Watching fat digestion.

During fat digestion a number of physicochemical events can be seen directly by light microscopy. Under simulated physiological conditions, hydrolysis of emulsified fat droplets by human pancreatic lipase in the presence of colipase and bile salt micelles proceeds with the sequential formation of two visible product phases. A lamellar liquid crystalline or crystalline phase containing calcium and ionized fatty acid forms first; this is followed by the production of a "viscous isotropic" phase composed predominantly of monoglycerides and protonated fatty acids.

Aspirin prevention of cholesterol gallstone formation in prairie dogs.

When prairie dogs (Cynomys ludovicianus) are fed a diet containing cholesterol, a marked increase in gallbladder mucin secretion parallels the evolution of cholesterol supersaturated bile. Gelation of mucin precedes the precipitation of cholesterol liquid and solid crystals and the development of gallstones. Aspirin given to prairie dogs inhibited mucin hypersecretion and gel accumulation and prevented gallstone formation without influencing the cholesterol content of supersaturated bile. This suggests that gallbladder mucin is a nucleation matrix for cholesterol gallstones.

Crustacean intestinal detergent promotes sterol solubilization.

Although crustacean tissue cholesterol content is high, Crustacea, like other arthropods; are incapable of cholesterol synthesis, and presumably are dependent for maintaining tissue cholesterol stores on the intestinal absorption of ingested sterol. A detergent, N-(N-dodecanoylsarcosyl)taurine, representative of a set of detergents synthesized by the crustacean hepatopancreas and secreted into the intestine, is capable of efficient cholesterol solubilization, and thus of promoting sterol absorption.

The physical chemistry of cholesterol solubility in bile. Relationship to gallstone formation and dissolution in man.

We determined the maximum solubilities of cholesterol in aqueous conjugated bile salt-egg lecithin-cholesterol systems as a function of several physical-chemical variables including those of physiological importance employing phase equilibria techniques. Equilibration rates are influenced by time and the method of sample preparation in that metastable supersaturation is readily induced at high bile salt: lecithin ratios, and equilibrium saturation by dissolution is achieved sluggisly at low bile salt:lecithin ratios. Equilibrium values for cholesterol saturation vary with the bile salt species, bile salt: lecithin ratio, temperature, ionic strength, and, in particular, with the total concentration of biliary lipids. Within physiological bile salt:lecithin ratios at 37 degreesC the influence of bile salt type and ionic strength is small, whereas the effects of bile salt:lecithin ratio and the total lipid concentration are major factors. We plotted on triangular coordinates a family of cholesterol solubility curves for each total lipid concentration (0.30--30 g/dl) and computed fifth-degree polynomial equations for each curve. With both the curves and the polynomial equations the "per cent cholesterol saturation" of fasting gallbladder and hepatic biles from patients with and without gallstones was calculated and both methods gave similar values. These results deomonstrate that by employing cholesterol saturation values appropriate to the total lipid concentration (range 0.2--24.9 g/dl) of individual biles, all cholesterol stone patients have supersaturated gallbladder biles, (mean, 132% [normal weight individuals], and 199% [morbidly obese individuals]). With controls and pigment stone patients the mean values were 95 and 98%, respectively, and in both approximately 50% of biles were supersaturated. Fasting hepatic biles were significantly more supersaturated than gallbladder biles (means 228--273%). Cholesterol monohydrate crystals were found in the majority of gallbladder (83%) and hepatic (58%) biles of cholesterol gallstone patients but were not observed in pigment stone patients or controls. We conclude that of the several factors in addition to the bile salt:lecithin ratios which can influence the cholesterol saturation of bile the total lipid concentration is the predominant determinant physiologically. Our results demonstrate that (a) metastable supersaturation is frequent in both normal and abnormal biles, (b) cholesterol gallstone patients have supersaturated gallbladder and hepatic biles without exception, and (c) the predominant driving force for cholesterol precipitation appears to be the absolute degree of cholesterol supersaturation.

Effects of taurodihydrofusidate, a bile salt analogue, on bile formation and biliary lipid secretion in the rhesus monkey.

Bile salts play a major role in bile formation and biliary lipid secretion. Sodium taurodihydrofusidate (TDHF), a derivative of the antibiotic fusidic acid, closely resembles bile salts in terms of structure, micellar characteristics, and capacity ot solubilize otherwise insolbule lipids. We have therefore studied the biliary secretion of this bile salt analogue and its influence on bile formation and biliary lipid secretion in primates. Alert, unanesthetized female rhesus monkeys prepared with a total biliary fistula were allowed to reach a steady bile salt secretion rate before each study. In three animals (group I),[14C]TDHF was infused intravenously. Most of the compound was secreted rapidly in bile chemically unchanged. The biliary secretion of this drug produced a twofold increase in bile flow; however, the bile salt output was markedly reduced during the infusion. In spite of this reduction, the phospholipid output remained essentially unchanged whereas the cholesterol output increased almost twofold. In five other animals (group II), the effect of TDHF on the bile salt secretion was further investigated by an intravenous infusion of [14C]taurocholate followed by a combined infusion of [14C]taurocholate and TDHF. When TDHF was added to the infusate, a reduction in the [14C]taurocholate output and a progressive rise in the plasma [14C]taurocholate concentration were observed in each animal. An analysis of the data in both groups indicates that (a) the most likely explanation to account for the decreased bile salt output is that the bile salt analogue, TDHF, interfered with bile salt secretion into the biliary canaliculi; (b) TDHF induces a greater secretion of biliary water than was observed with bile salts, an effect consistent with a stimulation of the bile salt-independent canalicular flow; (c) at similar 3alpha-hydroxysteroid secretion rates TDHF caused a significant increase in cholesterol secretion compared to that induced by bile salt. This finding suggests that TDHF affects cholesterol metabolism or secretion in a way distinct from bile salts. Thus, the solubilization of biliary lipids in mixed micelles, although essential, is only one of the factors which determine their secretion into bile.

Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones.

Because mucin glycoproteins may be important in the pathophysiology of gallstones, we studied the relationship among biliary lipids, gallbladder mucin secretion, and gallstone formation in cholesterol-fed prairie dogs. Organ culture studies of gallbladder explants revealed that the incorporation of [(3)H]glucosamine into tissue and secretory gallbladder glycoproteins was significantly increased at 3, 5, 8, and 14 d of feeding. Peak secretion of labeled mucin occurred at 5 d, when total tissue and secreted glycoprotein production was fivefold greater than control. Gel filtration of the secreted glycoprotein on Sepharose 4B indicated that the majority of radioactivity was present in a macromolecule of > 1 million molecular weight. The increased secretion of gallbladder mucin was organ specific, in that [(3)H]glucosamine incorporation into glycoproteins of stomach and colon was unaffected by cholesterol feeding. Similarly, the incorporation of [(3)H]mannose into gallbladder membrane glycoproteins was not altered by cholesterol feeding. The rate of glycoprotein synthesis and secretion returned to normal upon withdrawal of the cholesterol diet, and ligation of the cystic duct before cholesterol feeding prevented gallbladder mucin hypersecretion. Both results indicate that the stimulus to mucin secretion was a constituent of bile. Gallbladder bile after 5 d contained cholesterol in micelles, liquid crystals, and crystals, whereas hepatic bile remained a single micellar phase throughout cholesterol feeding. For this reason the cholesterol-saturation indices of gallbladder bile were compared in both homogenized and centrifuged samples. The micellar phase of gallbladder bile was appreciably less saturated than homogenized bile at 5 and 8 d, which reflects the continuous nucleation of cholesterol in the gallbladder. Purified human gallbladder mucin gels were shown to induce nucleation of lecithin-cholesterol liquid crystals from supersaturated hepatic bile. These in turn gave rise to cholesterol monohydrate crystals within 18 h. Control supersaturated hepatic bile could not be nucleated by the addition of other proteins, and was stable for days upon standing. These results suggest that the increase in cholesterol content of bile in cholesterolfed prairie dogs stimulates gallbladder mucus hypersecretion, and that gallbladder mucus gel is a nucleating agent for biliary cholesterol.

Effects of bile and bile salts on growth and membrane lipid uptake by Giardia lamblia. Possible implications for pathogenesis of intestinal disease.

We have shown previously that ox and pig bile accelerate in vitro growth of Giardia lamblia. We have now investigated the possible mechanisms by which mammalian biles promote parasite growth. Growth effects of (a) ox, pig, guinea pig, and human biles, (b) pure bile salts, and (c) egg and soybean lecithins were studied in the presence of a lecithin-containing growth medium. Individually, dilute native bile and pure sodium taurocholate (TC), glycocholate (GC), and taurodeoxycholate (TDC) promoted parasite growth; growth was most marked with biles of high phospholipid content, with biles enriched in more hydrophobic bile salts (ox approximately equal to human greater than pig greater than guinea pig) and with micellar concentrations of GC and submicellar concentrations of TC and TDC. By measuring uptake of radiolabeled biliary lipids from bile and bile salt-supplemented growth medium, we showed that the parasite consumed bile lipids, with the rank order lecithin greater than bile salts. Apparent net uptake of cholesterol was considered to be due to exchange, since net loss of cholesterol from the growth medium was not detected. Although bile and bile salt-stimulated parasite growth was associated with enhanced lecithin uptake, reduction in generation time was observed at low bile and bile salt concentrations when lecithin uptake was similar to bile free controls. Thus, bile salts may stimulate Giardia growth initially by a mechanism independent of enhanced membrane phospholipid uptake. However, since Giardia has no capacity to synthesize membrane lipid, biliary lecithin may be a major source of phospholipid for growth of this parasite.

Filamentous, helical, and tubular microstructures during cholesterol crystallization from bile. Evidence that cholesterol does not nucleate classic monohydrate plates.

Precipitation of cholesterol in gallbladder bile is believed to produce platelike cholesterol monohydrate crystals directly. We report complementary time-lapse microscopic studies of cholesterol crystallization from model bile that reveal initial assembly of filamentous cholesterol crystals covered by a monomolecular layer of lecithin. Over a few days, the filaments evolved through needle, helical, and tubular microstructures to form classical platelike cholesterol monohydrate crystals. Similar crystallization phenomena were observed in human gallbladder biles from cholesterol but not pigment stone patients. Synchrotron x-ray diffraction of the earliest filaments suggested a cholesterol monohydrate polymorph or admixture with an anhydrous cholesterol precursor. However, density gradient centrifugation of filamentous crystals revealed that their density was 1.032 g/ml, consistent with anhydrous cholesterol. Conventional x-ray diffraction of transitional crystalline forms was consistent with pure cholesterol monohydrate crystals, as were the equilibrium platelike crystals. These novel findings suggest that crystalline cholesterol in bile may not be completely mature or hydrated initially, but undergoes a series of transformations to become thermodynamically stable monohydrate plates. These observations have important implications for understanding the control of cholesterol crystallization in bile, as well as explaining putative crystal cytotoxicity during gallstone formation.

Influence of bile salts on molecular interactions between sphingomyelin and cholesterol: relevance to bile formation and stability.

Bile salts enhance secretion of cholesterol into bile and its subsequent solubilization with phosphatidylcholine in mixed micelles. Sphingomyelin, a major structural lipid of the hepatocyte canalicular membrane, and disaturated phosphatidylcholines are known to impede nucleation of solid cholesterol crystals in supersaturated model systems. To understand these effects physico-chemically, we compared the influence of bile salts on interactions of cholesterol with natural sphingomyelins, as well as with dipalmitoyl and egg yolk phosphatidylcholines using various in vitro systems. Submicellar bile salts enhanced significantly bidirectional transfer of dehydroergosterol (a fluorescent cholesterol analog) between sphingomyelin and egg yolk phosphatidylcholine vesicles in the rank order taurocholate < tauroursodeoxycholate < taurodeoxycholate. Quasielastic light scattering of serially diluted sphingomyelin-taurocholate mixtures (1:1 molar ratio, 3 g/dl) revealed metastable temperature-dependent transitions between globular micelles, rod-shaped micelles and vesicles, suggesting that phase transitions under these experimental conditions were metastable only at temperatures below 37 degrees C. Ternary phase diagrams of all sphingomyelins and dipalmitoyl phosphatidylcholine with cholesterol and taurocholate (37 degrees C, 3 g/dl, 0.15 M NaCl) were identical. Compared to systems containing egg yolk phosphatidylcholine, the 1-phase micellar zone and 2- and 3-phase solid cholesterol crystal-containing zones were reduced markedly while the 2-phase zone with stable cholesterol-sphingomyelin liquid crystals was greatly expanded. Our results suggest that the high affinity of cholesterol for sphingomyelin is lost in the presence of bile salts. Our findings may be relevant to secretion of cholesterol into bile and to its inability to crystallize in the hepatocyte canalicular lumen or its surrounding membranes.

Pan-sulfation of bile salts markedly increases hydrophilicity and essentially abolishes self- and hetero-association with lecithin.

In chronic liver disease, partially and to a lesser extent completely (pan-)sulfated common bile salts are synthesized, yet little information is available concerning their physical-chemical characteristics. We studied solution properties of pan-sulfated common free, taurine and glycine-conjugated bile salts, and the interactions of taurodeoxycholate di-sulfate (TDC-S) with lecithin. By reverse-phase HPLC, pan-sulfated glycine and taurine-conjugated bile salts were very hydrophilic, with hydrophobic indices 1.7 to 2.5 units lower than their non-sulfated congeners. In contrast to non-sulfated species, pan-sulfated free and glycine-conjugated bile salts produced simple potentiometric titration curves without precipitation of bile salt below the pK'A of the carboxylic acids. By quasi-elastic light scattering, critical micellar concentrations of TDC-S fell from 28 mM in 0.15 M NaCl to 3 mM in 4.0 M NaCl, a value slightly higher than that of TDC. TDC-S formed very small micelles (hydrodynamic radii approx. 11A) that, in contrast to TDC, did not grow with increases in bile salt (7-66 mM) or NaCl (0.15-2.0 M) concentrations. TDC-S formed mixed micelles with lecithin in 0.15 M NaCl, but with a micellar zone drastically reduced compared with that of the non-sulfated congener. However, in 4 M NaCl, the micellar zone of TDC-S expanded and approached that of the non-sulfated parent compound. Therefore, under physiological conditions, pan-sulfation of common bile salts should largely eliminate their capacity to form mixed micelles with membrane lipids.

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate.

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.

Insulin administered intranasally as an insulin-bile salt aerosol. Effectiveness and reproducibility in normal and diabetic subjects.

Efficacy and reproducibility of insulin administered intranasally as an insulin-deoxycholate 1% (w/v) aerosol to normal and diabetic subjects were assessed by measurements of blood glucose and serum insulin levels. Following administration of 0.5 U insulin/kg with the unconjugated bile salt to fasting volunteers (N = 29), peak serum insulin levels of 103 +/- 49 microU/ml above baseline were observed at 10 min. Blood glucose concentration began to fall by 10 min, reaching 54 +/- 14% of control levels by 30 min, and returning to baseline by 60-80 min. Blood glucose response and peak serum insulin levels were reproducible when the same aerosol dose was repeatedly administered to the same subjects; however, intersubject variations were noted. By comparing serum insulin levels after i.v. and nasal routes of administration, nasal insulin absorption was approximately 10% as efficient as intravenous insulin. Dose response studies revealed that peak serum insulin concentrations were a linear function of the administered dose. In subjects with type I and type II diabetes mellitus, serum insulin levels increased in a manner similar to controls, and resulted in a prompt reduction of blood glucose concentration. However, in contrast to normal subjects, the duration of the glucose response was more prolonged, lasting as long as 5 h. Nasal administration of insulin as an aerosol with bile salts or bile salt analogs should be further evaluated as a possible nonparenteral approach to insulin therapy.

Quantitative trait loci mapping for cholesterol gallstones in AKR/J and C57L/J strains of mice.

Quantitative trait locus (QTL) mapping was used to locate genes that determine the difference in cholesterol gallstone disease between the gallstone-susceptible strain C57L/J and the gallstone-resistant strain AKR/J. Gallstone weight was determined in 231 male (AKR x C57L) F(1) x AKR backcross mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butterfat for 8 wk. Mice having no stones and mice having the largest stones were genotyped at approximately 20-cM intervals to find the loci determining cholesterol gallstone formation. The major locus, Lith1, mapped near D2Mit56 and was confirmed by constructing a congenic strain, AK. L-Lith1(s). Another locus, Lith2, mapped near D19Mit58 and was also confirmed by constructing a congenic strain AK.L-Lith2(s). Other suggestive, but not statistically significant, loci mapped to chromosomes 6, 7, 8, 10, and X. The identification of these Lith genes will elucidate the pathophysiology of cholesterol gallstone formation.

Pathogenesis of gallstones.

Gallstones form as a result of many disorders. Unphysiologic supersaturation, generally from hypersecretion of cholesterol, is essential for the formation of cholesterol gallstones. The other common abnormalities of the hepatobiliary system in gallstone patients are accelerated nucleation, gallbladder hypomotility, and the accumulation of mucin gel. An attempt is made here to relate hypersecretion of cholesterol and biliary supersaturation to the molecular basis of the associated phenomena. Supersaturation of bile with calcium hydrogen bilirubinate, the acid calcium salt of unconjugated bilirubin, is essential for pigment gallstone formation, but its magnitude remains undefined in model systems. Nucleation and the precipitation of calcium hydrogen bilirubinate with the polymerization of the pigment in the gallbladder, together with the deposition of the inorganic salts, calcium carbonate and phosphate, result in black pigment gallstone formation. On the basis of ex vivo muscle studies, gallbladder hypomotility is unlikely in patients with black pigment stones but is invariably present in patients with cholesterol stones. Pigment supersaturation in the gallbladder is the result of hepatic hypersecretion of bilirubin conjugates in hemolytic disorders and possibly enterohepatic cycling of unconjugated bilirubin in nonhemolytic states. Less common is bile salt hyposecretion from impaired synthesis in constitutional disorders and cirrhosis, and uncompensated interruption of the enterohepatic circulation in ileal dysfunction syndromes. Bile salt deficiency causes incomplete solubilization of unconjugated bilirubin and impaired binding of calcium ions. Stasis and anaerobic bacterial infection are responsible for brown pigment stones, which usually form in the bile ducts. In addition to the precipitation of calcium hydrogen bilirubinate that remains unpolymerized, there is also the deposition of the calcium salts of saturated fatty acids and free bile acids, both of which are the result of bacterial enzymatic hydrolysis of biliary lipids.

Mapping cholesterol gallstone susceptibility (Lith) genes in inbred mice.

The individual risk for developing cholesterol gallstones in response to specific environmental factors is determined by complex genetics involving multiple genes. In this review, we introduce inbred mice as a model to localise and identify the murine genes that harbour cholesterol gallstone susceptibility alleles (Lith genes). These genes are associated with increased risk of gallstone formation when mice are fed a lithogenic diet containing cholesterol and cholic acid. We summarise the steps involved in localising the chromosomal regions that harbour Lith genes, focusing particularly on the initial step known as quantitative trait locus mapping, which employs breeding crosses of gallstone-susceptible and gallstone-resistant inbred mouse strains. Subsequent steps to narrow the chromosomal regions of the quantitative trait loci and identify the underlying Lith genes are outlined, with particular reference to the examples of Lith1 and Lith2, the first discovered quantitative trait loci associated with murine cholesterol cholelithiasis. We have now reported five quantitative trait loci for murine cholelithogenesis, which are officially named Lith1 through Lith5. Once the genes underlying these quantitative trait loci and other chromosomal loci from ongoing mouse crosses are identified and confirmed, the 'road-map' for discovery of orthologous human LITH genes will be available and, thereafter, their putative roles in cholesterol gallstone formation can be tested in selected human populations.

Effects of sodium taurodihydrofusidate on nasal absorption of insulin in sheep.

To investigate the utility of a novel adjuvant, sodium taurodihydrofusidate (STDHF), as an enhancer of mucosal permeation of drugs, experiments involving intranasal insulin:STDHF administration in sheep were performed. Rabbit erythrocyte lysis assays were employed to assess the relative membrane lytic activity of STDHF, as well as that of its glycine-conjugated analogue, compared with a nonionic detergent and a common bile salt. Equivalent weight concentrations of the fusidates were found to be 5- to 10-fold less lytic than the bile salt and at least 100-fold less lytic than the nonionic detergent laureth-9. Provided the concentration of STDHF was greater than its critical micellar concentration, formulations of insulin with STDHF greatly enhanced intranasal insulin absorption. Optimal nasal insulin absorption was attained at a molar ratio of STDHF to insulin of 5:1. In addition, intranasal absorption was linearly related to insulin dose. Compared with intravenous administration, the mean bioavailability of intranasal insulin was 16.4%. Interovine variability was low, with a coefficient of variation of 14% for 12 animals. It was found that intranasal absorption of sodium insulin was not significantly different from that of zinc insulin. However, formulations of both crystalline insulin preparations were absorbed more efficiently than a formulation prepared using commercially available solutions of U-500 insulin. The results taken together indicate that STDHF is an excellent enhancer of insulin absorption from the nasal mucosa.

The Louisiana State University Comprehensive Epilepsy Program: procedures and outcomes.

The first comprehensive epilepsy surgery center in Louisiana was established in 1990 at the Louisiana State University Medical Center in New Orleans by the Departments of Neurology and Neurosurgery. The center performs a wide variety of diagnostic tests essential for the medical and surgical treatment of epilepsy including EEG and video monitoring, quantitative hippocampal MRI volumetry, ictal SPECT brain scanning, intracranial evoked potential and subdural stimulation functional mapping, neuropsychological evaluations, and intracarotid amobarbital (Wada) language and memory localization. Surgical interventions include (1) the placement of subdural strip and grid electrodes, depth electrodes, and foramen ovale electrodes, (2) temporal lobectomies, and (3) frontal, temporal, parietal, and occipital lobe resections. From August 1990 through October 1995 41 patients with medically intractable seizures underwent neurosurgical procedures for epilepsy. Thirty-five patients had resective surgery, while six had only intracranial monitoring by subdural or intracerebral electrodes. The surgical outcomes thus far compare favorably with those of other established centers in North America.

Pathogenesis of gallstones.

Gallstones are composed principally of cholesterol monohydrate crystals (cholesterol stones) or the acid salt of calcium bilirubinate (pigment stones). Cholesterol stones and the black variety of pigment gallstones form in sterile gallbladder bile whereas brown pigment gallstones form in infected bile. Biliary supersaturation is the principal pathophysiological defect and is hepatic in origin. Supersaturation results from excessive secretion of cholesterol or bilirubin conjugates, the precursors of unconjugated bilirubin, and/or, deficient secretion of bile salt and lecithin, the solubilizers of these otherwise insoluble lipids. As has now being clarified for cholesterol stones, an imbalance in pro- and antinucleating biliary proteins, hypersecretion of gallbladder mucin and gallbladder dysmotility possibly from cholesterol "toxicity" to sarcolemma, all interact to promote nucleation. Crystallisation results in suspension of cholesterol crystals or bilirubinate salts in gallbladder mucin gel and is known as "biliary sludge". It is believed today that this stage is essential for evolution of both cholesterol and pigment stones. Brown pigment gallstones form principally in the bile ducts. These stones result from infection of the biliary tree, most commonly due to obstruction from migrating gallbladder stones. Chemical compositions of brown and black pigment stones are different: In black stones, calcium bilirubinate is polymerized and oxidatively degraded but in brown stones, calcium bilirubinate is present as the unpolymerised salt. Brown stones differ also from black stones in containing calcium fatty acid soaps, a result of bacterial phospholipase A1 hydrolysis of biliary lecithin. Both types of pigment gallstones may contain crystalline inorganic calcium salts especially carbonate (gallbladder stones) and phosphate (bile ducts stones). Since a molecular understanding of the multiple defects that lead to cholesterol and pigment gallstones is becoming a reality, the future holds much promise for gallstone prevention.

Roles of cholesterol and bile salts in the pathogenesis of gallbladder hypomotility and inflammation: cholecystitis is not caused by cystic duct obstruction.

BACKGROUND AND PURPOSE: A large number of human and animal studies have challenged the hypothesis that cystic duct obstruction by gallstones causes cholecystitis. These studies suggest that lithogenic bile that can deliver high cholesterol concentrations to the gallbladder wall causes hypomotility and creates a permissive environment that allows normal concentrations of hydrophobic bile salts to inflame the mucosa and impair muscle function inhibiting gallbladder emptying. High concentrations of cholesterol increase its diffusion rates through the gallbladder wall where they are incorporated into the sarcolemmae of muscle cells by caveolin proteins. High caveolar cholesterol levels inhibit tyrosine-induced phosphorylation of caveolin proteins required to transfer receptor-G protein complexes into recycling endosomes. The sequestration of these receptor-G protein complexes in the caveolae results in fewer receptors recycling to the sarcolemmae to be available for agonist binding. Lower internalization and recycling of CCK-1 and other receptors involved in muscle contraction explain gallbladder hypomotility. PGE2 receptors involved in cytoprotection are similarly affected. Cells with a defective cytoprotection failed to inactivate free radicals induced by normal concentrations of hydrophobic bile salts resulting in chronic inflammation that may lead to acute inflammation. Ursodeoxycholic acid salts (URSO) block these bile salts effects thereby preventing the generation of free radicals in muscle cells in vitro and development of cholecystitis in the ligated common bile duct in guinea pigs in vivo. Treatment with URSO improves muscle contraction and reduces the oxidative stress in patients with symptomatic cholesterol gallstones by lowering cholesterol concentrations and blocking the effects of hydrophobic bile salts on gallbladder tissues.

Disruption of gallbladder smooth muscle function is an early feature in the development of cholesterol gallstone disease.

UNLABELLED: BACKGROUND; Decreased gallbladder smooth muscle (GBSM) contractility is a hallmark of cholesterol gallstone disease, but the interrelationship between lithogenicity, biliary stasis, and inflammation are poorly understood. We studied a mouse model of gallstone disease to evaluate the development of GBSM dysfunction relative to changes in bile composition and the onset of sterile cholecystitis. METHODS: BALB/cJ mice were fed a lithogenic diet for up to 8 weeks, and tension generated by gallbladder muscle strips was measured. Smooth muscle Ca(2+) transients were imaged in intact gallbladder. KEY RESULTS: Lipid composition of bile was altered lithogenically as early as 1 week, with increased hydrophobicity and cholesterol saturation indexes; however, inflammation was not detectable until the fourth week. Agonist-induced contractility was reduced from weeks 2 through 8. GBSM normally exhibits rhythmic synchronized Ca(2+) flashes, and their frequency is increased by carbachol (3 µm). After 1 week, lithogenic diet-fed mice exhibited disrupted Ca(2+) flash activity, manifesting as clustered flashes, asynchronous flashes, or prolonged quiescent periods. These changes could lead to a depletion of intracellular Ca(2+) stores, which are required for agonist-induced contraction, and diminished basal tone of the organ. Responsiveness of Ca(2+) transients to carbachol was reduced in mice on the lithogenic diet, particularly after 4-8 weeks, concomitant with appearance of mucosal inflammatory changes. CONCLUSIONS & INFERENCES: These observations demonstrate that GBSM dysfunction is an early event in the progression of cholesterol gallstone disease and that it precedes mucosal inflammation.