Gallbladder Bile Samples Research Articles

Enterohepatic circulation of nanoplastics induced hyperplasia, epithelial-mesenchymal transition, and neutrophil extracellular traps in gallbladder

Increasing concerns surround nanoplastic’s health risks owing to global exposure emphasize clear understanding of their dynamic distribution and organ-specific molecular effects. We assessed the health risks associated with nanoplastics (100 nm) following oral ingestion. Using a fluorescent tracking system, we demonstrated their recyclability through gastrointestinal tract-liver-gallbladder axis, with specific accumulation in the gallbladder. Pathological alterations in the gallbladder and single-cell RNA sequencing data indicated that short-term (three weeks) exposure induces gallbladder epithelial hyperplasia, whereas long-term exposure (six weeks) induces progressive hyperplasia, epithelial-mesenchymal transition (EMT), and fibrosis. Nanoplastic exposure facilitates neutrophil extracellular trap (NET) formation. Various nanoplastics were identified in human gallbladder bile samples using scanning electron microscopy (SEM) and Raman spectroscopy. Consistently, epithelial hyperplasia and neutrophil infiltration were observed in the gallbladder tissues, alongside nanoplastic detection in the bile. Our findings offer insights into the understanding of the enterohepatic-biliary recycling route of nanoplastics and their potential toxic consequences.

Protein Phosphatase 1 Regulatory Subunit 3 Beta rs4240624 Genotype Is Associated With Gallstones and With Significant Changes in Bile Lipidome

Gallstone disease (GSD) associates with significant morbidity and mortality. Decreased secretion of bile acids has been suggested as a driving factor for GSD. Recently, we linked the protein phosphatase 1 regulatory subunit 3 beta (PPP1R3B) rs4240624 genotype to decreased bile acid levels in bile. In this study, we investigated whether these individuals had an increased risk for GSD as well as the differences in the lipid composition of the gallbladder bile of these individuals compared to controls and patients with GSD. Bile acids, cholesterol, and phospholipid levels in gallbladder bile samples were enzymatically measured in 46 patients (34 female, age 45.7 ± 9.8 years, BMI 41.3 ± 4.4 kg/m2) who underwent elective laparoscopic Roux-en-Y gastric bypass. The lipidome of gallbladder bile was analyzed using high-performance liquid chromatography-mass spectrometry. Gallstone status was evaluated using abdominal ultrasonography before the surgery. The G allele of PPP1R3B rs4240624 was significantly associated with GSD in patients with obesity. We validated this association in the UK Biobank. Bile lipidomics demonstrated that 13 of the 17 minor lipid classes measured were higher in individuals with the G allele. The concentrations of bile acids, cholesterol, and phospholipids, as well as the cholesterol saturation index, were lower in patients with GSD than in those without gallstones. GSD had an effect similar to that of PPP1R3B genotype on minor lipids. The PPP1R3B rs4240624 genotype is associated with gallstones and with changes in gallbladder bile similar to those observed in patients with gallstones, suggesting that the PPP1R3B genotype contributes to the risk of gallstones by altering the bile lipidome.

Regional Differences in Bile Acid Composition in Gallbladder Bile

Background: Chemical and structural analyses of gallstones (GS) from the Indian subcontinent has shown that the formation of GS type is dependent on regional and dietary factors. Aim of the Study: The aim is to determine the proportion of primary and secondary bile acids in gallbladder (GB) bile in patients with GS from South and North India using high-performance liquid chromatography (HPLC). Materials and Methods: Standards for primary and secondary bile acids were prepared and concentrations were determined by reversed-phase C18 HPLC column. Thirty-three GB bile samples from southern India and 28 samples from northern states of India were analyzed for differences in the proportion of primary and secondary bile acids. Ethics Committee of Gleneagles Global Health City, Chennai, approved the study. Statistical Analysis: concentration of bile acids (in mmol/L) were expressed as median and range. Chi-square test and Mann–Whitney U-test were applied. A P < 0.05 was considered as significant. Results: The median concentrations of cholic acid (CA) (P = 0.005) and its derivative deoxycholic acid (DCA) (P < 0.006) were significantly high in GB bile samples from South India with no differences in the concentration of chenodeoxycholic acid between the two samples. Furthermore, samples from North India had a significantly higher proportion of lithocholic acid (LCA) and low DCA compared to samples from South India. Conclusion: Primary bile acid CA and its derivative is high in GB bile from South; the proportion of hepatotoxic LCA is significantly high with low concentrations of DCA in bile samples from North India.

Clinical characteristics and management of acute cholecystitis after cardiovascular surgery.

Acute cholecystitis (AC) is a severe complication after cardiovascular surgery (CS). The purpose of this study was to delineate the clinical picture of AC after CS to propose an optimal treatment strategy. We retrospectively reviewed the records of 88 patients who underwent cholecystectomy for grade II or III AC between 2008 and 2019 (AC after CS: Group CS, n=37; AC without CS: Group non-CS, n=51). The proportion of grade III AC in Group CS was significantly higher than that in Group non-CS (73% vs 41%, P=.005). Furthermore, the incidences of acalculous (81% vs 39%) and gangrenous (86% vs 59%) AC were significantly higher in Group CS (P<.05 for both). In Group CS, 11 patients had had percutaneous drainage preceding surgery, for whom cholecystectomy within 3days was eventually necessary because their general condition was exacerbated. The incidence of a positive culture from the gallbladder bile and blood samples of Group CS were significantly higher (P<.05 for both); multidrug-resistant bacteria were detected at an especially high rate. However, the morbidity rate was comparable, and zero mortality was achieved in both groups. Timely surgical intervention without hesitation is recommended for AC after CS.

Protein Phosphatase 1 Regulatory Subunit 3B Genotype at rs4240624 Has a Major Effect on Gallbladder Bile Composition.

The protein phosphatase 1 regulatory subunit 3B (PPP1R3B) gene is a target of farnesoid X receptor (FXR), which is a major regulator of bile acid metabolism. Both PPP1R3B and FXR have been suggested to take part in glycogen metabolism, which may explain the association of PPP1R3B gene variants with altered hepatic computed tomography attenuation. We analyzed the effect of PPP1R3B rs4240624 variant on bile acid composition in individuals with obesity. The study cohort consisted of 242 individuals from the Kuopio Obesity Surgery Study (73 men, 169 women, age 47.6 ± 9.0 years, body mass index 43.2 ± 5.4 kg/m2) with PPP1R3B genotype and liver RNA sequencing (RNA‐seq) data available. Fasting plasma and gallbladder bile samples were collected from 50 individuals. Bile acids in plasma did not differ based on the PPP1R3B rs4240624 genotype. However, the concentration of total bile acids (109 ± 55 vs. 35 ± 19 mM; P = 1.0 × 10−5) and all individual bile acids (also 7α‐hydroxy‐4‐cholesten‐3‐one [C4]) measured from bile were significantly lower in those with the AG genotype compared to those with the AA genotype. In addition, total cholesterol (P = 0.011) and phospholipid (P = 0.001) levels were lower in individuals with the AG genotype, but cholesterol saturation index did not differ, indicating that the decrease in cholesterol and phospholipid levels was secondary to the change in bile acids. Liver RNA‐seq data demonstrated that expression of PPP1R3B, tankyrase (TNKS), Homo sapiens chromosome 8 clone RP11‐10A14.5 (AC022784.1 [LOC157273]), Homo sapiens chromosome 8 clone RP11‐375N15.1 (AC021242.1), and Homo sapiens chromosome 8, clone RP11‐10A14 (AC022784.6) associated with the PPP1R3B genotype. In addition, genes enriched in transmembrane transport and phospholipid binding pathways were associated with the genotype. Conclusion: The rs4240624 variant in PPP1R3B has a major effect on the composition of gallbladder bile. Other transcripts in the same loci may be important mediators of the variant effect.

Gram staining of gallbladder bile samples is useful for predicting surgical site infection in acute cholecystitis patients undergoing an early cholecystectomy

Microbiological assessment of gallbladder bile is important for postoperative management in cholecystectomy for acute cholecystitis (AC). Gram staining is used as the first step in the assessment, in order to preliminarily detect bacteria in the bile sample. This study was conducted to evaluate the clinical significance of Gram staining results in the development of postoperative surgical site infection (SSI) in AC patients. A total of 428 AC patients, who underwent an early cholecystectomy with microbiological assessment of gallbladder bile, were enrolled in this retrospective study. The clinical usefulness of the Gram staining results was evaluated by univariate and multivariate regression analyses. Of the 428 patients, 298 patients (69.6%) were diagnosed with bile infection by the Gram staining method. The rate of SSI was higher in patients with bile infection (9.7%) than in those without the infection (0.8%). The multivariate analysis indicated that the bile infection diagnosed by Gram staining (odds ratio: 9.091; P=.033) was an independent factor to predict SSI development, along with open surgery. Gram staining diagnosis of bile infection in an early cholecystectomy for AC is useful for predicting postoperative SSI development, which should benefit postoperative management.

Differential proteomics analysis of bile between gangrenous cholecystitis and chronic cholecystitis

To establish human biliary protein expression profiles of gangrenous cholecystitis, chronic cholecystitis, and to discover differently expressed proteins for gangrenous cholecystitis by comparative proteomics, we gathered human gallbladder bile samples from gangrenous cholecystitis and chronic cholecystitis patients, respectively After removing the bile salts and lipid peptide fragments were identified by the iTRAQ-coupled LC-MS/MS technology,then identified in SwissProt with Mascot software. A total of 2251 proteins from chronic cholecystitis patients and 2180 proteins from gangrenous cholecystitis patients were identified. A total of 575 differential proteins were found between gangrenous cholecystitis and chronic cholecystitis, 159 proteins were over-expressed and 416 proteins were under-expressed in gangrenous cholecystitis. By bio-informatics analysis, in gangrenous cholecystitis, cell death, necrosis,immune response of neutrophils, apoptosis and degranulation of cells were activated; while cell survival, fatty acid metabolism, transport of molecular and proliferation of cells were inhibited, which might reflect the de-compensatory phase. Pathway analysis showed acute phase proteins were changed, indicating the role of the inflammatory response in the pathogenesis of gangrenous cholecystitis. Six acute phase proteins were found up-regulated,implying a close linkage to gangrenous gallbladder. Our study could be applicable in the biomarker discovery of gangrenous cholecystitis.

Detection of Salmonella human carriers in Colombian outbreak areas.

Salmonellosis, a zoonotic and foodborne disease, is a public health problem in developing countries. With the aim of identifying human carriers of Salmonella, a survey was performed in five regions of Colombia with reported salmonellosis outbreaks. The general population and cholecystectomy surgical patients were included in this study. Stool samples from 667 volunteers and gallbladder bile samples from 199 surgical patients were examined. Detection of Salmonella from cultured stool and bile samples was determined by polymerase chain reaction (PCR). Multiplex PCR and biochemical and serological tests were performed to identify the serovars of the isolates. Nine (1.35%) stool samples were positive for Salmonella: two S. Newport, two S. Anatum, one S. Sinstorf, and four Salmonella spp. A total of 11 gallbladder bile samples were positive: S. Enteritidis was isolated from 3 bile cultures (1.5%), and 8 samples (4%) were positive for Salmonella spp. Our results show the presence of Salmonella carriers in the inhabitants of regions with reported outbreaks and suggest that these carriers are potential sources of infection in endemic and epidemic cases. Carriers also suggest Salmonella zoonotic transmission, since broiler and beef cattle are hosts to the Salmonella serotypes isolated. It is important to establish the source of infection in regions where salmonellosis is endemic in order to control transmission.

Conjugated bile acids in gallbladder bile and serum as potential biomarkers for cholesterol polyps and adenomatous polyps.

To identify conjugated bile acids in gallbladder bile and serum as possible biomarkers for cholesterol polyps (CPs) and adenomatous polyps (APs). Gallbladder bile samples and serum samples were collected from 18 patients with CPs (CP group), 9 patients with APs (AP group), and 20 patients with gallstones (control group) from March to November, 2013. High performance liquid chromatography (HPLC) assay with ultraviolent detection was used to detect the concentration of 8 conjugated bile acids (glycocholic acid, GCA; taurocholic acid, TCA; glycochenodeoxycholic acid, GCDCA; taurochenodeoxycholic acid, TCDCA; glycodeoxycholic acid, GDCA; taurodeoxycholic acid, TDCA; taurolithocholic acid, TLCA; tauroursodeoxycholic acid, TUDCA) in bile samples and serum samples. The diagnostic efficacy of serum GCA, GCDCA and TCDCA was evaluated. These 8 conjugated bile acids in gallbladder bile and serum were completely identified within 10 minutes with good linearity (correlation coefficient: R&gt;0.9900; linearity range: 3.91-500 µg/mL). Among these conjugated bile acids, the levels of gallbladder bile GCDCA and TCDCA in the CP group were significantly higher than those in the AP group (p&lt;0.05). Furthermore, serum GCDCA and TCDCA as well as GCA were significantly higher in the AP group than the CP group (p&lt;0.05). Serum GCDCA alone (≤12 µg/mL) had relatively better diagnostic efficacy than the other conjugated bile acids. The levels of serum GCA, GCDCA and TCDCA may be valuable for differentiation of APs and CPs.

Long-term follow-up may be needed for pancreaticobiliary reflux in healthy adults

PurposeThe reflux of pancreatic enzymes into the biliary tract is associated with chronic inflammation and increases cellular proliferation of the biliary epithelium, leading to biliary carcinoma. The aim of this study is to detect the incidence of occult pancreaticobiliary reflux (OPBR) in patients who underwent elective cholecystectomy.MethodsForty-seven patients with symptomatic gallstones who underwent cholecystectomy were recruited for this study. The gallbladder bile samples were obtained from the specimen of gallbladder and the amylase level was measured. The immunohistochemistry of p53, SMAD4 and Ki-67 were performed for the detection of metaplasia and dysplasia.ResultsBiliary amylase was higher than the serum amylase in 10 patients (group A, 15,402.66 ± 33,592.43 IU/L; group B, 13.06 ± 18.12 IU/L). The mean age was 67.2 years in group A and 51.2 in group B (P < 0.01). The ratio of male to female was 1:2.3 and 1:1.8 in group A and B, respectively (P = 0.297). Eight patients in group A and thirteen patients in group B had inflammation (P = 0.014). The positive results of the Ki-67 test were exhibited in five cases in each group (P = 0.024).ConclusionResults from the study indicate that the age was older, degree of inflammation and positive rate of Ki-67 were higher when OPBR was suspected. In conclusion, the patients with OPBR would need long-term follow-up, because the OPBR can cause dysplasia and the reflux of pancreatic juice may be considered as a risk factor for extrahepatic bile duct carcinoma.

Calcium content of different compositions of gallstones and pathogenesis of calcium carbonate gallstones

Our aim was to investigate the calcium content of different gallstone compositions and the pathogenic mechanisms of calcium carbonate gallstones. Between August 2001 and July 2007, gallstones from 481 patients, including 68 calcium carbonate gallstones, were analyzed for total calcium content. Gallbladder bile samples from 33 cases and six controls were analyzed for pH, carbonate anion level, free-ionized calcium concentration and saturation index for calcium carbonate. Total calcium content averaged 75.6 %, 11.8 %, and 4.2 % for calcium carbonate, calcium bilirubinate and cholesterol gallstones. In 29.4 % of patients, chronic and/or intermittent cystic duct obstructions were caused by polypoid lesions in the neck region and 70.6 % were caused by stones. A total of 82 % of patients had chronic low-grade inflammation of the gallbladder wall and 18.0 % had acute inflammatory exacerbations. In the bile, we found the mean pH, mean carbonate anion, free-ionized calcium concentrations, and mean saturation index for calcium carbonate to be elevated in comparison to controls. From our study, we found chronic and/or intermittent cystic duct obstructions and low-grade GB wall inflammation lead to GB epithelium hydrogen secretion dysfunction. Increased calcium ion efflux into the GB lumen combined with increased carbonate anion presence increases SI_CaCO(3) from 1 to 22.4. Thus, in an alkaline milieu with pH 7.8, calcium carbonate begins to aggregate and precipitate.

Development of a real-time PCR assay for the detection of Clonorchis sinensis DNA in gallbladder bile and stone samples from patients with cholecystolithiasis

High prevalence of cholecystolithiasis in parts of East Asia has been postulated to be associated with Clonorchis sinensis infection. This study describes the development of a TaqMan-based real-time PCR assay for the detection of C. sinensis DNA in gallbladder bile and stone samples from patients with cholecystolithiasis. Primers and probe targeting the cytochrome c oxidase subunit 1 gene of mitochondrial DNA proved to be highly specific for C. sinensis and did not amplify other related heterogeneous DNA samples. The detection limit of this assay was 0.1 pg of adult C. sinensis genomic DNA. All of the egg-positive samples determined by microscopy yielded positive results by real-time PCR assay and that genetic testing of gallbladder stones using real-time PCR was considered as the most effective means for assessing C. sinensis infection status. This assay not only contributes to a greater understanding of stone pathogenesis but also benefits patients with cholecystolithiasis by facilitating effective diagnosis, treatment, and relapse prevention.

Osteopontin and integrin are involved in cholesterol gallstone formation

SummaryBackgroundThis study aimed to investigate the role of osteopontin and its receptor, integrin αv, in gallstone formation using human tissue specimens and a guinea pig lithogenic model.Material/MethodsThe nucleation role of osteopontin was determined in patients’ and normal gallbladder bile samples in vitro. Normal gallbladder was the control, and gallstone gallbladders were divided into group I (with normal epithelia) and group II (with degenerated epithelia) based on pathology change. Immunostaining, mRNA and protein expressions of osteopontin and integrin αv were analyzed. The animals were randomly divided into a lithogenic diet group and a normal diet group; the osteopontin mRNA expression in gallbladder and liver and osteopontin concentrations were determined.ResultsOsteopontin prolonged nucleation time and inhibited the pro-nucleating role induced by calcium in human bile in vitro. Immunostaining for osteopontin and integrin αv in human gallbladder tissues showed a higher reactivity in Group I than control group and Group II. The immunostaining in Group II was weaker than control group; similar results were observed for mRNA and protein expression of osteopontin and integrin αv. In the animal assay, the mRNA expression and concentration of osteopontin in gallbladder and liver gradually increased at initial stages and decreased in later stages. The concentrations of osteopontin in bile and serum of guinea pig showed similar trends.ConclusionsOur results suggest that osteopontin is involved in cholesterol gallstone formation, and the role of osteopontin might correlate with integrin αv and calcium.

Effect of twice-daily oral administration of hydrocortisone on the bile acids composition of gallbladder bile in dogs

To investigate the effects of twice-daily oral administration of hydrocortisone on the bile acids composition of gallbladder bile in dogs. 6 placebo-treated control dogs and 6 hydrocortisone-treated dogs. Dogs received hydrocortisone (median dose, 8.5 mg/kg) or a gelatin capsule (control group) orally every 12 hours for 84 days. Gallbladder bile samples were obtained via percutaneous ultrasound-guided cholecystocentesis from each dog before (day 0 [baseline]), during (days 28, 56, and 84), and after (days 28p, 56p, and 84p) treatment for differentiated quantification of unconjugated bile acids and taurine-conjugated and glycine-conjugated bile acids via high-performance liquid chromatography-tandem mass spectrometry. Treatment with hydrocortisone for 84 days resulted in significant and reversible increases in the concentrations of unconjugated bile acids (ie, cholic, chenodeoxycholic, and deoxycholic acids) and a significant and reversible decrease in the concentration of total taurine-conjugated bile acids, compared with baseline or control group values. Treatment with hydrocortisone had no effect on bile concentrations of glycine-conjugated bile acids. In dogs, hydrocortisone administration caused reversible shifts toward higher concentrations of the more hydrophobic unconjugated bile acids (chenodeoxycholic acid and deoxycholic acid) and toward lower concentrations of the amphipathic taurine-conjugated bile acids in gallbladder bile. These data suggest that similar bile acids changes could cause major alterations in gallbladder structure or function over time in hypercortisolemic dogs.

Gallbladder Inflammation is Associated with Increase in Mucin Expression and Pigmented Stone Formation

Mucin is a high molecular weight glycoprotein that plays an important role in protecting the gallbladder epithelium from the detergent effect of bile. However, it also participates in gallstone formation. There is little information about a possible relationship between gallbladder inflammation and mucin expression or gallbladder stones' characteristics. The aims of this study were to investigate stone characteristics and patterns of mucin expression in the gallbladder epithelium and bile of gallstone patients, in relation to inflammation. Gallbladder bile and tissue samples from 21 patients were obtained at surgery. Mucin content was evaluated by gel filtration on a Sepharose CL-4B column. Dot blot for bile mucin apoproteins and immunohistochemistry staining for gallbladder mucosal mucin apoproteins were performed with antibodies to MUC2, MUC3, MUC5AC, MUC5B and MUC6. Staining intensity score (0-3) was used for assessment of antigen expression and the level of inflammation. Gallstone cholesterol content was determined in 16 patients. MUC 5AC and MUC 5B were demonstrated in 95.4 and 100% of gallbladder bile samples, respectively. Immunohistochemistry staining with antibodies to MUC 2, MUC 3, MUC 5AC, MUC 5B and MUC 6 were positive in 0, 100, 85.7, 100 and 95.4% of the gallbladder mucosal samples, respectively. Pigmented brown stones were associated with a higher level of gallbladder inflammation. Mucin species expressed in gallbladder epithelium are MUC3, MUC5AC, MUC5B and MUC6. MUC5AC and MUC5B are secreted into bile. Inflammation of the gallbladder is accompanied by a higher level of MUC5AC expression and is associated with pigmented brown stones.

Psoriasis treated with ursodeoxycholic acid: three case reports

We report three Japanese patients with psoriasis vulgaris associated with nonalcoholic fatty liver disease in which the skin lesions dramatically resolved after treatment of the fatty liver disease with ursodeoxycholic acid (UDCA). According to the literature, arachidonic acid is released from phospholipid by phospholipase A(2) (PLA(2)) and is a precursor of eicosanoids, including prostaglandins, leucotrienes, and thromboxanes, which are potent inflammatory mediators. PLA(2) activity has been reported to be significantly raised in the serum and skin tissue of patients with psoriasis. UDCA has been reported to suppress the increased activity of group IIA PLA(2), a secretory low-molecular-weight PLA(2) (PLA(2)IIA), in HepG2 cells (a human hepatoblastoma-derived cell line) and in gallbladder and gallbladder bile samples from patients with cholesterol stones. Thus, UCDA may improve the skin lesions of patients with psoriasis by suppressing PLA(2)IIA activity.

An Animal Model of Black Pigment Gallstones Caused by Nanobacteria

Black pigment gallstones are often founded in gallbladder, but their pathogenesis is unclear. The present study was undertaken to study the role of nanobacteria in pigment gallstone formation in Japanese white rabbits. Nanobacteria were successfully cultured from 3 of 7 cholecystolithiasis patients gallbladder (without acute cholecystitis) bile samples and affirmed by Hoechst 33258 staining and specific immunostaining using monoclonal antibody. Nanobacteria were injected into rabbits' gallbladder. After 2 weeks follow-up, the incidence of black pigment gallstones in rabbits was significantly greater in nanobacteria-injected group (8/10) than that in DMEM-injected group (2/10) and that in hydroxyapatite-injected group (2/10). This study indicates that nanobacteria exists in gallbladder bile of cholelithiasis patients and causes the formation of black pigment gallstones in rabbits. For the first time, we successfully established an animal model of black pigment gallstones caused by nanobacteria. This will be helpful in further delineating the pathogenesis of black pigment gallstones.

Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice

As previously reported by us, mice with targeted disruption of the CYP8B1 gene (CYP8B1-/-) fail to produce cholic acid (CA), upregulate their bile acid synthesis, reduce the absorption of dietary cholesterol and, after cholesterol feeding, accumulate less liver cholesterol than wild-type (CYP8B1+/+) mice. In the present study, cholesterol-enriched diet (0.5%) or administration of a synthetic liver X receptor (LXR) agonist strongly upregulated CYP7A1 expression in CYP8B1-/- mice, compared to CYP8B1+/+ mice. Cholesterol-fed CYP8B1-/- mice also showed a significant rise in HDL cholesterol and increased levels of liver ABCA1 mRNA. A combined CA (0.25%)/cholesterol (0.5%) diet enhanced absorption of intestinal cholesterol in both groups of mice, increased their liver cholesterol content, and reduced their expression of CYP7A1 mRNA. The ABCG5/G8 liver mRNA was increased in both groups of mice, but cholesterol crystals were only observed in bile from the CYP8B1+/+ mice. The results demonstrate the cholesterol-sparing effects of CA: enhanced absorption and reduced conversion into bile acids. Farnesoid X receptor (FXR)-mediated suppression of CYP7A1 in mice seems to be a predominant mechanism for regulation of bile acid synthesis under normal conditions and, as confirmed, able to override LXR-mediated mechanisms. Interaction between FXR- and LXR-mediated stimuli might also regulate expression of liver ABCG5/G8.

Biliary Sludge Is Formed by Modification of Hepatic Bile by the Gallbladder Mucosa

We studied 22 patients with symptomatic microlithiasis to determine whether a contributory role of the gallbladder in the early stage of cholesterol gallstone formation exists. We compared the merits of different methods (ultrasonography and microscopy) and sources (hepatic or gallbladder) of bile samples for diagnosing microlithiasis. Paired hepatic and gallbladder bile samples were studied with polarizing microscopy. Nucleation time, bile salts, phospholipid, cholesterol, cholesterol saturation index (CSI), bilirubin, total protein, albumin and mucin concentration were measured. All patients had abdominal ultrasound examination. With polarizing microscopy as the standard, ultrasonography was positive in 13 patients (59%) and negative in 9 (41%). All gallbladder bile samples were positive for microlithiasis by microscopy. Only one hepatic bile sample was positive (P < .0001). There was a disproportional enrichment of total protein, albumin, and mucin (P < .05) in the gallbladder bile and a conversion of bilirubin diglucuronide to monoglucuronide (P < .01). Gallbladder samples had lower CSI but a faster nucleation time (P < .001), which correlates inversely with CSI, total protein, and mucin concentration. Biochemical composition and physical chemical behavior of hepatic bile are modified during residence in the gallbladder, contributing to sludge formation. Gallbladder bile has a lower calculated CSI, higher deconjugation of bilirubin, protein and mucin concentration and crystals were present. Hepatic bile samples are inappropriate for microscopic detection of microlithiasis.

Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers (Biophysical J., In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles. However, under certain (low phospholipid) conditions, transient intermediate polymorphs may form. These findings, documenting single-crystal analysis in bulk solution, provide an experimental approach to investigating factors influencing biliary cholesterol crystal nucleation and growth as well as other processes of nucleation and crystallization in liquid systems.