Cholesterol Crystallization In Model Biles Research Articles

Structural Related Effects of Natural Steroid Molecules on Cholesterol Crystallization in Model Bile and Ethanol

AbstractThe purpose of the present experiment is to study the different effects of natural steroid molecules (NSM) on the crystallization of cholesterol in model bile and ethanol. Three natural steroids (stigmasterol, β‐sitosterol, and ergosterol) were selected as model molecules. The supernatant and precipitated crystals collected from 95% ethanol and model bile were quantitatively analyzed by high‐performance liquid chromatography‐evaporative light scattering detection (HPLC‐ELSD), powder X‐ray diffraction, and differential scanning calorimetry, as well as scanning electron microscope, were conducted to understand the crystalline properties of a substance. Results show that Stigmasterol and β‐sitosterol can increase the solubility of cholesterol in specific mass ratios under 95% ethanol system, and lower the solubility of cholesterol in model bile. Ergosterol, on the other hand, has no significant effect on cholesterol crystallization. The reason for this phenomenon is that phytosterols are more similar in molecular structure to cholesterol and act as co‐precipitates with cholesterol in both systems.

Transgenic overexpression of Niemann-Pick C2 protein promotes cholesterol gallstone formation in mice

Niemann-Pick C2 (NPC2) is a lysosomal protein involved in the egress of low-density lipoprotein-derived cholesterol from lysosomes to other intracellular compartments. NPC2 has been detected in several tissues and is also secreted from the liver into bile. We have previously shown that NPC2-deficient mice fed a lithogenic diet showed reduced biliary cholesterol secretion as well as cholesterol crystal and gallstone formation. This study aimed to investigate the consequences of NPC2 hepatic overexpression on liver cholesterol metabolism, biliary lipid secretion, gallstone formation and the effect of NPC2 on cholesterol crystallization in model bile. We generated NPC2 transgenic mice (Npc2.Tg) and fed them either chow or lithogenic diets. We studied liver cholesterol metabolism, biliary lipid secretion, bile acid composition and gallstone formation. We performed cholesterol crystallization studies in model bile using a recombinant NPC2 protein. No differences were observed in biliary cholesterol content or secretion between wild-type and Npc2.Tg mice fed the chow or lithogenic diets. Interestingly, Npc2.Tg mice showed an increased susceptibility to the lithogenic diet, developing more cholesterol gallstones at early times, but did not show differences in the bile acid hydrophobicity and gallbladder cholesterol saturation indices compared to wild-type mice. Finally, recombinant NPC2 decreased nucleation time in model bile. These results suggest that NPC2 promotes cholesterol gallstone formation by decreasing the cholesterol nucleation time, indicating a pro-nucleating function of NPC2 in bile.

Pathways of cholesterol crystallization in model bile and native bile

Hypersecretion of hepatic cholesterol, chronic supersaturation of bile with cholesterol and rapid precipitation of cholesterol crystals in the gallbladder from cholesterol-enriched vesicles represent the primum movens in cholesterol gallstone formation. Physical–chemical factors and pathways leading to cholesterol crystallization can be investigated in artificial model biles and ex vivo in fresh human bile. Depending on modulatory factors (i.e., lipid concentration, bile salt or phospholipid species, humidity, mucins, etc.), cholesterol can precipitate in several forms (i.e., monohydrate, anhydrous) and habits (i.e., plate-like, needle-like, intermediate arcs, filaments, tubules, spirals). Careful analysis of biliary cholesterol crystals includes biochemical analysis of precipitated crystals, polarizing quantitative light microscopy, and turbidimetric methods. In this paper, recent concepts on cholesterol crystallization in artificial model biles as well as in human bile will be reviewed.

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.

Effects of bile salt and phospholipid species composition on cholesterol crystallization in model biles: Potential implications in gallstone prevention

Effects of bile salt and phospholipid species composition on cholesterol crystallization in model biles: Potential implications in gallstone prevention

Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition

Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn-2 position). Hydrophilic tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all decrease cholesterol crystal-containing zones in the equilibrium ternary phase diagram (van Erpecum, K. J., and M. C. Carey. 1997. Biochim. Biophys. Acta. 1345: 269–282) and thus could be valuable in gallstone prevention. We have now compared crystallization in cholesterol-supersaturated model systems (3.6 g/dl, 37°C) composed of various bile salts as well as egg yolk phosphatidylcholine (unsaturated acyl chains at sn-2 position), dipalmitoyl phosphatidylcholine, or sphingomyelin throughout the phase diagram. At low phospholipid contents [left two-phase (micelle plus crystal-containing) zone], tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all enhanced crystallization. At pathophysiologically relevant intermediate phospholipid contents [central three-phase (micelle plus vesicle plus crystal-containing) zone], tauroursodeoxycholate inhibited, but dipalmitoyl phosphatidylcholine and sphingomyelin enhanced, crystallization. Also, during 10 days of incubation, there was a strong decrease in vesicular cholesterol contents and vesicular cholesterol-to-phospholipid ratios (~1 on day 10), coinciding with a strong increase in crystal mass. At high phospholipid contents [right two-phase (micelle plus vesicle-containing) zone], vesicles were always unsaturated and crystallization did not occur. Strategies aiming to increase amounts of hydrophilic bile salts may be preferable to increasing saturated phospholipids in bile, because the latter may enhance crystallization. —Moschetta, A., G. P. vanBerge-Henegouwen, P. Portincasa, G. Palasciano, and K. J. van Erpecum. Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition.

Pathways of cholesterol crystallization in model biles by nephelometry, spectrophotometry & microscopy

Pathways of cholesterol crystallization in model biles by nephelometry, spectrophotometry & microscopy

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.

Effects of serpins on cholesterol crystallization in model bile

Background/Aims: The serine proteinase inhibitors (serpins), alpha-1-antitrypsin (AAT) and alpha-1-antichymotrypsin (ACT), are acute phase proteins synthesized by hepatocytes and excreted to some extent into bile. Their role in gallstone pathogenesis is unclear, and it was the aim of this study to determine their effect on cholesterol crystal growth rate in model bile. Methods: Purified AAT and ACT were added to model bile at concentrations from 0.5 to 500 μg/ml. Cholesterol crystal growth was analyzed daily by polarizing microscopy and spectrophotometrically at 650 nm. Serpin inhibitory activity was measured spectrophotometrically at 405 nm, and polymerization was studied on 7.5% SDS-PAGE under non-reducing conditions, by immunoelectrophoresis and Western blotting. Results: ACT added to model bile at a concentration of 0.5 μg/ml, inhibited cholesterol crystallization by 30%, had no influence at 5 μg/ml, and increased the crystallization rate 2–3 fold at concentrations of 50 and 500 μg/ml. AAT at a concentration of 0.5 μg/ml had a profound (50%) inhibitory effect on cholesterol crystal growth rate, lacked significant effect at both concentrations of 5 and 50 μg/ml, and showed stimulation of crystal growth up to 30% at a concentration of 500 μg/ml. Both serpins incubated in model bile polymerized and totally lost their inhibitory activity. Conclusions: Serpins can exhibit both inhibiting and promoting effects on the cholesterol crystallization rate in model bile, dependent on their concentrations. Since AAT and ACT are acute phase proteins, their concentrations may vary under certain pathological conditions, which may result in different actions of these serpins in gallstone formation.

Mucins and calcium phosphate precipitates additively stimulate cholesterol crystallization

Human biliary mucin and calcium binding protein (CBP) influence formation of both calcium salt precipitates and cholesterol crystals and colocalize in the center of cholesterol gallstones. We investigated how physiological concentrations of these proteins regulate cholesterol crystallization in model biles, supersaturated with cholesterol and calcium salts, mimicking pathological human bile. Using polarizing light microscopy and nephelometry to assess cholesterol crystallization, the influence of calcium ions and calcium phosphate precipitates in the absence and presence of mucin, CBP, and human serum albumin was determined. Calcium phosphate precipitates stimulated cholesterol crystallization more strongly than soluble calcium. Mucin also stimulated, and with soluble calcium or calcium phosphate precipitates additively increased, the cholesterol crystal mass. In the absence of mucin, only human serum albumin plus CBP, not these proteins individually, decreased the stimulating effect of calcium phosphate precipitates but not of soluble calcium. However, seeding of calcium phosphate precipitates in biles with mucins resulted in near complete cholesterol crystallization within one day whether CBP and HSA were or were not also present.▪ In conclusion, calcium salt precipitates plus human biliary mucins induce rapid and complete cr ystallization of cholesterol from model biles, little influenced by human biliary calcium binding proteins.—van den Berg, A. A., J. D. van Buul, G. N. J. Tytgat, A. K. Groen, and J. D. Ostrow. Mucins and calcium phosphate precipitates additively stimulate cholesterol crystallization.

Biliary secretory immunoglobulin A is a major constituent of the new group of cholesterol crystal-binding proteins

Background & Aims: Recently we described a new group of lectin-bound biliary proteins that bind to cholesterol crystals, modify crystal morphology, and inhibit cholesterol crystallization. The aim of the current study was to characterize and identify individual members of this group of cholesterol crystal-binding proteins. Methods: Crystal-binding proteins were purified from human gallbladder bile by lectin affinity chromatography and preparative gel electrophoresis. Purified crystal-binding proteins were characterized by using cholesterol crystal-growth assays, immunoblotting, and amino acid analysis. For comparison, identified biliary proteins were isolated from gallbladder bile by lectin affinity and immunoaffinity chromatography. Results: The individual crystal-binding proteins with molecular weights of 74, 63, and 28 kilodaltons inhibited cholesterol crystallization in a dose-dependent manner (2.5–10 μg/mL). Immunoblotting with specific antibodies and N-terminal amino acid sequences revealed that the 74-kilodalton crystal-binding protein is the secretory component, the 63-kilodalton protein is the heavy chain, and the 28-kilodalton protein is the light chain of human secretory immunoglobulin (Ig) A. Isolated biliary IgA showed a potent inhibitory effect on cholesterol crystallization in model bile even at levels less than physiological concentrations (1–100 μg/mL). Conclusions: Biliary secretory IgA is a major constituent of the previously described group of cholesterol crystal-binding proteins. Crystal-binding IgA may be an important modulator of crystal agglomeration into stones and stone growth in vivo. GASTROENTEROLOGY 1998;115:129-138

The effects of phospholipid molecular species on cholesterol crystallization in model biles: the influence of phospholipid head groups

Background/Aims: Variations in the molecular species of biliary phospholipids have been shown to exert major effects on cholesterol solubility and carriers in model and human biles. The aim of this study was to explore systematically the effects of various phospholipid head groups on the cholesterol crystallization process in model biles. Methods: Three different control model biles were prepared using varying proportions of egg lecithin, cholesterol and Na taurocholate. In the test biles, 20% of the egg lecithin was replaced with synthetic phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol or phosphatidylcholine, keeping the phospholipid acyl chains and other biliary lipids constant in each experiment. Results: Phosphatidylserine and phosphatidylglycerol significantly prolonged the crystal observation time, from 2 days to 10 and 6 days, respectively ( p<0.02), while phosphatidylethanolamine had little and phosphatidylcholine no effect. The crystal growth rate was significantly slowed down with 20% phospholipid replacement in the following order: phosphatidylglycerol > phosphatidylserine > phosphatidylethanolamine. The total crystal mass after 14 days, as measured by chemical analysis, was reduced by 59% with phosphatidylserine ( p<0.05), and by 73% with phosphatidylglycerol ( p<0.05); while phosphatidylethanolamine had little effect. The precipitable cholesterol crystal fractions after 14 days were significantly reduced with phosphatidylserine (54%) and phosphatidylglycerol (37%), but not with phosphatidylethanolamine or phosphatidylcholine. Conclusions: Variations in the head groups of biliary phospholipids may markedly slow down the cholesterol crystallization process in model biles.

Increased saturation of the fatty acids in the sn-2 position of phospholipids reduces cholesterol crystallization in model biles

Changes in the molecular structure of biliary phospholipids were shown to have major effects on cholesterol solubility, carriers and crystallization in human and model biles. This study investigated systematically the effects of varying saturation of the phosphatidylcholine (PC) sn-2 fatty acid on the cholesterol crystallization process in 3 different model biles. Twenty % of the egg PC (EPC) in these biles were replaced by synthetic PC's with 16:0–18:0, 16:0–18:1, or 16:0–18:2 fatty acyl chains. With 18:0 in the sn-2 position, the crystal observation time (COT) was prolonged from 2 days in the control EPC solution to 14 days ( P<0.05). The crystal growth rate (CGR) was reduced from 0.1 OD/day to unmeasurable levels, and the total crystal mass on day 14 decreased by 86%. The introduction of one (18:1), and two (18:2) double bonds in the sn-2 fatty acid rapidly reversed these effects. Ultracentrifugal analysis showed precipitable cholesterol as monohydrate crystals. In the 16:0–18:0 test solution, most of the precipitable cholesterol remained in the supersaturated multilamellar vesicles. Saturation of the biliary PC sn-2 fatty acyl chain prolongs the COT, slows the CGR, reduces the crystal mass, and extends cholesterol solubility in multilamellar vesicles. Desaturation of the sn-2 fatty acid reverses these effects.

Effects of serpins on cholesterol crystallization in model bile

Effects of serpins on cholesterol crystallization in model bile

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile

Background/Aims: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstones pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. Methods: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. Results: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. Conclusions: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.

A new method for the rapid measurement of cholesterol crystallization in model biles using a spectrophotometric microplate reader

Measurements of the cholesterol crystal observation time, and particularly the crystal growth rate in model biles, are important in biliary pathophysiology. The aim of this study was to develop a semi-automated method permitting multiple, simultaneous, and precise measurements of the crystal growth rate in model biles. Incubated model biles were mixed with a high concentration of NaTDC to solubilize non-crystalline turbidity and spectrophotometric measurements were performed. In parallel, samples were observed by light microscopy. The absorbance correlated linearly with the crystal mass and permitted quantitation of the crystal growth rate. Polarized light microscopy was more sensitive than spectrophotometry for determining the initial crystal observation time, while spectrophotometry was more precise and quantitative for measuring the crystal growth rate.

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.

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile: [formula omitted]. Dept of Gastroenterology, Tel-Aviv Medical Center and Tel-Aviv University, Israel and ∗INSERM U-130, Marseille, France

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile: [formula omitted]. Dept of Gastroenterology, Tel-Aviv Medical Center and Tel-Aviv University, Israel and ∗INSERM U-130, Marseille, France

Promotion of cholesterol crystal nucleation by immunoglobulins (IGS) is not dependent on hydrophobic/hydrophilic balance: [formula omitted]. Washington Univ. School of Med, St Louis, MO

In earlier work we showed that cholesterol monohydrate crystallization from model and native biles can involve filamentous cholesterol crystals, and other metastable intermediates which are covered by a layer of phosphatidylcholine (lecithin) molecules [Konikoff et al., J. Clin. Invest. 90 (1992) 1155]. The aim of the present study was to isolate the initial filamentous cholesterol crystals by density gradient centrifugation and microfiltration and to sequentially monitor their transformations into equilibrium plates within the mother liquor composed of a dilute (1.2 g/dl) bile salt-rich model bile (cholesterol/egg yolk lecithin/sodium taurocholate, 1.7/0.8/97.5 mol%). When assayed by dual radiolabeling at 37°C, total precipitated cholesterol in bile increased from zero at 2–4 h of incubation to 43% at 24 h, reaching a stable value by 48 h when 36% of total cholesterol had crystallized. Isopycnic sucrose density gradient centrifugation at 20°C separated early filamentous crystals from plate-like crystals and revealed densities compatible with anhydrous cholesterol (1.029 g/ml) and cholesterol monohydrate (1.048 g/ml), respectively. Rapid (1 h) density gradient centrifugation carried-out in time-lapse sequence disclosed that cholesterol crystallization involved initially low-density (1.01-1.03 g/ml) filamentous crystals, which reached a maximal concentration at 24 h and disappeared gradually by 156 h of incubation. Concomitantly, the concentrations of high-density (1.04-1.06 g/ml) plate-like cholesterol crystals increased reciprocally throughout the crystallization process suggesting a precursor-product relationship. Rates of crystal filament formation and transitions to thermodynamically stable plates accelerated curvilinearly with increases in temperature from 4 to 60°C, but the crystallization process per se remained unchanged. We conclude that metastable intermediate crystals during cholesterol precipitation from bile may involve either low-density anhydrous crystals or a new cholesterol polymorph that transforms slowly at physiologic temperature (37°C) into classic plate-like cholesterol monohydrate crystals. Clearly, cholesterol crystallization in model bile is more complex and heterogenous than hitherto believed and monitoring metastable intermediate forms, habits and possibly polymorphs should provide a better framework for studying the physical chemistry of nucleation and crystal growth in native bile as well as promoters and inhibitors of these processes.

Evidence that cholesterol antibodies exist in human gallbladder bile and may be promoters of nucleation: [formula omitted], Washington University School of Medicine, St Louis, MO

In earlier work we showed that cholesterol monohydrate crystallization from model and native biles can involve filamentous cholesterol crystals, and other metastable intermediates which are covered by a layer of phosphatidylcholine (lecithin) molecules [Konikoff et al., J. Clin. Invest. 90 (1992) 1155]. The aim of the present study was to isolate the initial filamentous cholesterol crystals by density gradient centrifugation and microfiltration and to sequentially monitor their transformations into equilibrium plates within the mother liquor composed of a dilute (1.2 g/dl) bile salt-rich model bile (cholesterol/egg yolk lecithin/sodium taurocholate, 1.7/0.8/97.5 mol%). When assayed by dual radiolabeling at 37°C, total precipitated cholesterol in bile increased from zero at 2–4 h of incubation to 43% at 24 h, reaching a stable value by 48 h when 36% of total cholesterol had crystallized. Isopycnic sucrose density gradient centrifugation at 20°C separated early filamentous crystals from plate-like crystals and revealed densities compatible with anhydrous cholesterol (1.029 g/ml) and cholesterol monohydrate (1.048 g/ml), respectively. Rapid (1 h) density gradient centrifugation carried-out in time-lapse sequence disclosed that cholesterol crystallization involved initially low-density (1.01-1.03 g/ml) filamentous crystals, which reached a maximal concentration at 24 h and disappeared gradually by 156 h of incubation. Concomitantly, the concentrations of high-density (1.04-1.06 g/ml) plate-like cholesterol crystals increased reciprocally throughout the crystallization process suggesting a precursor-product relationship. Rates of crystal filament formation and transitions to thermodynamically stable plates accelerated curvilinearly with increases in temperature from 4 to 60°C, but the crystallization process per se remained unchanged. We conclude that metastable intermediate crystals during cholesterol precipitation from bile may involve either low-density anhydrous crystals or a new cholesterol polymorph that transforms slowly at physiologic temperature (37°C) into classic plate-like cholesterol monohydrate crystals. Clearly, cholesterol crystallization in model bile is more complex and heterogenous than hitherto believed and monitoring metastable intermediate forms, habits and possibly polymorphs should provide a better framework for studying the physical chemistry of nucleation and crystal growth in native bile as well as promoters and inhibitors of these processes.