Cholesterol Monohydrate Crystals Research Articles

Properties of Mixtures of Cholesterol with Phosphatidylcholine or with Phosphatidylserine Studied by 13C Magic Angle Spinning Nuclear Magnetic Resonance

The behavior of cholesterol is different in mixtures with phosphatidylcholine as compared with phosphatidylserine. In 13C cross polarization/magic angle spinning nuclear magnetic resonance spectra, resonance peaks of the vinylic carbons of cholesterol are a doublet in samples containing 0.3 or 0.5 mol fraction cholesterol with 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) or in cholesterol monohydrate crystals, but a singlet with mixtures of cholesterol and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). At these molar fractions of cholesterol with POPS, resonances of the C-18 of cholesterol appear at the same chemical shifts as in pure cholesterol monohydrate crystals. These resonances do not appear in samples of POPS with 0.2 mol fraction cholesterol or with POPC up to 0.5 mol fraction cholesterol. In addition, there is another resonance from the cholesterol C18 that appears in all of the mixtures of phospholipid and cholesterol but not in pure cholesterol monohydrate crystals. Using direct polarization, the fraction of cholesterol present as crystallites in POPS with 0.5 mol fraction cholesterol is found to be 80%, whereas with the same mol fraction of cholesterol and POPC none of the cholesterol is crystalline. After many hours of incubation, cholesterol monohydrate crystals in POPS undergo a change that results in an increase in the intensity of certain resonances of cholesterol monohydrate in 13C cross polarization/magic angle spinning nuclear magnetic resonance, indicating a rigidification of the C and D rings of cholesterol but not other regions of the molecule.

Endoscopic Ultrasound-Guided Gallbladder Bile Aspiration in Idiopathic Pancreatitis Carries a Significant Risk of Bile Peritonitis

Background: Direct microscopic examination of bile for the presence of microlithiasis is often performed during the evaluation of patients with idiopathic pancreatitis. Bile sampled from the duodenum and/or the common bile duct may not represent gallbladder bile, and thus may be inadequate for the diagnosis of microlithiasis. Aim: We sought to determine the safety and efficacy of endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) of gallbladder bile in patients with idiopathic pancreatitis. Methods: Patients with idiopathic pancreatitis underwent EUS with a linear echoendoscope. After excluding potential causes of pancreatitis such as common bile duct stones or pancreatic lesions, the gallbladder was identified. The gallbladder lumen was entered using a 22-gauge FNA needle via the duodenal wall. Bile was aspirated and analyzed for the presence of cholesterol monohydrate crystal, calcium bilirubinate granules, calcium carbonate microspheroliths and mucin gel strands. Results: Three patients underwent EUS-guided FNA of gallbladder bile. Two of these patients developed bile peritonitis within one hour of the procedure prompting us to discontinue the study. One patient’s gallbladder bile contained microlithiasis. This patient had bile aspirated from the common bile duct via ERCP 3 weeks prior to EUS. However, analysis of that bile sample failed to show microlithiasis. Conclusion: Unfortunately, transduodenal EUS-guided FNA of gallbladder bile using a 22-gauge needle carries a significant risk of bile peritonitis.

Growth and micro-topographical studies of gel grown cholesterol crystals

Cholesterol (C27H46O) is the most abundant and best-known steroid in the animal kingdom. The in vitro crystallization of this important biomaterial has been attempted by few researchers. Here we are reporting crystallization of pure cholesterol monohydrate crystals in gel medium. It is found that the morphology of the crystals depends on various parameters. The effect of solvent has been studied in detail. The different morphologies observed are fibrous, needle, platelet, dendrite etc. Micro topographical studies have been made and it is found that the crystals grow, at least in the last stage, by spreading of layers. However, at initial stage microcrystals formed and developed into dendrite or needle forms. These one-dimensional crystals developed into platelets and finally thickened. Further studies reveal that micro impurities play a vital role in the development of these crystals as seen by dissolution figures on the crystals. These crystals are characterized by using the XRD and IR spectroscopic methods.

Stereoselective recognition of monolayers of cholesterol, ent-cholesterol, and epicholesterol by an antibody.

The interaction between a monoclonal antibody and four distinct monolayers with varying degrees of structural, chemical, and stereochemical similarity were studied and quantified. The antibody, raised and selected against cholesterol monohydrate crystals, interacts with cholesterol monolayers stereospecifically, but not enantiospecifically. Monolayers of ent-cholesterol molecules, which are chemically identical to cholesterol and whose structure is the exact mirror image of the cholesterol monolayer, interact with the antibody to the same extent as the cholesterol monolayers. The affinity of the antibody for both enantiomeric monolayers is extremely high. However, the antibody does not interact with monolayers of epicholesterol, which is an epimer of cholesterol: The hydroxy group in epicholesterol is in the 3alpha position rather than in the 3beta position, imposing a different angle between the hydroxy group and the rigid steroid backbone, and a different packing of the molecules. Monolayers of triacontanol, a long-chain primary aliphatic alcohol, interact with the antibody to a lesser extent than the cholesterol and ent-cholesterol monolayers, presumably due to the structural flexibility of the triacontanol molecule. The lack of chiral discrimination by the antibody is thus correlated to the level at which the chirality is exposed at the surface of the monolayers.

Cholesterol crystal binding of biliary immunoglobulin A: visualization by fluorescence light microscopy.

To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. Crystal binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography.Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)conjugated anti IgA at 37 degree. Samples were examined under polarizing and fluorescence light microscopy with digital image processing. Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC conjugated anti IgA antibodies. Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence. Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystal binding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.

Composition and immunofluorescence studies of biliary “sludge” in patients with cholesterol or mixed gallstones

Background/Aims: Gallbladder bile from patients with cholesterol or mixed gallstones frequently contains biliary “sludge”, a suspension of cholesterol monohydrate crystals and pigment granules embedded in mucin and proteins. The composition of biliary “sludge” and the preferential localization of mucin and proteins could be an indicator for its potential role in gallstone formation. Methods: Ultracentrifugation (100 000 g/1 h) was used to precipitate “sludge” from bile, and the concentration difference of its main components between native bile and ultracentrifuged bile samples was calculated. After purification of the sediment, immunolocalization was performed for the detection of mucin, IgA, albumin, aminopeptidase, and anionic polypeptide fraction using polyclonal and monoclonal antibodies. Results: The amount of sludge in gallbladder bile was 4.26 mg/ml±0.78 (mean±SEM) in patients with cholesterol and 2.51 mg/ml±0.39 in patients with mixed stones and cholesterol was the main component (48.9±4.6% and 44.4±7.1%). The sediment appeared as a mixture of vesicular aggregates and pigment particles which were linked by a gel matrix of mucin containing cholesterol crystals. While anionic polypeptide fraction and aminopeptidase were associated to pigments, IgA was uniformly spread in the crystalline parts of “core-like” structures, and albumin, when it was present, appeared as randomly located small spots. Conclusions: Our study demonstrates that the cholesterol content and the distribution pattern of mucin and different proteins is similar in the sediments of biliary “sludge” to that previously shown in cholesterol and mixed gallstones. This suggests that biliary “sludge” represents an early stage of gallstone formation in these patients.

Stereoselective interactions of a specialized antibody with cholesterol and epicholesterol monolayers.

The stereoselective recognition by monoclonal antibodies of two-dimensional monolayers of cholesterol spread at the air-water interface is presented. Using immunofluorescence, we show that one antibody, raised and selected against crystals of cholesterol monohydrate, specifically recognizes monolayers of cholesterol, but not monolayers of epicholesterol--its epimeric form. This demonstrates that stereoselective recognition also applies to protein-surface interactions.

Stimulation of gallbladder by intravenous infusion of amino acid: a new method to obtain duodenal bile for bile analyses.

Bile analysis is performed on duodenal bile obtained after stimulation of the gallbladder (GB) with cholecystokinin or its analog. Rapid amino acids (AA) infusion contracts the GB. Our objective was to assess qualitatively the adequacy of duodenal bile obtained following AA infusion. We prospectively studied 15 patients each with non ulcer dyspepsia (NUD) and gallstones (GS), and 17 patients with acute idiopathic pancreatitis (AIP). Duodenal bile was obtained after duodenal intubation and stimulation of GB by rapid AA infusion. The GB contractile response to AA infusion was assessed by functional ultrasonography. Bile was analyzed for lipids, nucleation time, cholesterol saturation index (CSI), and biliary crystals. Adequate duodenal bile could be collected in all but one patient with AIP and one with GS. The mean +/- SD percent ejection fraction of GB following AA infusion in patients with NUD, GS and AIP was 53.41 +/- 16.23, 52.06 +/- 16.07, and 43.37 +/- 14.16, respectively. Biliary microscopy showed the presence of cholesterol monohydrate crystals (CMC) in 7 of 16 patients with AIP, 12 of 14 patients with GS, and 1 of 15 patients with NUD. Mean CSIs in patients with NUD, GS, CMC-positive AIP, and CMC-negative AIP were 0.78, 1.97, 1.85, and 1.00, respectively. Nucleation time in patients with NUD and gallstones was 21.1 and 8.42 days. respectively. In conclusion, qualitatively adequate bile can be obtained for chemical and microscopic examination following rapid intravenous infusion of AA to stimulate the GB.

Cryoelectron Microscopy of a Nucleating Model Bile in Vitreous Ice: Formation of Primordial Vesicles

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23–31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10–20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.

Biliary sludge.

Biliary sludge was first described with the advent of ultrasonography in the 1970s. It is defined as a mixture of particulate matter and bile that occurs when solutes in bile precipitate. Its composition varies, but cholesterol monohydrate crystals, calcium bilirubinate, and other calcium salts are the most common components. The clinical course of biliary sludge varies, and complete resolution, a waxing and waning course, and progression to gallstones are all possible outcomes. Biliary sludge may cause complications, including biliary colic, acute pancreatitis, and acute cholecystitis. Clinical conditions and events associated with the formation of biliary sludge include rapid weight loss, pregnancy, ceftriaxone therapy, octreotide therapy, and bone marrow or solid organ transplantation. Sludge may be diagnosed on ultrasonography or bile microscopy, and the optimal diagnostic method depends on the clinical setting. This paper proposes a protocol for the microscopic diagnosis of sludge. There are no proven methods for the prevention of sludge formation, even in high-risk patients, and patients should not be routinely monitored for the development of sludge. Asymptomatic patients with sludge can be managed expectantly. If patients with sludge develop symptoms or complications, cholecystectomy should be considered as the definitive therapy. Further studies of the pathogenesis, natural history, and clinical associations of biliary sludge will be essential to our understanding of gallstones and other biliary tract abnormalities.

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.

Modification of Assembled Structures of Steroids Adsorbed on Apatite Ceramic Surface by Electrical Poling

AbstractAggregate structures of steroids adsorbed on electrically-poled hydroxyapatite (HAp) ceramics were investigated by X-ray diffractometry (XRD) and scanning electron microscopy. Effectivity of the poled HAp on modification of the steroid assembly modes was discussed by the arrangement estimations of the steroid molecules. The dense HAp ceramics were obtained from the pelletized HAp powder precipitated from calcium hydroxide suspension and phosphoric acid using conventional sintering at 1250°C under the saturated water vapor pressure. The HAp ceramic blocks were poled in a DC electrical field of 1.0 kVcm-1 with being heated at 300°C. The HAp ceramics were immersed in ethanol solution of steroids at 37°C. The steroid crystals were overgrown onto the HAp substrates by recrystallization method. In the case of cholesterol, the tabular crystals of cholesterol monohydrate and the needle-like crystals of anhydrate were overgrown on all of the HAp ceramic surfaces. The thickness of the cholesterol layers on the positively (p) poled HAp surfaces was considerably less than those of the negatively (n) poled and non-poled (0) surfaces. The remanent chaige of the HAp ceramics altered the morphology of vitamin D3 crystals deposited on the HAp surfaces. It was revealed that the electrically-poled HAp ceramic surfaces changed the aggregate structures and the crystal growth rates of steroids.

Pattern Recognition by Antibodies for Two-Dimensional Arrays of Molecules

Direct antibody recognition of supramolecular arrays of small molecules, rather than of an individual molecule, is demonstrated. Monoclonal antibodies, raised and selected against crystals of cholesterol monohydrate are shown by ELISA (enzyme-linked immunosorbent assay) and immunofluorescence imaging to specifically interact with monolayers of cholesterol at the air–water interface. This raises the possibility of producing antibodies against solid antigens and using them to tag geometrical arrays of molecules in, for example, membranes, which cannot be presented as such to the immune system.

Acute coronary syndromes: unstable angina and non-Q-wave myocardial infarction.

Ischemic heart disease includes a wide spectrum of conditions, ranging from silent ischemia and exertion-induced angina, through unstable angina, to acute MI. Unstable angina occupies the center of this spectrum, causing disability and risk greater than that of chronic stable angina but less than that of acute MI1 (Fig 1⇓). Although non–Q-wave MI for many years was considered prognostically similar to unstable angina, recent longitudinal studies indicate that it is similar to Q-wave infarction2 3 (Fig 2⇓). Figure 1. Cumulative 6-month mortality from ischemic heart disease. Diagnosis on admission to hospital (n=21 761; 1985 to 1992). From Duke Cardiovascular Database. Reproduced with permission from Reference 1. Figure 2. Top, Cumulative 1-year combined death or MI among patients with Q-wave and non–Q-wave MI treated with fibrinolysis. Reproduced with permission from Reference 2. Bottom, Risk of subsequent cardiac events in stable convalescing patients after first non–Q-wave and Q-wave MI. Reproduced with permission from Reference 3. The concept of unstable angina has emerged from observations of frequent symptoms preceding acute MI, followed by prospective documentation that unstable symptoms frequently culminated in acute MI. The syndrome was rapidly accepted as a well-defined clinical entity as specific clinical manifestations, pathophysiological mechanisms, laboratory findings, and treatment became better characterized. Unstable angina is currently one of the leading causes of hospital admission for CAD, and non–Q-wave MI accounts for >30% of admissions for acute MI.1 4 Yet, the diagnosis of unstable angina remains clinical, based on symptom recognition. The physician caring for patients with unstable angina is in a privileged position of recognizing rapidly evolving CAD and being able to intervene to prevent irreversible left ventricular damage and progression of CAD. Unstable angina is classically described as a heterogeneous disease, referring to a wide spectrum of clinical manifestations from stable angina to MI, of disease …

Imaging supramolecular aggregates in bile models and human bile

Investigation of cholesterol crystallization is essential for the understanding of gallstone formation. Previous work has revealed a variety of aggregates of different sizes and shapes prior to the appearance of “classical” plate—like cholesterol monohydrate crystals both in native biles and model systems. In this article, we review existing data based on various microscopic techniques and present data on microstructural pathways leading to cholesterol crystal formation in two different bile models and in native bile. In continuation of our recent investigation of the microstructures in nucleating human bile, we now present data suggesting that polymorphism is not limited to complex native bile, but also appears in two, simplified model systems. These studies employed cryo-transmission electron microscopy (cryo-TEM) and video-enhanced light microscopy, using Nomarski optics (VELM). Only the combined use of these two complementary, non-perturbing direct methods can cover the whole range of microstructures ranging from a few nanometers to several microns. Concentrated isotropic solutions of bile models, composed of cholesterol, lecithin and taurocholate, were diluted to induce cholesterol supersaturation and start an evolution of microstructures, leading to cholesterol crystallization. Initially, small spheroidal micelles were observed by cryo-TEM. Subsequently, uni-, oligo- and multilamellar vesicles, compatible with structures seen at the same time by VELM, appeared in coexistence with micelles. Thereafter, during a dynamic phase of cholesterol crystallization, filaments, tubular and helical microstructures, as well as classical plate-like cholesterol monohydrate crystals were noted by light microscopy. Eventually, large plate-like crystals were observed by VELM, while cryo-TEM revealed only small spheroidal micelles. The crystallization process in native human bile during ex vivo incubation was found to bear close resemblance to the findings in the model systems, further supporting the applicability of these systems to the exploration of microstructural aspects of nucleating human bile. Microsc. Res. Tech., 39:85–96, 1997. © 1997 Wiley-Liss, Inc.

Ground squirrel model for cholelithiasis: Role of epithelial glycoproteins

The cholesterol-fed Richardson's ground squirrel (Spermophilus richardsonii) has proven to be an effective animal model in which to study factors that influence cholesterol gallstone formation and associated alterations in the gallbladder epithelium. Ground squirrels of either sex, fed a 2% cholesterol-enriched diet, exhibit cholesterol monohydrate crystal precipitation within 24 hours and macroscopically visible cholesterol stones by 3 weeks. Data on bile chemistry, biliary cholesterol precipitation, and various mucosal alterations occurring prior to, during, and after stone formation were collected using sampling intervals from 6 hours to 20 weeks on the diet. The results indicate that mucin hypersecretion appears to be more closely related to the initiation of nucleation than does either bile calcium or pH. Mucus hypersecretion begins within 18 hours of diet initiation and continues throughout the 20 week experimental period. Apical excrescences became more common and were larger in size during the early stages of cholelithiasis. Administration of aspirin during the experimental period demonstrated an inhibition of mucin synthesis and release. Gallstones were not formed in these aspirin-treated animals. A lectin-binding panel for 10 epithelial glycoprotein-related sugars indicated the mucin secreted by the gallbladder epithelium of 7 day experimental animals differed from that of controls. The most obvious difference was the abolition of WGA binding in the experimental animals, suggesting an absence of sialic acid expression in the mucin during the lithogenic process. Ultrastructural histochemistry indicated that both sulphomucin and sialomucin were present in the secretory granules and within the surface mucus layer of both experimental and control animals. Experimental animals, however, exhibited a significant predominance for sulphomucin. This pattern varies from that typically seen in other regions of the gastrointestinal tract where sialomucins predominate during pathologic processes. Microsc. Res. Tech. 39:39–55, 1997. © 1997 Wiley-Liss, Inc.

Imaging supramolecular aggregates in bile models and human bile.

Investigation of cholesterol crystallization is essential for the understanding of gallstone formation. Previous work has revealed a variety of aggregates of different sizes and shapes prior to the appearance of "classical" plate-like cholesterol monohydrate crystals both in native biles and model systems. In this article, we review existing data based on various microscopic techniques and present data on microstructural pathways leading to cholesterol crystal formation in two different bile models and in native bile. In continuation of our recent investigation of microstructures in nucleating human bile, we now present data suggesting that polymorphism is not limited to complex native bile, but also appears in two, simplified model systems. These studies employed cryo-transmission electron microscopy (cryo-TEM) and video-enhanced light microscopy, using Nomarski optics (VELM). Only the combined use of these two complementary, non-perturbing direct methods can cover the whole range of microstructures ranging from a few nanometers to several microns. Concentrated isotropic solutions of bile models, composed of cholesterol, lecithin and taurocholate, were diluted to induce cholesterol supersaturation and start an evolution of microstructures, leading to cholesterol crystallization. Initially, small spheroidal micelles were observed by cryo-TEM. Subsequently, uni-, oligo- and multilamellar vesicles, compatible with structures seen at the same time by VELM, appeared in coexistence with micelles. Thereafter, during a dynamic phase of cholesterol crystallization, filaments, tubular and helical microstructures, as well as classical plate-like cholesterol monohydrate crystals were noted by light microscopy. Eventually, large plate-like crystals were observed by VELM, while cryo-TEM revealed only small spheroidal micelles. The crystallization process in native human bile during ex vivo incubation was found to bear close resemblance to the findings in the model systems, further supporting the applicability of these systems to the exploration of microstructural aspects of nucleating human bile.

Ground squirrel model for cholelithiasis: role of epithelial glycoproteins.

The cholesterol-fed Richardson's ground squirrel (Spermophilus richardsonii) has proven to be an effective animal model in which to study factors that influence cholesterol gallstone formation and associated alterations in the gallbladder epithelium. Ground squirrels of either sex, fed a 2% cholesterol-enriched diet, exhibit cholesterol monohydrate crystal precipitation within 24 hours and macroscopically visible cholesterol stones by 3 weeks. Data on bile chemistry, biliary cholesterol precipitation, and various mucosal alterations occurring prior to, during, and after stone formation were collected using sampling intervals from 6 hours to 20 weeks on the diet. The results indicate that mucin hypersecretion appears to be more closely related to the initiation of nucleation than does either bile calcium of pH. Mucus hypersecretion begins within 18 hours of diet initiation and continues throughout the 20 week experimental period. Apical excrescences became more common and were larger in size during the early stages of cholelithiasis. Administration of aspirin during the experimental period demonstrated an inhibition of mucin synthesis and release. Gallstones were not formed in these aspirin-treated animals. A lectin-binding panel for 10 epithelial glycoprotein-related sugars indicated the mucin secreted by the gallbladder epithelium of 7 day experimental animals differed from that of controls. The most obvious difference was the abolition of WGA binding in the experimental animals, suggesting an absence of sialic acid expression in the mucin during the lithogenic process. Ultrastructural histochemistry indicated that both sulphomucin and sialomucin were present in the secretory granules and within the surface mucus layer of both experimental and control animals. Experimental animals, however, exhibited a significant predominance for sulphomucin. This pattern varies from that typically seen in other regions of the gastrointestinal tract where sialomucins predominate during pathologic processes.

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile

Background/Aims: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstones pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. Methods: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. Results: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. Conclusions: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.

Effect of medicinal plants on the crystallization of cholesterol

One of the least desirable calcifications in the human body is the mineral deposition in atherosclerosis plaques. These plaques principally consist of lipids such as cholesterol, cholesteryl esters, phospholipids and triglycerides. Chemical analysis of advanced plaques have shown the presence of considerable amounts of free cholesterol identified as cholesterol monohydrate crystals. Cholesterol has been crystallized in vitro. The extracts of some of the Indian medicinal plants detailed below were used as additives to study their effect on the crystallization behaviour of cholesterol. It has been found that many of the herbs have inhibitory effect on the crystallization such as nucleation, crystal size and habit modification. The inhibitory effect of the plants are graded as Commiphora mughul > Aegle marmeleos > Cynoden dactylon > Musa paradisiaca > Polygala javana > Alphinia officinarum > Solanum trilobatum > Enicostemma lyssopifolium.