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

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

Effect of cholesterol nucleation-promoting activity on cholesterol solubilization in model bile

Human bile contains a factor with cholesterol nucleation-promoting activity that binds to concanavalin A-Sepharose. In this study we have investigated the effect of this activity on the dynamics of lipid solubilization in supersaturated model bile. A concanavalin A binding protein fraction of human bile was mixed with model bile and the effect on the distribution of cholesterol and phospholipid between mixed micelles and phospholipid/cholesterol vesicles was studied by means of density gradient ultracentrifugation. The nucleation-promoting activity containing fraction induced a transfer of cholesterol and phospholipid from the micellar to the vesicular phase. This led to a decrease in the density of the vesicular fraction. We have also studied the effect of promoting activity on the nucleation time of an isolated vesicle fraction. A decrease of the nucleation time of 10.7 +/- 1.3 to 2.3 +/- 0.3 days was observed. In conclusion, a concanavalin A binding protein fraction from human bile stimulated cholesterol nucleation via a double effect; it increased the amount of vesicular cholesterol and phospholipid, and it also directly induced nucleation of cholesterol from the vesicles.     

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.     

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

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

Contribution of vesicular and micellar carriers to cholesterol transport in human bile

A nonmicellar, bile salt-independent mode of cholesterol transport in human bile involving phospholipid vesicles was recently reported by our group. In the present study, we have investigated the relative contribution of the phospholipid vesicles and mixed bile salt-phospholipid micelles to cholesterol transport in human hepatic and gallbladder biles. The vesicles (ca 800 A diameter) were demonstrated by quasi-elastic light scattering (QELS) in fresh bile and after chromatography. Gel filtration under conditions that preserved micellar integrity demonstrated that biliary cholesterol was associated with both vesicles and micelles. At low bile salt concentration, the vesicular phase was predominant and most of the cholesterol was transported by it. With increasing bile salt concentrations, a progressive solubilization of the vesicles occurred with a concomitant increase in the amount of cholesterol transported by micelles. The vesicular carrier may be of particular biological significance for cholesterol solubilization in supersaturated biles.     

Comparative analysis of cholesterol transport in bile from patients with and without cholesterol gallstones

Aggregation of cholesterol-phospholipid vesicles in supersaturated biles precedes cholesterol crystal formation. In this study we examined the relationship between the percentage of cholesterol carried by vesicles and/or their composition and the propensity to form cholesterol crystals (nucleation time). Bile (common bile duct, gallbladder and T-tube) was obtained from patients with and without gallstones. Gel filtration chromatography resolved three peaks, a void volume vesicle, a smaller vesicle (identified by electron microscopy and of distinct composition compared to the larger void volume vesicle), and the mixed micelle. The void volume vesicle was present in 11 of 28 abnormal gallbladder biles, but in none of the 10 normal gallbladder biles. Despite this difference, no correlation between the nucleation time of whole bile with either the percentage of cholesterol carried by or cholesterol/phospholipid ratio of the void volume vesicle was found. Nucleation time was, however, found to correlate with the composition of the small-vesicular transport form. No significant difference in the composition or percentage of the small-vesicular form or the combined vesicular forms was found between normal and abnormal gallbladder biles, although the latter nucleated significantly more rapidly. Our results confirm the importance of vesicles in the nucleation process but suggest that other factors, not yet identified, appear to be responsible for the more rapid nucleation seen in abnormal gallbladder biles.     

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.     

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.     

Characterization of model bile using fluorescence energy transfer from dehydroergosterol to dansylated lecithin

Fluorescence energy transfer from dehydroergosterol (DHE) to dansylated lecithin (DL) was used to characterize lecithin-cholesterol vesicles in the presence of the bile salt, sodium taurocholate. At lipid concentrations approximating physiological levels, exposure of fluorescently labeled vesicles to the bile salt led to a dose-dependent increase in the DHE-to-DL fluorescence ratio during the first 24 h after mixing. The initial changes in the fluorescence ratio correlated well with conventional turbidity measurements that quantify partial micellization of vesicles as a function of bile salt loading. In addition, fluorescence energy transfer from DHE to DL revealed cholesterol enrichment of vesicles and re-vesiculation of micelles at bile salt loadings for which vesicles and micelles coexisted. Samples containing the cholesterol-enriched vesicle fraction exhibited further increases in the DHE-to-DL fluorescence ratio during a 4-week observation period but only after a significant lag period of several days. The lag period decreased with cholesterol loading, and the increase in the fluorescence ratio always preceded the appearance of microscopic, birefringent, either needlelike or platelike, cholesterol crystals, in samples that were initially supersaturated with cholesterol. Cholesterol crystals were not observed, and the fluorescence ratio did not increase, for any sample that was undersaturated with cholesterol.Taken together, these results suggest that the latter changes in fluorescence are the result of cholesterol nucleation. Fluorescence energy transfer from DHE to DL is therefore a promising technique for the characterization of model bile and, possibly, provides a direct measurement of cholesterol nucleation.     

Stability of mixed micellar bile models supersaturated with cholesterol.

The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.     

Biliary lipids, water and cholesterol gallstones

Cholesterol supersaturation, hydrophobic bile salts, pronucleating proteins and impaired gall-bladder motility may contribute to gallstone pathogenesis. We here show that both gallstone-susceptible C57L and gallstone-resistant AKR male inbred mice exhibit supersaturated gall-bladder biles during early lithogenesis, whereas bile-salt composition becomes hydrophobic only in susceptible C57L mice. In vitro, cholesterol crystallization occurs depending on relative amounts of lipids; excess cholesterol may exceed solubilizing capacity of mixed bile salt-phospholipid micelles, whereas excess bile salts compared with phospholipids leads to deficient cholesterol-storage capacity in vesicles. In vivo, bile lipid contents are mainly determined at the level of the hepatocyte canalicular membrane, where specific transport proteins enable lipid secretion [ABCG5/G8 (ATP-binding cassette transporter G5/G8) for cholesterol, MDR3 (multi-drug resistant 3) for phospholipid, BSEP (bile salt export pump)]. These transport proteins are regulated by farnesoid X and liver X nuclear receptors. After nascent bile formation, modulation of bile water contents in biliary tract and gall-bladder exerts critical effects on cholesterol crystallization. During progressive bile concentration (particularly in the fasting gall-bladder), cholesterol and, preferentially, phospholipid transfer occurs from cholesterol-unsaturated vesicles to emerging mixed micelles. The remaining unstable cholesterol-enriched vesicles may nucleate crystals. Various aquaporins have recently been discovered throughout the biliary tract, with potential relevance for gallstone formation.     

Distribution of phosphatidylcholine molecular species between mixed micelles and phospholipid-cholesterol vesicles in human gallbladder bile: dependence on acyl chain length and unsaturation.

The partitioning of phosphatidylcholine (PC) molecular species between mixed micelles and vesicles was studied in each of seven human gallbladder biles. Biles were fractionated by Sephacryl S-300 SF gel filtration chromatography, and PC species in the micellar and vesicular fractions were quantitated by high performance liquid chromatography. Micelles were enriched in species containing unsaturated acyl groups (e.g., 16:1-18:2, 18:1-18:2, and 18:1-18:3); vesicles were enriched in more highly saturated species (e.g., 16:0-16:1, 16:0-18:1, and 18:0-18:1). Separate multivariate analyses for each bile demonstrated that the distribution of PC species between vesicles and micelles was related to the degree of sn-1 and sn-2 unsaturation, and sn-1, but not sn-2, chain length. In addition, the tendency to partition into the micellar phase was particularly marked when unsaturation was present at both the sn-1 and sn-2 positions. When this interaction was included in the multivariate analyses, the regression models accounted for virtually all of the variation in PC partitioning (for each of the seven patients r2 = 0.92-0.98, P less than 0.03). These results suggest that the partitioning of PC species between micelles and vesicles is strictly determined by sn-1 chain length and the degree of unsaturation at both the sn-1 and sn-2 positions. In light of recent reports that fatty acyl composition influences the cholesterol content of vesicles and micelles in model biles, these results raise the possibility that diet-induced alterations in the phospholipid species and the relative proportions of biliary lipid particles may influence the cholesterol-carrying capacity of bile.     

Differential patterns of lipid-protein association in fast and slow cholesterol nucleating human gallbladder biles: implications for cholesterol nucleation from biliary lipid carriers

We compared the protein/lipid structure and Ch-nucleating capacity of individual lipid carriers in two groups of human gallbladder biles: 11 with Fast cholesterol nucleation (2.2 +/- 1.3 days) and 10 with Slow cholesterol nucleation (19.2 +/- 4.4 days). The groups had comparable cholesterol-saturation (1.31 vs. 1.28), total lipids (9.9 vs. 8.5 g/dl) and proteins (8.5 vs. 7.6 mg/ml). Bile was ultracentrifuged (2 h at 150,000 x g) and the resulting isotropic phase was incubated with [3H]Ch and [14C]lecithin and gel-chromatographed on a Superose 6 column with a buffer containing 7.0 mM sodium-taurocholate. Seven protein peaks were identified (280 nm and biochemistry), with the following molecular mass ranges (kDa): 1 (Void volume), 2 (155-205), 3 (50-79), 4 (20-29), 5 (6-15), 6 (3.5-6), 7 (2-3.5). Peaks 2 and 3 were identified as vesicles and micelles, respectively. Fast vs. Slow Ch nucleating biles had: (a) more (P less than 0.02) cholesterol coeluting with vesicles, (b) more (P less than 0.01) lecithin coeluting with low m.w. peaks (Nos. 5-6), (c) less (P less than 0.01) cholesterol and lecithin coeluting with micelles. An inverse correlation (P less than 0.001) was observed between the amount of proteins coeluting with the micellar peak and the cholesterol nucleation of both whole bile and isolated micellar fractions. A marked shift of cholesterol and lecithin from micelles to vesicles was apparent, in the whole bile, after cholesterol nucleation had occurred. Incubation and sequential analysis of isolated and radiolabeled micelles showed a progressive transfer of lecithin and cholesterol molecules to low molecular weight fractions and to vesicles before cholesterol nucleation. We conclude that pro-nucleating biliary vesicles develop from micelles, due to the phasing out and redistribution of micellar cholesterol and lecithin, which are probably induced by biliary proteins.     

Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation

Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by beta-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of beta-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without beta-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with beta-glucuronidase-free bile, (1) beta-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96+/-17.0 vs. 114+/-20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with beta-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without beta-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with beta-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of beta-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.     

Influence of total lipid concentration, bile salt:lecithin ratio, and cholesterol content on inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile.

We modified classic equilibrium dialysis methodology to correct for dialysant dilution and Donnan effects, and have systematically studied how variations in total lipid concentration, bile salt (taurocholate):lecithin (egg yolk) ratio, and cholesterol content influence inter-mixed micellar/vesicular (non-lecithin-associated) concentrations (IMC) of bile salts (BS) in model bile. To simulate large volumes of dialysant, the total volume (1 ml) of model bile was exchanged nine times during dialysis. When equilibrium was reached, dialysate BS concentrations plateaued, and initial and final BS concentrations in the dialysant were identical. After corrections for Donnan effects, IMC values were appreciably lower than final dialysate BS concentrations. Quasielastic light scattering was used to validate these IMC values by demonstrating that lipid particle sizes and mean scattered light intensities did not vary when model biles were diluted with aqueous BS solutions of the appropriate IMC. Micelles and vesicles were separated from cholesterol-supersaturated model bile, utilizing high performance gel chromatography with an eluant containing the IMC. Upon rechromatography of micelles and vesicles using an identical IMC, there was no net transfer of lipid between micelles and vesicles. To simulate dilution during gel filtration, model biles were diluted with 10 mM Na cholate, the prevailing literature eluant, resulting in net transfer of lipid between micelles and vesicles, the direction of which depended upon total lipid concentration and BS/lecithin ratio. Using the present methodology, we demonstrated that inter-mixed micellar/vesicular concentrations (IMC) values increased strongly (5 to 40 mM) with increases in both bile salt (BS):lecithin ratio and total lipid concentration, whereas variations in cholesterol content had no appreciable effects. For model biles with typical physiological biliary lipid compositions, IMC values exceeded the critical micellar concentration of the pure BS, implying that in cholesterol-supersaturated biles, simple BS micelles coexist with mixed BS/lecithin/cholesterol micelles and cholesterol/lecithin vesicles. We believe that this methodology allows the systematic evaluation of IMC values, with the ultimate aim of accurately separating micellar, vesicular, and potential other cholesterol-carrying particles from native bile.     

Phospholipid lamellae are cholesterol carriers in human bile

Cholesterol solubility and precipitation in bile are major factors in the pathogenesis of cholesterol gallstones. At present, mixed micelles and phospholipid vesicles are considered to be the only cholesterol carriers in bile. In this study we present evidence showing that phospholipid lamellae are major cholesterol carriers in human bile. Lamellae are a known aggregational form in pure phospholipid model systems. In the present study, lamellae were demonstrated by electron microscopy after negative staining and by small-angle X-ray diffraction in all human gallbladder bile samples examined. During diffraction experiments, cholesterol was found to crystallize from these lamellae. Cholesterol carriers in bile were separated by high-resolution chromatography and by prolonged ultracentrifugation. Lamellae were shown to solubilize most of the biliary cholesterol; vesicles solubilized a lesser amount; while micelles solubilized only a minor portion. Our data suggest that phospholipid aggregates are the main cholesterol carriers in bile. Bile salts may control the equilibrium between the various aggregational forms of cholesterol-carrying phospholipids.     

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.     

Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.     

Scavenger receptor class B type I reduces cholesterol absorption in cultured enterocyte CaCo-2 cells

Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesteryl esters from HDL as well as efflux of cellular free cholesterol to HDL. It is unclear whether the receptor is involved in intestinal cholesterol absorption. We addressed this issue by studying [3H]cholesterol flux in differentiated CaCo-2 cells incubated at their apical side with mixed taurocholate/phosphatidylcholine/cholesterol micelles. Biotinylation and HDL binding experiments showed predominant apical expression of endogenous and overexpressed SR-BI. Mixed micellar cholesterol saturation affected the magnitude and direction of cholesterol flux with significant net uptake only from supersaturated micelles and net efflux from unsaturated micelles. Incubation with micelles that depleted cellular cholesterol resulted in a decrease of SR-BI protein, whereas incubation with cholesterol-loading micelles resulted in a significant increase of SR-BI protein. Apical cholesterol uptake by CaCo-2 cells was increased in the presence of a SR-BI-blocking antibody and by partial inhibition of SR-BI expression with small inhibitory RNA. Adenovirus-mediated overexpression of apical SR-BI did not affect cholesterol uptake but stimulated apical cholesterol efflux, even to supersaturated mixed micelles. Partial inhibition of SR-BI with small inhibitory RNA reduced apical cholesterol efflux. Our data argue against a direct role for SR-BI in micellar cholesterol uptake. However, SR-BI might be involved in cholesterol absorption by facilitating cholesterol efflux to micelles.     

The induction of lamellar stacking by cholesterol in lecithin-bile salt model systems and human bile studied by synchrotron X-radiation

Small angle X-ray scattering (SAXS) with synchroton radiation was used to investigate interactions among lipid particles in lecithin-bile salt model systems and in native gallbladder biles. In model systems in the absence of cholesterol, isotropic, continuous spectra were found, indicating the absence of periodic structures. In the presence of excess cholesterol, interaction in the form of lamellar stacking was detected by the appearance of discrete diffraction peaks. In the supersaturated cholesterol region of the commonly accepted phase diagram [1], where cholesterol crystals were expected, we found lamellar stacking. The high proportion of cholesterol to bile salts seems to be the common denominator of these models. The lamellar stacking was also found in native unprocessed bile. This effect of cholesterol on lipid structure has not been previously described. Lamellar stacking may contribute to cholesterol solubilization. Its influence on the kinetics of cholesterol crystallization is presently unknown.