Bile Constituents Research Articles

Studying the intracellular bile acid concentration and toxicity in drug-induced cholestasis: Comprehensive LC-MS/MS analysis with human liver slices.

Drug-induced cholestasis (DIC) is a leading cause of drug-induced liver injury post-drug marketing, characterized by bile flow obstruction and toxic bile constituent accumulation within hepatocytes. This study investigates the toxicity associated with intracellular bile acid (BA) accumulation during DIC development. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, we examined intracellular BA concentrations in human precision-cut liver slices (PCLS) following the administration of cyclosporin A and chlorpromazine, both with and without an established BA mixture. Our findings indicate toxicity of cyclosporin A upon BA addition, while chlorpromazine's toxicity remained unaffected. Although neither drug led to the accumulation of all BAs intracellularly, BA mixture addition resulted in the accumulation of unconjugated BAs associated with DIC, such as deoxycholic acid (DCA) and cholic acid (CA). Additionally, cyclosporin A increased taurolithocholic acid (TLCA) concentrations. In the absence of the BA mixture, a decrease in conjugated BAs was observed, suggesting inhibition of BA metabolism by cholestatic drugs and warranting further investigation. The evident increase in CA and DCA for both drugs (and TLCA for cyclosporin A), despite not exacerbating toxicity with chlorpromazine, suggests these increases may be related to DIC development and possible toxicity. In conclusion, the current human PCLS model is appropriate for investigating and detecting essential contributors to DIC and can be used in future studies elucidating DIC ex vivo.

Analysis of various ATP-binding cassette transporters revealed quantification of ABCB4 as a potential diagnostic tool in primary sclerosing cholangitis (PSC).

ATP-binding cassette transporters are important proteins in regulating bile constituent transport between hepatocytes and the bile canalicular system. Dysfunctional transporters lead to accumulation of toxic bile components within hepatocytes or the biliary system, known as cholestasis, resulting in liver damage. It has been previously reported that two particular ATP-binding cassette transporters, ABCB4 and ABCB11, have altered expression in patients with primary sclerosing cholangitis (PSC). Interested in further analysis of expression patterns of ATP-binding cassette transporters in PSC patients, we investigated liver samples from 201 patients, including 43 patients with PSC and 51 patients with primary biliary cholangitis patients (PBC). In addition to ABCB4 and ABCB11, we also included other ATP-binding cassette transporters, to determine if upregulation of ABCB4 and ABCB11 is specifically found in the liver of patients with PSC. Retrospectively, formalin-fixed and paraffin-embedded liver biopsies, resections, and explants were selected to investigate the expression of ABCB1, ABCB4, ABCB11, ABCG5/8, and FXR1 using nanoString nCounter and immunohistochemistry for validation of differently expressed transporters seen in PSC liver samples in comparison to non-PSC liver specimens. Strikingly, ABCB4 was the only ATP-binding cassette transporter showing increased gene and protein expression in hepatocytes of PSC livers when compared to non-PSC liver specimens. Furthermore, ABCB4 protein expression also correlated with disease stage in PSC. Our study concluded that altered ABCB4 expression is specifically seen in liver specimens of PSC patients. Therefore, quantitative ABCB4 analysis may be an additional useful tool for the histopathological diagnosis of PSC to distinguish this entity from other cholangiopathies.

Interim results from an ongoing, open-label, single-arm trial of odevixibat in progressive familial intrahepatic cholestasis

Background & AimsPEDFIC 2, an ongoing, open-label, 72-week study, evaluates odevixibat, an ileal bile acid transporter inhibitor, in patients with progressive familial intrahepatic cholestasis. MethodsPEDFIC 2 enrolled and dosed 69 patients across two cohorts; all received odevixibat 120 μg/kg per day. Cohort 1 comprised children from PEDFIC 1, and cohort 2 comprised new patients (any age). We report data through 15 July 2020, with Week 24 of PEDFIC 2 the main time point analysed. This represents up to 48 weeks of cumulative exposure for patients treated with odevixibat from the 24-week PEDFIC 1 study (cohort 1A) and up to 24 weeks of treatment for those who initiated odevixibat in PEDFIC 2 (patients who received placebo in PEDFIC 1 [cohort 1B] or cohort 2 patients). Primary endpoints for this prespecified interim analysis were change from baseline to Weeks 22–24 in serum bile acids (sBAs) and proportion of positive pruritus assessments (≥1-point drop from PEDFIC 2 baseline in pruritus on a 0–4 scale or score ≤1) over the 24-week period. Safety monitoring included evaluating treatment-emergent adverse events (TEAEs). ResultsIn cohort 1A, mean change from PEDFIC 1 baseline to Weeks 22–24 of PEDFIC 2 in sBAs was -201 μmol/L (p <0.0001). For cohort 1B and cohort 2, mean changes from odevixibat initiation to weeks 22–24 in sBAs were -144 and -104 μmol/L, respectively. The proportion of positive pruritus assessments in the first 24-week period of PEDFIC 2 was 33%, 56%, and 62% in cohorts 1A, 1B, and 2, respectively. Most TEAEs were mild or moderate. No drug-related serious TEAEs occurred. ConclusionsOdevixibat in patients with progressive familial intrahepatic cholestasis was generally well tolerated and associated with sustained reductions in sBAs and pruritus. Clinical Trials RegistrationThis study is registered at ClinicalTrials.gov (NCT03659916). Impact and ImplicationsDisrupted bile flow is a hallmark feature of patients with progressive familial intrahepatic cholestasis and can result in build-up of bile constituents in the liver with spill over into the bloodstream; other effects that patients can experience include extremely itchy skin, and because not enough bile reaches the gut, patients can have problems digesting food, which may lead to poor growth. Odevixibat is an orally administered medication that shunts bile acids away from the liver. The current study, called PEDFIC 2, suggested that odevixibat can improve the problematic signs and symptoms of progressive familial intrahepatic cholestasis and was generally safe for patients.

A3907, a systemic ASBT inhibitor, improves cholestasis in mice by multiorgan activity and shows translational relevance to humans.

Cholestasis is characterized by intrahepatic accumulation of bile constituents, including bile acids (BAs), which promote liver damage. The apical sodium-dependent BA transporter (ASBT) plays an important role in BA reabsorption and signaling in ileum, bile ducts, and kidneys. Our aim was to investigate the pharmacokinetics and pharmacological activity of A3907, an oral and systemically available ASBT inhibitor in experimental mouse models of cholestasis. In addition, the tolerability, pharmacokinetics, and pharmacodynamics of A3907 were examined in healthy humans. A3907 was a potent and selective ASBT inhibitor in vitro. In rodents, orally administered A3907 distributed to the ASBT-expressing organs, that is, ileum, liver, and kidneys, and dose dependently increased fecal BA excretion. A3907 improved biochemical, histological, and molecular markers of liver and bile duct injury in Mdr2-/- mice and also had direct protective effects on rat cholangiocytes exposed to cytotoxic BA concentrations in vitro . In bile duct ligated mice, A3907 increased urinary BA elimination, reduced serum BA levels, and prevented body weight loss, while improving markers of liver injury. A3907 was well tolerated and demonstrated target engagement in healthy volunteers. Plasma exposure of A3907 in humans was within the range of systemic concentrations that achieved therapeutic efficacy in mouse. The systemic ASBT inhibitor A3907 improved experimental cholestatic disease by targeting ASBT function at the intestinal, liver, and kidney levels, resulting in marked clearance of circulating BAs and liver protection. A3907 is well tolerated in humans, supporting further clinical development for the treatment of cholestatic liver diseases.

Transporters for Bile Formation in Physiology and Pathophysiology.

The liver fulfills many vital functions for the body, among them bile formation and detoxification. Bile salts are organic anions, are the major constituents of bile and are at high concentrations cytotoxic. Detoxification exposes the liver to many harmful compounds. This function is therefore potentially damaging to the liver. Impaired bile formation may lead to hepatic accumulation of bile salts and subsequently to liver disease. Diagnosis of liver diseases involves the measurement of so-called liver function parameters. This overview aims to characterize and summarize the role of organic anion transporters in bile formation at the protein level under normal physiologic conditions and in liver function tests used for diagnosing liver diseases in pathophysiologic situations.

Screening of gall bladder cancer through infrared analysis of bile and examination of varied bile constituent composition by the disease

To demonstrate the infrared (IR)-based bile analysis as a reliable screening tool for gall bladder (GB) cancer, we analyzed a sample set of 37 diverse bile samples (five normal, 18 GB polyp, six hepatocellular carcinoma (HCC), and eight GB cancer subjects). Bile samples of normal subjects (control) and HCC patients were newly included to examine if IR-based bile analysis could be expanded to identify HCC. Concentrations of three bile acids and eight bile salts in the aqueous phase samples were determined in parallel and lipidomic analysis of nine lipid classes in the organic phase samples was performed using liquid chromatography-mass spectrometry. Concentrations of bile salts were lower and relative abundances of bile salts were dissimilar between GB cancer samples and remained group samples. Also, the levels of lipids such as phosphatidylcholines and phosphatidylethanolamines were again lower and their relative abundances in the organic phase of GB cancer samples were different from those of other samples. IR spectral features of the aqueous, organic, and amphiphilic aggregate phases were individually characteristic, while not descriptive enough for the thorough identification of GB cancer. Nonetheless, since they were mutually complementary to represent different metabolites in bile, the use of three phase-merged spectra was synergetic to yield the superior discrimination of GB cancer.

An integrated strategy combining metabolomics and machine learning for the evaluation of bioactive markers that differentiate various bile.

Animal bile is an important component of natural medicine and is widely used in clinical treatment. However, it is easy to cause mixed applications during processing, resulting in uneven quality, which seriously affects and harms the interests and health of consumers. Bile acids are the major bioactive constituents of bile and contain a variety of isomeric constituents. Although the components are structurally similar, they exhibit different pharmacological activities. Identifying the characteristics of each animal bile is particularly important for processing and reuse. It is necessary to establish an accurate analysis method to distinguish different types of animal bile. We evaluated the biological activity of key feature markers from various animal bile samples. In this study, a strategy combining metabolomics and machine learning was used to compare the bile of three different animals, and four key markers were screened. Quantitative analysis of the key markers showed that the levels of Glycochenodeoxycholic acid (GCDCA) and Taurodeoxycholic acid (TDCA) were highest in pig bile; Glycocholic acid (GCA) and Cholic acid (CA) were the most abundant in bovine and sheep bile, respectively. In addition, four key feature markers significantly inhibited the production of NO in LPS-stimulated RAW264.7 macrophage cells. These findings will contribute to the targeted development of bile in various animals and provide a basis for its rational application.

Aflatoxin B1 Induces Gut-Inflammation-Associated Fecal Lipidome Changes in F344 Rats

Abstract Aflatoxin B1 (AFB1) induced intestinal epithelial damage in rodent models, which indicates that long-term exposure to AFB1 may cause chronic gut disorders. In this study, we tested the hypothesis that AFB1-induced adverse effects on gut is mediated by gut-microbiota, which is partially reflected by the changes of fecal microbiome and metabolome. F344 rats were orally exposed to AFB1 of 0, 5, 25, and 75 µg kg−1 body weight for 4 weeks and fecal samples were collected. An ion-fragmentation-spectrum-based metabolomics approach was developed to investigate the fecal microbiota-associated metabolic changes in fecal samples. We found that AFB1 inhibited the hepatic and intestinal metabolism of bile constituents. As compared with the controls, bile acid synthesis-associated cholesterols in rats treated with 25 µg kg−1 (the middle-dose group) were significantly decreased in the fecal samples, for example, lathosterol (45% reduction), cholesterol ester (21% reduction), chenodeoxycholic acid (20% reduction), dihydroxycholesterol (55% reduction), hydroxycholesterol (20% reduction), and 5-cholestene (29% reduction). Although disease-associated lipids were not detectable in the feces of the control group, they were found in AFB1-treated groups, including diglyceride, monoacylglyceride, 19,20-dihydroxy-docosapentaenoic acid, and phosphatidylethanolamine. Metabolisms of carbohydrates and production of short-chain fatty acids were remarkedly decreased in all treated groups. Moreover, an inflammatory-bowel-disease (IBD)-associated taxonomic structure of fecal microbiota was observed as ∼25% Lachnospiraceae, ∼25% Ruminococcaceae, and &amp;lt;1% Lactobacillales, which was similar to the composition pattern found in IBD patients. These results suggest that AFB1-induced disruption on gut-microbiota, partially reflected by fecal microbiome and metabolome, may play important roles in the pathogenesis of chronic gut disorders.

Robust and Comprehensive Targeted Metabolomics Method for Quantification of 50 Different Primary, Secondary, and Sulfated Bile Acids in Multiple Biological Species (Human, Monkey, Rabbit, Dog, and Rat) and Matrices (Plasma and Urine) Using Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) Analysis.

Bile acids (BAs) are biomolecules synthesized in the liver from cholesterol and are constituents of bile. The in-vivo BA pool includes more than 50 known diverse BAs which are unconjugated, amino acid conjugated, sulfated, and glucuronidated metabolites. Hemostasis of bile acids is known to be highly regulated and an interplay between liver metabolism, gut microbiome function, intestinal absorption, and enterohepatic recirculation. Interruption of BA homeostasis has been attributed to several metabolic diseases and drug induced liver injury (DILI), and their use as potential biomarkers is increasingly becoming important. Speciated quantitative and comprehensive profiling of BAs in various biomatrices from humans and preclinical animal species are important to understand their significance and biological function. Consequently, a versatile one single bioanalytical method for BAs is required to accommodate quantitation in a broad range of biomatrices from human and preclinical animal species. Here we report a versatile, comprehensive, and high throughput liquid chromatography-high resolution mass spectrometry (LC-HRMS) targeted metabolomics method for quantitative analysis of 50 different BAs in multiple matrices including human serum, plasma, and urine and plasma and urine of preclinical animal species (rat, rabbit, dog, and monkey). The method has been sufficiently qualified for accuracy, precision, robustness, and ruggedness and addresses the issue of nonspecific binding of bile acids to plastic for urine samples. Application of this method includes comparison for BA analysis between matched plasma and serum samples, human and animal species differences in BA pools, data analysis, and visualization of complex BA data using BA indices or ratios to understand BA biology, metabolism, and transport.

Progressive Familial CD10 Deficient Ductopenic Disorder; Hitherto an Unnamed Entity! - A Case Report

Inherited liver disorders are group of genetic diseases that cause early liver involvement many of them progressing to early cirrhosis. Of the familial cholestatic disorders most widely studied are progressive familial intrahepatic cholestatic disorders. These disorders are caused by defects in enzymes involved with the formation and excretion of bile constituents. They are, however, not associated with ductopenia. We herein report the first case of a familial cholestatic disorder in three female siblings resident of Lahore, Pakistan presenting as chronic cholestasis all progressing to cirrhosis before 10 years of age. The first two female siblings underwent liver transplants for chronic cholestasis. The third sibling underwent liver biopsy for the evaluation of cholestasis and later liver transplant for same. There are no other associated cardiac or skeletal anomalies in any of the sisters. The findings of biopsy and explant tissue were similar in all three sibling sisters. There are features of advanced fibrosis, significant ductopenia, bile ductular reaction at the porto-parenchymal interface, cholestasis, increased copper stores on rhodanine stain no loss of bile salt export pump, and multi-drug resistant 3 protein (MDR3), and the absence of CD10 from canaliculi. The findings raised differentials for progressive familial intrahepatic cholestasis (PFIC) type 3, Alagille syndrome, and variant of familial cholestatic disorder. PFIC 3 causes cholestasis, but the presence of MDR3 stain, ductopenia, and deficient CD10 are not seen in PFIC 3. Familial ductopenic disorders have been identified in the adult population called as idiopathic adulthood ductopenia and had autosomal dominant pattern of inheritance. The argument against Alagille syndrome is the absence of any other syndromic features of Alagille and autosomal recessive mode of inheritance. These findings led us to conclude if there is a need to redefine a new entity of progressive familial CD10 deficient ductopenic disorder. The findings though limited by genetic studies give way for further research on the subject.

Silymarin mitigates bile duct obstruction-induced cholemic nephropathy.

Bile duct obstruction or cholestasis can occur by several diseases or xenobiotics. Cholestasis and the accumulation of the bile constituents in the liver primarily damage this organ. On the other hand, extrahepatic organs are also affected by cholestasis. The kidney is the most affected tissue during cholestatic liver injury. Cholestasis-associated renal injury is known as cholemic nephropathy (CN). Several lines of evidence specify the involvement of oxidative stress and mitochondrial impairment in the pathogenesis of CN. The current study aimed to assess the role of silymarin as a potent antioxidant on CN-induced oxidative stress and mitochondrial dysfunction in the kidney. Bile duct ligated (BDL) rats were treated with silymarin (10 and 100mg/kg, oral) for seven consecutive days. A significant increase in reactive oxygen species (ROS), lipid peroxidation, protein carbonylation, and oxidized glutathione (GSSG) levels were evident in the kidney of BDL animals. Moreover, reduced glutathione (GSH) content and total antioxidant capacity were significantly decreased in the kidney of cholestatic rats. Mitochondrial depolarization, decreased mitochondrial dehydrogenases activity, mitochondrial permeabilization, and depleted ATP stores were detected in the kidney mitochondria isolated from BDL animals. Kidney histopathological alterations, as well as serum and urine levels of renal injury biomarkers, were also significantly different in the BDL group. It was found that silymarin treatment significantly ameliorated CN-induced renal injury. The antioxidant effects of silymarin and its positive impact on mitochondrial indices seem to play a significant role in its renoprotective effects during cholestasis.

Taurine attenuates the damage of lupus nephritis mouse via inactivation of the NF-κB pathway

Taurine is an organic amino acid and a major constituent of bile. With its contribution to various cellular functions, it has demonstrated therapeutic effects in a wide range of diseases. Since there is a lack of literature investigating taurine as a treatment for lupus nephritis (LN), here we examined the potential of taurine as a treatment for LN. Experiments were carried out using MRL/lpr mice as a model of LN, and C57BL/6 mice were used as negative controls. At 12 weeks old, MRL/lpr mice were divided into four groups and treated with 0, 50 and 100 mg/kg body weight taurine for 5 days. Enalapril is used as a positive control drug. All animals were sacrificed after treatment. LN-induced damage was assessed by proteinuria, blood urea nitrogen (BUN) and serum creatinine (CRE) levels. The degree of inflammation was assessed by inducible nitric oxide synthase (iNOS), interleukin-4 (IL-4), IL-10, and tumor necrosis factor-α (TNF-α) levels. The degree of oxidative stress was assessed by malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione peroxidase (Gpx) levels. Hematoxylin and eosin (HE) staining and TUNEL staining assessed histopathological damage and apoptosis, respectively. The levels of Bcl-2, Bax, caspase-3, caspase-9, and NF-κB p65 were detected by western blot. The data indicated that taurine administration improved kidney functions, reversed cell death, suppressed oxidative stress, and importantly, adjusted the immune response of LN mice to a more balanced state. These results provide a novel strategy for LN therapy, which may overcome the disadvantages of traditional immunosuppression and hormone treatments with greater efficacy and fewer side effects.

Disturbed gut microbiota and bile homeostasis in Giardia-infected mice contributes to metabolic dysregulation and growth impairment.

Although infection with the human enteropathogen Giardia lamblia causes self-limited diarrhea in adults, infant populations in endemic areas experience persistent pathogen carriage in the absence of diarrhea. The persistence of this protozoan parasite in infants has been associated with reduced weight gain and linear growth (height-for-age). The mechanisms that support persistent infection and determine the different disease outcomes in the infant host are incompletely understood. Using a neonatal mouse model of persistent G. lamblia infection, we demonstrate that G. lamblia induced bile secretion and used the bile constituent phosphatidylcholine as a substrate for parasite growth. In addition, we show that G. lamblia infection altered the enteric microbiota composition, leading to enhanced bile acid deconjugation and increased expression of fibroblast growth factor 15. This resulted in elevated energy expenditure and dysregulated lipid metabolism with reduced adipose tissue, body weight gain, and growth in the infected mice. Our results indicate that this enteropathogen's modulation of bile acid metabolism and lipid metabolism in the neonatal mouse host led to an altered body composition, suggesting how G. lamblia infection could contribute to growth restriction in infants in endemic areas.

Possible Protective Effect of Onion Supplementation on Hepatic Functional and Structural Alterations Induced by Cholestasis

Abstract Background Cholestasis is the obstruction or the reduction in bile flow that results in intrahepatic accumulation of bile constituents, which progresses to develop liver pathology. Common bile duct ligation (BDL) in rodents is an experimental model of cholestasis that has been carried out in research for many years. BDL model of cholestatic liver injury involves other mechanisms, including oxidative stress, inflammation, and fibrogenesis. Antioxidant, antiinflammatory or antiapoptotic properties gained much interest for the amelioration of liver dysfunction. Aim the aim of this study is to assess the possible protective effects of onion supplementation on hepatic structural and functional alterations induced by BDL in rats, which reflect the effects of cholestasis resulting from intrahepatic accumulation of bile. Methods Thirty adult female Wistar rats were randomly and equally allocated into three groups: (1) control group, (2) BDL group; subjected to ligation of the common bile duct and (3) Onion-supplemented BDL groups (O-BDL). Both control and BDL groups received distilled water (solvent for onion powder) daily by gavage for 4 weeks. Onion-supplemented BDL group (O-BDL); subjected to ligation of the common bile duct and then received 500 mg/kg of onion powder dissolved in distilled water, daily by gavage for 4 weeks. At the end of the experimental period, plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin, total proteins, total antioxidant capacity (TAC), tumor necrosis factor-α (TNF-α), and hepatic tissue level of malondialdehyde (MDA) and transforming growth factor-β (TGF-β) were measured for all groups. In addition, histopathological examination of liver tissue samples was performed for the three groups. Results Plasma levels of ALT, AST, ALP, direct bilirubin, TNF-α and hepatic tissue levels of MDA and TGF-β were significantly increased and TAC was significantly decreased in the BDL group compared to the control group. In addition, altered architecture was detected in hepatic tissue samples of BDL group. Onion supplementation significantly decreased the plasma levels of ALT, AST, ALP, direct bilirubin, TNF-α and hepatic tissue levels of MDA and TGF-β in the O-BDL group when compared to the BDL group. Total proteins level was not significantly different among all the studied groups. In addition in O-BDL group, histopathological examination of liver revealed near normal structure of hepatic tissue. Conclusion BDL induces hepatic structural alterations and functional disturbances. Onion supplementation inhibits inflammation and oxidative insults that associate BDL, and subsequently protects against BDL-induced liver injury.

Expanded complement of Niemann-Pick type C2-like protein genes in Clonorchis sinensis suggests functions beyond sterol binding and transport

BackgroundThe parasitic flatworm Clonorchis sinensis inhabits the biliary tree of humans and other piscivorous mammals. This parasite can survive and thrive in the bile duct, despite exposure to bile constituents and host immune attack. Although the precise biological mechanisms underlying this adaptation are unknown, previous work indicated that Niemann-pick type C2 (NPC2)-like sterol-binding proteins might be integral in the host-parasite interplay. Expansions of this family in some invertebrates, such as arthropods, have shown functional diversification, including novel forms of chemoreception. Thus, here we curated the NPC2-like protein gene complement in C. sinensis, and predicted their conserved and/or divergent functional roles.MethodsWe used an established comparative genomic-bioinformatic approach to curate NPC2-like proteins encoded in published genomes of Korean and Chinese isolates of C. sinensis. Protein sequence and structural homology, presence of conserved domains and phylogeny were used to group and functionally classify NPC2-like proteins. Furthermore, transcription levels of NPC2-like protein-encoding genes were explored in different developmental stages and tissues.ResultsTotals of 35 and 32 C. sinensis NPC2-like proteins were predicted to be encoded in the genomes of the Korean and Chinese isolates, respectively. Overall, these proteins had low sequence homology and high variability of sequence alignment coverage when compared with curated NPC2s. Most C. sinensis proteins were predicted to retain a conserved ML domain and a conserved fold conformation, with a large cavity within the protein. Only one protein sequence retained the conserved amino acid residues required in bovine NPC2 to bind cholesterol. Non-canonical C. sinensis NPC2-like protein-coding domains clustered into four distinct phylogenetic groups with members of a group frequently encoded on the same genome scaffolds. Interestingly, NPC2-like protein-encoding genes were predicted to be variably transcribed in different developmental stages and adult tissues, with most being transcribed in the metacercarial stage.ConclusionsThe results of the present investigation confirms an expansion of NPC2-like proteins in C. sinensis, suggesting a diverse array of functions beyond sterol binding and transport. Functional explorations of this protein family should elucidate the mechanisms enabling the establishment and survival of C. sinensis and related flukes in the biliary systems of mammalian hosts.

EVALUATION OF TYPES OF GALL STONES AND FACTORS AFFECTING IT.

Introduction: Gallstone (GS) is an important cause of morbidity all over the world. Gallstones are becoming increasingly common and are seen in all age groups. When there is an imbalance in the chemical constituents of bile that result in precipitation of one or more of the components gall stones are formed. In majority of the cases they do not cause symptoms, and about 10% to 20% will eventually become symptomatic within 5 years and 20 years of diagnosis. Gallstones contain mainly cholesterol, bilirubin, and calcium salts, with some amounts of protein and other materials. Pure cholesterol crystals are generally soft, and protein contributes to the strength of cholesterol stones. Pigment stones account for 30 % of gallstones and are divided into black pigment and calcium bilirubinate stones.&#x0D; Material and Methods: Demographic characteristics of the patients participating in the study such as age, sex, occupation, lifestyle habits such as alcohol consumption , smoking, sports activity , medication if any used, any diseases, Body mass index (BMI), milk consumption, liver diseases and dietary habits was obtained. Patients were divided into two groups depending upon the type of gall stone either cholesterol or pigment stone.&#x0D; Results: A total of 76 patients were included in the present study who underwent cholecystectomy surgery. Age group of more than 51 years showed maximum number of stones in both the groups. Maximum numbers of stones were seen in patients having family history of stones. Patients who were overweight and obsess were having higher incidence of gall stone as compared to normal weight and slim population. Formation of stones in female was more as compared to males.&#x0D; Conclusion: Various factors can be considered for the formation of gall stones of which gender, increasing age, genetic factors, dilatory habits, and sedentary lifestyle can be considered as the contributing factors for formation of gall stones and most of them can be prevented by proper education.&#x0D; Keywoerds: GS, BMI, liver disease, smoking, alcohol consumption.

Possible Protective Effect of Onion Supplementation on Hepatic Functional and Structural Alterations Induced by Cholestasis

AbstractBackground: Cholestasis is the obstruction or the reduction in bile flow that results in intrahepatic accumulation of bile constituents, which progresses to develop liver pathology. Common bile duct ligation (BDL) in rodents is an experimental model of cholestasis that has been carried out in research for many years. BDL model of cholestatic liver injury involves other mechanisms, including oxidative stress, inflammation, and fibrogenesis. Antioxidant, anti-inflammatory or antiapop-totic properties gained much interest for the amelioration of liver dysfunction.Aim of Study: The aim of this study is to assess the possible protective effects of onion supplementation on hepatic struc-tural and functional alterations induced by BDL in rats, which reflect the effects of cholestasis resulting from intrahepatic accumulation of bile.Material and Methods: Thirty adult female Wistar albino rats were randomly and equally allocated into three groups: (1) Control group, (2) BDL group; subjected to ligation of the common bile duct and (3) Onion-supplemented BDL groups (O-BDL). Both control and BDL groups received distilled water (solvent for onion powder) daily by gavage for 4 weeks. Onion-supplemented BDL group (O-BDL); subjected to ligation of the common bile duct and then received 500mg/kg of onion powder dissolved in distilled water, daily by gavage for 4 weeks. At the end of the experimental period, plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin, total proteins, total antioxidant capacity (TAC), tumor necrosis factor-a (TNF-a), and hepatic tissue level of malondialdehyde (MDA) and transforming growth factor-b1 (TGF-b1) were measured for all groups. In addition, his-topathological examination of liver tissue samples was per-formed for the three groups.Results: Plasma levels of ALT, AST, ALP, direct bilirubin, TNF-a and hepatic tissue levels of MDA and TGF-b1 were significantly increased and TAC was significantly decreased in the BDL group compared to the control group. In addition, altered architecture was detected in hepatic tissue samples of BDL group. Onion supplementation significantly decreased the plasma levels of ALT, AST, ALP, direct bilirubin, TNF-a and hepatic tissue levels of MDA and TGF-b1 in the O-BDL group when compared to the BDL group. Total proteins level was not significantly different among all the studied groups. In addition in O-BDL group, histopathological exam-ination of liver revealed near normal structure of hepatic tissue.Conclusion: BDL induces hepatic structural alterations and functional disturbances. Onion supplementation inhibits inflammation and oxidative insults that associate BDL, and subsequently protects against BDL-induced liver injury.

Inflammation and Cell Death During Cholestasis: The Evolving Role of Bile Acids.

Cholestasis results in blockage of bile flow whether the point of obstruction occurs extrahepatically or intrahepatically. Bile acids are a primary constituent of bile, and thus one of the primary outcomes is acute retention of bile acids in hepatocytes. Bile acids are normally secreted into the biliary tracts and then released into the small bowel before recirculating back to the liver. Retention of bile acids has long been hypothesized to be a primary cause of the associated liver injury that occurs during acute or chronic cholestasis. Despite this, a surge of papers in the last decade have reported a primary role for inflammation in the pathophysiology of cholestatic liver injury. Furthermore, it has increasingly been recognized that both the constituency of individual bile acids that make up the greater pool, as well as their conjugation status, is intimately involved in their toxicity, and this varies between species. Finally, the role of bile acids in drug-induced cholestatic liver injury remains an area of increasing interest. The purpose of this review is to critically evaluate current proposed mechanisms of cholestatic liver injury, with a focus on the evolving role of bile acids in cell death and inflammation.

Chemical profiling for bile acid derivatives in yak bile

Bile acids( BAs),the major constituents of bile,are also known to be potential biomarkers of various diseases,especially liver disease. The systematic analysis of BAs is believed to be of great importance towards the clarification of the effective material basis for bile-type medicines,and the diagnosis and therapy of related diseases as well. As a part of systematic study on bile-type medicine ongoing in our group,this study lays emphasis on the isomer discrimination,and the improvement of analytical method of BAs. Further,this method was subsequently applied to elucidate in depth the chemical profile of BAs in yak bile. Regarding isomer discrimination for BAs,we constructed relative response-collision energy curves( RRCECs) by high performance liquid chromatographyion trap-time of flight-mass spectrometry( HPLC-IT-TOF-MS) in combination with high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry( HPLC-Qtrap-MS). As a result,both the optimum collision energy( OCE) and CE_(50) exhibited great correlations with structural characteristics,thus enabling the isomer distinguishing,such as unconjugated BAs,glycine-conjugated BAs,and taurine-conjugated BAs. According to information provided by mass spectrometry,the comparison of OCE and CE_(50),retention time matching,combined with reference substances and database retrieval,a total of 30 bile acid derivatives were observed and identified in yak bile. The newly developed method could serve as a feasible tool for the in-depth characterization of BAs in bile and biological samples.

Analysis of the fingerprint profile of bioactive constituents of traditional Chinese medicinal materials derived from animal bile using the HPLC–ELSD and chemometric methods: An application of a reference scaleplate

Traditional Chinese medicinal materials derived from animal bile are widely applied in clinical therapy for thousands of years in several Southeast Asian countries. Although the constituents are similar, these crude drugs exhibit different pharmacological activities; bile acids are the main bioactive constituent. Depending on the source, the price of these crude drugs differs significantly. Therefore, a reliable fingerprint method is needed to analyze and distinguish these crude drugs with a similar composition. In this milieu, we aimed to establish a fingerprint chromatography method that can separate and detect several bile acids simultaneously. A high-performance liquid chromatography separation method was established with evaporative light scattering detection to detect the analytes. The main bioactive constituents of pig bile, cattle bile, sheep bile, bear bile, and three types of cow bezoar were analyzed using the proposed method. The fingerprint chromatography profile of 35 samples were obtained and analyzed using chemometric methods. Considering the differences among samples, a reference scaleplate method was used in the peak alignment procedure. Unsupervised methods (hierarchical cluster analysis and principle component analysis) and supervised methods (K-nearest neighbor, partial least squares discriminant analysis, support vector machine discriminant analysis, and soft independent modeling of class analogy) were used in the chemometric analysis. The results indicated that the fingerprint chromatograms of the seven crude drugs had fingerprint specificity and that they can be well distinguished. In addition, the reference scaleplate method using the chromatogram of a mixed standard solution is practically applicable for peak alignment in the analysis of samples with a large difference in chromatographic peaks. Overall, 17 bile acids can be separated and detected simultaneously using this method and some frequently used TCMMs derived from animal bile can be distinguished accurately.