Supersaturated Model Bile Research Articles

Lack of a liver‐specific apolipoprotein (apo)A‐V in bile promotes cholesterol gallstone formation by disrupting biliary cholesterol homeostasis in mice

BackgroundApoA‐V is synthesized exclusively by the liver and secreted into the circulation, involving in plasma lipoprotein and lipid metabolism. Although its concentration in human plasma is extremely low (114–258 ng/mL), 1,000 to 10,000‐fold lower than that of apoB‐100 and apoA‐I, respectively, human and mice lacking functional apoA‐V suffer from hypertriglyceridemia. Because our new results found that apoA‐V is also secreted into bile, we hypothesize that it plays an important role in regulating biliary cholesterol metabolism, and its deficiency reduces cholesterol solubility in bile, promoting cholesterol crystallization and gallstone formation.MethodsTo explore the lithogenic mechanisms of biliary apoA‐V deficiency, male apoA‐V knockout (KO) and wild‐type (WT) mice (n=10 per group) were fed a lithogenic diet for 4 wk. Gallstones and cholesterol crystallization in bile were examined by physical‐chemical methods. Hepatic lipid output was determined by biliary secretion studies. Gene expression and protein content were analyzed by qRT‐PCR, Western blots, and ELISA.ResultsCompared to the chow diet, feeding the lithogenic diet significantly (P<0.01) reduced hepatic apoA‐V mRNA and protein levels by 55% and its bile concentrations from 45.7±12.4 ng/mL to 16.7±6.0 ng/mL in WT mice. ApoA‐V was associated only with vesicles, but not micelles, in bile of WT mice. Biliary cholesterol, but not phospholipid or bile salt, output was higher (P<0.05) in KO mice (29.0±8.5 μmol/h/kg) than in WT mice (20.4±5.5 μmol/h/kg), leading to cholesterol‐supersaturated bile. The deficiency of apoA‐V in bile reduced biliary cholesterol solubility and promoted cholesterol crystallization through the liquid crystalline pathway by enhancing biliary unilamellar vesicle aggregation and fusion to form unstable multilamellar vesicles in KO mice. After 50 ng/mL of apoA‐V protein isolated from WT mice was added to supersaturated model bile, cholesterol crystallization was significantly delayed than that added the protein at 0 ng/mL. This implies that apoA‐V plays a key role in regulating cholesterol solubility in bile by modulating the physical state of cholesterol carriers. In addition, the lack of apoA‐V impaired gallbladder emptying and refilling function, thus promoting the accumulation of excess mucin gel and the formation of biliary sludge, a precursor of gallstones. These abnormalities enhanced the growth and agglomeration of solid cholesterol crystal into microlithiasis. After 4 wk of feeding, gallstone prevalence was higher (P<0.05) in KO mice (60%) than in WT mice (25%).ConclusionsThese findings clearly demonstrate that the liver‐specific protein apoA‐V plays a critical role in the regulation of biliary cholesterol metabolism and transport, and its lack in bile dramatically enhances cholelithogenesis by disrupting biliary cholesterol homeostasis. Increasing biliary apoA‐V may be a novel and efficient approach for the prevention of cholesterol gallstones.Support or Funding InformationThis work was supported in part by research grants DK101793, DK106249 and AA03557, all from the National Institutes of Health (US Public Health Service).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of Ca2+ on cholesterol crystallization from supersaturated model biles

The quaternary system of bile salt (BS)–lecithin (L)–cholesterol (Ch)–water has been mainly employed to simulate the in vitro formation of cholesterol gallstones. In this work, a quinary system of bile salt (BS)–lecithin (L)–cholesterol (Ch)–inorganic salt (Ca2+)–water was developed to study the nucleation of cholesterol crystals from a thermodynamic standpoint. The accurate concentration of each component in the freshly prepared biles and after incubation was analyzed by high performance liquid chromatography (HPLC). The micelles, vesicles and crystals formed in the biles were characterized by quasielastic light scattering (QLS) and polarizing microscope, respectively. The results show that, under different concentrations of above five components at 37°C the model biles can finally disperse as one phase (micelles), two phases (micelles and vesicles, micelles and crystals) or three phases (micelles, vesicles and crystals). The mean diameter of BS–L micelles in the one phase region is about 5.7nm, whereas that of Ch–L vesicles in the phase region of micelles and vesicles is about 362.4nm. In addition, the introduction of Ca2+ will decrease the solubility of cholesterol in model biles and thus will shorten the nucleation time of cholesterol crystals, which indicates that Ca2+ plays an important role in the pathogenesis of cholesterol gallstones.

Quantitative analysis of cholesterol nucleation with time in supersaturated model bile

The amount of cholesterol (Ch) crystals formed in supersaturated taurochenodeoxycholate (TCDC) - lecithin (L) solutions of the same Ch saturation index (CSI) but at different Ch thermodynamic activities (Ch AT) was quantified at different time intervals. The initial Ch nucleation rate (i.e., amount of Ch crystals formed with respect to time) in a Ch AT=1.73 and TCDC to L molar ratio (TCDC:L)=5.1 system was faster than that in a Ch AT=1.42 and TCDC:L=3.4 system. Shaking could enhance the early appearance of Ch crystals and cause the fast initial Ch nucleation rates for the TCDC:L=5.1 and the TCDC:L=3.4 systems. The final Ch nucleation rates were faster than the initial Ch nucleation rates for the TCDC:L=5.1 and the TCDC:L=3.4 systems. According to a light scattering analysis of vesicle concentration in supersaturated TCDC–L solutions, vesicles provide nucleation sites only in the Ch nucleation process and the vesicle concentration may not be an important factor for the Ch nucleation rate. A model of a mixed TCDC–L micelle releasing Ch molecules together with the surface area of Ch crystals formed was used in the interpretation of the Ch nucleation.

Effects of Aramchol on in vitro bile cholesterol crystallization and bile acid detergency

AbstractThe hydrophilic bile acid ursodeoxycholic acid may dissolve cholesterol gallstones and is beneficial in cholestatic liver diseases. The C20 fatty acid‐bile acid conjugate arachidyl amido cholanoic acid (Aramchol) could be a more effective option. We therefore studied its effects on cholesterol crystallization and on bile salt‐induced cytotoxicity. Effects of Aramchol at therapeutically relevant concentrations on crystallization in supersaturated model biles (by microscopy and chemical measurement), on the ternary cholesterol‐taurocholate‐phosphatidylcholine phase diagram, and on micelle ↔ vesicle transitions (by serial dilution or by incubation of cholesterol‐phosphatidylcholine vesicles with taurocholate) were evaluated. Effects on bile salt‐induced cytotoxicity were determined in erythrocytes and CaCo2 cells. Incorporation of Aramchol in model biles did not change micellar cholesterol solubilization, induced a small rightward shift of crystal‐containing zones of the ternary phase diagram, exerted no appreciable effects on vesicle ↔ micelle transitions and had only minor effects on cholesterol crystallization. Bile salt‐induced cytotoxicity was increased by Aramchol in all models. Since Aramchol does not affect cholesterol crystallization, its previously reported beneficial effects in animal gallstone models should relate to other mechanisms. Since Aramchol increases bile salt detergency, it is not likely to be beneficial in cholestatic liver disease.

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 β-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of β-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 β-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 β-glucuronidase-free bile, (1) β-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 β-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without β-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with β-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 β-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.

Effects of hydrophobic and hydrophilic bile salt mixtures on cholesterol crystallization in model biles

The hydrophilic bile salt ursodeoxycholate is frequently used to dissolve cholesterol gallstones. We have now quantitated crystallization as a function of bile salt hydrophobicity, phospholipid content, cholesterol saturation and total lipid concentration (TLCo). Methods: Crystallization in supersaturated model biles with low phospholipid contents (left two-phase-micelles and crystal-containing-zone) was assessed during 21 days by microscopy and chemical measurement of crystal mass. For model biles with higher phospholipid contents (central three-phase-micelles, vesicles and crystal-containing-zone), lipid distribution into various phases was determined by combined ultracentrifugation–filtration–dialysis methodology (Biochim. Biophys. Acta 1532 (2001) 15–27). Results: In the left two-phase zone, crystal numbers and masses were highest in case of more hydrophilic bile salt composition (TUDC 100%>TC/TUDC 70%/30%>TC 100%>TC/TDC 70%/30%>TDC 100%) and decreased with increasing phospholipid contents, lower TLCo and lower cholesterol saturation index (CSI). In contrast, in the presence of vesicles (three-phase zone), crystallization decreased at increasing bile salt hydrophilicity, with concomitant increased vesicular cholesterol solubilization. Conclusions: Presence of vesicular phases is a prerequisite for inhibition of cholesterol crystallization by tauroursodeoxycholate.

Cholesterol crystallite nucleation in supersaturated model biles from a thermodynamic standpoint

A rapid, silicone polymer film uptake method was used to determine the cholesterol (Ch) thermodynamic activity ( A T) in taurocholate (TC)–lecithin (L) and taurochenodeoxycholate (TCDC)–L model biles supersaturated with Ch. Also, time-dependent quasielastic light scattering (QLS) measurements and microscopic observations were made to determine the nature of particle species and the Ch nucleation times. In all cases in which Ch–L vesicles were present, a linear relationship between the logarithm of Ch nucleation times and Ch A T was found. These findings support that Ch A T is the appropriate parameter that represents the Ch nucleation tendency and that vesicles are catalytic sites in the Ch nucleation process. When Ca 2+, a nucleation promoter ion, was present in the supersaturated model biles, the increased values of Ch A T quantitatively correlated with shorter Ch nucleation times. These latter findings further demonstrate that Ch A T is the dominant factor in explaining the Ch nucleation tendencies in supersaturated model biles.

Less hydrophobic phosphatidylcholine species simplify biliary vesicle morphology, but induce bile metastability with a broad spectrum of crystal forms

Cholesterol crystallization in bile is affected by phosphatidylcholine (PtdCho) hydrophobicity. The aim of the present study was to determine whether PtdCho species modulate the metastable—labile limit and equilibrium solubility of cholesterol in the micellar phase of bile, thereby altering the distribution of cholesterol to biliary lipid carriers and thus influencing cholesterol crystallization. Supersaturated model bile (with a cholesterol saturation index of 2.0 and a total lipid concentration of 10g/dl) was prepared with various PtdCho/(bile salt+PtdCho) ratios (0.1–0.5) using egg yolk or soya bean PtdCho. Subsequently, the following features were determined: metastable—labile limit, equilibrium solubility of cholesterol, metastable zone, and cholesterol crystallization process. Less hydrophobic PtdCho species destabilized bile cholesterol to induce rapid crystallization, because of a broad integrated metastable zone, whereas more hydrophobic species stabilized bile cholesterol with a less integrated metastable zone and thus retarded cholesterol crystallization. Cholesterol crystallization was accelerated by a decrease in the PtdCho/(bile salt+PtdCho) ratio, whereas the final nucleated crystal mass was increased by an increase in this ratio. With decreasing hydrophobicity of the PtdCho species, the intermixed micellar/vesicular concentration of bile salts decreased in association with less formation of vesicles and increased formation of micelles, and a variety of crystal forms were detected. In conclusion, PtdCho species directly influenced the cholesterol crystallization process in model bile by remodelling the bile mesophase, and also had an indirect influence by altering the balance between bile salt micelles and vesicles.

Less hydrophobic phosphatidylcholine species simplify biliary vesicle morphology, but induce bile metastability with a broad spectrum of crystal forms

Cholesterol crystallization in bile is affected by phosphatidylcholine (PtdCho) hydrophobicity. The aim of the present study was to determine whether PtdCho species modulate the metastable-labile limit and equilibrium solubility of cholesterol in the micellar phase of bile, thereby altering the distribution of cholesterol to biliary lipid carriers and thus influencing cholesterol crystallization. Supersaturated model bile (with a cholesterol saturation index of 2.0 and a total lipid concentration of 10 g/dl) was prepared with various PtdCho/(bile salt+PtdCho) ratios (0.1-0.5) using egg yolk or soya bean PtdCho. Subsequently, the following features were determined: metastable-labile limit, equilibrium solubility of cholesterol, metastable zone, and cholesterol crystallization process. Less hydrophobic PtdCho species destabilized bile cholesterol to induce rapid crystallization, because of a broad integrated metastable zone, whereas more hydrophobic species stabilized bile cholesterol with a less integrated metastable zone and thus retarded cholesterol crystallization. Cholesterol crystallization was accelerated by a decrease in the PtdCho/(bile salt+PtdCho) ratio, whereas the final nucleated crystal mass was increased by an increase in this ratio. With decreasing hydrophobicity of the PtdCho species, the intermixed micellar/vesicular concentration of bile salts decreased in association with less formation of vesicles and increased formation of micelles, and a variety of crystal forms were detected. In conclusion, PtdCho species directly influenced the cholesterol crystallization process in model bile by remodelling the bile mesophase, and also had an indirect influence by altering the balance between bile salt micelles and vesicles.

Does bilirubin play a role in the pathogenesis of both cholesterol and pigment gallstone formation?: Direct and indirect influences of bilirubin on bile lithogenicity

Bilirubin is found in the center of cholesterol gallstones, but its pathogenic role in their formation is unknown. Bilirubin causes a disproportionate reduction of biliary lipid secretion without affecting bile salt secretion in association with a change of biliary lecithin species, which modulates the cholesterol crystallization process. Therefore, the present study investigated whether bilirubin can influence the cholesterol crystallization procedure, and the mechanism(s) of any such action. Supersaturated model bile was prepared (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 9.0 g/dl, and cholesterol saturation index of 1.8), and cholesterol crystallization was monitored over time using a spectrophotometer and video-enhanced differential contrast microscopy in the absence or presence of bilirubin (at a final concentration of 10 μM, 20 μM, 40 μM, and 100 μM). Bilirubin enhanced the onset of cholesterol crystallization by 50%, whereas the crystal growth rate and final crystal mass were reduced at a high concentration of bilirubin. Taken together, these results suggest that bilirubin influences the cholesterol crystallization process, by either a direct interaction with biliary lipids that alters metastability, an indirect alteration of the bile salt-micellar lipid holding capacity, or both. Thus, bilirubin may play a role in the pathogenesis of both cholesterol and pigment gallstones.

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.

Is a role of phospholipase A 2 in cholesterol gallstone formation phospholipid species-dependent?

Phospholipase A 2 plays a role in cholesterol gallstone formation by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. This study investigated its effects on cholesterol crystallization in model bile systems. Supersaturated model bile solutions with different cholesterol saturation indexes (1.2, 1.4, and 1.6) were prepared using cholesterol, taurocholate, and egg yolk phosphatidylcholine, soybean phosphatidylcholine, palmitoyl-oleoyl phosphatidylcholine, or palmitoyl-linoleoyl phosphatidylcholine. Then the effect of digestion of phosphatidylcholine by phospholipase A 2 on bile metastability was assessed by spectrophotometry and video-enhanced differential contrast microscopy. Addition of phospholipase A 2 caused the release of free fatty acids in a time-dependent manner. Cholesterol crystallization was enhanced by an increased crystal growth rate in model bile containing hydrophilic species such as soybean or palmitoyl-linoleoyl phosphatidylcholine, consisting predominantly of polyunsaturated fatty acids. Because phospholipase A 2 enhanced cholesterol crystallization in bile containing hydrophilic phosphatidylcholine species, but not hydrophobic phosphatidylcholine species, release of polyunsaturated fatty acids by hydrolysis may be responsible for such enhancement. Therefore, the role of phospholipase A 2 in cholesterol gallstone formation depends on the phospholipid species present in bile, so that phospholipid species selection during hepatic excretion is, in part, crucial to the cholesterol stone formation.

Measurement of cholesterol gallstone growth in vitro

Methods to study growth of gallstones in the laboratory have not been reported. We here present such a method. Human cholesterol gallstones were harvested from patients with multiple nearly identical stones. The gallstones were washed and added to supersaturated model biles and the formation of cholesterol crystals and the increases in mass of human cholesterol gallstones were studied concurrently, over a period of weeks, using nephelometry and a microbalance, respectively. All stones incubated in model biles supersaturated with cholesterol increased in mass. Increases in the degree of supersaturation of cholesterol in the model biles resulted in increased growth of stones. The mass increases, the growth rates, and the spatial orientation of accreted crystalline cholesterol differed among various stone types. The kinetics and structures of stone growth were similar when the stones were incubated in supersaturated, native, human gallbladder biles. The structure of accreted cholesterol was the same as found on the surface of some human gallstones that were harvested during apparent active growth in situ. This simple method allows accurate measurements of stone growth in vitro, in patterns that mimic stone growth in vivo, and is useful for studies on the relationships of gallstone growth and the kinetics of cholesterol crystallization. —van den Berg, A. A., J. D. van Buul, J. D. Ostrow, and A. K. Groen. Measurement of cholesterol gallstone growth in vitro.

Role of phospholipase A2 in cholesterol gallstone formation is associated with biliary phospholipid species selection at the site of hepatic excretion: indirect evidence.

Phospholipase A2 plays a role in cholesterol gallstone development by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. Lysolecithin and polyunsaturated free fatty acids are known to stimulate the synthesis and/or secretion of gallbladder mucin via a prostanoid pathway, leading to enhancing cholesterol crystal nucleation and growth, and therefore, the action of phospholipase A2 is associated, in part, with bile phospholipid fatty acid. To clarify this hypothesis, we evaluated the effect on bile lipid metastability in vitro of replacing phospholipids with lysolecithin and various free fatty acids. Supersaturated model biles were created with an identical composition (cholesterol saturation index, 1.8; egg yolk lecithin, 34 mM; taurocholate, 120 mM; cholesterol, 25 mM) except for 5%, 10%, or 20% replacement of egg yolk lecithin with a combination of palmitoyl-lysolecithin and a free fatty acid (palmitate, stearate, oleate, linoleate, or arachidonate), followed by time-sequentially monitoring of vesicles and cholesterol crystals using spectrophotometer and video-enhanced differential contrast microscopy. Replacement with hydrophilic fatty acids (linoleate and arachidonate) reduced vesicle formation and promoted cholesterol crystallization, whereas an enhanced cholesterol-holding capacity was evident after replacement with hydrophobic fatty acids (palmitate and stearate). These results indicate that the effect of phospholipase A2 on bile lithogenecity is modulated by the fatty acid species in bile phospholipids, and therefore, that the role of phospholipase A2 in cholesterol gallstone formation is dependent, in part, on biliary phospholipid species selection at the site of hepatic excretion.

Influence of cholesterol crystallization effector proteins on vesicle fusion in supersaturated model bile.

In lithogenic bile, cholesterol-rich vesicles rapidly aggregate and fuse to eventually form cholesterol crystals. This process is modulated by cholesterol crystallization effector substances. In this study, we developed a method for quantitative assessment of vesicle fusion and used it to partly characterize the mechanisms of action of cholesterol crystallization effector proteins. Cholesterol:phospholipid (1:1) liposomes were prepared and labelled with octadecyl rhodamine B chloride (R18). Fusion of these liposomes was detected by the increase of R18 fluorescence after incubation with various proteins, such as albumin, concanavalin-A bound glycoprotein, immunoglobulins, apolipoprotein A-I and apolipoprotein B (all at 100 microg/mL). Fusion of cholesterol/phospholipid liposomes was increased by 16 and 14% in the presence of concanavalin-A bound glycoprotein and immunoglobulins, respectively, and decreased by 21 and 9% after addition of apolipoprotein A-I and apolipoprotein B, respectively. The effect of each protein on vesicle fusion was correlated with its hydrophobicity. These results suggest that nucleation effector proteins modulate the stability of vesicles and, thus, affect cholesterol crystallization. Such modulation is based upon protein-vesicle association, which defines the physico-chemical metastability of vesicular cholesterol.

Partial replacement of bile salts causes marked changes of cholesterol crystallization in supersaturated model bile systems

Partial replacement of bile salts causes marked changes of cholesterol crystallization in supersaturated model bile systems

Partial replacement of bile salts causes marked changes of cholesterol crystallization in supersaturated model bile systems

Cholesterol crystallization is a key step in gallstone formation and is influenced by numerous factors. Human bile contains various bile salts having different hydrophobicity and micelle-forming capacities, but the importance of lipid composition to bile metastability remains unclear. This study investigated the effect of bile salts on cholesterol crystallization in model bile (MB) systems. Supersaturated MB systems were prepared with an identical composition on a molar basis (taurocholate/phosphatidylcholine/cholesterol, 152 mM:38 mM: 24 mM), except for partial replacement of taurocholate (10, 20, and 30%) with various taurine-conjugated bile salts. Cholesterol crystallization was quantitatively estimated by spectrophotometrically measuring crystal-related turbidity and morphologically scanned by video-enhanced microscopy. After partial replacement of taurocholate with hydrophobic bile salts, cholesterol crystallization increased dose-dependently without changing the size of vesicles or crystal morphology and the rank order of crystallization was deoxycholate>chenodeoxycholate>cholate (control MB). All of the hydrophilic bile salts (ursodeoxycholate, ursocholate and beta-muricholate) inhibited cholesterol precipitation by forming a stable liquid-crystal phase, and there were no significant differences among the hydrophilic bile-salt species. Cholesterol crystallization was markedly altered by partial replacement of bile salts with a different hydrophobicity. Thus minimal changes in bile-salt composition may dramatically alter bile lipid metastability.

Role of bile salt hydrophobicity in distribution of phospholipid species to carriers in supersaturated model bile solutions

Phospholipid species modulate cholesterol-holding capacity and, therefore, regulate bile metastability. In this study, we investigated the effect of bile salt hydrophobicity on the distribution of phospholipids among lipid particles in supersaturated model bile solutions (total lipid concentration, 9 g/dL; taurocholate/phospholipid ratio 3.0, cholesterol saturation index 1.3), by using gel permeation chromatography. With an increase of bile salt hydrophobicity in the elution buffer, the uptake of cholesterol and phospholipids into bile salt micelles was increased, associated with an increased cholesterol/phospholipid molar ratio of the vesicles. In contrast, there was an inverse correlation between the hydrophobicity of the phospholipid species in the vesicles and that of bile salts in the elution buffer, suggesting that hydrophobic bile salts induced preferential uptake of hydrophobic phospholipids into bile salt micelles, while less hydrophobic phospholipids, with a relatively low cholesterol-holding capacity, remained in the vesicles. These data indicate that bile salt hydrophobicity regulates vesicular cholesterol metastability by modulating the hydrophobicity of phospholipids in vesicles, as well as the lipid distribution among various biliary lipid particles.

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.

Effect of β-cyclodextrin dietary supplementation on biliary proteins and their resulting cholesterol nucleating activity in pigs

We explored the possibility that the biliary protein fraction may support part of the variation in the nucleating activity previously measured in gallbladder biles of pigs. Eighteen gallbladder aspirates freshly obtained from three dietary groups (0, 5, or 10% β-cyclodextrin) of six pigs were chromatographed to purify their total protein fraction. Proteins were quantified, and analysed through electrophoresis and immunoblotting or enzyme-linked immunosorbent assay for albumin, and five putative effectors of cholesterol crystallisation, mucins, immunoglobulin A, 130 kDa, apolipoprotein A-I, and anionic polypeptide fraction. Each total protein fraction was also assayed for its ability to influence cholesterol precipitation, when added to supersaturated model bile. The current data provided evidence that the cholesterol crystallisation-promoting activity of biliary proteins in model biles increased with the β-cyclodextrin dietary content. This occurred without any significant change in the total biliary protein content, but was associated with a significant decrease in the concentration of albumin and apolipoprotein A-I, resulting in changes in the overall balance of proteins in bile. Comparison of these results with the crystallisation figures previously obtained from the corresponding native biles led us to conclude that biliary proteins might influence the outcome of the crystallisation process, namely the final crystal concentration at equilibrium, but would not systematically represent a major driving force for determining the velocity of crystal formation in native bile of pigs.