P.Robert C. Harvey

Nucleation of cholesterol from vesicles isolated from bile of patients with and without cholesterol gallstones

Bile was obtained from patients with and without cholesterol gallstones at surgery. Biliary vesicles were separated from micelles by gel filtration. The cholesterol/phospholipid ratio in vesicles was much higher than in micelles. Cholesterol crystals nucleated from vesicular fractions, but nucleation from the micellar fractions was slow or did not occur at all. Cholesterol nucleated from vesicles obtained from bile of control patients as rapidly (2.4 days +/- 0.7) as from patients with stones (2.4 days +/- 0.9) and there was no difference in the vesicular cholesterol/phospholipid ratio. The effect of alteration of the bile salt environment was studied by changing the concentration of sodium cholate in the eluting buffer. At low concentrations (5 mM) only vesicles were eluted from the column. These vesicles had a relatively low cholesterol/phospholipid ratio and cholesterol nucleated slowly from these vesicles. At higher concentrations the proportion of micelles increased. The proportion of vesicles decreased progressively but their cholesterol/phospholipid ratio increased and the nucleation time fell. These studies demonstrate that cholesterol nucleates from vesicles in the absence of micelles, that control vesicles are not protected by tightly bound antinucleating substances and that exposure of vesicles to micelles strips relatively more phospholipid than cholesterol from the vesicular fraction, resulting in vesicles with higher cholesterol/phospholipid ratios and shorter nucleation times.

High protein and total lipid concentration are associated with reduced metastability of bile in an early stage of cholesterol gallstone formation

Previous studies from this laboratory suggested that high gallbladder protein concentrations as well as excessive dehydration of bile might reduce the normal metastability of human gallbladder bile. This study attempted to identify persons in an early stage of stone formation, when there are crystals but no stones, and to determine the composition of bile under these conditions of reduced metastability. Two hundred twenty-seven patients were studied, 96 without gallstones. Twenty-three of 96 control patients had cholesterol crystals in their bile. Total protein concentration, total lipid concentration, and cholesterol saturation index were greater in control patients with crystals in bile. To determine whether or not cholesterol saturation index alone could account for the presence of crystals, control patients with cholesterol saturation index above the median value of 1.04 were studied. In this case there was no difference in cholesterol saturation index between the 19 crystal-positive (1.27) and 29 crystal-negative patients (1.26), but the difference in total protein and lipid concentrations persisted. Total protein and total lipid concentrations were even higher in crystal-positive sediments containing large numbers of crystals. Sludge seen by ultrasonography was more common in patients with crystal-positive sediments. High protein and lipid concentrations are associated with reduced metastability of bile.

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.

Evidence of the existence of a soluble mediator of cold preservation injury.

A study was designed to determine whether soluble mediators of injury are released during cold preservation. A first set of livers consisting of three groups was stored in cold Euro-Collins solution. These were a control group stored for 10 min (group 1), an experimental group stored for 16 hr (group 2), and an “antiprotease” group to which a cocktail of antiproteases had been added, which was also stored for 16 hr (group 3). The preservation solution in these livers was washed out at the end of preservation, and this effluent was concentrated and infused into a second set of livers that were all cold-stored for 4 hr. Then, the second-set livers were either perfused-fixed at 4°C with universal fixative or reperfused at 37°C for 180 min in the isolated perfused rat liver (IPRL). Morphometric assessment of sinusoidal lining cells (SLC) on light and electron microscopy showed an increased degree of microcirculatory injury in livers preserved with concentrates from livers of the experimental group. On light microscopy, only 2.2± 0.4% (mean ± SD) of the SLC had a normal flattened morphology compared with 11.9±2.0% in the control group, and 10.7±2.3% of the SLC appeared completely detached from the underlying hepatocytes compared with 2.6±0.8% in the control group, the differences being statistically significant (P<0.05). This injury was prevented by the addition of antiproteases to EC solution. Similar results were obtained in the IPRL model, in which a number of typical changes related to cold preservation injury were noted in livers preserved with concentrates from the experimental group. Compared with controls, livers preserved with concentrates from the experimental group had early and significant alterations in markers of microcirculatory injury, including a reduction in portal flow and an increase in creatinine kinase-BB isoenzyme release, followed by an increase in perfusate transaminases, LDH, and a decrease in bile production. Again the injuries were largely prevented by the addition of antiproteases. There were no differences among groups in the degree of white cell and platelet adherence during reperfusion. Experiments using UW solution showed similar results, indicating that the soluble mediator(s) is not specific for a particular preservation solution. These observations are consistent with the hypothesis that soluble mediators are produced during the hypothermic period, and are responsible for a significant part of cold preservation injury, and that proteolytic reactions are involved in this type of injury.

Fluorometric assay of protein in native human bile

Proteins in human native bile samples were determined by a fluorometric assay. Results were compared to biliary proteins quantified by the Lowry and Bradford techniques. The mean protein concentrations in bile as determined by the Lowry, Bradford and fluorometric assays were respectively 7.26 +/- 4.52 (SD), 2.9 +/- 1.42, and 2.12 +/- 1.28 mg/ml (n = 27). Bilirubin was shown to significantly interfere with the Lowry and Bradford assays but not the fluorometric assay. Bile salts remaining in the trichloroacetic acid (TCA) precipitate did not interfere with the fluorometric assay. No cholesterol or phospholipid could be detected in the TCA preparation prior to protein analysis. Proteolytic digestion of proteins in native bile was shown to occur at 37 degrees C and to a lesser extent at 22 degrees C. The fluorometric protein assay is an easy and accurate method to quantitate proteins in native human bile.

Quantitation of phospholipid vesicles and their cholesterol content in human bile by quasi-elastic light scattering

The proportion of biliary cholesterol carried by phospholipid vesicles may be an important determinant of the lithogenicity of bile. The distribution of biliary cholesterol between vesicles and other aggregational forms is often determined by gel filtration under standard conditions. The aim of this study was to measure the proportion of biliary cholesterol in vesicles in native unprocessed bile and to compare it with values obtained by chromatography. A modified quasi-elastic light-scattering method was used to measure vesicular cholesterol in whole bile. It was suitable only for lightly pigmented biles with a relatively monodisperse population of vesicles. In ten human biles examined, the proportion of cholesterol in vesicles by gel filtration was 40 +/- 8.1% (mean +/- S.D.) by chemical measurement, and 38 +/- 7.2% by [3H]cholesterol estimation. Quasi-elastic light-scattering measurements of these biles produced vesicular cholesterol values of 36 +/- 9.4%. Chromatography may affect lipid particles in bile. Nevertheless, it provides a relatively accurate measurement of biliary cholesterol in vesicles.

Lectin binding characteristics of a cholesterol nucleation promoting protein

Gallbladder bile supersaturated with cholesterol is not the only requirement for the formation of cholesterol gallstones ]1,2]. Biliary proteins are now recognized as important factors in the pathogenesis of cholesterol gallstone disease. Nucleating proteins [3-61 as well as anti-nucleating proteins [7] have been identified in gallbladder biles. Anti-nucleating proteins are believed to result in the metastability of cholesterol supersaturated control biles. On the other hand, nucleating proteins have been suggested to be responsible for the rapid nucleation of cholesterol from the bile of gallstone patients. Recently Groen et al. [6] have reported that a biliary protein that binds to Concanavalin A has nucleation influencing activity. In this study we investigated the potency of this Con A binding protein. We have also further characterized this nucleation promoting protein by studying its ability to bind wheat germ lectin (WGA).

Pathogenesis of Cholesterol Gallstones

Cholesterol gallstone disease is extremely common. Three major stages are recognized for stone formation, namely bile that becomes supersaturated with cholesterol, cholesterol nucleation leading to crystal formation and finally retention of the crystals in the gallbladder resulting in stone formation. Supersaturation is common but nucleation into crystals probably requires protein nucleating factors. Impaired motility of the gallbladder causes crystal retention and is probably very important in stone formation.

Chicken mucin-cross-reactive antigen

We have previously described a chicken heterophile antigenic determinant (CHAD-1) shared by Mycobacterium smegmatis and chicken tissues. We then demonstrated that CHAD-1 is present on several chicken glycoproteins and that its immunoreactive domains are highly branched asparagine-linked oligosaccharides terminating in N-acetylglucosamine residues. In the present study, we have shown that CHAD-1 is also expressed by mucin purified to homogeneity from a soluble mucus of chicken intestine. Another antigen found on chicken mucin is a chicken mucin-cross-reactive antigen (CMCRA). Antisera to this antigen were produced by immunization of rabbits with an enriched preparation of CHAD-1 isolated from the bursa of Fabricius. These antisera were absorbed with Mycobacterium smegmatis (to block the anti-CHAD-1 antibody) and with chicken serum, and then used for immunoperoxidase staining of chicken tissue sections for CMCRA. The latter antigen was detected in most medullary cells of the bursa, in epithelial cells and Hassals corpuscles of the thymus, and in mucus-producing cells of the intestine, esophagus, trachea, and bronchi. Using Western immunoblot analysis, we demonstrated that CMCRA is expressed by a number of polypeptides extracted from bursal lymphoid cells. These polypeptides could not be detected in extracts of thymus, spleen, peripheral blood or bone marrow mononuclear cells.