Cholesterol Nucleation Research Articles

SAT009 Investigating The Role Of Hepatic Small Heterodimer Partner In Regulating Gallstone Formation

Abstract Disclosure: N.M. Dang: None. S. Anakk: None. Nuclear receptor, Small Heterodimer partner (Shp) regulates bile acid homeostasis by inhibiting transcription of Cholesterol 7 alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme involved in converting cholesterol to bile acids, within the liver. Bile acid regulation prevents the formation of gallstones as cholesterol is excreted into bile along with bile acids and phospholipids. How hepatic Shp affects the development of gallstones and the mechanisms underlying these effects remain unclear. Here we used a liver-specific knockout mouse model and a lithogenic diet model to uncover potential liver-to-gallbladder crosstalk that contributes to gallstone development. Previously, we had shown that the knockout of Shp alters bile acid composition, resulting in higher percentages of secondary bile acid, deoxycholic acid. This increased the hydrophobic index of hepatic bile acids and also implicated a trend for a subsequent increase in the hydrophobicity of biliary bile. Higher hydrophobicity is sufficient to increase susceptibility to developing gallstones by promoting cholesterol nucleation. Of note, the expression of transporter genes Abcg5 and Aqp1 were altered in the gallbladder of hepatic Shp knockout mice, indicating a potential change in their bile composition. The enterokine, Fgf15 is known to stimulate gallbladder filling through its interaction with its receptor, Fgfr4. Recently, the Fgf15-Fgfr4 axis was shown to prevent degradation of the SHP protein such that impeding this axis led to higher Cyp7a1 transcript, increased bile acid levels, and altered gallbladder filling. Taken together, these findings suggest that hepatic Shp acts as a central node that regulates bile acid transport, homeostasis, and composition within the gall bladder and thus can prevent gallstone development. Elucidating Shp-mediated liver-to-gallbladder crosstalk is important for understanding the contribution of liver dysfunction towards gallbladder diseases and gallstones. Presentation: Saturday, June 17, 2023

Effects of Biliary Phospholipids on Cholesterol Crystallization and Growth in Gallstone Formation.

The prevalence of cholesterol gallstone disease is increasing, primarily due to the global epidemic of obesity associated with insulin resistance, and this trend leads to a considerable healthcare, financial, and social burden worldwide. Although phospholipids play an essential role in maintaining cholesterol solubility in bile through both mixed micelles and vesicles, little attention has been paid to the impact of biliary phospholipids on the pathogenesis of cholesterol gallstone formation. A reduction or deficiency of biliary phospholipids results in a distinctly abnormal metastable physical-chemical state of bile predisposing to supersaturation with cholesterol. Changes in biliary phospholipid concentrations influence cholesterol crystallization by yielding both liquid crystalline and "anhydrous" crystalline metastable intermediates, evolving into classical parallelogram-shaped cholesterol monohydrate crystals in supersaturated bile. As a result, five distinct crystallization pathways, A-E, have been defined, mainly based on the prime habits of liquid and solid crystals in the physiological or pathophysiological cholesterol saturation of gallbladder and hepatic bile. This review concisely summarizes the chemical structures and physical-chemical properties of biliary phospholipids and their physiological functions in bile formation and cholesterol solubility in bile, as well as comprehensively discusses the latest advances in the role of biliary phospholipids in cholesterol crystallization and growth in gallstone formation, largely based on the findings from clinical and animal studies and in vitro experiments. The insights gleaned from uncovering the cholelithogenic mechanisms are expected to form a fundamental framework for investigating the hitherto elusive events in the earliest stage of cholesterol nucleation and crystallization. This may help to identify better measures for early diagnosis and prevention in susceptible subjects and effective treatment of patients with gallstones.

Effect of nicotine on cholesterol gallstone formation in C57BL/6J mice fed on a lithogenic diet.

Gallstones are diseases of the biliary system caused by cholesterol supersaturation and/or deficiency in bile salts in bile. Early studies have shown that symptomatic gallstones are primarily a disease of non-smokers, raising the possibility that nicotine can prevent gallstone formation. The present study investigated the effect of nicotine on the formation of cholesterol gallstone in C57BL/6J mice. C57BL/6J mice (eight-weeks-old) were fed a normal or lithogenic diet (basic feed 82.45%, fat 15.8%, cholesterol 1.25% and sodium cholate 0.5%) and divided into five groups: normal diet (ND); ND + high dose nicotine (H); lithogenic diet (LD); LD + low dose nicotine (L) and LD + nicotine (H). They were treated with or without nicotine injection for 10 weeks. Nicotine treatment did not change the rate of cholesterol gallstone formation. There was no difference in TNFα, IL-1β and IL-6 among the five groups. The LD group showed the highest cholesterol levels and there was significant suppression of the total cholesterol, low-density lipoprotein-cholesterol and total bile acid levels in the serum of the nicotine-treated mice. Quantitative PCR showed nicotine altered few bile acid metabolism-related genes expression in liver tissue and significantly altered cholesterol-metabolism genes in gallbladder tissue. Hematoxylin and eosin staining and western blotting showed that protein levels of farnesoid X receptor (FXR) and megalin in the gallbladder increased in the lithogenic-diet mice, which was significantly suppressed in the nicotine-treated mice. In vitro studies using gallbladder epithelial cells showed that chenodeoxycholic acids increased megalin expression, which could be attenuated by nicotine. Nicotine could regulate bile acid metabolism via the FXR-megalin/cubilin pathways, which potentially contribute to cholesterol nucleation and subsequent gallstone formation.

Adeno-associated virus-based caveolin-1 delivery via different routes for the prevention of cholesterol gallstone formation

BackgroundHepatic caveolin-1 (CAV1) is reduced in cholesterol gallstone disease (CGD). Mice with CAV1 deficiency were prone to develop CGD. However, it remains unknown whether restored hepatic CAV1 expression prevents the development of CGD.MethodsC57BL/6 mice were injected with adeno-associated virus 2/8 (AAV2/8) vectors carrying the CAV1 gene (AAV2/8CAV1) via intravenous (i.v.) or intraperitoneal (i.p.) route and then subjected to a lithogenic diet (LD) for 8 weeks. Uninjected mice were used as controls. The functional consequences of rescuing CAV1 expression by either i.v. or i.p. AAV2/8CAV1 treatment for CGD prevention and its subsequent molecular mechanisms were examined.ResultsCAV1 expression was reduced in the liver and gallbladder of LD-fed CGD mice. We discovered that AAV2/8CAV1 i.p. delivery results in higher transduction efficiency in the gallbladder than tail vein administration. Although either i.v. or i.p. injection of AAV2/8CAV1 improved liver lipid metabolic abnormalities in CGD mice but did not affect LD feeding-induced bile cholesterol supersaturation. In comparison with i.v. administration route, i.p. administration of AAV2/8CAV1 obviously increased CAV1 protein levels in the gallbladder of LD-fed mice, and i.p. delivery of AAV2/8CAV1 partially improved gallbladder cholecystokinin receptor (CCKAR) responsiveness and impeded bile cholesterol nucleation via the activation of adenosine monophosphate-activated protein kinase (AMPK) signaling, which induced a reduction in gallbladder mucin-1 (MUC1) and MUC5ac expression and gallbladder cholesterol accumulation.ConclusionCGD prevention by i.p. AAV2/8CAV1 injection in LD-fed mice was associated with the improvement of gallbladder stasis, which again supported the notion that supersaturated bile is required but not sufficient for the formation of cholesterol gallstones. Additionally, AAV treatment via the local i.p. injection offers particular advantages over the systemic i.v. route for much more effective gallbladder gene delivery, which will be an excellent tool for conducting preclinical functional studies on the maintenance of normal gallbladder function to prevent CGD.

Cholesterol crystals activate NLRP3 inflammasomes and promote gallstone formation by increasing mucin secretion

ABSTRACT Cholesterol crystals participate in cholesterol nucleation; however, the role of cholesterol crystals in gallstone development is unknown. Mucin secretion contributes to increased size of gallstones. Cholesterol crystals activate inflammasomes and participate in many sterile inflammation related human diseases. We investigated the role of cholesterol crystals and mucins in sterile inflammation and gallstone enlargement. We found that expression of mucin 5AC (MUC5AC), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) and interleukin-1b (IL-1b) was increased significantly in tissues adjacent to gallstones. Experiments in vitro showed that cholesterol crystals promote MUC5AC secretion; they also increase expression of NLRP3, NLR family CARD domain-containing 4 (NLRC4), apoptosis-associated speck-like protein containing caspase recruitment domain (ASC), and cleaved caspase-1 in biliary epithelial cells. Inhibition of Inflammasomes by NLRP3, ASC or caspase-1 small interfering RNAs reduced MUC5AC secretion. Also, the IL-1 receptor antagonist, IL1RA, and caspase-1 inhibitor, Ac-YVAD, both inhibited MUC5AC secretion induced by cholesterol crystals. We found that inflammasome activation participates in cholesterol crystal induced mucin secretion and gallstone development.

Polymorphism, Structure, and Nucleation of Cholesterol·H2O at Aqueous Interfaces and in Pathological Media: Revisited from a Computational Perspective.

We revisit the important issues of polymorphism, structure, and nucleation of cholesterol·H2O using first-principles calculations based on dispersion-augmented density functional theory. For the lesser known monoclinic polymorph, we obtain a fully extended H-bonded network in a structure akin to that of hexagonal ice. We show that the energy of the monoclinic and triclinic polymorphs is similar, strongly suggesting that kinetic and environmental effects play a significant role in determining polymorph nucleation. Furthermore, we find evidence in support of various O–H···O bonding motifs in both polymorphs that may result in hydroxyl disorder. We have been able to explain, via computation, why a single cholesterol bilayer in hydrated membranes always crystallizes in the monoclinic polymorph. We rationalize what we believe is a single-crystal to single-crystal transformation of the monoclinic form on increased interlayer growth beyond that of a single cholesterol bilayer, interleaved by a water bilayer. We show that the ice-like structure is also relevant to the related cholestanol·2H2O and stigmasterol·H2O crystals. The structure of stigmasterol hydrate both as a trilayer film at the air–water interface and as a macroscopic crystal further assists us in understanding the polymorphic and thermal behavior of cholesterol·H2O. Finally, we posit a possible role for one of the sterol esters in the crystallization of cholesterol·H2O in pathological environments, based on a composite of a crystalline bilayer of cholesteryl palmitate bound epitaxially as a nucleating agent to the monoclinic cholesterol·H2O form.

Process intensification of cooling crystallization of cholesterol from acetone solution using CO2 gas bubbles: Experiments and modeling

The present work investigates dependence of heterogeneous nucleation of solids on the gas bubble-solution interface in a process termed as "Precipitation by Gas-Bubbling" (PGB), through experiments and modeling. This process involves bubbling of CO2 gas at ambient condition through a solution of cholesterol in acetone, which is simultaneously cooled by an external coolant. The process parameters selected are coolant temperature, initial solution concentration, and CO2 flow rate. The flowing CO2 gas bubbles facilitate reduction in metastable zone width and induction time for cholesterol precipitation, rendering more efficient surface-mediated crystallization, thereby enabling process intensification of cooling crystallization. Faster precipitation of cholesterol at higher CO2 flow rate, i.e., higher CO2 bubble-solution interfacial area, demonstrates gas bubble-solution interface acting as a substrate for heterogeneous nucleation. Accordingly a mechanism for heterogeneous nucleation of cholesterol is proposed and validated by developing a model for prediction of final average particle size. The kinetic model parameters, namely, the pre-exponential factor as per the classical nucleation theory, and the crystal growth coefficient are regressed by comparing the predicted values of average particle size with the corresponding experimental data, and are found to be proportional to the surface area of gas bubbles and in turn on CO2 flow rate.

Distinctive Weak Interactions Underlie Diverse Nucleation and Small-Angle Scattering Behavior of Aqueous Cholesterol, Cholesteryl Hemisuccinate, and Glycocholic Acid.

Increased total cholesterol is a major cause of serious heart ailments leading to an estimated 3 million deaths annually throughout the world. Understanding the flocculation behavior of small lipids is thus quintessential. Nucleation, small-angle scattering, and dynamical behavior of lipids and analogues like cholesterol (CHL), cholesteryl hemisuccinate (CHM), and glycocholic acid (GHL) are studied in water by molecular dynamics simulation. The study shows a distinct aggregation behavior of these physiologically relevant molecules owing to a systematic gradation in their non-bonding interactions with solvents and near neighbors. Spontaneous self-assemblies formed during simulation are observed to have different stability, aggregation patterns, and dynamics depending crucially on the nature of the hydrophobic/hydrophilic tails. With increasing hydrophilicity, in the order CHL < CHM < GHL, the aggregates become breakable and less compact, often interposed by water molecules in the interstitial spaces between the lipids. Small-angle scattering data obtained from our simulations provide insights toward the structural integrity and shape of the aggregates formed. Unique features are noticed while following the time evolution of the packing of the nucleated assemblies from the solution phase in terms of local density and molecular orientation. As hydrophilicity increases from CHL to GHL, the packing becomes progressively erratic with diverse angles between the molecular vectors. Surface electrostatic potential calculation indicates drastic increase in positive surface charge from CHL to CHM, which has strong implication in water and ion transport through membranes. These observations can be further correlated to comprehend the flocculation of cholesterol and bile acids in the human body.

Gallstone formation analysis using the particle appearance, the particle binding to calcium ions, and the cholesterol nucleation with time in supersaturated taurocholate–lecithin–calcium ion solutions

AbstractWe conducted a time‐dependent study of cholesterol (Ch) nucleation to investigate the effect of calcium ions in the moderate supersaturated bile. In taurocholate/lecithin (TC/L) bile at a TC to L ratio of 5.1, the presence of calcium ions enhanced the nucleation rate of Ch. Contrarily, we observed the delayed nucleation of Ch after ~30 days in TC/L bile at a ratio of 2.0, regardless of the calcium ions. From the physical chemistry standpoint, the cholesterol activity (ChAT) is believed to be the driving force for Ch nucleation together with the sufficient nucleation sites. Hence, the micellar formation models along with the binding of TC monomers to calcium ions interpreted the time‐dependent results. Furthermore, a quasielastic light–scattering technique detected the formation of micelles and vesicles. In conclusion, the presence of calcium ions in TC/L bile at a high ratio enhances the vesicle appearance for nucleation sites and the high ChAT values for fast nucleation rate of Ch. However, an increase in the L concentration (i.e., low ratio bile) plays a significant role in the prevention of Ch gallstone formation, compared to the decrease in calcium ion concentration.

Role of In-Situ Generated CO2 Bubbles in Heterogeneous Nucleation of Solid Solutes in the Precipitation by Pressure Reduction of Gas-Expanded Liquid (PPRGEL) Process

Precipitation by pressure reduction of a gas-expanded liquid (PPRGEL) entails dissolution of subcritical CO2 in the solution of an organic solvent and a solid solute resulting in a gas-expanded liquid (GEL). Depressurization of a GEL leads to evolution of CO2 bubbles along with a large decrease in temperature facilitating solid precipitation. An illustrative system comprising CO2 (1), acetone (2), and cholesterol (3) is considered. It is proposed that CO2 bubbles generated from the depressurized GEL provide a substrate for heterogeneous nucleation of cholesterol. The size of nucleated CO2 bubbles and their rate of generation with time are predicted. The effective size of the CO2 bubbles ranges over 200–500 μm and their generation rate is ∼106–107 #/s. Comparing these with the corresponding solute nuclei size and nucleation rates, and considering the GEL–solid interfacial energetics, we conclude that the CO2 bubbles provide the required surface area for solid nucleation.

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.

Influence of dietary tender cluster beans (Cyamopsis tetragonoloba) on biliary proteins, bile acid synthesis and cholesterol crystal growth in rat bile

Tender cluster beans (CBs; Cyamopsis tetragonoloba) are observed to possess anti-lithogenic potential in experimental mice. Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile in addition to supersaturation of cholesterol. This study aimed at evaluating the influence of CB on biliary glycoproteins, low molecular weight (LMW) and high molecular weight (HMW) proteins, cholesterol nucleation time, and cholesterol crystal growth in rat hepatic bile. Groups of rats were fed for 10weeks with 0.5% cholesterol to render the bile lithogenic. Experimental dietary interventions were: 10% freeze-dried CB, 1% garlic powder or their combination. Incorporation of CB into HCD decreased the cholesterol saturation index in bile, increased bile flow and biliary glycoproteins. Dietary CB prolonged cholesterol nucleation time in bile. Electrophoresis of biliary proteins showed the presence of high concentration of 27kDa protein which might be responsible for the prolongation of cholesterol nucleation time in the CB fed group. Proteins of 20kDa and 18kDa were higher in CB treated animals, while the same were less expressed in HCD group. Biliary proteins from CB fed animals reduced cholesterol crystal growth index which was elevated in the presence of proteins from HCD group. Cholesterol-7α-hydroxylase and cholesterol-27-hydroxylase mRNA expression was increased in CB treated animals contributing to the bile acid synthesis. Thus, the beneficial anti-lithogenic effect of dietary CB which primarily is due to reduced cholesterol saturation index was additionally affected through a modulation of the nucleating and anti-nucleating proteins that affect cholesterol crystallization.

Cholesterol nucleation-promoting potency of retinol binding protein

Objective To detect the nucleation-promoting potency of retinol binding protein (RBP) in the simulated bile system,and observe the process of cholesterol nucleation and crystallization.Methods Small and synthetic bile systems were synthesized.RBP (RBP group),α1-acid glycoprotein (AAG,AAG group) and albumin ( Alb,Alb group) were added into the Small bile respectively.The derived indices of nucleation time,crystal growth rate and final concentration of crystal were studied by Busch method.Alb ( control group) and RBP (experimental group) were added into the Small and synthetic bile systems respectively.The nucleation time was detected by polarizing microscopy according to the Holan method and the nucleation-promoting potency was calculated according to the Holzbach method. The morphological dynamics of cholesterol nucleation and crystallization were observed under a light microscope.All data were analyzed by using the analysis of variance or the t test.Results The derived indices of nucleation time,crystal growth rate and final concentration of crystal were 0.66,1.29 and 1.01 in the AAG group,and 0.73,1.02,0.95 in the RBP group,respectively.In the Small bile system,the average nucleation time of cholesterol monohydrate crrystals were ( 12.2 ± 1.2 )days in the control group and (8.2 ± 1.5)days in the experimental group,respectively,with a significant difference between the 2 groups (t =2.97,P ＜ 0.05 ).The nucleation activity of RBP was 0.67 in the Small bile system.In the synthetic bile system,the average nucleation time of cholesterol monohydrate crystals were (9.5 ± 1.1 ) days in the control group and (7.5 ± 1.1 )days in the experimental group,respectively,with a significant difference between the 2 groups ( t =2.35,P ＜ 0.05 ).The nucleation activity of RBP was 0.79 in the synthetic bile system.The process of cholesterol nucleation and crystallization was divided into 4 stages,and the split phenomenon appeared in the bile system at day 21.Conclusion RBP is a pro-nucleation factor in simulated bile system. Key words: Cholelithiasis; Cholesterol; Nucleating proteins

Resolving the Structure of Ordered Domains in Single Hydrated Lipid Bilayers by New X-Ray Diffraction Methodology

Saturated lipids spontaneously form rigid domain in lipid membranes. X-ray scattering from these domains indicate that their sizes vary between 2-60nm and that the lipids within are well ordered. This research characterizes the lipid packing and phase behavior of such domains in bilayers composed of sphingomyelin, ceramide, DPPC, POPC and cholesterol.Over the past 30 years, X-ray studies were performed on either lipid monolayers, or multilayers. Several attempts have been made over the past 20 years, to perform experiments on single lipid bilayers, all of which have failed due to the strong scattering of the bilayer's surrounding water. In order to overcome this challenge we have developed a new system, which enables measurements of single hydrated lipid bilayers using synchrotron radiation.1This research provided, for the first time, knowledge on the interactions between the opposing lipids of each leaflet at the core of the bilayer. Comparisons with lipid monolayers made of the same lipid composition showed that the phase behavior of the ordered domains differs greatly, indicating strong correlation between the opposing leaflets of the bilayer.2Large quantities of cholesterol monohydrate crystals are found in atherosclerotic plaques. Studies have shown that these crystals are an early cause of inflammation. We managed to characterize the conditions under which cholesterol molecules nucleate at the lipid bilayer. Results demonstrate that cholesterol nucleation depends on the lipid composition.2,3The study was awarded the 2011 Barenholz Prize for basic research on sphingolipids.1. Ziblat, R.; Leiserowitz, L.; Addadi, L., Angewandte Chemie-International Edition 2011, Accepted for publication.2. Ziblat, R.; Leiserowitz, L.; Addadi, L., J Am Chem Soc 2010, 132, (28), 9920-9927.3. Ziblat, R.; Kjaer, K.; Leiserowitz, L.; Addadi, L., Angewandte Chemie-International Edition 2009, 48, (47), 8958-8961.

Effect of dietary fenugreek seeds on biliary proteins that influence nucleation of cholesterol crystals in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallising and anticrystallising factors present in bile. Dietary fenugreek seed has been recently observed to possess anti-lithogenic potential in experimental mice. In the current animal study, we evaluated the effect of dietary fenugreek on the compositional changes in the bile, particularly its effect on glycoproteins, low-molecular-weight (LMW) and high-molecular-weight (HMW) proteins, cholesterol nucleation time and cholesterol crystal growth. Groups of Wistar rats were fed for 10 weeks with diets: (1) basal control (C), (2) C + fenugreek (12%), (3) high cholesterol diet (HCD) and (4) HCD + fenugreek (12%). Feeding of HCD containing 0.5% cholesterol for 10 weeks rendered the bile lithogenic. Incorporation of fenugreek into HCD decreased the cholesterol content (70.5%), total protein (58.3%), glycoprotein (27.5%), lipid peroxides (13.6%) and cholesterol saturation index (from 1.98 to 0.75) in bile, increased the bile flow rate (19.5%), prolonged the cholesterol nucleation time and reduced the vesicular form of cholesterol (65%), which was accompanied with an increase in smaller vesicular form (94%). There was an increase in biliary phospholipid (33%) and total bile acid (49%) contents in the HCD + fenugreek group as compared with the HCD group. Electrophoretic separation of biliary LMW proteins showed the presence of a high concentration of 28-kDa protein, which might be responsible for the prolongation of cholesterol nucleation time in the fenugreek-fed groups. These findings indicate that the beneficial anti-lithogenic effect of dietary fenugreek, which primarily is due to reduction in the cholesterol content in bile, was additionally affected through a modulation of the nucleating and anti-nucleating proteins, which, in turn, affect cholesterol crystallisation.

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.

Changing treatments for bacterial meningitis

The relations between the concentration of deoxycholic acid (DCA), the cholesterol saturation index, and the nucleation time in gall bladder bile were measured to determine the role of DCA in bile in the pathogenesis of cholesterol gall stone disease. Bile was obtained from patients with cholesterol gall stones (n = 30), subjects without gall stones (n = 35), and patients with pigment gall stones (n = 9). Three of 30 cholesterol gall stone patients and 10 of 35 gall stone free subjects were treated with antibiotics by mouth to decrease the concentration of bile DCA and determine the effect of DCA on biliary lithogenecity. Both the percentage and concentration of DCA in bile were similar in patients with and without cholesterol gall stones despite significant differences in their cholesterol saturation indices and nucleation times. Neither the percentage nor the concentration of DCA in bile correlated with either the cholesterol saturation index or the nucleation time. Analysis of subgroups with matching cholesterol saturation indices showed no correlation between the proportion of DCA in the bile and the cholesterol nucleation time. The proportion of DCA in bile was decreased by antibiotic treatment, but this had no effect on the cholesterol saturation index or nucleation time. These results suggest that DCA in bile is not responsible for biliary cholesterol saturation or cholesterol nucleation time.

Effect of dietary garlic and onion on biliary proteins and lipid peroxidation which influence cholesterol nucleation in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallizing and anticrystallizing factors present in bile. Dietary garlic and onion have been recently observed to possess anti-lithogenic potential in experimental mice. In this investigation, the role of biliary proteins from rats fed lithogenic diet or garlic/onion-containing diet in the formation of cholesterol gallstones in model bile was studied. Cholesterol nucleation time of the bile from lithogenic diet group was prolonged when mixed with bile from garlic or onion groups. High molecular weight proteins of bile from garlic and onion groups delayed cholesterol crystal growth in model bile. Low molecular weight (LMW) proteins from the bile of lithogenic diet group promoted cholesterol crystal growth in model bile, while LMW protein fraction isolated from the bile of garlic and onion groups delayed the same. Biliary LMW protein fraction was subjected to affinity chromatography using Con-A and the lectin-bound and unbound fractions were studied for their influence on cholesterol nucleation time in model bile. Major portion of biliary LMW proteins in lithogenic diet group was bound to Con-A, and this protein fraction promoted cholesterol nucleation time and increased cholesterol crystal growth rate, whereas Con-A unbound fraction delayed the onset of cholesterol crystallization. Biliary protein from garlic/onion group delayed the crystallization and interfered with pronucleating activity of Con-A bound protein fraction. These data suggest that apart from the beneficial modulation of biliary cholesterol saturation index, these Allium spices also influence cholesterol nucleating and antinucleating protein factors that contribute to their anti-lithogenic potential.

Gender Differences in Cholesterol Nucleation in Native Bile: Estrogen Is a Potential Contributory Factor

The incidence of gallstone disease is two to three times higher in women than in men, and female sex hormones, particularly estrogens, have been implicated as contributory factors. Cholesterol nucleation is the initial step in gallstone pathogenesis and proceeds from cholesterol-rich phospholipid vesicles. The aim of this study was to investigate if there is a difference in cholesterol nucleation rates in male and female bile and whether estrogen influences nucleation rates by interacting with cholesterol-rich regions known as "lipid rafts" that exist within the cholesterol-phospholipid vesicles of the bile. Cholesterol nucleation from native prairie dog bile and the interaction of estrogens with lipid rafts in model bile solutions were investigated using Förster resonance energy transfer (FRET). Female native bile samples showed a greater reduction in energy transfer than did male native bile, indicating that cholesterol nucleation occurred more readily in female bile than in male bile. Model bile experiments demonstrated that the addition of estrogen has a significant effect, either cholesterol nucleation or raft disruption, but only in samples containing cholesterol-rich rafts. These results suggest that estrogen interacts with cholesterol-rich rafts in vesicles within bile to promote cholesterol nucleation and predispose females to gallstone formation.

Biliary lipids and cholesterol gallstone disease

Biliary lipids are a family of four dissimilar molecular species consisting of a mixture of bile salts (substituted cholanoic acids), phospholipids, mostly (>96%) diacylphosphatidylcholines, unesterified cholesterol, and bilirubin conjugates known trivially as lipopigments. The primary pathophysiological defect in cholesterol gallstone disease is hypersecretion of hepatic cholesterol into bile with less frequent hyposecretion of bile salts and/or phospholipids. Several other gallbladder abnormalities contribute and include hypomotility, immune-mediated inflammation, hypersecretion of gelling mucins, and accelerated phase transitions; there is also reduced intestinal motility that augments "secondary" bile salt synthesis by the anaerobic microflora. Cholesterol nucleation is initiated when unilamellar vesicles of cholesterol plus biliary phospholipids fuse to form multilamellar vesicles. From these "plate-like" cholesterol monohydrate crystals, the building blocks of macroscopic stones are nucleated heterogeneously by mucin gel. Multiple Lith gene loci have been identified in inbred mice, paving the way for discovery of an ever-increasing number of LITH genes in humans. Because of the frequency of the metabolic syndrome today, insulin resistance and LITH genes all interact with a number of environmental cholelithogenic factors to cause the gallstone phenotype. This review summarizes current concepts of the physical-chemical state of biliary lipids in health and in lithogenic bile and outlines the molecular, genetic, hepatic, and cholecystic factors that underlie the pathogenesis of cholesterol gallstones.