Cholesterol Monohydrate Crystal Formation Research Articles

Mechanistic Aspects of the Evolution of 3D Cholesterol Crystallites in a Supported Lipid Membrane via a Quartz Crystal Microbalance with Dissipation Monitoring.

The irreversible formation of cholesterol monohydrate crystals within biological membranes is the leading cause of various diseases, including atherosclerosis. Understanding the process of cholesterol crystallization is fundamentally important and could also lead to the development of improved therapeutic strategies. This has driven several studies investigating the effect of the environmental parameters on the induction of cholesterol crystallite growth and the structure of the cholesterol crystallites, while the kinetics and mechanistic aspects of the crystallite formation process within lipid membranes remain poorly understood. Herein, we fabricated cholesterol crystallites within a supported lipid bilayer (SLB) by adsorbing a cholesterol-rich bicellar mixture onto a glass and silica surface and investigated the real-time kinetics of cholesterol crystallite nucleation and growth using epifluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) monitoring. Microscopic imaging showed the evolution of the morphology of cholesterol crystallites from nanorod- and plate-shaped habits during the initial stage to mostly large, micron-sized three-dimensional (3D) plate-shaped crystallites in the end, which was likened to Ostwald ripening. QCM-D kinetics revealed unique signal responses during the later stage of the growth process, characterized by simultaneous positive frequency shifts, nonmonotonous energy dissipation shifts, and significant overtone dependence. Based on the optically observed changes in crystallite morphology, we discussed the physical background of these unique QCM-D signal responses and the mechanistic aspects of Ostwald ripening in this system. Together, our findings revealed mechanistic details of the cholesterol crystallite growth kinetics, which may be useful in biointerfacial sensing and bioanalytical applications.

The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism.

Cholesterol crystallization from mixtures of unesterified cholesterol with phospholipids and cholesterol esters is believed to be a key event in atherosclerosis progression. Not much is understood, however, about the influence of the lipid environment on cholesterol crystallization. Here we study cholesterol monohydrate crystal formation from mixed bilayers with palmitoyl-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin. We show that disordered phospholipids and sphingomyelin stabilize the formation of crystal plates of the triclinic cholesterol monohydrate polymorph, whereas saturated glycerolipids stabilize helical and tubular crystals of the metastable monoclinic polymorph. We followed the subsequent transformation of these helical crystals into the stable triclinic plates. Discovering the relations between membrane lipid composition and cholesterol crystal polymorphism may provide important clues to the understanding of cholesterol crystal formation in atherosclerosis.

Thyroid dysfunction, either hyper or hypothyroidism, promotes gallstone formation by different mechanisms

We have investigated comprehensively the effects of thyroid function on gallstone formation in a mouse model. Gonadectomized gallstone-susceptible male C57BL/6 mice were randomly distributed into three groups each of which received an intervention to induce hyperthyroidism, hypothyroidism, or euthyroidism. After 5 weeks of feeding a lithogenic diet of 15% (w/w) butter fat, 1% (w/w) cholesterol, and 0.5% (w/w) cholic acid, mice were killed for further experiments. The incidence of cholesterol monohydrate crystal formation was 100% in mice with hyperthyroidism, 83% in hypothyroidism, and 33% in euthyroidism, the differences being statistically significant. Among the hepatic lithogenic genes, Trβ was found to be up-regulated and Rxr down-regulated in the mice with hypothyroidism. In contrast, Lxrα, Rxr, and Cyp7α1 were up-regulated and Fxr down-regulated in the mice with hyperthyroidism. In conclusion, thyroid dysfunction, either hyperthyroidism or hypothyroidism, promotes the formation of cholesterol gallstones in C57BL/6 mice. Gene expression differences suggest that thyroid hormone disturbance leads to gallstone formation in different ways. Hyperthyroidism induces cholesterol gallstone formation by regulating expression of the hepatic nuclear receptor genes such as Lxrα and Rxr, which are significant in cholesterol metabolism pathways. However, hypothyroidism induces cholesterol gallstone formation by promoting cholesterol biosynthesis.

Rafting for gallstones by slowing mass transit

Rafting for gallstones by slowing mass transit

T-Cell Function Is Critical for Murine Cholesterol Gallstone Formation

The formation of cholesterol gallstones is a complex process involving contributions from genes and environmental factors. Although gallbladder inflammation is believed to be common during cholelithogenesis, the role of immunologic factors is unknown. The role of adaptive immunity in cholesterol cholelithogenesis was analyzed utilizing immunocompetent Helicobacter spp.-infected and -uninfected BALB/c and congenic immunodeficient Rag2(-/-) (Rag) mice. Lymphocyte transfer studies were performed to determine which cellular subset was responsible for cholesterol gallstone formation. Also, gallbladder inflammation was quantified to determine the nature of the inflammatory response associated with cholelilithogenesis. When fed a lithogenic diet for 8 weeks, wild-type mice developed significantly more cholesterol gallstones (27%-80% prevalence) than Rag mice ( approximately 5%, P < .05). Helicobacter spp.-infected BALB/cJ mice displayed statistically significant increases in cholesterol gallstone prevalence compared with uninfected mice (81% vs. 39%; P < .05). Transfer of splenocytes or T lymphocytes to Rag2(-/-) mice increased stone prevalence markedly (26% and 40% respectively; P < .05), whereas transfer of B cells was not appreciably cholelithogenic (13%). The adaptive immune response increased the expression of gallbladder Muc genes and accumulation of mucin gel. In addition, T cells and cholesterol monohydrate crystals induced proinflammatory gene expression in the gallbladder, which likely contributes to gallbladder dysfunction. These studies indicate that T cells are critical in murine cholesterol cholelithogenesis. Furthermore, cholesterol monohydrate crystals induce expression of proinflammatory cytokines in a T-cell-dependent fashion. Acquired immunity and inflammation are likely to be crucial factors in cholesterol gallstone pathogenesis, rather then merely the result of cholelithogenesis.

Helicobacter pyloriand cholesterol gallstone formation in C57L/J mice: a prospective study

Recently, we demonstrated that cholesterol gallstone-prone C57L/J mice rarely develop gallstones unless they are infected with certain cholelithogenic enterohepatic Helicobacter species. Because the common gastric pathogen H. pylori has been identified in the hepatobiliary tree of cholesterol gallstone patients, we wanted to ascertain if H. pylori is cholelithogenic, by prospectively studying C57L infected mice fed a lithogenic diet. Weanling, Helicobacter spp.-free male C57L mice were either infected with H. pylori SS1 or sham dosed. Mice were then fed a lithogenic diet (1.0% cholesterol, 0.5% cholic acid, and 15% dairy triglycerides) for 8 wk. At 16 wk of age, mice were euthanatized, the biliary phenotype was analyzed microscopically, and tissues were analyzed histopathologically. H. pylori infection did not promote cholesterol monohydrate crystal formation (20% vs. 10%), sandy stone formation (0% for both), or true gallstone formation (20%) compared with uninfected mice fed the lithogenic diet (10%). Additionally, H. pylori failed to stimulate mucin gel accumulation in the gallbladder or alter gallbladder size compared with uninfected animals. H. pylori-infected C57L mice developed moderate to severe gastritis by 12 wk, and the lithogenic diet itself produced lesions in the forestomach, which were exacerbated by the infection. We conclude that H. pylori infection does not play any role in murine cholesterol gallstone formation. Nonetheless, the C57L mouse develops severe lesions of both the glandular and nonglandular stomach in response to H. pylori infection and the lithogenic diet, respectively.

Effect of gallbladder mucin on the crystallization of cholesterol in bile

Mucin is supposed to accelerate the crystallization of cholesterol in model bile while studies in native human gallbladder bile revealed conflicting results. Therefore, we determined the relation of mucin concentration and cholesterol crystal observation time in gallbladder bile of 73 patients with cholesterol and mixed and 21 patients with pigment stones. In addition, bile samples of 20 patients with cholesterol gallstones were supplemented with either 0 (control) or 0.5-4.0 mg/ml purified bovine mucin or human mucin isolated from gallbladder bile, to study the effect of variable mucin concentrations on the crystallization of cholesterol. Rapid nucleating biles (</= 4 days, n = 59) showed higher mucin concentrations (0.73 +/- 0.1 mg/ml vs 0.43 +/- 0.07 mg/ml) than biles with longer (> 4 days, n = 35) cholesterol crystal observation times (P < 0.05), but no correlation between mucin concentration and cholesterol crystal observation time was observed. Supplementation experiments with bovine purified mucin (up to 4.0 mg/ml) showed no significant effect on the total amount of newly formed cholesterol crystals within 21 days. However, higher amounts of newly formed cholesterol crystals were seen in bile samples supplemented with human mucin in comparison to negative controls. Our results demonstrate a dose-dependent effect of human but not of bovine gallbladder mucin on the formation of cholesterol monohydrate crystals in gallbladder bile of patients with cholesterol stones. Therefore, studies of cholesterol crystallization in model bile systems may be valuable but should always be confirmed in native gallbladder bile as the more physiological effector system.

Rapid formation of cholesterol crystals in gallbladder bile is associated with stone recurrence after laparoscopic cholecystotomy.

Laparoscopic cholecystotomy (LCT) with subsequent extraction of gallstones and primary closure of the gallbladder has been introduced as an alternative therapy for patients with cholecystolithiasis and preserved gallbladder function. However, stone recurrence has to be considered as a major drawback that might be related to lithogenic factors of gallbladder bile or the composition of gallbladder stones. Therefore, these were studied in relation to stone recurrence within an observation period of 1 to 5 years (median, 3.6 years) in 50 patients after LCT. The concentrations of total and individual bile acids, phospholipids, cholesterol, total lipids, mucin, protein, and the cholesterol saturation indices in gallbladder bile were not significantly different between 10 patients with and 40 patients without stone recurrence. However, the crystal observation time was significantly (P < .02) shorter (range, 1-2 days; median, 1.5) in the bile of patients with stone recurrence compared to those without (range, 1-21 days, median 3.5). Moreover, all 10 stone recurrences were observed in the 28 patients with a crystal observation time in the bile of less than or equal to 2 days (approximate annual risk: 12%-15%), and no recurrences were observed in the 22 patients with a crystal observation time greater than 2 days (P < .0001) or in patients with pigment stones. The rapid formation of cholesterol monohydrate crystals in bile seems to be the major risk factor for recurrent stones after LCT. These are most likely cholesterol stones and, therefore, are amenable to oral bile-acid prevention or treatment.

Reactive oxygen metabolites promote cholesterol crystal formation in model bile: Role of lipid peroxidation

In animal models of gallstone disease inflammatory alterations of the gallbladder mucosa are regularly found before the first appearence of cholesterol monohydrate crystals in bile. At sites of inflammation granulocytes generate reactive oxygen metabolites (ROM). The aim of our study was to investigate whether ROM may influence the cholesterol monohydrate crystal formation in supersaturated model bile. Superoxide anions (O2), hydrogen peroxide (H 20 2), and hydroxyl radicals ('OH) were generated by the interaction of Fe 3+-EDTA with ascorbic acid (Asc). The influence of ROM on cholesterol crystal formation was studied by measurement of the nucleation time. To check whether lipid peroxidation was induced by the ROM generation, production of malondialdehyde equivalents was measured in bile with the thiobarbituric assay. Furthermore, the lipid pattern of bile after ROM exposure was analyzed by thin layer chromatography. Addition of Fe 3+-EDTA/Asc to model bile markedly decreased the cholesterol nucleation time (NT) ( p < 0.01), caused a significant increase in malondialdehyde equivalents ( p < 0.001) and induced the generation of 4-hydroxy-2,3-trans-nonenal (4-HNE). In an attempt to identify a specific oxygen metabolite responsible for the alterations in bile, the effects of various oxygen radical scavengers were tested. Desferal, which prevents 'OH generation by chelation of ferrous iron, completely protected bile against Fe 3+-EDTA/Asc-induced decrease in NT ( p < 0.001), increase in lipid peroxidation ( p < 0.001) and generation of 4-HNE. Our results indicate that formation of cholesterol crystals in model bile is enhanced by ROM. Hydroxyl radical induced lipid peroxidation appears to be the mechanism responsible for the crystallisation promoting activity of ROM.

Cholesterol nucleates rapidly from mixed micelles in the prairie dog

Current theory suggests that the nucleation of cholesterol in human bile requires the aggregation and fusion of cholesterol-enriched phospholipid vesicles. This theory is based on observations which do not exclude the precipitation of cholesterol from mixed micelles. The present study examines the role of mixed micelles and vesicles in the formation of cholesterol monohydrate crystals in the prairie dog. The intermicellar bile salt concentration of prairie dog gallbladder bile was determined using equilibrium dialysis. Model bile equivalent to gallbladder bile from cholesterol-fed prairie dogs was used as dialysant yielding the intermicellar (dialysate) concentration of 9 mM. Cholesterol carriers in gallbladder bile from 11 cholesterol-fed animals were then separated by Sephacryl S200 gel filtration chromatography using eluant buffer containing the intermicellar bile salt concentration. Gel filtration chromatography of fresh bile demonstrated that 100% of cholesterol was carried in the mixed micellar fraction with no vesicles observed in any of the 11 animals. The gallbladder bile nucleation time was 2.0 ± 0.3 days for the cholesterol-fed animals. Gel filtration chromatography immediately after nucleation again revealed a single mixed micellar peak. These data indicate that cholesterol is carried exclusively in and nucleates rapidly from mixed micelles in the cholesterol-fed prairie dog and that cholesterol-phospholipid vesicles are not required in this process.

Formation of cholesterol monohydrate crystals in macrophage-derived foam cells.

In a previous study using the J774 macrophage foam cells, we quantitated the accumulation of unesterified (free) cholesterol derived from cholesteryl ester hydrolysis in lysosomes, after phagocytic uptake of cholesteryl ester droplets. In the present study, we examined whether the accumulation of free cholesterol in lysosomes leads to the formation of cholesterol monohydrate crystals by analyzing the lipid composition of low density lysosome fractions isolated from cholesteryl ester-loaded macrophages after a 24-h incubation. Phase diagrams of the constituent lipids in the lipid-filled lysosomes predicted the formation of cholesterol monohydrate crystals. The formation of cholesterol monohydrate crystals was observed in cholesteryl ester-loaded macrophages after a 48-h incubation by polarizing light microscopy. The crystals had a density of 1.04 g/ml and the morphology of cholesterol monohydrate crystals with an acute edge angle of about 80 degrees. The crystals appeared as needles as well as plates and melted only when heated to greater than 85 degrees C. The physical properties of these crystals are characteristic of cholesterol monohydrate. In our studies, crystal formation was observed even when cells had active acyl-CoA:cholesterol acyltransferase or when cholesterol efflux was stimulated. Electron microscopy and acid phosphatase cytochemistry of lysosomes in cholesteryl ester-loaded cells confirmed that cholesterol crystal formation occurred within lipid-loaded lysosomes. Time-lapse video microscopic studies revealed that most of the cells containing cholesterol monohydrate crystals not only remain viable but also have the capacity to translocate single crystals within cells. The data demonstrate that lysosomal accumulation of free cholesterol in macrophages after phagocytic uptake and hydrolysis of cholesteryl ester droplets leads to the formation of cholesterol monohydrate crystals within lipid-filled lysosomes. Such a process may lead to deposition of free cholesterol and cholesterol monohydrate crystals in macrophage foam cells during the progression of atherosclerosis.

Gallstone cholesterol content is related to apolipoprotein E polymorphism

Background: The genetically determined phenotypes of apolipoprotein E are related to variations in lipoprotein levels and in the enterohepatic metabolism of cholesterol and bile acids. The present study was designed to elucidate the role of apolipoprotein E polymorphism in gallstone formation. Methods: Apolipoprotein E phenotype was determined in 169 consecutive cholecystectomy patients and in 200 controls. The cholesterol content of the gallstones (n = 169), the presence of cholesterol monohydrate crystals of fresh gallbladder bile (n = 142), and the nucleation time (n = 35) were also analyzed. Results: The median cholesterol content of the gallstones was higher in the apolipoprotein E4 category (phenotypes E4/4 and E4/3, 97%) than in the E3 (E3/3, 78%) and E2 patients (E2/2 and E2/3, 76%, P = 0.0003). In E4 patients, cholesterol crystals were found immediately after surgery in 27 of 40 (68%), whereas in E3 and E2 groups in 36 of 88 (41%), and 4 of 14 (29%) of the patients (P = 0.0001). The median nucleation time in E4 patients (2.5 days) was shorter than in patients with E3 (5.5 days) or E2 (6.0 days) (P = 0.0016). Conclusions: These data indicate that apolipoprotein E polymorphism affects cholesterol content of cholelithiasis. We suggest that this phenomenon is mediated by the altered formation of cholesterol monohydrate crystals in different apolipoprotein E phenotypes.

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.

Comparison of gall bladder bile and endoscopically obtained duodenal bile.

In 10 patients with gall stone disease (eight women, two men; mean (SD) age 47.4 (13) years), bile was obtained by endoscopic aspiration after stimulation of the gall bladder with ceruletid and also by fine needle puncture of the gall bladder under local anaesthetic. The total lipid concentration of the puncture bile samples was mean (SD) 11.9 (4.7) g/dl, significantly higher than the endoscopic bile samples (3.9 (3.3) g/dl, p less than 0.001). Total bile acids, phospholipids, and biliary cholesterol (expressed in mol%) and cholesterol saturation index showed no significant differences between the two types of samples. The glycocholic acid concentration in the endoscopically obtained bile (27.7 (6.6) mol% v 23.3 (5.4) mol%; p less than 0.01) was significantly higher than the puncture bile samples. Puncture bile exhibited a significantly shorter nucleation time (3.5 (3.3) days v 19.6 (11.9) days; p less than 0.001). For determination of the nucleation time, endoscopic bile aspiration after gall bladder stimulation with ceruletid led to adequately concentrated samples in 50% of the study subjects. Cholesterol monohydrate crystal formation in native bile was observed in six samples of puncture bile and in three samples of the endoscopically obtained bile. The presence of cholesterol crystals and the determination of nucleation time in the puncture bile were the best discriminants between cholesterol and pigment gall stones and correlated well with computed tomogram analysis.

Effects of dietary fish oil on biliary phospholipids and prostaglandin synthesis in the cholesterol‐fed pairie dog

Cholesterol gallstone formation in the prairie dog is accompanied by an increase in the percentage of biliary phospholipids containing arachidonic acid, and an increase in gallbladder prostaglandin (PG) synthesis, but the pathogenetic significance of these changes is unclear. Dietary supplementation with eicosapentaenoic acid (EPA), an omega-3 fatty acid which is commonly found in fish oil, decreases prostaglandin synthesis in some tissues by replacing arachidonic acid, and by competitively inhibiting prostaglandin synthesis. We studied the effect of dietary fish oil on gallbladder PG synthesis, and the relative abundance of various molecular species of phosphatidylcholines and phosphatidylethanolamines in bile and gallbladder epithelium in the cholesterol-fed prairie dog. Prairie dogs were maintained for 4 weeks on one of four diets: i) control, ii) cholesterol-supplemented (0.34%), iii) menhaden oil (50 g/kg chow), or iv) cholesterol plus menhaden oil. Supplementation with menhaden oil resulted in a replacement of arachidonic and linoleic acids with EPA and docosahexaenoic acids in the phospholipids of bile and gallbladder mucosa. In cholesterol-fed animals, supplementation with menhaden oil prevented increased gallbladder PG synthesis. Menhaden oil also reduced the incidence of cholesterol monohydrate crystals among cholesterol-fed animals (9/20 with cholesterol plus menhaden oil vs 21/22 with cholesterol alone), but the improvement could not clearly be attributed to decreased PG synthesis since supplementation with menhaden oil also increased the total phospholipid concentration in bile, and decreased the degree of cholesterol saturation. These results demonstrate that dietary supplementation with omega-3 fatty acids significantly influences biliary phospholipids, and decreases the incidence of cholesterol monohydrate crystal formation in this animal model.

Effect of hyocholic acid on the prevention and dissolution of biliary cholesterol crystals in mice

Gallstone prevention and dissolution were studied in a mouse model of cholesterol cholelithiasis using hyocholic acid (3 alpha, 6 alpha, 7 alpha-trihydroxy-5 beta-cholanic acid). Addition of hyocholic acid, 0.1 or 0.3%, in the lithogenic diet (1% cholesterol + 0.5% cholic acid) prevented the formation of cholesterol monohydrate crystals in 70 and 90% of cases, respectively. On the other hand, chow diet supplemented with 0.1 or 0.3% hyocholic acid dissolved cholesterol crystals in lithiasic mice in, respectively, 80 and 100% of cases within 12 days. In both protocols, biles were largely supersaturated with cholesterol; lecithin-cholesterol lamellar liquid crystals were responsible for the transport of the excess cholesterol content. The percentage of hydrophilic bile salts (hyocholic acid, hyodeoxycholic acid, beta-muricholic acid) in bile, although moderate (15-50% of total bile salts), appears to induce such liquid crystalline dispersion. This study demonstrates that the balance between hydrophilic and hydrophobic bile salts plays a major role in the prevention and dissolution of cholesterol crystals. It is also shown that the desaturation of biliary cholesterol is not a prerequisite for gallstone dissolution.

Vesicular cholesterol in bile. Relationship to protein concentration and nucleation time

A study was done to determine whether the nucleation time was related to the amount of cholesterol carried in vesicles. Bile was obtained from cholesterol gallstone patients and controls. Gel-exclusion chromatography was used to separate vesicles and micelles in the native bile using an eluting buffer containing 10 mM sodium cholate. The percent of total cholesterol carried in vesicles in gallbladder bile of stone patients was significantly greater than that in control patients. Total cholesterol concentration in gallbladder bile of stone patients was significantly greater than in controls. This difference was due to the fact that vesicular cholesterol concentration was significantly greater in the gallbladder bile of stone patients compared to controls. Micellar cholesterol concentrations were similar in the two groups. Nucleation time was related significantly to vesicular cholesterol concentration in correlation analysis and, as previously shown, so was total protein concentration. This study supports the importance of vesicular cholesterol in solid crystal formation and demonstrates for the first time that the rate of cholesterol monohydrate crystal formation is directly related to the amount of cholesterol transported in vesicles.

Cholesterol gallstone pathogenesis: a study of potential nucleating agents for cholesterol crystal formation in bile.

Cholesterol monohydrate crystal formation was measured quantitatively in model bile solutions, which were supersaturated with cholesterol, by a radiochemical method and qualitatively in human gallbladder bile by polarizing microscopy. Various agents, which have been postulated to act as nucleating factors for cholesterol crystal and gallstone formation, were added to bile and their effect on the appearance of cholesterol crystals was determined. These agents included calcium salts found in gallstones (calcite, aragonite, apatite, bilirubinate), Escherichia coli bacteria, pigment residues from cholesterol gallstones, bilirubin and several mucin preparations. Human gallbladder bile, which was collected from patients with and without cholesterol gallstones, was also mixed with model bile to examine whether nucleating or anti-nucleating factors were present. None of the agents tested markedly and consistently promoted cholesterol monohydrate crystal formation in model or human bile, except seed crystals of cholesterol monohydrate which were used as a control. Human gallbladder bile from obese patients without gallstones delayed the appearance of cholesterol crystals in model bile solutions, whereas gallbladder bile from gallstone patients did not. These results do not provide experimental support for the hypothesis that calcium salts and pigment material found in gallstones, or gallbladder mucin at concentrations less than 10 mg/ml, act as nucleating agents for cholesterol crystal and stone formation. The difference between gallbladder biles from patients with and without gallstones in their propensity to form cholesterol crystals may be due to the presence of an anti-nucleating factor in normal bile.

Role of gallbladder mucin in pathophysiology of gallstones.

A critical step in the formation of cholesterol gallstones in nucleation (i.e., the formation of cholesterol monohydrate crystals from supersaturated bile). The rate of nucleation of cholesterol depends upon a critical balance between pronucleating and antinucleating factors in bile. Mucin, a high molecular weight glycoprotein secreted by the gallbladder and biliary duct epithelium, is a pronucleating agent in experimental and human gallstone disease. Gallbladder mucin shares with other epithelial mucins the ability to bind lipids and bile pigment. The hydrophobic binding sites in the polypeptide core of mucin may provide a favorable environment for nucleation of cholesterol monohydrate from supersaturated bile. In nearly all animal models of cholelithiasis, mucin hypersecretion is prominent. The stimulus for gallbladder mucin hypersecretion appears to be a component of lithogenic bile. Prostaglandins regulate mucin release in gallbladder epithelium in vitro and probably in vivo. In the cholesterol-fed prairie dog, blockage of mucin release with aspirin inhibits gallstone formation. These findings suggest that inhibition of mucin release may prevent cholesterol stone formation during high-risk periods or after dissolution therapy with bile salts.

Cholesterol crystal formation and growth in model bile solutions.

Cholesterol monohydrate crystal formation was studied in supersaturated model bile solutions, containing unlabeled cholesterol, sodium cholate and soybean phosphatidylcholine, and tracer amounts of [3H]cholesterol. Solutions were either seeded with cholesterol crystals to initiate growth, or not seeded to allow self-nucleation and subsequent crystal growth to occur. Crystal growth at 37 degrees C was measured by two methods. First, radioactive cholesterol crystals were isolated by filtration, and the mass of cholesterol that had precipitated was calculated. In unseeded solutions, there was a long lag period before crystal growth was detected. This lag time was decreased by increases in the cholesterol concentration, temperature, and lipid concentration. In seeded solutions, crystal growth also was dependent on the cholesterol concentration, temperature, and lipid concentration. The second method used to measure crystal growth involved the Coulter Counter. At 37 degrees C, reproducible results were not obtained using unseeded solutions due to blocking of the counter aperture with large crystals. In seeded solutions, crystal growth could be measured as an increase in total particle volume. However, comparison of growth rate estimates from the Coulter Counter with those obtained radiochemically revealed poor agreement between the two methods. It is probable that the Coulter Counter is inaccurate in measuring the volume of cholesterol monohydrate crystals due to their anisometric shape.