Supersaturated Model Bile Research Articles

Partial replacement of bile salts causes drastic alteration in bile metastability in supersaturated model bile systems: Relation to bile salt micelle-forming capacity

Partial replacement of bile salts causes drastic alteration in bile metastability in supersaturated model bile systems: Relation to bile salt micelle-forming capacity

Monitoring cholesterol crystallization from lithogenic model bile by time-lapse density gradient ultracentrifugation

Background/Aims: Cholesterol crystallization in a dilute, bile salt-rich model bile is a multiphase process in which early filamentous crystals gradually transform to classical cholesterol monohydrate plates. The pertinence of similar transformations in more complex model systems or native bile is, however, unclear. The aim of the present study was to characterize and monitor cholesterol crystallization in a model bile of physiological relevance. Methods: A supersaturated model bile was prepared with a lipid composition (18 mM cholesterol, 37 mM lecithin, 120 mM taurocholate) that was derived from analyzing 10 gallbladder biles from cholesterol gallstone patients. Cholesterol crystallization was followed by light and electron microscopy, and sequential density gradient analysis of cholesterol-containing precipitates. Results: During cholesterol crystallization a reproducible sequence of events was recorded. First (T<18 h), cholesterol-rich vesicular and multilamellar structures (density 1.005–1.015 g/ml) were observed. Later, (T > 60 h) filamentous, helical, tubular (density 1.015–1.04 g/ml) and plate-like (density 1.04–1.06 g/ml) cholesterol crystals appeared. The concentration of crystals increased gradually, while bilayer structures became desaturated with cholesterol and disappeared, and early crystal forms were replaced by plates. Eventually (T > 25 days) only classical plate-like cholesterol monohydrate crystals were present. Exposure of cholesterol-containing precipitates to micellar (100 mM) deoxycholate dissolved the bilayer structures but not the crystals. Conclusions: These data demonstrate that cholesterol crystallization in a physiologically relevant model bile is a multiphase process consisting of a sequence of transitions from vesicular and multilamellar structures to early crystal forms and to classical plate-like cholesterol monohydrate crystals. These transitions are associated with increasing density and decreasing phospholipid content of cholesterol precipitates. We suggest that time-lapse density gradient ultracentrifugation is a useful method for investigating and quantitating the process of cholesterol cyrstallization and factors that influence this process in bile.

Method for quantitative assessment of transformation of non-micellar cholesterol carriers in model bile systems.

Aggregation and fusion of non-micellar particulate species, such as unilamellar vesicle and phospholipid lamellae, are believed to precede the nucleation of cholesterol crystals in bile. However, little is known about the time sequence relationship between transformation of non-micellar particles and the initial appearance of cholesterol crystals, as no adequate technique is available for assessing such transformations quantitatively. We have developed a novel method for quantitatively estimating vesicle transformation in supersaturated model bile systems, using a spectrophotometric technique to determine the time sequence relationship between such transformations and cholesterol crystal nucleation. We also investigated the potency of a given effector substance on this transformation. This method permits simultaneous quantitative determination of vesicle aggregation and of cholesterol crystal growth. Maximal vesicular aggregation as determined from turbidity, coincided with initiation of cholesterol crystal nucleation. The addition of divalent cations, Ca2+ and Mg2+, to the model bile solutions promoted vesicle aggregation and cholesterol crystal nucleation and growth. In contrast, apolipoproteins A-1 and A-2 retarded such processes. These data were highly reproducible and reliable. The method described is easy to perform, provides reproducible results and permits the determination of the potency of effector substances on vesicle transformation and on the nucleation of cholesterol crystals.

Lecithin hydrophobicity modulates the process of cholesterol crystal nucleation and growth in supersaturated model bile systems

The present study was performed to determine whether the degree of lecithin hydrophobicity regulates bile metastability and, therefore, affects the process of cholesterol crystallization. Supersaturated model bile (MB) solutions were prepared with an identical composition on a molar basis (taurocholate/lecithin/cholesterol, 73:19.5:7.5; total lipid concentration 9 g/dl) except for the lecithin species; egg yolk phosphatidylcholine, soybean phosphatidylcholine, 1-palmitoyl-2-linoleoyl-sn-phosphatidylcholine, dilinoleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine. Each MB solution was incubated and sequentially examined. Video-enhanced contrast microscopy demonstrated that the rate of vesicular aggregation and fusion correlated with the degree of lecithin hydrophobicity, and that the rate of cholesterol crystal nucleation correlated with the degree of lecithin hydrophilicity. In MBs containing less hydrophobic lecithin, needle-like crystals developed and transformed into mature plate-like crystals, whereas classical plate-like crystals were consistently observed in MBs composed of hydrophobic lecithin. Laser-diffraction particle size analysis demonstrated that the increase in lecithin hydrophobicity enlarged the vesicle dimension, enhancing its cholesterol-holding capacity. Correlation between vesicular cholesterol packing density and lecithin hydrophobicity suggests that the process of bile cholesterol nucleation and growth is regulated, in part, by acyl chain unsaturation in lecithin. Since the composition of biliary lecithins is responsive to dietary manipulations, this study provides new insights into the prevention of cholesterol gallstones.

Does the HMG-CoA reductase inhibitor pravastatin influence nucleation of cholesterol crystals in supersaturated model bile?

To assess whether the presence in bile of HMG-CoA reductase inhibitors, which are secreted predominantly into the bile, influences biliary lithogenicity. Physiologic biliary concentrations of the hydrophilic HMG-CoA reductase inhibitor pravastatin were added to supersaturated model bile (cholesterol saturation index 1.4) and vesicles, the latter with and without the concomitant addition of the nucleation-promoting bile salt taurodeoxycholate. Nucleation time, defined as the number of days after which cholesterol monohydrate crystals are visible by phase contrast microscopy in filtered samples of model bile and vesicles, was assessed. The addition of pravastatin 0.01-1 mg/ml did not influence the nucleation time of supersaturated model bile (mean nucleation time without pravastatin: 8.3 +/- 2.2 days (SD), with pravastatin 1 mg/ml 9.3 +/- 0.4 days and pravastatin 0.01 mg/ml 7.6 +/- 2.3 days). The addition of similar concentrations of pravastatin to vesicle fractions alone did not influence nucleation time (> 20 days), nor could it prevent the nucleation-promoting effect of taurodeoxycholate (nucleation time with or without pravastatin 1 day). The results from this in-vitro study indicate that the presence of pravastatin in bile may not influence gallbladder bile lithogenicity. It can be hypothesized that this also applies to other HMG-CoA reductase inhibitors.

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

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

A new subgroup of lectin-bound biliary proteins binds to cholesterol crystals, modifies crystal morphology, and inhibits cholesterol crystallization.

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.

Influence of bile salt molecular species on cholesterol crystallization from supersaturated model biles

Time-sequential enzymatic determination of cholesterol (CH) crystals harvested by ultrafiltration, and concomitant polarizing light microscopy observations corroborated the striking importance of the bile salts (BS) species in determining CH crystals formation rate from supersaturated model biles incubated in vitro. The more hydrophilic tauroursodeoxycholate, taurohyocholate, glycohyocholate, taurohyodeoxycholate, glycohyodeoxycholate and glyco-3α, hydroxy-6 oxo-5ß-cholanate inhibited CH precipitation through the formation of a stabilized liquid-crystalline phase. In contrast, in all hydrophobic systems (taurine (T) and glycine (G) conjugates of cholate (C), deoxycholate (DC) and chenodeoxycholate (CDC)), CH crystals precipitated with time. When crystallized CH concentrations were plotted vs. time, the figures showed a sigmoidal pattern, consistent with the transition from metastable systems to stable equilibrium states. Over the equilibration period, the nucleation kinetics (as inferred from enzymatic measurements) and all crystallization events (as microscopically observed) were both shifted in time, depending on the BS species: they were earliest in CDC systems, then in DC systems, and finally in C systems. In the latter, the delay was clearly due to the formation of a transient labile liquid-crystalline phase. G-conjugation also induced a significant delay in CH precipitation, compared to T-conjugation. At last, maximum crystallized CH concentrations at equilibrium were in the decreasing order: C > CDC > DC and T-conjugates > G-homologues. All data are discussed in connection with BS hydrophobicities, with predictions from the phase equilibria of aqueous biliary lipid systems and with new insights into CH crystal habits.

Effect of deoxycholate on immunoglobulin G concentration in bile: Studies in humans and pigs

Because an increase in biliary deoxycholate levels seems to be a risk factor for cholesterol gallstone formation, we determined the relationship between deoxycholate levels and levels of the pronucleating protein, immunoglobulin G (Ig) in human gallbladder bile. Patients with cholesterol gallstones had a higher concentration of biliary IgG compared with a pigmented stone group and control patients. This was associated with the simultaneous presence of two conditions in the cholesterol stone group, supersaturated bile and a high deoxycholate/cholate ratio. The other patient groups met only one of the two conditions. Next, animal studies were performed to determine if model biles mimicking the two conditions could affect IgG secretion by the gallbladder. Gallbladders were exposed in vivo and then in an Ussing chamber to model biles. The voltage clamp technique was used to monitor functional integrity of the preparation. Three different model biles were tested: (1) taurodeoxycholate (TDC), 80%; taurocholate (TC), 20%; and cholesterol saturation index (CSI), 1.2; (2) TDC, 20%; TC, 80%; and CSI, 1.2; and (3) TDC, 80%; TC, 20%; and CSI, 0.6. IgG concentrations became significantly higher in group 1 than in the other two groups. The concentration of mucous glycoprotein was also significantly greater in group 1 when compared with group 2. Plasma cells were increased in number in mucosal and submucosal layers in group 1. We conclude that cholesterol supersaturated model bile with high content of TDC induces gallbladder epithelial alterations, which increase the luminal concentration of IgG and mucous glycoprotein. (Hepatology 1995;21:215-222).

Effect of deoxycholate on immunoglobulin G concentration in bile: Studies in humans and pigs

Because an increase in biliary deoxycholate levels seems to be a risk factor for cholesterol gallstone formation, we determined the relationship between deoxycholate levels and levels of the pronucleating protein, immunoglobulin G (Ig) in human gallbladder bile. Patients with cholesterol gallstones had a higher concentration of biliary IgG compared with a pigmented stone group and control patients. This was associated with the simultaneous presence of two conditions in the cholesterol stone group, supersaturated bile and a high deoxycholate/cholate ratio. The other patient groups met only one of the two conditions. Next, animal studies were performed to determine if model biles mimicking the two conditions could affect IgG secretion by the gallbladder. Gallbladders were exposed in vivo and then in an Ussing chamber to model biles. The voltage clamp technique was used to monitor functional integrity of the preparation. Three different model biles were tested: (1) taurodeoxycholate (TDC), 80%; taurocholate (TC), 20%; and cholesterol saturation index (CSI), 1.2; (2) TDC, 20%; TC, 80%; and CSI, 1.2; and (3) TDC, 80%; TC, 20%; and CSI, 0.6. IgG concentrations became significantly higher in group 1 than in the other two groups. The concentration of mucous glycoprotein was also significantly greater in group 1 when compared with group 2. Plasma cells were increased in number in mucosal and submucosal layers in group 1. We conclude that cholesterol supersaturated model bile with high content of TDC induces gallbladder epithelial alterations, which increase the luminal concentration of IgG and mucous glycoprotein.

Degree of fatty acyl chain unsaturation in biliary lecithin dictates cholesterol nucleation and crystal growth

To clarify factors involved in the formation of cholesterol gallstones, we studied the relationship between the degree of fatty acyl chain unsaturation of biliary lecithin and bile metastability. We used supersaturated model bile solutions (molar taurocholate /lecithin/cholesterol ratio (73:19.5:7.5), total lipid concentration 9 g/dl) that contained equimolar egg yolk or soybean lecithins or a sn-1 palmitoyl, sn-2 linoleoyl phosphatidylcholine. Gel permeation Chromatographie studies showed that the vesicular cholesterol distribution and dimension were inversely related to the degree of unsaturation of the lecithin species, estimated by reverse phase, high-performance liquid chromatography. Differential interference contrast microscopy and assay of cholesterol crystal growth showed that a higher degree of fatty acyl chain unsaturation of the lecithin species was associated with a faster nucleation time and rate of crystal growth. Our results suggest that vesicular lecithins containing more unsaturated fatty acyl chains bind less tightly to cholesterol than lecithins containing predominantly saturated fatty acids, and that the biliary lecithin species dictates, in part, the nucleation and growth of cholesterol crystals in bile.

Bile salt-induced cholesterol crystal formation from model bile vesicles: a time course study.

Precipitation of cholesterol crystals from vesicles is an important step in the pathogenesis of cholesterol gallstones. Little is known, however, about the kinetics and the mechanisms involved in cholesterol crystallization. Therefore, the time course of cholesterol crystal precipitation and lipid exchange between vesicles and micelles were monitored in a model bile system. Vesicles obtained from supersaturated model bile (cholesterol saturation index (CSI) 1.4; 10 g/dl) by KBr density gradient ultracentrifugation, were incubated with various bile salts: deoxycholate (DC), chenodeoxycholate (CDC), cholate (C), ursodeoxycholate (UDC), and their respective taurine and glycine conjugates. Vesicle integrity was assessed in a leakage-assay of carboxyfluorescein-loaded vesicles (0-15 min) and by the change in optical absorbance at 340 nm of a vesicle solution (0-50 min). Fluorescence increased within 1 min after addition of bile salt, and was stable within 5-10 min. After addition of bile salt, absorbance fell immediately and stabilized within 30 min. Fluorescence and absorbance were dependent on bile salt hydrophobicity and concentration. At several time points after addition of bile salt to vesicles (from 1 to 72 h), the extent of cholesterol nucleation was determined semiquantitatively and incubation mixtures were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, de novo formed mixed micelles, and cholesterol crystals. Nucleation occurred within 0.5 h after exposure of vesicles to the hydrophobic bile salts DC or CDC, and the cholesterol/phospholipid (c/p) ratio of the vesicles showed a transient rise from 1.45 to 3-4 (at t = 0.5 h) that coincided with the appearance of mixed micelles. Then the vesicular c/p ratio decreased to 0.6-0.8 (at t = 24 h) concomitantly with increasing precipitation of cholesterol crystals. In the case of UDC, the most hydrophilic bile salt used, < 5% micellization, no nucleation, and a constant vesicular c/p ratio were observed. We conclude that under the conditions used in the present model study, the kinetics of cholesterol crystallization are governed by the hydrophobicity of the added bile salts and their capacity to form mixed micelles. The results emphasize the pivotal role of time, and the dynamic aspects of the processes involved in cholesterol crystal formation.

The effects of bile salt hydrophobicity on model bile vesicle morphology

The addition of bile salts to vesicles supersaturated with cholesterol induces cholesterol precipitation, an important step in the formation of cholesterol gallstones. To investigate the effects of bile salt hydrophobicity on vesicle morphology, vesicles obtained from supersaturated model bile by density gradient ultracentrifugation, were incubated with mixtures of deoxycholate (DC) and ursodeoxycholate (UDC) with a constant total bile salt concentration of 30 mM but with a varying hydrophobicity index ranging from −0.31 (UDC alone) up to + 0.72 (DC alone) depending on the composition of the mixture. Five days after addition of bile salts to vesicles, cholesterol precipitation was determined microscopically and incubation samples were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, mixed micelles, and cholesterol crystals. Structure and size of the isolated residual vesicles were studied by freeze fracture electron microscopy. The control, and samples exposed to hydrophilic bile salt mixtures, consisted of unilamellar vesicles of which more than 75% had a diameter of 50–80 nm. After addition of increasingly hydrophobic bile salt mixtures, multilamellar vesicles with progressively greater diameters (up to 1300 nm) were found, suggesting that vesicle fusion and aggregation took place and might hence be important in the cholesterol precipitation process. Accordingly, crystallization was positively correlated with bile salt hydrophobicity. We conclude that cholesterol crystallization from vesicles depends on the hydrophobicity of the bile salts added, and apparently occurs from fused or aggregated vesicles of extended magnitude and with a multilamellar constitution.

The effect of bile acid hydrophobicity on nucleation of several types of cholesterol crystals from model bile vesicles

Nucleation of cholesterol crystals is thought to occur from cholesterol-phospholipid vesicles. We tested the hypothesis that bile acids are necessary for nucleation of cholesterol crystals. Model bile vesicles were prepared by KBr density ultracentrifugation of supersaturated model bile and mixed with one of the following bile acids: ursodeoxycholate, taurocholate, cholate, chenodeoxycholate or deoxycholate in final concentrations of 3, 30 and 100 mM. Vesicles were also mixed with various combinations of ursodeoxycholate and deoxycholate. Nucleation was assessed semi-quantitatively with polarizing microscopy. After 5 days, samples were again subjected to ultracentrifugation. Addition of 3 and 30 mM taurocholate, cholate, chenodeoxycholate and deoxycholate to vesicles induced nucleation. The extent of nucleation increased significantly with increasing bile acid hydrophobicity: deoxycholate > chenodeoxycholate > cholate > taurocholate (p < 0.05). At 100 mM bile acid this order was reversed (p < 0.05) because most of the cholesterol was solubilized in micelles as shown by ultracentrifugation after 5 days. Percentages of vesicular cholesterol decreased with increasing hydrophobicity: deoxycholate < chenodeoxycholate < cholate < taurocholate (p < 0.05). Ursodeoxycholate did not induce nucleation. At least seven cholesterol crystal shapes could be distinguished and all crystal types could be found after addition of various combinations of ursodeoxycholate+deoxycholate. We conclude that in this model: (a) bile acid species play an important role in the precipitation of cholesterol crystals from model bile vesicles; (b) the more hydrophobic bile acids induce more cholesterol crystal precipitation; and (c) the hydrophobicity of bile acids influences cholesterol crystal morphology.

Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 μg ml . Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.

Bovine gallbladder mucin accelerates cholesterol monohydrate crystal growth in model bile

Background: Gallbladder mucin accelerates cholesterol crystal nucleation, an early step in the pathogenesis of gallstones. To examine the role of gallbladder mucin in postnucleation gallstone maturation, the influence of mucin on cholesterol monohydrate crystal growth was studied in a novel model system. Methods: Cholesterol crystals of a uniform size were incubated in model biles at 37 °C with varying cholesterol saturation indices. Crystal size was quantitated by measuring the width and length of individual crystals under polarizing light microscopy and calculating average crystal area. Results: Crystal growth was dependent on the degree of cholesterol supersaturation of bile. Bovine gallbladder mucin (0.5–8 mg/mL) accelerated crystal growth in supersaturated model bile in a concentration- and time-dependent fashion compared with control incubations with bovine serum albumin or model bile alone (P < 0.05). Cholesterol crystal growth was accompanied by a progressive decrease in cholesterol saturation and an increase in total cholesterol crystal mass. Crystal growth was also accompanied by a decrease in total crystal number, suggesting net transfer of cholesterol to larger crystals. Conclusions: The acceleration of cholesterol crystal growth by gallbladder mucin may be of pathophysiological importance in the postnucleation maturation of cholesterol gallstones.

Isolation of mucin from human hepatic bile and its induced effects on precipitation of cholesterol and calcium carbonatein vitro

Biliary mucin was isolated from human hepatic bile, and its induced effects on the appearance time of cholesterol monohydrate crystals (nucleation time) and on the precipitation of calcium carbonate were studied in vitro to examine the possible significance of mucin for ductular gallstone formation. Mucin was isolated by gel filtration on Sepharose CL-4B and a subsequent CsCl density gradient ultracentrifugation. Mucin thus obtained had a high purity as shown by a high-molecular-weight band on SDS-polyacrylamide gel electrophoresis and by the compatible amino acid composition with mucin purified from the gallbladder. The mucin at as low a concentration as 100 micrograms/ml significantly shortened the cholesterol nucleation time in the supersaturated model bile, mimicking human hepatic bile. On the other hand, the addition of mucin inhibited calcium carbonate precipitation in vitro. Taking account of that both cholesterol and calcium salts are major constituents of ductular gallstones, we conclude that biliary mucin is likely to play an important regulating role in the formation of ductular stones.

Fatty acid composition of lecithin is a key factor in bile metastability in supersaturated model bile systems

We studied the effect of fatty acid saturation of biliary lecithin on bile metastability, determined by nucleation time, using model bile solutions with identical lipid compositions except for the lecithin species (total lipid concentration, 9 g/dl; cholesterol, 12 mM; lecithin, 31 mM, bile salts, 116 mM). Gel permeation Chromatographic studies revealed that nonmicellar cholesterol distribution was inversely related to the degree of unsaturation of the lecithin species. Differential interference contrast microscopy and cholesterol crystal growth assay showed that a lower degree of saturation of the lecithin species was associated with a faster nucleation time and crystal growth rate. These results suggest that vesicular lecithin containing more unsaturated fatty acyl chains binds less tightly to cholesterol as compared with lecithin containing predominantly saturated fatty acids and that the biliary lecithin species modulates cholesterol crystal nucleation in bile. Also, the high ratio of cholesterol to lecithin (more than 1.0) was found in the crystal forming model biles, although the vesicle aggregation was not always observed prior to the cholesterol crystal formation. These findings indicated that there are different processes in cholesterol crystal nucleation, with or without vesicle aggregation, and that such processes depend, in part, on lecithin species in vesicles.

Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles

Cholesterol is transported both in unilamellar phosphatidylcholine vesicles and in bile salts-mixed micelles in native bile. The vesicular carrier of biliary lipids apparently has a well defined protein profile with a potent cholesterol crystallization-promoting activity. This study was conducted to identify and further characterize these vesicular proteins and to test the effect of isolated vesicular proteins on the cholesterol crystal formation in supersaturated model bile. The results confirmed that proteins are a constant component of highly purified biliary vesicles both in hepatic and gallbladder bile. Immunoglobulins (IgA, IgG and IgM) and albumin are associated to the purified hepatic biliary vesicles. Furthermore, four different hydrophobic glycoproteins with a molecular mass of 130, 114, 86, and 62–67 kDa were isolated. These glycoproteins showed no reactivity with anti-human whole serum or anti-immunoglobulin antibodies, suggesting that these proteins are biliary-specific. Isolated 130, 114 and 62–67 kDa vesicular glycoproteins significantly decreased the cholesterol nucleation time in artificial model bile. We concluded that some, but not all, vesicular-bound hydrophobic glycoproteins have cholesterol pronucleating activity and they may be involved in the pathogenesis of cholesterol gallstone disease.

Apolipoprotein A-I stabilizes phospholipid lamellae and thus prolongs nucleation time in model bile systems: an ultrastructural study

To explore the mechanisms whereby apolipoprotein A-I inhibits the nucleation of cholesterol crystals, we performed an ultrastructural study using supersaturated model bile systems. Vesicles, micelles and phospholipid lamellae were consistently separated by gel permeation chromatography either in the absence or presence of apolipoprotein A-I. Furthermore, apolipoprotein A-I coeluted with phospholipid lamellae. A sequential study using transmission electron microscopy revealed that phospholipid lamellae without apolipoprotein A-I showed a rapid transformation, with formation of multilamellae and fusion followed by microcrystal nucleation. In contrast, lamellae with apolipoprotein A-I showed little transformation. In conclusion, apolipoprotein A-I stabilizes the phospholipid lamellae, thereby inhibiting the nucleation of cholesterol crystals in supersaturated model bile systems.