Nucleation Of Cholesterol Monohydrate Crystals Research Articles

Comparative Proteomic Analysis of Gallbladder Bile Proteins Related to Cholesterol Gallstones

BackgroundNucleation of cholesterol monohydrate crystals following the aggregation and fusion of cholesterol-enriched vesicles is a critical procedure in the formation of cholesterol gallstone. Biliary proteins play important roles in the process. It is inefficient to screen pro-nucleating or anti-nucleating proteins with routine physiochemical techniques, by which we discovered several pro-nucleating proteins.Methodology/Principal FindingsBased on comparative proteomic technologies, we investigated the differentially expressed proteins between the cholesterol gallstone and control groups, and between the vesicular phase and micellar phase. There are 401±75 spots detected on the cholesterol gallstone group and 389±94 spots on the control group gels, 120±24 spots detected on vesicular phase and 198±37 on micellar phase gels, and accordingly 22 and 8 differentially expressed proteins were identified successfully, respectively. Three of them, HSA, Profilin and Retinol Binding Protein, were validated by Western blot.Conclusion/SignificanceSome of the identified proteins are in good agreement with proteins reported to be involved in the gallstone formation before. The information from this study might provide some important clues to uncover the key proteins involved in the formation of cholesterol gallstone.

Mucin-vesicle interactions in model bile: Evidence for vesicle aggregation and fusion before cholesterol crystal formation

Nucleation of cholesterol monohydrate crystals from bile is a critical step in the formation of cholesterol gallstones. Measurement of nucleation in model bile systems and the characteristics of the initial nucleus have proven elusive. In this study we have used three separate physical chemical techniques to examine vesicle aggregation and fusion, including dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescent biochemical assays. These assays enabled us to quantify the effect of biliary proteins, such as gallbladder mucin, on vesicle fusion and aggregation. In the absence of mucin, fusion is a relatively slow process occurring over 24 hours, whereas physiological concentrations of mucin are able to accelerate almost complete fusion of vesicles within 6 hours. Vesicle fusion and aggregation as characterized by TEM result in the formation of aggregates of multilamellar vesicles and giant fusion bodies associated with a background of mucin. These mucin-vesicle aggregate bodies may represent true nuclei and precede cholesterol monohydrate crystal nucleation. In future studies, these vesicle fusion assays can be used to quantitatively examine the effect of putative pro- and anti-nucleating proteins on the earliest steps of cholesterol crystal nucleation.

Mucin-vesicle interactions in model bile: Evidence for vesicle aggregation and fusion before cholesterol crystal formation

Nucleation of cholesterol monohydrate crystals from bile is a critical step in the formation of cholesterol gallstones. Measurement of nucleation in model bile system and the characteristics of the initial nucleus have proven elusive. In this study we have used three separate physical chemical techniques to examine vesicle aggregation and fusion, including dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescent biochemical assays. These assays enabled us to quantify the effect of biliary proteins, such as gallbladder mucin, on vesicle fusion and aggregation. In the absence of mucin, fusion is a relatively slow process occurring over 24 hours, whereas physiological concentrations of mucin are able to accelerate almost complete fusion of vesicles within 6 hours. Vesicle fusion and aggregation as characterized by TEM result in the formation of aggregates of multilamellar vesicles and giant fusion bodies associated with a background of mucin. These mucin-vesicle aggregate bodies may represent true nuclei and precede cholesterol monohydrate crystal nucleation. In future studies, these vesicle fusion assays can be used to quantitatively examine the effect of putative pro- and anti-nucleating proteins on the earliest steps of cholesterol crystal nucleation.

Dietary fish oil inhibits cholesterol monohydrate crystal nucleation and gallstone formation in the prairie dog

Epidemiologic studies suggest that populations consuming a diet rich in fish oil have lower rates of both atherosclerotic heart disease and gallstones. The mechanisms underlying this inhibitory effect on cholesterol gallstone formation remain unclear. We therefore studied the effect of dietary fish oil on bile composition and cholesterol precipitation in an animal model of gallstone disease. Adult male prairie dogs were fed a standard control diet (n = 12) or a lithogenic 1.2% cholesterol diet (n = 16). One half of the animals in each group had their diet supplemented with concentrated fish oil. After 14 days animals receiving the cholesterol diet all developed biliary cholesterol monohydrate crystals and gallstones. When fish oil was added to this high cholesterol diet, solid cholesterol crystal precipitation and gallstone formation were completely inhibited. This inhibition of gallstone formation was accompanied by a significant decrease in biliary calcium and total protein concentration. Microscopic cholesterol liquid crystals were evident in the bile of all of the animals fed the cholesterol plus fish oil diet. Dietary fish oil also significantly prolonged cholesterol monohydrate crystal observation time in animals receiving the lithogenic diet. These data suggest that dietary fish oil exerts a potent antilithogenic effect on cholesterol gallstone disease and may induce a stable liquid crystalline phase retarding nucleation.

Identification of a 130-kilodalton human biliary concanavalin a binding protein as aminopeptidase N

Background/Aims: Human gallbladder bile contains a group of nonmucin glycoproteins that binds to the lectin concanavalin A (con A) and has been reported to promote cholesterol monohydrate crystal nucleation, an event preceding the formation of gallstones. Several of these proteins, including a 130-kilodalton protein, have been isolated and shown to promote nucleation in vitro. The aim of this study was to identify this and other major biliary con A binding glycoproteins. Methods: Gallbladder bile was chromatographed on con A agarose, and the eluted proteins were electrophoresed, blotted, and subjected to amino-terminal sequence analysis. Results: The major con A binding proteins were identified as aminopeptidase N (a 130-kilodalton protein), α2 macroglobulin, hemopexin, immunoglobulin heavy chains, and the β chain of haptoglobin. After further purification, aminopeptidase N was found to be enzymatically active and to promote cholesterol crystallization at its approximate physiological concentration in bile. Conclusions: It is likely that aminopeptidase N is the previously characterized 130-kilodalton biliary crystallization promoting protein. Aminopeptidase N is probably released from the biliary canalicular membrane by the detergent activity of bile salts and may be one factor that promotes cholesterol crystallization in the gallbladder.

Bile concentration promotes nucleation of cholesterol monohydrate crystals by increasing the cholesterol concentration in the vesicles

Cholesterol in bile is solubilized in mixed micelles and cholesterol-phospholipid vesicles. Biliary cholesterol supersaturation and increased concentration of bile in the gallbladder promotes nucleation of cholesterol monohydrate crystals and gallstone formation, possibly by creating unstable vesicles with a high cholesterol/phospholipid ratio. In the present study super-saturated and unsaturated biles (cholesterol saturation index (CSI) 1.4 and 0.8 respectively) were prepared with concentrations typical of gallbladder and more dilute hepatic bile (total lipid concentration (TLCo) 10 and 2.5 g dl-1 respectively). The distribution of cholesterol between vesicles and micelles, and vesicular cholesterol/phospholipid ratio were studied using ultracentrifugation and gel-permeation chromatography. The nucleation time of cholesterol crystals was determined in whole model bile, and in the vesicular and micellar peak fractions. Increased CSI and bile dilution led to an increased proportion of cholesterol solubilized in vesicles. The concentration of bile did not influence vesicular cholesterol/phospholipid ratio. The vesicular cholesterol/phospholipid ratio found in gel-permeation chromatography experiments was similar at high and low CSI, whereas the ratio was significantly higher in supersaturated than in unsaturated biles in ultracentrifugation studies. Nucleation of cholesterol crystals from whole model bile was more rapid at the higher bile concentration and higher cholesterol saturation. Nucleation time in whole model bile correlated significantly with nucleation time in the corresponding vesicular peak fraction obtained by gel-permeation chromatography (r = 0.58: P < 0.01) and with the cholesterol concentration in this vesicular peak (r = -0.77; P < 0.002) but not with vesicular peak cholesterol/phospholipid ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of cholesterol in human gallbladder bile in relation to nucleation of cholesterol monohydrate crystals

To examine the hypothesis that the chemical activity of cholesterol molecules reflects the amount of cholesterol phasing out from bile associating with cholesterol monohydrate crystal nucleation, the cholesterol activities in human gallbladder biles from cholesterol gallstone patients, either untreated or treated with ursodeoxycholic acid, and from gallstone-free patients were determined in relation to the nucleation time and vesicular lipid composition. The cholesterol activity (nmol/disc/h) determined by the polyethylene disc uptake method was higher in the untreated gallstone group than the gallstone-free group ( P < 0.05) and the ursodeoxycholic acid treated group ( P < 0.01). The cholesterol activity correlated negatively with the nucleation time ( P < 0.01) and positively with both the vesicular cholesterol concentration ( P < 0.05) and the cholesterol/phospholipid ratio in vesicles ( P < 0.05). After the separation of vesicles from micelles by gel filtration, the cholesterol activity in the vesicular phase was found to be similar to that in the micellar phase. Interestingly, both the activities of cholesterol in the vesicular and micellar phases were significantly higher in the untreated gallstone group than in the gallstone-free group ( P <0.05). These results suggest that cholesterol activity represents the amount of thermodynamically unstable cholesterol in bile.

Inhibition of prostaglandin synthesis fails to prevent gallbladder mucin hypersecretion in the cholesterol-fed prairie dog

Gallstone formation in the cholesterol-fed prairie dog is preceded by an increase in mucin secretion by the gallbladder epithelium, and mucin hypersecretion is believed to promote cholesterol gallstone formation by accelerating the nucleation of cholesterol monohydrate crystals. Some studies have suggested that gallbladder mucin hypersecretion is mediated by increases in gallbladder prostaglandin synthesis, but other observations are difficult to reconcile with this view. An organ culture technique was used to measure mucin secretion in normal prairie dog gallbladder in response to exogenous prostaglandins and agents that increased or decreased endogenous prostaglandin production. Incubation with indomethacin produced a concentration-dependent inhibition of endogenous prostaglandin synthesis with virtually complete inhibition at 10−5 mol/L indomethacin. However, indomethacin had no effect on gallbladder mucin secretion at concentrations as high as 10−5 mol/L, and significant inhibition of mucin secretion was only found at 10−4 mol/L indomethacin, a concentration that also produced a significant increase in lactate dehydrogenase release from cultured explants. Incubation of gallbladder explants with the calcium ionophore A23187 significantly stimulated endogenous prostaglandin synthesis in a concentration-dependent manner, increasing synthesis of prostaglandins E and F to as much as 278% ± 20% and 335% ± 21% of basal values, respectively; however, the same concentrations of A23187 did not stimulate mucin secretion. Incubation of gallbladder explants in the presence of exogenous prostaglandin E2 or prostaglandin F2a in concentrations as high as 10−6 mol/L also did not stimulate mucin secretion. Prairie dogs fed a lithogenic 1.2% cholesterol diet showed a significant increase in gallbladder mucin secretion after 1 week (117.5 ± 10.2% of control, P < 0.05), and 4 of 5 had formed cholesterol monohydrate crystals after 3 weeks. Long-term treatment with indomethacin, 1.2 mg · kg−1 · day−1, failed to inhibit gallbladder mucin hypersecretion (129.2 ± 10.7% of control after 1 week) or cholesterol monohydrate crystal formation (3/5) in cholesterol-fed prairie dogs. Furthermore, incubation of explants with 10−5 mol/L indomethacin failed to prevent in vitro mucin hypersecretion in cholesterol-fed animals. These findings suggest that prostaglandins do not regulate gallbladder mucin secretion in the prairie dog, and it is unlikely that increases in gallbladder prostaglandin synthesis are responsible for mediating gallbladder mucin hypersecretion during cholelithiasis in the prairie dog.

Bovine gallbladder mucin promotes cholesterol crystal nucleation from cholesterol-transporting vesicles in supersaturated model bile.

This study examined the ability of purified gallbladder mucin to accelerate the nucleation of cholesterol monohydrate crystals from the cholesterol-transporting particles in supersaturated model bile. Mixed lipid micelles and cholesterol-phosphatidylcholine vesicles in supersaturated model bile were separated by Sephadex G-200 column chromatography. Mixed lipid micelles prepared by column chromatography had a low cholesterol-phosphatidylcholine ratio (0.30) and did not spontaneously nucleate cholesterol monohydrate crystals. In contrast, vesicles prepared by column chromatography had a cholesterol-phosphatidylcholine ratio of 1.00 and nucleated cholesterol crystals rapidly (P less than 0.001). Nucleation of cholesterol crystals was significantly accelerated in a concentration- and time-dependent manner by purified bovine gallbladder mucin in cholesterol containing vesicles, but not in mixed lipid micelles (P less than 0.001). A rapid filtration binding assay demonstrated significant binding of cholesterol and phosphatidylcholine in vesicles to gallbladder mucin but only minimal binding of cholesterol and phosphatidylcholine in mixed micelles. These data indicate that gallbladder mucin binds cholesterol and phosphatidylcholine in vesicles and accelerates the nucleation of cholesterol monohydrate crystals from these cholesterol-transporting particles in supersaturated model bile.

Association between cholesterol-phospholipid vesicles and cholesterol crystals in human gallbladder bile.

Rapid aggregation of cholesterol-phospholipid vesicles in gallbladder bile seems to be the first event in the production of cholesterol crystals, a prerequisite for cholesterol gallstone formation. We examined the amount of these vesicles in 33 human gallbladder biles in relation to biliary lipid composition and to the presence of cholesterol crystals. Biliary microscopy detected cholesterol crystals in all 19 biles from patients with cholesterol gallstones but in none of 14 biles from patients with pigment stones. Gel chromatography was used to separate vesicles and micelles in the native bile with an eluting buffer containing 10 mM sodium cholate to prevent disruption of micellar lipids. Cholesterol, phospholipid and bile salt concentrations were measured in every fraction collected. Bile acid, phospholipid, cholesterol and total lipid concentrations were not significantly different in samples with and without cholesterol crystals. The cholesterol saturation index (1.4 +/- 0.11 vs. 1.0 +/- 0.08) was significantly (p less than 0.01) higher in biles with crystals than without crystals. Gel filtration revealed a vesicular peak in addition to micellar fraction in 18 (23.1 +/- 3.2% of total cholesterol) of the 19 biles with crystals but only in three (15.7 +/- 2.4% of total cholesterol) of 14 biles without crystals. There was no relation between biliary lipid concentration or the cholesterol saturation index and the percentage of vesicular cholesterol in biles with or without crystals. The close association of vesicles and crystals in human gallbladder bile supports the contention that vesicles are important in the initial nucleation of cholesterol monohydrate crystals.

Human gallbladder mucin binds biliary lipids and promotes cholesterol crystal nucleation in model bile

The binding of phosphatidylcholine and cholesterol in model bile to human gallbladder mucin was studied by means of a rapid filtration binding assay and sucrose density gradient ultracentrifugation. Numerous low affinity binding sites for phosphatidylcholine and cholesterol were present on gallbladder mucin. Binding of phosphatidylcholine and cholesterol to mucin increased as a function of cholesterol saturation index. Proteolytic digestion of mucin disaggregated the native mucin polymer and removed hydrophobic domains on the mucin peptide core that bind l-anilino-8-naphthalenesulfonic acid. Proteolytic digestion also resulted in a 91% and 78% decrease, respectively, in the binding of phosphatidylcholine and cholesterol to mucin. The ability of trypsin-treated and native mucin to promote the nucleation of cholesterol monohydrate crystals was compared in a model bile. The incidence of cholesterol monohydrate crystal nucleation with native mucin was significantly greater at 3 days than with trypsin-treated mucin or controls (P less than 0.001). After 3, 6, and 9 days of incubation, samples containing native mucin contained significantly more crystals than controls or trypsin-digested mucin samples (P less than 0.0001 for each). These data indicate that highly purified human gallbladder mucin binds phosphatidylcholine and cholesterol in model bile. Furthermore, this study demonstrates that structural integrity of the native mucin polymer and hydrophobic domains on the peptide core are essential for the nucleation of cholesterol monohydrate crystals by mucin in model bile.

Rapid vesicle formation and aggregation in abnormal human biles: A time-lapse video-enhanced contrast microscopy study

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.

Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile.

We explored the influence of several compositional factors considered capable of influencing the nucleation time of model biles supersaturated in cholesterol. In addition to the classical techniques, e.g., electron microscopy and quasielastic light scattering, employed for size measurement and structural assessment, we employed a novel technique, i.e., video-enhanced microscopy, for particle evaluation in these polydisperse systems which often may simultaneously contain isolated small vesicles, their complex aggregates, and small cholesterol monohydrate crystals. The factors we studied included dilution, degree of cholesterol supersaturation, bile salt/lecithin molar ratio, and Ca2+ concentration. Dilution markedly raised the degree of cholesterol saturation, prolonged nucleation time for cholesterol monohydrate crystals, and favored formation of metastable small unilamellar vesicles. Increasing the degree of cholesterol supersaturation as an independent variable in more concentrated systems both shortened the nucleation time and favored spontaneous formation of a relatively small number of isolated vesicles. A decrease in bile salt/lecithin molar ratio within the physiologically relevant range was accompanied by a prolonged nucleation time and favored spontaneous vesicle formation. Large numbers of small unilamellar vesicles were observed even in concentrated model bile solutions (total lipids: 20 g/dl) when the bile salt/lecithin molar ratio was 1.9 or less. At physiological concentrations, Ca2+ promoted nucleation of cholesterol monohydrate crystals only in vesicle-containing solutions. Taken together, the following conclusions can be drawn. First, spontaneous vesicle formation in dilute systems prolongs solid cholesterol crystal nucleation. It can thus provide a supplementary non-micellar mode of cholesterol transport in micellar systems of supersaturated human bile. Second, dilution, degree of cholesterol supersaturation, and a decrease in bile salt/lecithin ratio prolong cholesterol crystal nucleation time and favor spontaneous vesicle formation. With increasing calcium concentrations, opposite effects are observed. Third, the presence of vesicles may help to account for the frequently observed and otherwise unexplained remarkable degree of metastable supersaturation and prolonged metastability (delayed nucleation time) for cholesterol in human bile.

Human gallbladder mucin accelerates nucleation of cholesterol in artificial bile

The gallbladder bile of patients with cholesterol gallstones is characterized by two abnormalities: (a) supersaturation with cholesterol and (b) accelerated nucleation of cholesterol monohydrate crystals. We studied the ability of purified human gallbladder mucin to nucleate artificial bile in vitro. Human gallbladder mucin at concentrations of 2 and 4 mg/ ml accelerated the nucleation time of cholesterol crystals in model bile. The mean number of cholesterol crystals in artificial bile incubated for 10 days with 4 mg/ml of human gallbladder mucin was 2327/ mm3 (p < 0.01) vs. control of 51/mm3. The number of crystals found in model bile was dependent on the concentration of human gallbladder mucin (2–16 mg/ml) and the time of incubation (4–14 days). Human gallbladder mucin was associated with an increase in the number of liquid crystals after 4 days of incubation, which then decreased in number as solid cholesterol monohydrate crystals formed. Nucleation by human gallbladder mucin was significantly increased only with cholesterol saturation indices >1.0, and in biles containing 10% but not 3% total lipid by weight. Pooled human gallbladder mucin from gallbladders with and without stones both increased nucleation significantly when compared with controls. Increased nucleation of saturated model bile was also observed with purified monkey cervical and bovine gallbladder mucin, but not with porcine gastric mucin. These observations provide further evidence that human gallbladder mucin may contribute to cholesterol gallstone formation in humans by accelerating nucleation of cholesterol monohydrate crystals from supersaturated gallbladder bile.

Nucleation of cholesterol monohydrate crystals from hepatic and gall-bladder bile of patients with cholesterol gall stones.

Nucleation time and cholesterol saturation index of hepatic and gall-bladder bile were measured in 16 patients with cholesterol gall stones to determine whether a gall bladder or liver defect was responsible for the rapid nucleation time of gall-bladder bile in such patients. Although hepatic bile was consistently more saturated than gall-bladder bile, the in vitro nucleation time of gall-bladder bile was more rapid. Dilution of gall-bladder bile to hepatic bile concentrations did not affect nucleation time. The results indicate that the gall bladder plays an important role in the production of the rapidly nucleating bile which is found in patients with cholesterol gall stones, and that this role is not simply concentration of bile by the gall bladder. Normal and abnormal gall-bladder biles were also compared in a larger group of patients. The view that there is a nucleation defect in cholesterol cholelithiasis which is independent of cholesterol saturation was confirmed. Subgroups of normal and gall-stone population were defined by the nucleation time and saturation index. Results suggested that solitary stones may be produced under different conditions than multiple stones. Some putative nucleating factors were examined but none was found to distinguish between normal and gall-stone bile.