accurately measured, generally parallels the concentrations of non-absorbed biliary solutes, reflecting the efficiency of fluid absorption. Measurement of biliary proteins by the conventional fluorescamine method is unreliable in clinical studies in which intergroup differences in pigment content are commonly present.

GALLSTONES may grow from microspheroliths, non-crystalline associations of cholesterol, pigment or calcium carbonate1,2; these spherical structures have been observed to aggregate and crystallize, but their composition and nature have never been well defined.