Microbially conjugated bile salts found in human bile activate the bile salt receptors TGR5 and FXR.

BACKGROUND: Bile salts of hepatic and microbial origin mediate interorgan cross talk in the gut-liver axis. Here, we assessed whether the newly discovered class of microbial bile salt conjugates (MBSCs) activate the main host bile salt receptors (Takeda G protein-coupled receptor 5 [TGR5] and farnesoid X receptor [FXR]) and enter the human systemic and enterohepatic circulation. METHODS: N-amidates of (chenodeoxy) cholic acid and leucine, tyrosine, and phenylalanine were synthesized. Receptor activation was studied in cell-free and cell-based assays. MBSCs were quantified in mesenteric and portal blood and bile of patients undergoing pancreatic surgery. RESULTS: MBSCs were activating ligands of TGR5 as evidenced by recruitment of Gsα protein, activation of a cAMP-driven reporter, and diminution of lipopolysaccharide-induced cytokine release from macrophages. Intestine-enriched and liver-enriched FXR isoforms were both activated by MBSCs, provided that a bile salt importer was present. The affinity of MBSCs for TGR5 and FXR was not superior to host-derived bile salt conjugates. Individual MBSCs were generally not detected (ie, < 2.5 nmol/L) in human mesenteric or portal blood, but Leu-variant and Phe-variant were readily measurable in bile, where MBSCs comprised up to 213 ppm of biliary bile salts. CONCLUSIONS: MBSCs activate the cell surface receptor TGR5 and the transcription factor FXR and are substrates for intestinal (apical sodium-dependent bile acid transporter) and hepatic (Na+ taurocholate co-transporting protein) transporters. Their entry into the human circulation is, however, nonsubstantial. Given low systemic levels and a surplus of other equipotent bile salt species, the studied MBSCs are unlikely to have an impact on enterohepatic TGR5/FXR signaling in humans. The origin and function of biliary MBSCs remain to be determined.

Oil spill in an amazon blackwater environment: Biochemical and physiological responses of local fish species.

The accidental spill of petroleum asphalt cement (PAC) in São Raimundo (SR Harbor, located on the Rio Negro (Manaus, Amazonas, Brazil) was monitored through the analysis of polyciclic aromatic hydrocarbons (PAHs) in water and a set of biomarkers in fishes (exposure biomarkes: PAHs-type metabolites concentrations in bile; the activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in liver. Effect biomarkers: lipid peroxidation concentration (LPO) in liver, acetylcholinesterase activity in brain, and genotoxic DNA damage in erythrocytes). Two fish species, Acarichthys heckelii and Satanoperca jurupari, were collected 10, 45, and 90 days after the PAC spill in São Raimundo. At the same time, fish were collected from the Tupé Sustainable Development Reserve (Tupé) which served as a reference area. The sampling periods were related to the rising waters of the natural flood pulse of the Rio Negro. Higher concentrations of PAHs in water were observed at 10 and 45 days and returned to the values of TP 90 days after the PAC spill, a period in which harbor waters rose about 0.2 m. Unlike the PAHs in water, biomarker responses in both fish species significantly increased following the PAC spill in SR. Hepatic ethoxyresorufin-O-deethylase (EROD), PAH-like metabolites in bile, and erythrocyte DNA damage increases, together with inhibition of acetylcholinesterase (AChE) activity in the brain were the most evident responses for both fish species. The calculated pyrolytic index showed mixed sources of PAHs (petrogenic and pyrolytic). The applied PCA-FA indicated important relationships between dissolved organic carbon (DOC) and PAHs concentrations in water, where DOC and PAHs concentrations contributed to biomarkers responses for both fish species in all collection periods.

An integrated microfluidic system for automatic detection of cholangiocarcinoma cells from bile.

Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the epithelial cells lining the bile ducts. Due to its location deep within the body and nonspecific symptoms in the early stages, it is often diagnosed at the advanced stage, thus leading to worse prognosis. Circulating tumor cells within liquid biopsies (i.e. blood) have been considered as promising biomarkers for CCA diagnosis, though current methods for profiling them are not satisfactory in terms of sensitivity and specificity. Herein we developed a new cancer cell probing and immuno-tracking assay known as "CAPTURE", which was performed on an integrated microfluidic system (IMS) to automate CCA diagnosis from bile with a sample amount of only 1 mL. The assay utilized magnetic beads surface-coated with two affinity reagents, a nucleic acid aptamer (HN16) and a glycosaminoglycan (SCH 45-mix), for capturing cancer cells in bile; the "gold standard" anti-epithelial cell adhesion molecule was used as a comparison. In a single-blind test of 54 CCA-positive (+) and 102 CCA-negative (-) clinical samples, sensitivities and specificities of 96 and 80%, respectively, were documented with the CAPTURE assay on-bench. An IMS composed of a centrifugal module for sample pretreatment and a CAPTURE module for cell capture and staining was integrated with a new "vertical integration module" for detecting cancer cells from bile without human intervention. Furthermore, a novel micro-tier structure within the centrifugal module was designed to block passage of gallbladder stones with diameters >1 mm, thereby preventing their interference during the subsequent CAPTURE assay. Improved sensitivity and specificity (100 & 83%, respectively) by using three affinity reagents were achieved on the IMS when using 26 clinical bile samples, confirming its clinical bio-applicability for CCA diagnosis. This approach could be therefore used for early-stage CCA diagnostics, ideally enabling effective treatment, as well as reducing potential for relapse.

Bile liquid biopsy in biliary tract cancer.

Biliary tract cancers are heterogeneous in etiology, morphology and molecular characteristics thus impacting disease management. Diagnosis is complex and prognosis poor. The advent of liquid biopsy has provided a unique approach to more thoroughly understand tumor biology in general and biliary tract cancers specifically. Due to their minimally invasive nature, liquid biopsy can be used to serially monitor disease progression and allow real-time monitoring of tumor genetic profiles as well as therapeutic response. Due to the unique anatomic location of biliary tract cancer, bile provides a promising biologic fluid for this purpose. This review focuses on the composition of bile and the use of these various components, ie, cells, extracellular vesicles, nucleic acids, proteins and metabolites as potential biomarkers. Based on the disease characteristics and research status of biliary tract cancer, considerable effort should be made to increase understanding of this disease, promote research and development into early diagnosis, develop efficient diagnostic, therapeutic and prognostic markers.

Comparative analysis of bile metabolic profile in patients with biliary obstruction complicated by Clonorchis sinensis infection.

Background: Clonorchiasis is an important foodborne parasitic disease. However, eggs of Clonorchis sinensis (C. sinensis) cannot be detected in feces during biliary obstruction. Moreover, many diseases can cause biliary obstruction, such as gallstones, adenocarcinoma, cholangiocarcinoma and Ascaris lumbricoides infection. Therefore, it is of great significance to distinguish between patients of biliary obstruction and biliary obstruction with C. sinensis infection. Methods: A total of 48 biliary obstruction patients were enrolled, including 23 infected with C. sinensis (C. sinensis) (OB+C.s) and 25 non-infected subjects (OB). The bile samples were collected by endoscopic retrograde cholangiopancreatography and analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Additionally, multivariate statistical analysis methods were employed to identify differential metabolites. Next, bile amino acid levels were determined by targeted metabolomics analysis. Result: A total of 146 and 132 significant metabolites were identified in electrospray ionization (ESI)+ and ESI- modes, respectively. The levels of amino acids (asparagine, glutamate, ornithine) and polyamines (spermidine and spermine) were significantly changed. Targeted analysis showed that the levels of amino acids (such as L-arginine, L-glutamine, L-lysine, L-propionic, and L-tyrosine) were lower in OB+C.s patients compared to those in OB patients. Marked metabolic pathways were involved in "Glutathione metabolism", "Caffeine metabolism", "Alanine, aspartate and glutamate metabolism", "Arginine and proline metabolism", "Purine metabolism", "Beta-Alanine metabolism", and "D-glutamine and D-glutamate metabolism". Conclusion: These results show that there were significant differences between OB+C.s and OB patients, especially in amino acids. The metabolic signature and perturbations in metabolic pathways may help to better distinguish OB+C.s and OB patients.

Feasibility of Raman spectroscopic identification of gall bladder cancer using extracellular vesicles extracted from bile.

Extracellular vesicles (EVs), which are heterogeneous membrane-based vesicles with bilayer cell membrane structures, could be versatile biomarkers for the identification of diverse diseases including cancers. With this potential, this study has attempted the Raman spectroscopic identification of gall bladder (GB) cancer by directly measuring the EV solution extracted from human bile without further sample drying. For this purpose, bile samples were obtained from four normal individuals and 21 GB polyp, eight hepatocellular carcinoma (HCC), and five GB cancer patients, and EVs were extracted from each of the bile samples. The Raman peak shapes of the EVs extracted from the GB cancer samples, especially the relative intensities of peaks in the 1560-1340 cm-1 range, were dissimilar to those of the samples from the normal, GB polyp, and HCC groups. The intensity ratios of peaks at 1537 and 1453 cm-1 and at 1395 and 1359 cm-1 of the GB cancer samples were lower and higher, respectively, than those of the samples of the remaining three groups. The differences of peak intensity ratios were statistically significant based on the Mann-Whitney U test. DNA/RNA bases, amino acids, and bile salts contributed to the spectra of EVs, and their relative abundances seemed to vary according to the occurrence of GB cancer. The varied metabolite compositions and/or structures of EVs were successfully demonstrated by the dissimilar peak intensity ratios in the Raman spectra, thereby enabling the discrimination of GB cancer.

Identification of gallbladder cancer by direct near-infrared measurement of deuterated chloroform-extracted organic phase from human bile.

A simple near-infrared (NIR) spectroscopic scheme enabling direct measurement of organic phase extracted from human bile with no spectral interference from the extraction solvent was demonstrated for identification of gallbladder (GB) cancer. This scheme is used to recognize the different lipid contents in bile samples from GB cancer patients using NIR spectroscopy for disease identification. To this end, the extraction solvent should provide an absorption-free NIR region to observe peaks of related metabolite. For this purpose, deuterated chloroform (CDCl3) is uniquely suited as an extraction medium because it has few absorption peaks in the 4380-4100 cm-1 range, where intense peaks for lipids and cholesterol are located. This exploratory study used 37 bile samples (obtained from five normal subjects and nine GB polyp, 11 gallstone, six hepatocellular carcinoma (HCC), and six GB cancer patients). The transmission NIR spectra of the organic phases extracted using CDCl3 in a commercial glass vial were directly measured. The peak intensities of the GB cancer samples were lower than those of the other samples, and the differences were statistically significant, with a confidence interval greater than 99.0%. The lower lipid and cholesterol contents in the organic phases of the GB cancer samples were effectively identified in the corresponding NIR spectra. Therefore, the proposed NIR scheme is simpler and faster than the previous infrared (IR) measurement approach that requires solvent drying to highlight the buried metabolite peaks under a solvent absorption band.

Correlations between oxidative DNA damage and formation of hepatic tumours in two flatfish species from contaminated environments.

Many species in aquatic environments face increased exposure to oncogenic pollution due to anthropogenic environmental change which can lead to higher cancer prevalence. The mechanistic relationship connecting environmental pollution and cancer is multi-factorial and poorly understood, and the specific mechanisms are so far still uncharacterized. One potential mediator between pollutant exposure and cancer is oxidative damage to DNA. We conducted a study in the field with two flatfish species, European flounder (Platichthys flesus L.) and common dab (Limanda limanda L.) with overlapping distribution and similar ecological niche, to investigate if the link between oncogenic pollutants and cancer described in ecotoxicological literature could be mediated by oxidative DNA damage. This was not the case for flounders as neither polycyclic aromatic hydrocarbon (PAH) bile metabolites nor metallic trace element concentrations were related to oxidative DNA damage measurements. However, dabs with higher PAH concentrations did exhibit increased oxidative damage. High oxidative DNA damage also did not predict neoplasm occurrence, rather, healthy individuals tended to have higher oxidative damage measurements compared to fishes with pre-neoplastic tumours. Our analyses showed that flounders had lower concentrations of PAH bile metabolites, suggesting that compared to dab this species is less exposed or better at eliminating these contaminants.

Phosphatidylcholine in bile-derived small extracellular vesicles as a novel biomarker of cholangiocarcinoma.

BACKGROUND: Owing to the lack of definite diagnostic modalities, it is challenging to distinguish malignant cases of cholangiocarcinoma (CCA), which often causes biliary tract obstruction, from benign ones. Here, we investigated a novel lipid biomarker of CCA in bile-derived small extracellular vesicles (sEVs) and developed a simple detection method for clinical application. METHODS: Bile samples from seven patients with malignant diseases (hilar CCA = 4, distal CCA = 3) and eight patients with benign diseases (gallstones = 6, primary sclerosing cholangitis = 1, autoimmune pancreatitis = 1) were collected through a nasal biliary drainage tube. sEVs were isolated via serial ultracentrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting (with CD9, CD63, CD81, and TSG101). Comprehensive lipidomic analysis was performed using liquid chromatography-tandem mass spectrometry. Using a measurement kit, we further confirmed whether lipid concentrations could be used as a potential CCA marker. RESULTS: Lipidomic analysis of bile sEVs in the two groups identified 209 significantly increased lipid species in the malignant group. When focusing on lipid class, phosphatidylcholine (PC) level was 4.98-fold higher in the malignant group than in the benign group (P = 0.037). The receiver operating characteristic (ROC) curve showed a sensitivity of 71.4%, a specificity of 100%, and an area under the curve (AUC) of 0.857 (95% confidence interval [CI]:0.643-1.000). Using a PC assay kit, the ROC curve showed a cutoff value of 16.1 µg/mL, a sensitivity of 71.4%, a specificity of 100%, and an AUC of 0.839 (95% CI: 0.620-1.000). CONCLUSION: PC level in sEVs from human bile is a potential diagnostic marker for CCA and can be assessed by a commercially available assay kit.

First evidence of neonicotinoid insecticides in human bile and associated hepatotoxicity risk.

Neonicotinoids (NEOs) are widely applied in agricultural lands and are widespread in different environments, accelerating threats to ecosystems and human health. A number of in vitro/in vivo studies have reported adverse effects of NEOs on mammalian health, but the link between NEO exposure and toxic effects on human liver remains unclear. We randomly recruited 201 participants and quantified eight commercialized NEOs in bile. High frequency and concentration of detection indicate low degradation of human liver on NEOs. The main NEOs are nitenpyram and dinotefuran, which contribute to about 86% of the total residual levels of eight NEOs, due to the highest solubility in bile and are not degraded easily in liver. In contrast, imidacloprid and thiacloprid are major compounds in human blood, according to previous studies, suggesting that individual NEOs behave differently in blood and bile distribution. There was no statistical difference in NEO residues between cancer and non-cancer participants and among the different participant demographics (e.g., age, gender, and body mass index). The serum hematological parameters -bile acid, total bilirubin, cholesterol and alkaline phosphatase -were positively correlated with individual NEO concentrations, suggesting that NEO exposure affects liver metabolism and even enterohepatic circulation. The study first examined the NEO residues in human bile and provided new insights into their bioavailability and hepatoxicity risk.

Identification and validation of volatile organic compounds in bile for differential diagnosis of perihilar cholangiocarcinoma.

Early and differential diagnosis of perihilar cholangiocarcinoma (PHCCA) is highly challenging. This study aimed to evaluate whether volatile organic compounds (VOCs) in bile samples could be emerging diagnostic biomarkers for PHCCA. We collected 200 bile samples from patients with PHCCA and benign biliary diseases (BBD), including a 140-patient training cohort and an 60-patient test cohort. Gas chromatography-ion mobility spectrometry (GC-IMS) was used for VOCs detection. The predictive models were constructed using machine learning algorithms. Our analysis detected 19 VOC substances using GC-IMS in the bile samples and resulted in the identification of three new VOCs, 2-methoxyfuran, propyl isovalerate, and diethyl malonate that were found in bile. Unsupervised hierarchical clustering analysis supported that VOCs detected in the bile could distinguish PHCCA from BBD. Twelve VOCs defined according to 32 signal peaks had significant statistical significance between BBD and PHCCA, including four up-regulated VOCs in PHCCA, such as 2-ethyl-1-hexanol, propyl isovalerate, cyclohexanone, and acetophenone, while the rest eight VOCs were down-regulated. ROC curve analysis revealed that machine learning models based on VOCs could help diagnosing PHCCA. Among them, SVM provided the highest AUC of 0·966, with a sensitivity and specificity of 93·1% and 100%, respectively. The diagnostic model based on different VOC spectra could be a feasible method for the differential diagnosis of PHCCA.

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.

Metabolic profiling of soyasaponin Bb in rat plasma, urine, bile, and feces after intragastric administration.

Soyasaponin Bb is one of the bioactive oleanolic acid-type triterpenoid saponins mainly isolated from soybean. It possesses significant antithrombosis, hypolipidemic, anticancer, and antioxidant activities. However, the metabolic profiles of soyasaponin Bb are still unknown. The present study investigated the metabolites of soyasaponin Bb in plasma, bile, urine, and feces samples after intragastric administration using ultra-performance liquid chromatography coupled with quadrupole-time-of-flight-mass spectrometry, and its possible metabolic pathways were subsequently proposed. Using the metabolite profiling strategy, 11 metabolites were first identified from urine, plasma, bile, and feces of rats after intragastric administration of soyasaponin Bb. Hydroxylation and hydrolysis were the major metabolic pathways of soyasaponin Bb in rat. The results expand our knowledge of the metabolism of soyasaponin Bb, which could provide valuable information for better comprehension of future pharmacological research.

Feasibility for SERS-based discrimination of gallbladder cancer from polyp by indirect recognition of components in bile.

The aqueous solution extracted from raw bile juice is composed primarily of bile salts, with lower levels of bilirubin and its derivatives. Among them, the bilirubin and bilirubin-derived metabolites are the only surface-enhanced Raman scattering (SERS)-active components. An analytical scheme indirectly responsive and able to utilize all bile components, including SERS-inactive bile salts, was explored for SERS-based discrimination of gallbladder (GB) polyp and GB cancer. Initially, the surface of a SERS substrate (Au nanodendrite on Ni foil (AuND@NiF)) was covered with an alkanethiol molecule to generate a SERS signal and attract bile components by mutual interaction. For more effective recognition of bile components, 4 independent substrates covered with 4 different alkanethiols with various functional groups (SH(CH2)2CH3, SH(CH2)2NH2, SH(CH2)2COOH, and SH(CH2)2OH) were prepared. The SERS peaks of each substrate clearly varied on interaction with pure bile components as well as aqueous bile samples, and the SERS peaks in each substrate were individually characteristic. When the principal component (PC) scores of spectra obtained using the SH(CH2)2CH3- and SH(CH2)2OH-covered substrates were combined, the k-Nearest Neighbor-based discrimination accuracy was 100%, superior to those (90.6-96.9%) using individual substrates. The use of complementary bile component-induced spectral information provided by the two substrates was responsible for accurate discrimination. On the other hand, when bare AuND@NiF recognizing only SERS-active bilirubin derivatives was used, discrimination was unsatisfactory (accuracy: 75.0%).

Roles of ursodeoxycholic acid in the bile biochemistry and metabolomics in patients with choledocholithiasis: a prospective study.

INTRODUCTION: Recurrence after the endoscopic treatment of common bile duct stones (CBDS) is related to bile metabolism and bile compositions. Ursodeoxycholic acid (UDCA) has been proved effective in reducing the recurrence of CBDS. However, the detailed effects of UDCA on bile metabolism are still not extensively explored. OBJECTIVES: This study aimed to analyze the role of UDCA in patients with choledocholithiasis (CDC) from the perspective of biochemistry and metabolomics. METHODS: A total of 89 patients with CDC who underwent endoscopic retrograde cholangiopancreatography were prospectively examined and randomly assigned to control and UDCA groups. The biochemical detections (cholesterol, bilirubin, and so on) were performed on the collected bile. Moreover, the metabolomics analysis was conducted based on bile from 20 patients in the UDCA group. RESULTS: The bile levels of cholesterol and endotoxins significantly decreased after UDCA treatment. Regarding bile metabolomics, the levels of 25 metabolites changed significantly after UDCA treatment. The pathway enrichment analysis showed that the UDCA addition evoked a common response related to phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; arachidonic acid metabolism; and terpenoid backbone biosynthesis. CONCLUSIONS: UDCA treatment within a short time interval (7 days) did not improve the circulating laboratory values in patients with CDC who had undergone endoscopy surgery. However, relevant decreases in the bile levels of cholesterol and endotoxin were observed. UDCA evoked a common response related to lipid metabolism and amino acid metabolism, which probably reduced the bile level of cholesterol, protected hepatocytes, and corrected the abnormality of lipid metabolism caused by CDC.

Noxious Combination of Tobacco Smoke Nitrosamines with Bile, Deoxycholic Acid, Promotes Hypopharyngeal Squamous Cell Carcinoma, via NFκB, In Vivo.

Tobacco smoking is the most known risk factor for hypopharyngeal cancer. Bile reflux has recently been documented as an independent risk factor for NFκB-mediated hypopharyngeal squamous cell carcinoma. However, the carcinogenic effect of tobacco smoke on the hypopharynx and its combination with bile has not yet been proven by direct evidence. We investigated whether in vivo chronic exposure (12-14 weeks) of murine (C57Bl/6J) hypopharyngeal epithelium to tobacco smoke components (TSC) [N-nitrosamines; 4-(N-Methyl-N-Nitrosamino)-1-(3-pyridyl)-1-butanone (0.2 mmol/L), N-nitrosodiethylamine (0.004 mmol/L)], as the sole drinking fluid 5 days per week, along with topically applied (two times/day) bile [deoxycholic acid (0.28 mmol/L)], can accelerate a possible TSC-induced neoplastic process, by enhancing NFκB activation and the associated oncogenic profile, using histologic, IHC, and qPCR analyses. We provide direct evidence of TSC-induced premalignant lesions, which can be exacerbated by the presence of bile, causing invasive carcinoma. The combined chronic exposure of the hypopharynx to TSC with bile causes advanced NFκB activation and profound overexpression of Il6, Tnf, Stat3, Egfr, Wnt5a, composing an aggressive phenotype. We document for the first time the noxious combination of bile with a known risk factor, such as tobacco smoke nitrosamines, in the development and progression of hypopharyngeal cancer, via NFκB, in vivo. The data presented here encourage further investigation into the incidence of upper aerodigestive tract cancers in smokers with bile reflux and the early identification of high-risk individuals in clinical practice. This in vivo model is also suitable for large-scale studies to reveal the nature of inflammatory-associated aerodigestive tract carcinogenesis and its targeted therapy. PREVENTION RELEVANCE: Early assessment of bile components in refluxate of tobacco users can prevent the chronic silent progression of upper aerodigestive tract carcinogenesis. This in vivo model indicates that bile reflux might have an additive effect on the tobacco-smoke N-nitrosamines effect and could be suitable for large-scale studies of diagnostic and therapeutic interventions.

Combination of LIBS-based elemental analysis and near-infrared molecular fingerprinting of bile juice to enhance identification of gallbladder cancer.

Laser-induced breakdown spectroscopy (LIBS) and near-infrared (NIR) spectroscopy, enabling the measurement of raw bile directly without sample pretreatment, were cooperatively combined to enhance the discrimination of gallbladder cancer (GBC) from other diseases of gallstone and gallbladder (GB) polyp. Since elemental contents and metabolite compositions of bile vary according to the pathological conditions of pancreaticobiliary patients, the use of complementary information could be synergetic to improve disease identification accuracy. The ratios of Mg and Na peak areas (AMg/ANa) and Na and K peak areas (ANa/AK) in the LIBS spectra of GBC samples were different from those of the remaining samples. Also, the intensity ratios of main NIR peaks differed in GBC. Nonetheless, the use of only element peak area ratio or NIR peak intensity ratio was not sufficient to clearly discriminate GBC. On the other hand, when the ANa/AK values and second NIR principal component scores were combined, the discrimination of GBC from normal/gallstone/GB polyp was substantially enhanced owing to incorporation of both complementary GBC-discriminant spectroscopic signatures.

Rapid screening and identification of metabolites of murpanicin in rats by UHPLC/Q-TOF-MS/MS combined with diagnostic fragment ions (DFIs) and multiple mass defect filter.

Murpanicin is a main coumarin with obvious anti-inflammatory effect isolated from Murraya species. The primary aim of this research is to identify and characterize the possible metabolites of murpanicin in rats. The metabolites generated in the plasma, urine, bile, and feces of rats were identified and characterized employing ultra-high performance liquid chromatography coupled with quadrupole-time of flight tandem mass spectrometry (UHPLC/Q-TOF-MS/MS) based on diagnostic fragment ions (DFIs) and multiple mass defect filter (MMDF). A total of 67 metabolites were identified, among which 55 are phase I and 12 are phase II reacted products. The plausible structures of the metabolites and the probable metabolic pathways were deduced based on the diagnostic fragment ions, mass ppm error, and mass fragmentation pattern, as well as the MetabolitePilot™ software. The majority of phase I metabolites were generated by demethylation, deethylation, dehydrogenation, hydroxylation, and reduction, while phase II metabolites were mainly generated by glucuronidation and sulfation. Moreover, some rare phase II metabolic pathways, such as N-acetylcysteine conjugation, cysteine conjugation, and S-cysteine conjugation were also observed. In conclusion, our study first expounded the metabolites of murpanicin in rats and provided reference for further clarification of the in vivo therapeutic material basis of murpanicin and other 8-prenylcoumarin derivatives. Moreover, UHPLC/Q-TOF-MS combined with MMDF and DFIs has been proved to be an effective method for rapid identification of the homolog-gathered natural products and their metabolites.

Natural bear bile powder suppresses neuroinflammation in lipopolysaccharide-treated mice via regulating TGR5/AKT/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Tang Dynasty classics Dietetic Material Medica and the Ming Dynasty classics Compendium of Materia Medica records, bear bile powder (BBP) has been used to treat a variety of diseases, such as febrile seizures, the pathogenesis of which is associated to neuroinflammation. However, the mechanism of BBP on alleviating neuroinflammation remains unclear. AIMS OF THE STUDY: Microglia can be activated by peripheral lipopolysaccharide (LPS) and play an important role in the pathogenesis of neuroinflammation. The purpose of this study is to investigate the effects and mechanism of BBP in inhibiting LPS-induced microglia inflammation in vitro and in vivo. MATERIALS AND METHODS: The anti-microglia inflammatory effects and mechanism of BBP were assessed in LPS-treated BV2 microglial cells and in LPS-treated mice. The mRNA expression levels of the inflammatory factor and the protein expressions of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), takeda G-protein coupled receptor 5 (TGR5), nuclear factor-κB (NF-κB), inhibitor of NF-κB (IκBɑ), protein kinase B (AKT) in BV2 cells, mouse hippocampus and cortex were detected. The NF-κB transcription activity and NF-κB nuclear translocation were observed. RESULTS: Our findings showed that BBP reduces branched process retraction and NO in LPS-treated BV2 cells, inhibits the protein expression of ionized calcium binding adaptor molecule 1 in the hippocampus of LPS-treated mice. Moreover, we observed that BBP decreases tumor necrosis factor α, interleukin (IL)-6 and IL-1ß mRNA levels, deceases iNOS and COX-2 protein levels, increases TGR5 protein levels, suppresses the phosphorylation of AKT, NF-κB and IκBɑ protein in microglia both in vitro and in vivo. Further, we found that triamterene, the inhibitor of TGR5, abolishes the effects of BBP in LPS- treated BV2 cells. CONCLUSION: BBP inhibits LPS-induced microglia activation, and the mechanism of its action is partly through TGR5/AKT/NF-κB signaling pathway.

Early and accurate detection of cholangiocarcinoma in patients with primary sclerosing cholangitis by methylation markers in bile.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is associated with increased risk of cholangiocarcinoma (CCA). Early and accurate CCA detection represents an unmet clinical need as the majority of patients with PSC are diagnosed at an advanced stage of malignancy. In the present study, we aimed at establishing robust DNA methylation biomarkers in bile for early and accurate diagnosis of CCA in PSC. APPROACH AND RESULTS: Droplet digital PCR (ddPCR) was used to analyze 344 bile samples from 273 patients with sporadic and PSC-associated CCA, PSC, and other nonmalignant liver diseases for promoter methylation of cysteine dioxygenase type 1, cannabinoid receptor interacting protein 1, septin 9, and vimentin. Receiver operating characteristic (ROC) curve analyses revealed high AUCs for all four markers (0.77-0.87) for CCA detection among patients with PSC. Including only samples from patients with PSC diagnosed with CCA ≤ 12 months following bile collection increased the accuracy for cancer detection, with a combined sensitivity of 100% (28/28) and a specificity of 90% (20/203). The specificity increased to 93% when only including patients with PSC with longtime follow-up (> 36 months) as controls, and remained high (83%) when only including patients with PSC and dysplasia as controls (n = 23). Importantly, the bile samples from the CCA-PSC ≤ 12 patients, all positive for the biomarkers, included both early-stage and late-stage CCA, different tumor growth patterns, anatomical locations, and carbohydrate antigen 19-9 levels. CONCLUSIONS: Using highly sensitive ddPCR to analyze robust epigenetic biomarkers, CCA in PSC was accurately detected in bile, irrespective of clinical and molecular features, up to 12 months before CCA diagnosis. The findings suggest a potential for these biomarkers to complement current detection and screening methods for CCA in patients with PSC.