concanavalin-a and Cholelithiasis

Ursodeoxycholic acid prevents gallstone formation in selected patients. The aim of this study was to examine whether decreased concentration and nucleation-promoting activity of various proteins contribute to this beneficial effect.. Gallbladder bile of 13 patients with cholesterol gallstones treated with ursodeoxycholic acid (10 mg/kg(-1)/day(-1)) and of 13 untreated patients were compared.. Total protein concentration in gallbladder bile (2.8 +/- 0.6 vs. 6.7 +/- 1.3 mg/mL; P=0.008) and concanavalin A-binding fraction (0.16 +/- 0.03 vs. 0.42 +/- 0.07 mg/mL; P=0.003) were strongly decreased by ursodeoxycholic acid therapy. Significant decreases were also found for gallbladder bile alpha1-acid glycoprotein, haptoglobin, immunoglobulin (Ig) A, IgG, gamma-glutamyl transpeptidase, and aminopeptidase N but not for IgM, mucin, or beta-glucuronidase. Decreases were most pronounced for proteins of canalicular membrane origin. Gallbladder bile total protein correlated with cholesterol saturation index (r=0.54; P=0.0047) but not with bile salt hydrophobicity index. Crystallization-promoting activity of the concanavalin A-binding fraction (assessed by nephelometry and microscopic examination) was also significantly decreased by ursodeoxycholic acid.. Ursodeoxycholic acid strongly decreases levels of various proteins and nucleation-promoting activity in bile.

Topics: Adult; Analysis of Variance; Bile; Bile Acids and Salts; Cholagogues and Choleretics; Cholelithiasis; Cholesterol; Concanavalin A; Crystallization; Female; Gallbladder; Humans; Linear Models; Male; Microscopy, Polarization; Middle Aged; Mucins; Phosphatidylcholines; Proteins; Ursodeoxycholic Acid

Contrary to hitherto published results, the authors provided evidence of significant pronucleation activity in the protein fraction which is not linked to concanavaline A. Delipidation or proteolysis markedly reduce the pronucleation activity of this fraction. Albumin was identified as the main protein in this fraction. The lipid-protein complex formed by albumin and lipids had a high pronucleation and crystallization activity in relation to cholesterol. Calcium ions increased the crystalization activity. Complexes formed by proteins and lipids can be vectors of the main pronucleation activity in bile. In investigations of the main cholesterol fraction the authors provided evidence that only part of so-called pronucleation proteins is linked to vesicles--i.e. IgM, IgA and biliary glycoprotein BGP I and II. The authors assume that only proteins firmly linked to vesicles can participate in the process of cholesterol crystallization. Biliary glycoprotein BGP I and II was present in vesicles and when added into a model bile it presented a high pronucleation activity. Biliary glycoprotein is a new hitherto not identified pronucleation protein in bile.

Topics: Bile; Cholelithiasis; Cholesterol; Concanavalin A; Crystallization; Humans

This study explores the pathophysiologic effects of soluble biliary glycoproteins in comparison to mucin gel and cholesterol content on microscopic crystal and liquid crystal detection times as well as crystallization sequences in lithogenic human biles incubated at 37 degrees C. Gallbladder biles from 13 cholesterol gallstone patients were ultracentrifuged and microfiltered (samples I). Total biliary lipids were extracted from portions of samples I, and reconstituted with 0.15 m NaCl (pH 7.0) (samples II). Portions of samples II were supplemented with purified concanavalin A-binding biliary glycoproteins (final concentration = 1 mg/mL) (samples III), or mucin gel (samples IV), respectively, isolated from the same cholesterol gallstone biles. Samples V consisted of extracted biliary lipids from uncentrifuged and unfiltered bile samples reconstituted with 0.15 m NaCl (pH 7.0). Analytic lipid compositions of samples I through IV were identical for individual biles but, as anticipated, samples V displayed significantly higher cholesterol saturation indexes. Detection times of cholesterol crystals and liquid crystals were accelerated in the rank order of samples: IV > V > I = II = III, indicating that total soluble biliary glycoproteins in pathophysiologic concentration had no appreciable effect. Crystallization sequences (D. Q-H. Wang and M. C. Carey. J. Lipid Res. 1996. 37: 606-630; and 2539-2549) were similar among samples I through V. Crystal detection times and numbers of solid cholesterol crystals were accelerated in proportion to added mucin gel and the cholesterol saturation of bile only. For pathophysiologically relevant conditions, our results clarify that mucin gel and cholesterol content, but not soluble biliary glycoproteins, promote cholesterol crystallization in human gallbladder bile.

Topics: Bile; Cholelithiasis; Cholesterol; Chromatography, Affinity; Concanavalin A; Crystallization; Gallbladder; Glycoproteins; Humans; Lipids; Mucins; Solubility; Time Factors; Ultracentrifugation

The concanavalin A-bound glycoproteins in human gallbladder bile have recently been demonstrated to be strong promoters of cholesterol crystal nucleation. In the present study, we investigated the mechanism(s) whereby such promoters affect cholesterol crystal nucleation and/or growth, and compared these mechanisms with those of another promoter, calcium ion. Concanavalin A-bound glycoproteins were isolated from the Helix pomatia-unbound fraction of gallbladder bile from stone-free patients, and determined by electrohoresis to consist of six subclasses (MW 143, 98, 80, 58, 50, and 40 kDa). A cholesterol crystal growth assay showed that concanavalin A-bound glycoproteins both accelerated nucleation time and increased growth rate, whereas calcium ion affected nucleation time only. In the presence of both concanavalin A-bound glycoproteins and calcium ion, both cholesterol nucleation and growth were markedly enhanced. A gel permeation chromatographic study revealed that concanavalin A-bound glycoproteins shifted a considerable amount of cholesterol from micelles to vesicles, whereas calcium ion did not. These results suggest that concanavalin A-bound glycoproteins promote cholesterol crystal nucleation and growth, partly by shifting cholesterol from stable micelles to metastable nonmicellar fractions in bile. In contrast, calcium ion promotes these processes by other mechanisms and, therefore, enhances the effect of concanavalin A-bound glycoproteins.

Topics: Bile; Calcium; Cholelithiasis; Cholesterol; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; In Vitro Techniques; Phospholipids

Several proteins present in human bile have been reported to promote cholesterol crystallization and thus are potentially important in the formation of cholesterol crystals as the initial stage in gallstone pathogenesis. To be physiologically relevant, such proteins must either be present in high concentration in bile or have a potent promoting activity. The current study explored several of the more abundant but unexamined biliary proteins based upon their also having sufficiently high serum concentrations that antibodies were available for both their isolation and quantitation.. Protein purification was accomplished by immunoaffinity chromatography of bile followed by delipidation. Con A affinity chromatography of bile was used to obtain the bound fraction, a portion of which was delipidated. Crystallization-promoting activity of both the purified proteins and Con A-bound glycoprotein fractions (CABG) was measured by a photometric crystal growth assay. A competitive antibody-capture ELISA assay was developed to measure concentrations of alpha 1-antitrypsin, transferrin, and haptoglobin in native bile.. At their relevant physiological concentrations, biliary haptoglobin (15 micrograms/ml) had a crystallization-promoting activity twice that of the biliary IgM (75 micrograms/ml) used as a reference standard (P < 0.05). Biliary transferrin (20 micrograms/ml) had only modest promoting activity (P < 0.05). Biliary alpha 1-antitrypsin (50 micrograms/ml), by contrast, showed no promoting activity. Delipidation of the CABG fraction decreased its promoting activity by 75%. Biliary haptoglobin accounts for about 30% of delipidated total CABG-promoting activity.. Biliary haptoglobin at its physiological concentration has a highly potent crystallization-promoting activity and thus becomes a candidate for major attention in understanding gallstone pathogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha 1-Antitrypsin; Antibody Specificity; Bile; Cholelithiasis; Cholesterol; Chromatography, Affinity; Concanavalin A; Crystallization; Enzyme-Linked Immunosorbent Assay; Haptoglobins; Humans; Transferrin

Total protein, mucin, and specific nonmucin glycoproteins are proposed pronucleating agents in cholesterol gallstone pathogenesis. However, characterization of specific nonmucin glycoproteins in patients with and without gallstones is unknown. Furthermore, nonmucin glycoproteins may be qualitatively different in patients with and without gallstones. Total protein and total and specific nonmucin glycoproteins were studied in gallbladder bile of 43 patients with cholesterol gallstones and 13 patients without gallstones. Patients with cholesterol gallstones had higher concentrations of both total protein and nonmucin glycoproteins than that observed in control patients (P < 0.05). SDS gel electrophoresis of nonmucin glycoproteins demonstrated an 84-kDa protein that was present significantly more often in patients with cholesterol gallstones (87% vs 8%, P < 0.05). Proposed 130- and 42-kDa pronucleating and 120-kDa anti-nucleating nonmucin glycoproteins were present in similar percentages in gallstone and control bile. Moreover, gallbladder bile of patients with the 84-kDa protein nucleated 50% faster than model bile and > 100% faster than that of patients without this protein (P < 0.05). The currently described gallbladder pronucleating and anti-nucleating proteins are found with equal frequency in cholesterol gallstone and control patients. However, an 84-kDa protein is found more commonly in gallstone patients and was associated with a shortened crystal observation time. Thus, this glycoprotein may be important in cholesterol gallstone pathogenesis.

Topics: Adult; Aged; Aged, 80 and over; Bile; Cholelithiasis; Cholesterol; Concanavalin A; Crystallization; Electrophoresis, Polyacrylamide Gel; Female; Gallbladder; Glycoproteins; Humans; Male; Middle Aged; Reference Values

Biliary proteins inhibiting or promoting cholesterol crystallization are assumed to play a major role in cholesterol gallstone pathogenesis. We now report a new group of biliary proteins that bind to cholesterol crystals, modify crystal morphology, and inhibit cholesterol crystallization. Various glycoprotein mixtures were extracted from abnormal human gallbladder bile using lectin affinity chromatography on concanavalin A, lentil, and Helix pomatia columns and were added to supersaturated model bile. Independent of the protein mixtures added, from the cholesterol crystals harvested, the same four GPs were isolated having molecular masses of 16, 28, 63, and 74 kD, respectively. Each protein was purified using preparative SDS-PAGE, and influence on cholesterol crystallization in model bile was tested at 10 microg/ml. Crystal growth was reduced by 76% (GP63), 65% (GP16), 55% (GP74), and 40% (GP28), respectively. Thus, these glycoproteins are the most potent biliary inhibitors of cholesterol crystallization known so far. Evidence that the inhibiting effect on cholesterol crystallization is mediated via protein-crystal interaction was further provided from scanning electron microscopy studies. Crystals grown in presence of inhibiting proteins showed significantly more ordered structures. Incidence of triclinic crystals and regular aggregates was shifted from 30 to 70% compared with controls. These observations may have important implications for understanding the role of biliary proteins in cholesterol crystallization and gallstone pathogenesis.

Topics: Analysis of Variance; Animals; Bile; Carrier Proteins; Cholelithiasis; Cholesterol; Chromatography, Affinity; Concanavalin A; Crystallization; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Helix, Snails; Humans; Isoelectric Focusing; Lectins; Molecular Weight; Plant Lectins; Protein Binding

In recent years, the great achievement in the etiology of cholelithiasis is the discovery of pro- and anti-nucleating factors in human bile. Using Con-A affinity chromatography combined with Sephadex G-200 molecular sieve technique, we have isolated and purified a strong pronucleating glycoprotein in gallbladder bile. Its molecular weight is 70KD, the quantity ratio of neutral hexose to protein in this glycoprotein is 15.88%. One molecule has about 640 amino acid residues. Its N-terminal amino acid sequence is N-Ser-His-Gly-Met-Arg-Gln-Tyr-Tyr-Met-Lys. The pathogenic and biological significance of this glycoprotein in the pathogenesis of gallstone were discussed.

Topics: Amino Acid Sequence; Bile; Cholelithiasis; Chromatography, Affinity; Concanavalin A; Glycoproteins; Humans; Molecular Sequence Data; Molecular Weight

The relationship between protein concentrations and the nucleation activity of bile in cholesterol gallstone patients has already been investigated. Nucleation promoters are mucins and concanavalin A (Con-A)-extractable glycoproteins. Nucleation inhibitors are apolipoproteins. We wanted to investigate whether a change in concentration of apolipoprotein A-I (Apo A-I) or Con-A in the bile of cholesterol stone carriers is dependent on the nucleation time.. Total protein was measured by fluorescence photometry, and Con-A-extractable glycoproteins were separated by their affinity to lectins and measured by photometry. Apolipoproteins were measured by radioactive competitive protein binding assay.. The protein concentrations in our bile samples were 2.41 +/- 1.08 mg/ml for the whole group, 2.73 +/- 1.07 mg/ml for a nucleation time less than 3 days, and 2.04 +/- 1.00 for a longer nucleation time. The concentration of the Con-A fraction accounted for 0.289 +/- 0.096 mg/ml, 0.306 +/- 0.081 mg/ml, and 0.274 +/- 0.109, respectively. The Apo A-I concentration was 52 +/- 64 micrograms/ml; 50 +/- 56 micrograms/ml for a nucleation time less than 3 days and 85 +/- 133 micrograms/ml for a longer nucleation time.. Obviously, individual protein fractions have an effect on the nucleation behaviour of gallbladder bile in cholesterol gallstone patients.

Topics: Apolipoprotein A-I; Bile; Cholelithiasis; Cholesterol; Concanavalin A; Female; Humans; Male; Middle Aged; Radioligand Assay

Concanavalin A (Con A)-binding glycoproteins accelerate the rate of cholesterol crystal formation as a prelude to gallstone formation. Immunoglobulins (IgM, IgA, and IgG), aminopeptidase N (APN), phospholipase C (pcPLC), and alpha 1-acid glycoprotein from this Con A fraction have all been proposed as candidate promoters. We immunopurified each of the six putative promoters and examined their comparative effects by adding equal amounts to a cholesterol crystal growth assay. The effects of immunoabsorptive removal of each of the specific candidate promoters from native bile were also compared. In additional studies, the potency of these proteins was in the following order: IgM > IgA = AAG > IgG. APN and pcPLC showed no effect on cholesterol crystal growth at their apparent physiological concentrations. In subtractive experiments, only a minor loss (< 10%) of net promoting activity from that of the whole Con A-bound fraction was observed after immunoabsorptive removal of pcPLC, APN, or immunoglobulins. Total removal of AAG, however, showed a far greater loss (/33%) of the net promoting activity. These data indicate that AAG accounts for the greatest portion of net biliary Con A-bound promoting activity derived from currently defined and well-identified glycoproteins. However, more than 60% of total Con A-binding promoting activity remains unaccounted for, indicating the presence of other important and still unidentified promoters in human bile.

Topics: Aminopeptidases; Bile; CD13 Antigens; Chemical Fractionation; Cholelithiasis; Cholesterol; Concanavalin A; Crystallization; Humans; Immunoglobulins; Immunosorbent Techniques; Orosomucoid; Type C Phospholipases

The gallbladder bile of patients with cholesterol gallstones contains pronucleating proteins which accelerate precipitation of cholesterol crystals from bile. In this study we have improved the purification procedure developed earlier for these nucleating proteins and have now identified the nature of these proteins. Gallbladder bile from patients with cholesterol gallstones was applied to concanavalin A affinity columns. The ConA-binding glycoprotein fractions containing the nucleating proteins were then separated by FPLC (fast protein liquid chromatography) using a Superose 12 gel filtration column. Nucleating activity was detected in the high molecular weight (FPLC-1) as well as in the low molecular weight fractions (FPLC-3). Investigation of the high molecular weight fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution and amino acid sequencing suggested that these proteins were immunoglobulins. Immunostaining of Western blots with specific monoclonal antibodies identified the presence of immunoglobulin (Ig) M and IgA in the FPLC-1 fraction. These immunoglobulins were further purified by affinity chromatography employing an antibody exchanger (ABx) column which specifically binds immunoglobulins. There was no reduction in the cholesterol nucleating activity in the Abx-bound fraction compared to FPLC-1. Additional studies showed that the FPLC-1 fraction was significantly more potent than the ConA glycoproteins from either rapid and slow nucleating biles. Also the number of crystals formed was significantly greater in the FPLC-1 fraction isolated from cholesterol gallstone biles than from the FPLC-1 fraction from control patient biles. Commercially obtained IgM and IgA had no effect on nucleation, but IgM isolated from the serum of patients with Waldenstrom's macroglobulinemia did accelerate the nucleation of cholesterol. We conclude that the IgM and possibly IgA are pronucleating proteins and may be important in the pathogenesis of cholesterol gallstones in man.

Topics: Bile; Blotting, Western; Cholelithiasis; Cholesterol; Concanavalin A; Glycoproteins; Humans; Immunoglobulins; In Vitro Techniques; Solubility

Cholesterol crystallization-promoting factors probably play an important role in the pathogenesis of gallstone disease. We have isolated one of the factors involved by using lectin-affinity chromatography. A potent promoting activity binds to concanavalin A-Sepharose. The activity is heat labile and sensitive to digestion by glycosidase but remarkably insensitive to proteases. The concanavalin A-binding pronucleator affects cholesterol solubilization in model bile in two ways. It induces a shift of cholesterol and phospholipid from the micellar to the vesicular phase but also interacts directly with cholesterol-phospholipid vesicles. The concanavalin A-binding protein fraction contains at least two different promoting factors with gel permeation molecular weights of about 150 kD and 5 kD, respectively. The higher molecular weight activity could be assigned to a protein with an apparent molecular weight of 130 kD. Concanavalin A-binding-promoting activity was present in bile from both patients with and without stones, indicating that it is a normal constituent of bile. However, the activity was strongly increased in bile from patients with multiple cholesterol gallstones, suggesting that it could play a key role in gallstone formation in these patients.

Topics: Cholelithiasis; Chromatography; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Gallbladder; Glycoproteins; Humans; Liver; Models, Biological; Molecular Weight; Reference Values

Topics: Bile; Cholelithiasis; Cholesterol; Concanavalin A; Crystallization; Dose-Response Relationship, Drug; Glycoproteins; Humans; Proteins; Wheat Germ Agglutinins