A systematic study on 33 gallbladder stones resembling adult Clonorchis sinensis worms.

Limited studies provide direct evidence of Clonorchis sinensis adults in the early stage of gallbladder stone formation. Our current research systematically studied 33 gallbladder stones resembling adult worms and shed light on the definite connection of C. sinensis infection with concomitant cholelithiasis. A total of 33 gallbladder stones resembling adult C. sinensis worms were systematically analysed. Fourier transform infrared spectroscopy, scanning electron microscopy and X-ray energy spectrometry were used to analyse the composition and microstructure. Meanwhile, a histopathological examination of the stone was carried out. The 33 gallbladder stones resembling adult C. sinensis worms included nine calcium carbonate (CaCO3) stones, 12 bilirubinate stones and 12 mixed stones. Clonorchis sinensis eggs were found in 30 cases, including all CaCO3 and mixed stones. Parasite tissues were detected in 12 cases, which were mainly CaCO3 stones or bilirubinate-CaCO3 mixed stones. The outer layer of stones was wrapped with 12.88% calcium salt, as revealed by X-ray energy spectrometry, while surprisingly, many C. sinensis eggs were found in the inner part of these stones. Based on our current findings, we concluded that calcification and packaging occurred after C. sinensis adult entrance into the gallbladder, subsequently leading to the early formation of CaCO3 or bilirubinate-CaCO3 mixed gallbladder stones. This discovery highlights definite evidence for C. sinensis infection causing gallbladder stones.

Free fatty acids and triglyceride change in the gallbladder bile of gallstone patients with pancreaticobiliary reflux.

BACKGROUND: Pancreaticobiliary reflux (PBR) causes chronic inflammation of the gallbladder mucosa and changes in the bile components, which are known to promote gallstone formation. This study aimed to investigate the bile biochemistry changes in gallstone patients with PBR and provide new clues for research on the involvement of PBR in gallstone formation. METHODS: Patients undergoing surgery for gallstones between December 2020 and May 2021 were eligible for inclusion. The bile biochemistry (including amylase, lipase, triglyceride, cholesterol, free fatty acids [FFAs], alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], and γ-glutamyl transferase [γ-GT]) of the included gallstone patients was analysed to determine correlations with PBR. RESULTS: In this study, 144 gallstone patients who underwent surgery were enrolled. Overall, 15.97 % of the patients had an increased bile amylase level, which was associated with older age and significantly higher bile levels of ALP, lipase, triglyceride, and FFAs. Positive correlations were observed between amylase and lipase, triglyceride, FFAs levels in the gallbladder bile. However, the bile levels of triglyceride, FFAs, and lipase were positively correlated with each other only in the PBR group and showed no significant correlation in the control (N) group. In addition, elevated bile FFAs levels were found to be an independent risk factor for gallbladder wall thickening. CONCLUSIONS: In conclusion, PBR-induced increase in FFAs and triglyceride in the gallbladder bile is a cause of gallstone formation, and an increase in bile ALP suggests the presence of cholestasis in PBR.

Multi-analyses of gallstones and correlation between their properties with the laboratory results.

This study explores the morpho-structure of gallstones (GSs) removed from 36 patients in NW Romania and correlate it with the laboratory results of the patients. GSs were analyzed by SEM-EDS, X-ray diffraction and IR, UV-Vis and X-ray photoelectron spectroscopy. The laboratory studies consisted in hematological, coagulation, biochemistry, immunological and tumor markers tests. The morphological and structural investigations allowed to classify the GS in five different types and to establish their mechanism of formation. Only macroscopic evaluation, SEM microscopy, FTIR and UV-Vis spectroscopy give different easily noticeable information for each GS type. EDS, XPS and XRD diffraction are recommended to distinguish pigment and carbonate stones from the other GS types and a carefully examination is needed to establish the differences between the pure cholesterol, the mixed cholesterol and the composite cholesterol stones, due to the high similarities. The variation of specific markers cannot differentiate the patients with pure cholesterol GS from those with mixed cholesterol and pigment GS and those with mixed cholesterol from those with composite cholesterol stones. Seven laboratory parameters (RDW-CV, MPV, PCT, GLUC-HK, WBC, PT, GPT) are the key indicators for the GS disease and trend to present generally higher values than normal.

Experimental verification of factors influencing calcium salt formation based on a survey of the development of ceftriaxone-induced gallstone-related disorder.

Ceftriaxone (CTRX) forms salts with calcium (Ca) in the gall bladder and bile duct, and induces the formation of gallstones. In this study, factors of CTRX-induced gallstone formation were extracted from the results of a retrospective survey using the Japanese Adverse Drug Event Report (JADER), and the causal relationship between the factors and gallstone formation was investigated. From JADER, 136 patients who developed 'gallstone-related disorder' with CTRX as a suspected drug were extracted. The incidence of gallstone-induced adverse effects was high in patients treated with CTRX at a dose exceeding the normal daily dose and in children younger than 10 years old, suggesting that CTRX at a high level is a factor for gallstone formation. Thus, after mixing CTRX and Ca2+ at different concentrations under different pH condition, the number of particles in the solutions was measured using a Coulter counter. As a result, the number of minute particles significantly increased at all pH values when Ca2+ and CTRX were mixed at a concentration of 10 mEq/L or higher and 1.5 g/L or higher, respectively. At pH 6.5 or 7.0, visible crystals were detected when 25 mEq/L of Ca2+ and 2.0 g/L of CTRX were mixed. Based on these findings, attention should be sufficiently paid to the development of 'gallstone-related disorder' in pediatric patients and in patients treated with CTRX at a dose exceeding the normal dose. Furthermore, gallstone formation and growth may be promoted when CTRX and Ca2+ coexist at high concentrations under low pH conditions.

[Mechanism of Calculus Bovis Sativus in inhibiting hepatocyte lipid deposition based on serum pharmacology].

The aim of this paper was to investigate the molecular mechanism of Calculus Bovis Sativus( CBS) in alleviating lipid accumulation in vitro by serum pharmacology. The CBS-containing serum of mice was obtained by serum pharmacology method to evaluate its effect on the proliferation of LO2 hepatocytes. The lipid reducing effects of CBS-containing serum through Nrf2 was evaluated by fructose-induced LO2 hepatocyte steatosis model,nuclear factor erythroid 2 related factor 2( Nrf2) agonist oltipraz combined intervention,cell oil red O staining and intracellular triglyceride( TG) content. The effects of CBS-containing serum on lipid peroxidation and hepatocytes apoptosis were evaluated by reactive oxygen species( ROS) and apoptosis assay,respectively. Real-time quantitative polymerase chain reaction( PCR) was used to detect the relative expression of lipid synthesis-related genes and apoptosis-related genes.RESULTS:: showed that CBS drug-containing serum had no significant effect on LO2 hepatocyte proliferation. As compared with the model group,CBS-containing serum could effectively reduce the formation of lipid droplets in fructose-induced LO2 hepatocytes,significantly reduce intracellular TG and ROS levels,and significantly reduce hepatocyte apoptosis rate( P < 0. 05). As compared with the model group,carbohydrate responsive element binding protein( ChREBP),sterol regulatory element binding protein-1 c( SREBP-1 c),fatty acid synthase( FAS),acetyl-CoA carboxylase 1( ACC1),stearoyl-CoA desaturase 1( SCD1),Bax and caspase-3 mRNA levels were significantly reduced in CBS drug-containing serum treatment group( P<0. 05). All of the above effects could be reversed by oltipraz.In conclusion,CBS-containing serum can significantly inhibit the fructose-induced LO2 liver fat deposition,and the mechanism may be related to reducing intracellular ROS level through the Nrf2 pathway and improving intracellular peroxidation state to reduce apoptosis.

Comparison of Virtual Unenhanced Images Derived From Dual-Energy CT With True Unenhanced Images in Evaluation of Gallstone Disease.

OBJECTIVE: The aim of this study was to compare gallstones on virtual unenhanced images and true unenhanced images acquired with dual-energy CT (DECT). MATERIALS AND METHODS: We enrolled 112 patients with right upper quadrant pain and clinically suspected acute cholecystitis or gallstone who underwent DECT--including unenhanced, arterial, and portal phases. Eighty-three gallstones with composition proven by semiquantitative Fourier transform infrared spectroscopy from 45 patients who had undergone cholecystectomy (40 cholesterol gallstones from 21 patients, 43 calcium gallstones from 24 patients) were included. CT images were retrospectively evaluated for stone size, contrast-to-noise ratio (CNR) of gallstone to bile, and visibility and density of gallstones for each image set. The visibility of each type of stone was compared with a paired t test. RESULTS: Both cholesterol and calcium stones measured smaller on virtual unenhanced images than on true unenhanced images, yielding a lower sensitivity of virtual unenhanced images for detecting small gallstones. Mean CNR of cholesterol stones was 2.45 ± 1.32 versus 1.67 ± 1.55 (p < 0.032) and that of calcium stones was 10.59 ± 7.15 and 14.11 ± 9.81 (p < 0.001) for virtual unenhanced and true unenhanced images, respectively. For calcium stones, two readers found 43 of 43 (100%) on true unenhanced images; one reader found 41 of 43 (95%) and the other, 37 of 43 (86%) on virtual unenhanced images. For cholesterol stones, one reader found 20 of 40 (50%) and the other 19 of 40 (47%) on true unenhanced images versus 34 of 40 (85%) and 30 of 40 (75%), respectively, on virtual unenhanced images. The visibility of cholesterol stones was higher on virtual unenhanced images, but that of calcium stones was lower. CONCLUSION: Virtual unenhanced images at DECT allow better visualization of cholesterol gallstones, but true unenhanced images allow better visualization of calcium and small gallstones.

Preparation, in Vitro and in Vivo Evaluations of Compound Calculus Bovis Sativus and Ornidazole Film.

The aim of this study was to develop and to investigate a film of compound Calculus Bovis Sativus (CBS) and ornidazole film. A uniform mucoadhesive film was herein successfully obtained by a film-forming solusion containing insoluable drug. This film, as a valid adjunct for the treatment of oral mucosal ulcer, consisted of two main drugs (CBS, ornidazole) and three polymers (hydroxypropyl methyl cellulose, chitosan, poly(vinyl alcohol) (PVA)). The film was prepared with the film-forming suspension, using casting-solvent evaporation technique. The drug content, release behavior, swelling index and mucoadhesive properties of the film were detected. Then the effects of the prepared film on a glacial acetic acid-induced oral mucosal ulceration model of rabbits were evaluated. Moreover, the in vivo release of bilirubin and ornidazole in saliva were also detected in the oral mucosae of healthy volunteers. The films showed favorable in vitro drug release behaviors and swelling properties. Mucosal wounds in the animals were significantly relieved. With the films well tolerated, the salivary concentrations of ornidazole were maintained above the minimum inhibitory concentration against CBS for about 2 h. The compound CBS and ornidazole film functioned better than the film only containing CBS and ornidazole did. Therefore, it is a potentially efficient drug delivery system for the treatment of oral ulcers.

Analysis of heterogeneous gallstones using laser-induced breakdown spectroscopy (LIBS) and wavelength dispersive X-ray fluorescence (WD-XRF).

Laser-induced breakdown spectroscopy (LIBS) is an emerging analytical technique with numerous advantages such as rapidity, multi-elemental analysis, no specific sample preparation requirements, non-destructiveness, and versatility. It has been proven to be a robust elemental analysis tool attracting interest because of being applied to a wide range of materials including biomaterials. In this paper, we have performed spectroscopic studies on gallstones which are heterogeneous in nature using LIBS and wavelength dispersive X-ray fluorescence (WD-XRF) techniques. It has been observed that the presence and relative concentrations of trace elements in different kind of gallstones (cholesterol and pigment gallstones) can easily be determined using LIBS technique. From the experiments carried out on gallstones for trace elemental mapping and detection, it was found that LIBS is a robust tool for such biomedical applications. The stone samples studied in the present paper were classified using the Fourier transform infrared (FTIR) spectroscopy. WD-XRF spectroscopy has been applied for the qualitative and quantitative analysis of major and trace elements present in the gallstone which was compared with the LIBS data. The results obtained in the present paper show interesting prospects for LIBS and WD-XRF to study cholelithiasis better.

Cholesterol gallstone disease: focusing on the role of gallbladder.

Gallstone disease (GSD) is one of the most common biliary tract diseases worldwide in which both genetic and environmental factors have roles in its pathogenesis. Biliary cholesterol supersaturation from metabolic defects in the liver is traditionally seen as the main pathogenic factor. Recently, there have been renewed investigative interests in the downstream events that occur in gallbladder lithogenesis. This article focuses on the role of the gallbladder in the pathogenesis of cholesterol GSD (CGD). Various conditions affecting the crystallization process are discussed, such as gallbladder motility, concentrating function, lipid transport, and an imbalance between pro-nucleating and nucleation inhibiting proteins.

Metastable and equilibrium phase diagrams of unconjugated bilirubin IXα as functions of pH in model bile systems: Implications for pigment gallstone formation.

Metastable and equilibrium phase diagrams for unconjugated bilirubin IXα (UCB) in bile are yet to be determined for understanding the physical chemistry of pigment gallstone formation. Also, UCB is a molecule of considerable biomedical importance because it is a potent antioxidant and an inhibitor of atherogenesis. We employed principally a titrimetric approach to obtain metastable and equilibrium UCB solubilities in model bile systems composed of taurine-conjugated bile salts, egg yolk lecithin (mixed long-chain phosphatidylcholines), and cholesterol as functions of total lipid concentration, biliary pH values, and CaCl2 plus NaCl concentrations. Metastable and equilibrium precipitation pH values were obtained, and average pKa values of the two carboxyl groups of UCB were calculated. Added lecithin and increased temperature decreased UCB solubility markedly, whereas increases in bile salt concentrations and molar levels of urea augmented solubility. A wide range of NaCl and cholesterol concentrations resulted in no specific effects, whereas added CaCl2 produced large decreases in UCB solubilities at alkaline pH values only. UV-visible absorption spectra were consistent with both hydrophobic and hydrophilic interactions between UCB and bile salts that were strongly influenced by pH. Reliable literature values for UCB compositions of native gallbladder biles revealed that biles from hemolytic mice and humans with black pigment gallstones are markedly supersaturated with UCB and exhibit more acidic pH values, whereas biles from nonstone control animals and patients with cholesterol gallstone are unsaturated with UCB.

Cholesterol gallstones and bile host diverse bacterial communities with potential to promote the formation of gallstones.

The prevalence of cholesterol gallstones has increased in recent years. Bacterial infection correlates with the formation of gallstones. We studied the composition and function of bacterial communities in cholesterol gallstones and bile from 22 cholesterol gallstone patients using culture-dependent and culture-independent methods. Altogether fourteen and eight bacterial genera were detected in cholesterol gallstones and bile, respectively. Pseudomonas spp. were the dominant bacteria in both cholesterol gallstones and bile. As judged by diversity indices, hierarchical clustering and principal component analysis, the bacterial communities in gallstones were different from those in bile. The gallstone microbiome was considered more stable than that of bile. The different microbial communities may be partially explained by differences in their habitats. We found that 30% of the culturable strains from cholesterol gallstones secreted ß-glucuronidase and phospholipase A2. Pseudomonas aeruginosa strains showed the highest ß-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones.

Two-part zero-inflated negative binomial regression model for quantitative trait loci mapping with count trait.

Poisson regression models provide a standard framework for quantitative trait locus (QTL) mapping of count traits. In practice, however, count traits are often over-dispersed relative to the Poisson distribution. In these situations, the zero-inflated Poisson (ZIP), zero-inflated generalized Poisson (ZIGP) and zero-inflated negative binomial (ZINB) regression may be useful for QTL mapping of count traits. Added genetic variables to the negative binomial part equation, may also affect extra zero data. In this study, to overcome these challenges, I apply two-part ZINB model. The EM algorithm with Newton-Raphson method in the M-step uses for estimating parameters. An application of the two-part ZINB model for QTL mapping is considered to detect associations between the formation of gallstone and the genotype of markers.

Determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations by liquid chromatography with mass spectrometry.

So far, the components responsible for the neuroprotective effects of Calculus bovis are unclear. Cholesterol, one of the major components in Calculus bovis, is easily oxidized into oxysterols, which possess direct or indirect neuroprotective effects proved by our and others' previous studies. Therefore, a liquid chromatography with mass spectrometry method coupled with ultrasonic extraction and solid-phase extraction was developed for the determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations. Chromatographic separation was achieved on a C18 column with isocratic elution at a flow rate of 1 mL/min. The established method showed good linearity (R(2) > 0.998), sensitivity with low limits of detection (0.06-0.39 µg/g), acceptable precisions (relative standard deviations ≤ 7.4%), stability (relative standard deviations ≤ 5.9%), and satisfactory accuracy (92.4-102.9%) for all analytes identified by different retention times, which could be applied for the determination of oxysterols. Five kinds of oxysterols proved to function as neuroprotectants were detected at different concentrations. Among them, 7ß-hydroxycholesterol and cholestane-3ß,5α,6ß-triol were rather abundant in the samples. It could be concluded that the potential neuroprotective components in Calculus bovis may be these oxysterols.

Identification of cholesterol gallstone with two-dimensional gel electrophoresis and mass spectrometry.

BACKGROUND AND RATIONALE: We aimed to provide novel information to better understand the molecular mechanisms underlying gallstones formation and explore the potential protein markers for gallstones progression. The gallbladder tissues were collected from 20 patients with cholesterol gallstone and 10 liver transplant donors from November 2010 to April 2011. The proteomics were compared between gallstone patients and controls by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were identified and validated by western blotting and real-time PCR. RESULTS: Total 19 protein spots were found to be different between two groups and 11 proteins were identified, among which 4 ones (such as Peroxiredoxin 3/Prdx3) were down-regulated and 7 (such as Tropomyosin 4/TPM4, Transgelin/SM22, Transthyretin/ TTR) were up-regulated in gallstone group. Results of western blotting and RT-PCR were consistent with the 2-DE results. CONCLUSION: The differentially expressed proteins of TTR, TPM4, SM22 and Prdx3 may play key roles in gallstone formation and may be markers for gallstone progression.

Metals in Human Gall, Bladder, and Kidney Stones Based on an Electron Microprobe Investigation.

Several particles of copper accompanied by a few particles of nickel, lead, and a compound composed of selenium containing minor Ni, Si, Cu, and Co were found in human gall, kidney, and bladder stones. The investigated particles occur as tiny grains, <10 µm in size, that are irregularly dispersed in the stones. Therefore, they were studied by scanning electron microscopy and qualitatively analyzed by energy dispersive system. One grain of copper contained a small amount of Ni and Zn, and some grains of nickel proved to contain Cr as trace element. Most of the discovered metals formed a single-phase grain. However, a few grains found in two gallstones were associated with inclusions of calcium and apatite. Based on the results presented in this contribution, we argue that most of the studied metals can be classified as endogenous particles, i.e., directly precipitated from the same fluids that formed their host human stones. This observation suggests that the precipitation and accumulation of metals in some human stones can be considered an efficient way to eliminate them from the human body.

[Substitution of antelope horn in Danqi Piantan capsule with artificial bezoar in vitro].

The in vitro cell culture experiment was conducted to study the effect of Danqi Piantan capsule (DPC) and DPC dislodge the antelope horn with artificial bezoar double (DPCBD) on nerve regeneration and blood vessel regeneration and preliminarily investigate the possibility of substituting antelope horn in DPC with artificial bezoar. In this experiment, rats were randomly divided into 5 groups: the blank serum control group, the model group, DPC groups (0.306 g x kg(-1) x d(-1), the same below), DPC remove of antelope horn (DPCRA) groups and DPCBD groups. Brain microvascular endothelial cells cultured in vitro (BMEC), astrocytes and neural stem cells (NSC) were co-cultured to simulate neurovascular unit, label neurons with microtubule associated protein III (ß-tubulin III) antibody and lable astrocytes with glial fibrillary acidicprotein (GFAP). ELISA was used for the detection of the content of BMEC lactate dehydrogenase instrument method (LDH), the inverted phase contrastmicroscope was adopted to observe the formation of BMEC tube like structure, the number of leukocytes and leukocytes adherent to BMEC were counted under the microscope, the expression levels of ß-tubulin III and the ratio of GFAP positive cells was detected with inimmunofluorescence, and RT-PCR method was used to detect NGF, BDNF, VEGF and VEGFr-2 mRNA. According to the result, compared with the model group, both DPC and DPCBD can reduce LDH leakage, promote the formation of BMEC tube like structure, inhibit leukocytes and their adhesion to BMEC, increase the ß-tubulin III positive cell differentiation proportion (P < 0. 01), reduce the proportion of GFAP positive cells (P < 0.01), increase the expressions of co-cultured NGF, VEGF, BDNF and VEGFr-2 mRNA to a certain extent, with the most significant difference on NGF and VEGF mRNA expressions (P < 0.05) and the same efficacy in both groups. DPCRA groups showed less impact on all indexes than that of DPCBD and DPC groups. The same efficacy of DPCBD and DPC on nerve regeneration and angiogenesis suggested that antelope horn in DPC can be substituted by artificial bezoar.

Nonalcoholic fatty liver disease and cholesterol gallstones: which comes first?

Nonalcoholic fatty liver disease (NAFLD) and gallstone disease (GD) are both highly prevalent in the general population and are associated with obesity, insulin resistance, dyslipidemia, and high dietary cholesterol intake. Insulin resistance is a key feature of both NAFLD and GD. Hepatic insulin resistance provides a crucial link between the metabolic syndrome, NAFLD, and increased cholesterol gallstone susceptibility. Hepatic insulin resistance is not only associated with accumulation of hepatic fat but also has a crucial role in supersaturation and excessive production of bile salts. It is not yet clear whether NAFLD is a precursor of GD or whether the presence of GD possibly indicates the presence of long-standing features of metabolic syndrome that accelerates the progression of NAFLD. Recent reports suggested the association between gallstones and nonalcoholic steatohepatitis and liver fibrosis. Importantly, both NAFLD and GD are both associated with high incidence of cardiovascular disease (CVD) and mortality. Emerging evidence suggests a potential benefit of statin therapy in NAFLD and GD. Further research is needed to determine (i) how the presence of NAFLD and GD is associated with CVD (ii) and whether the presence of GD in association with NAFLD increases the risk of liver fibrosis, and (iii) the impact of therapy of NAFLD in the incidence of GD.

Can the type of gallstones be predicted with known possible risk factors?: A comparison between mixed cholesterol and black pigment stones.

BACKGROUND: Pathogenesis of gallstones (GS) is multifactorial and multiple genetic and environmental factors have been identified in different populations for different types of GS with varying prevalence. However the role of the each aetiological factor on the formation of mixed cholesterol and black pigment GS has not being addressed adequately. Hence in this study we attempted to compare known possible risk factors for mixed cholesterol and black pigment GS among two groups of patients with two types of GS. METHODS: The study was done on a cohort of patients with symptomatic GS admitted to the Teaching Hospital Peradeniya, Sri Lanka over a period of 18 months. Clinical and epidemiological data and physical parameters of the patients were recorded and surgically removed GS were analyzed chemically and physically to identify the type of GS. In addition lipid profile was done in all the patients with normal serum bilirubin levels. RESULTS: A total of 86 patients were included in the study. Mixed cholesterol GS was significantly common among females than males (χ2 test, p = 0.029). Mixed cholesterol GS was commonly seen among patients belonging to Moor ethnicity (χ2 test, p = 0.009). Majority of patients with mixed cholesterol GS had body mass index above 25 kg/m2 (χ2 test, p = 0.018). Black pigment GS were significantly common among patients with type II diabetes mellitus (Fisher's exact test, p = 0.035). Further all the patients with chronic haemolytic anaemia and alcoholic cirrhosis had black pigment GS. Age, family history, Fasting Blood Glucose, dyslipidaemia, lipid profile, parity and use of oral contraceptive pills in females, smoking and alcohol intake in males did not differ significantly among patients in the two groups. CONCLUSION: Gender, ethnicity and body mass index can be used to predict the formation of mixed cholesterol GS and black pigment GS.

Quantitative phase analysis and molecular structure of human gallstones using synchrotron radiation X-ray diffraction and FTIR spectroscopy.

Human gallstones are the most common disorder in the biliary system, affecting up to 20 % of the adult population. The formation of gallstones is primarily due to the supersaturating of cholesterol in bile. In order to comprehend gallstone disease in detail, it is necessary to have accurate information about phase identification and molecular structure. Different types of gallstone samples were collected from the Middle East area after surgical operations including; cholesterol, pigment, and mixed gallstones. To estimate the basic information about the stone formation and the pathophysiology of cholelithiasis as well as to classify the collected human gallstones, attenuated total reflection Fourier transform Infrared spectrometry (ATR-FTIR) was used to analyze the different gallstone structures in the wavenumber range from 400 to 4000 cm-1. Calcium bilirubinate was specified by the bands at 1662 cm-1, 1626 cm-1, and 1572 cm-1, while cholesterol rings were designated by the bands at 1464, 1438, 1055, and 1022 cm-1. It can be assumed that all samples consist of mixed gallstones based on the doublets at 1375 cm-1 and 1365 cm-1. The levels of calcium bilirubin and various minerals varied among the analyzed samples, indicating the heterogeneity in their composition and suggesting potential implications for gallstone formation. Based on the quantitative phase analysis using synchrotron radiation X-ray diffraction (SR-XRD), two phases of anhydrous cholesterol as a major content and one phase of monohydrate cholesterols as trace content represent the main components of most of the gallstones. Additional phases of calcium carbonate in the form of calcite, vaterite, aragonite, and bilirubinate were also quantified. According to the outcomes of the FTIR and the SR-XRD measurements, there exists a statistical correlation between the different types of chemical constituents of the gallstones.

Gallbladder microbial species and host bile acids biosynthesis linked to cholesterol gallstone comparing to pigment individuals.

Gallstones are crystalline deposits in the gallbladder that are traditionally classified as cholesterol, pigment, or mixed stones based on their composition. Microbiota and host metabolism variances among the different types of gallstones remain largely unclear. Here, the bile and gallstone microbial species spectra of 29 subjects with gallstone disease (GSD, 24 cholesterol and 5 pigment) were revealed by type IIB restriction site-associated DNA microbiome sequencing (2bRAD-M). Among them (21 subjects: 18 cholesterol and 3 pigment), plasma samples were subjected to liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics. The microbiome yielded 896 species comprising 882 bacteria, 13 fungi, and 1 archaeon. Microbial profiling revealed significant enrichment of Cutibacterium acnes and Microbacterium sp005774735 in gallstone and Agrobacterium pusense and Enterovirga sp013044135 in the bile of cholesterol GSD subjects. The metabolome revealed 2296 metabolites, in which malvidin 3-(6''-malonylglucoside), 2-Methylpropyl glucosinolate, and ergothioneine were markedly enriched in cholesterol GSD subjects. Metabolite set enrichment analysis (MSEA) demonstrated enriched bile acids biosynthesis in individuals with cholesterol GSD. Overall, the multi-omics analysis revealed that microbiota and host metabolism interaction perturbations differ depending on the disease type. Perturbed gallstone type-related microbiota may contribute to unbalanced bile acids metabolism in the gallbladder and host, representing a potential early diagnostic marker and therapeutic target for GSD.