Unconjugated Bile Acids Research Articles

Changes in bile acid subtypes and long-term successful weight-loss in response to weight-loss diets: The POUNDS lost trial.

Primary bile acids (BAs) are synthesized in the liver and secondary BAs result from intestinal microbial activity. Different subtypes of BAs may be involved in regulating adiposity and energy homeostasis. We examined how changes in circulating BA subtypes induced by weight-loss diets were associated with improvements in adiposity, regional fat deposition and energy metabolism among overweight and obese adults. The study included 551 subjects who participated in a 2-year weight-loss diet intervention trial. Circulating 14 BA subtypes (primary and secondary unconjugated BAs and their taurine-/glycine-conjugates) were measured at baseline and 6months. Associations of changes in BAs with changes in weight, waist circumference, resting energy expenditure (REE), body fat composition and fat distribution were evaluated. Greater decreases in primary BAs (cholate and chenodeoxycholate) and secondary BAs (deoxycholate and lithocholate) and their conjugates (except for glycolithocholate) were associated with more decreases in weight and waist circumference at 6months (P-after-false-discovery-rate-correction [PFDR ]<.05). We found that changes in glycocholate and glycoursodeoxycholate were consistently associated with reductions of general and central adiposity, REE, whole-body fat and visceral adipose tissue (PFDR <.05). Further, the initial (6-month) changes in BA subtypes were differently predictive of successful weight loss over 2years. The decreases in primary and secondary BA subtypes after eating low-calorie weight-loss diets were significantly associated with improving adiposity, fat accumulation and energy metabolism, suggesting that specific BA subtypes would be predictive of long-term successful weight loss and individuals' response to the treatment of weight-loss diets.

Si-Wu-Tang ameliorates fibrotic liver injury via modulating intestinal microbiota and bile acid homeostasis

BackgroundFibrotic liver injury is a progressive scarring event, which may permanently affect liver function and progress into devastating end-stage liver diseases due to the absence of effective therapies. Si-Wu-Tang (SWT), a traditional Chinese medicine formula used in clinic to treat gynecological disorders for centuries, has been investigated in recent preliminary findings for its role in alleviating chronic liver diseases. Here we aim to elucidate the therapeutic effects and possible mechanisms of SWT against fibrotic liver injury.MethodsUHPLC-MS/MS was performed to investigate the chemical characterization of SWT. After intragastrically administered with carbon tetrachloride (CCl4) every 3 days for 1-week, C57BL/6 mice were orally administered with SWT (5.2, 10.4 and 20.8 g/kg) once daily for 3 weeks along with CCl4 challenge. Liver function was determined by the measurement of serum biomarkers, hematoxylin and eosin (H&E) and Masson’s trichrome staining. Intestinal inflammatory infiltration and the disruption of intestinal barrier were examined by H&E and E-cadherin immunohistochemical staining. The microbial composition of intestinal content was determined by 16S rRNA sequencing. Serum bile acids (BAs) profiling was analyzed by LC–MS/MS. Simultaneously, the expression of genes of interest was determined by qPCR and western blot.ResultsSWT exhibited remarkable therapeutic effects on CCl4-induced liver fibrosis, as indicated by improved collagen accumulation in livers, intestinal barrier injury and hepatic and intestinal inflammatory response. Results of 16S rRNA sequencing revealed that SWT treatment strikingly restructured intestinal microbiota in fibrotic mice by increasing the relative abundances of Bacteroides and Lachnoclostridium and decreasing the relative abundances of Alistipes and Rikenellaceae. UHPLC-MS/MS data suggested that SWT altered the composition of BAs in circulation as evidenced by increased unconjugated BAs like cholic acid and chenodeoxycholic acid but decreased conjugated BAs including taurocholic acid and taurodeoxycholic acid, compared to that in CCl4 mice. Notably, SWT efficiently improved the imbalance of BA homeostasis in livers caused by CCl4 via activating farnesoid X receptor (FXR)-fibroblast growth factor 15 enterohepatic and FXR-small heterodimer partner hepatic pathways.ConclusionSWT decreased inflammatory response, reconstructed gut microbiota-mediated BA homeostasis as well as activated FXR pathways, which eventually protected against CCl4-induced fibrotic liver injury.

Polyelectrolytes Formulated with Primary Unconjugated Bile Acid Optimised Pharmacology of Bio-Engineered Implant

Introduction. Several studies have shown that different biomaterials and hydrogels comprising various bile acids such as chenodeoxycholic acid (CDCA), as well as excipients such as poly-(styrene)-sulphonate (PSS) and poly-(allyl)-amine (PAA), exhibited positive biological effects on encapsulated viable pancreatic β-cells. Hence, this study aimed to investigate whether incorporating CDCA with PSS and PAA will optimise the functions of encapsulated pancreatic islets post-transplantation in Type 1 diabetes (T1D). Methods. Mice were made T1D, divided into two equal groups, and transplanted with encapsulated islets in PSS-PAA (control) or with CDCA-PSS-PAA (treatment) microcapsules. The effects of transplanted microcapsules on blood glucose, inflammation and the bile acid profile were measured post-transplantation. Results and Conclusion. Compared with control, the treatment group showed better survival rate, improved glycaemic control, and lower inflammatory profile, illustrated by ↓ interleukin 1-β, interleukin-6, interleukin-12, and tumour-necrosis factor-α, and ↓ levels of the bile acid, as well as lithocholic acid in the plasma, liver, large intestine and faeces. The results suggest that CDCA incorporation with PSS-PAA microcapsules exerted beneficial effects on encapsulated islets and resulted in enhanced diabetes treatment, post-transplantation, at the local and systemic levels.

The Effects of Primary Unconjugated Bile Acids on Nanoencapsulated Pharmaceutical Formulation of Hydrophilic Drugs: Pharmacological Implications.

IntroductionIn a recent study, in our laboratory, primary unconjugated bile acids, commonly found in humans, chenodeoxycholic acid (CDCA), have been shown to improve stability of nanoencapsulated lipophilic drugs and improve their release profile after oral administration likely via electrokinetic stabilisation. Hence, this study aimed to examine the effects of CDCA on exerting similar effects on hydrophilic drugs.MethodsVarious CDCA-based formulations were produced for the orally administered hydrophilic drug, metformin. Analyses of these formulations included electrokinetic potentials, topography, drug and CDCA formulation contents, nano size distribution, heat-induced deformation and outer-core expansion indices, release profiles, shell-resistance ratio, and thermal and chemical indices. With the drug’s main target being pancreatic beta-cells, the formulations’ effects on cell viability, functions and inflammatory profiles were also investigated.Results and ConclusionsCDCA-based metformin formulations exhibited improved stability and release profiles via thermal, chemical and electrokinetic effects, which were formulation-dependent suggesting potential applications of CDCA in the oral targeted delivery of hydrophilic drugs.

Parallel derivatization strategy for comprehensive profiling of unconjugated and glycine-conjugated bile acids using Ultra-high performance liquid chromatography-tandem mass spectrometry

Bile acids (BAs) are steroidal compounds that play important roles in the occurrence and development of liver injury. However, comprehensive characterization of BAs was rarely reported due to the limitations of both standards access and detection sensitivity. In this study, a parallel derivatization strategy was established for the sensitive and comprehensive profiling of unconjugated and glycine-conjugated BAs by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Two structural analogues 2-hydrazinyl-4,6-dimethylpyrimidine (DMP) and 2-hydrazinylpyrimidine (DP) were used as the parallel derivatization reagents for BAs labeling, facilitating the improvements of both detection sensitivities and chromatographic performances. The derivatization reactions can be completed in 20 min at room temperature, with derivatization efficacy higher than 99 %. Through derivatization, the sensitivity of BAs increased dozens or hundreds of times compared to their non-derivatized forms. Due to the structural similarities of derivatized BAs, general MS parameters can be forged for the analysis of DMP and DP labeled BAs. In addition, the DP labeled BAs were incorporated into the DMP derivatized biological samples for both the discovery and comprehensive characterization of BAs. Retention time shift (RTS) and peak area ratio (PAR) induced by the parallel DMP and DP labeled BAs were used for the rapid identification of BAs from complex biological samples. More than 200 BAs were profiled in rat serum using this parallel derivatization strategy. Further, the new strategy was successfully implemented in BAs profiling of serum samples from tripterysium glycosides-induced liver injury rat model. The disturbance of the BA metabolism network was further interpreted.

Association between circulating bile acid alterations and nonalcoholic steatohepatitis independent of obesity and diabetes mellitus.

Bile acid (BA) dysregulation is related to not only metabolic diseases but also nonalcoholic fatty liver disease (NAFLD). We investigated whether circulating BA levels are altered according to the histological severity of NAFLD independent of metabolic derangements. Global metabolic profiling and targeted BA analysis using sera collected from biopsy-proven no-NAFLD (n=67), nonalcoholic fatty liver (NAFL) (n=99), and nonalcoholic steatohepatitis (NASH, n=75) subjects were performed sequentially. Circulating metabolome analysis integrated with the hepatic transcriptome was performed to elucidate the mechanistic basis of altered circulating BA profiles after stratification by obesity (body mass index≤25kg/m2 ). Circulating BA alterations were also validated in an independent validation cohort (29 no-NAFLD, 70 NAFL and 37 NASH). Global profiling analysis showed that BA was the metabolite significantly altered in NASH compared to NAFL. Targeted BA analysis demonstrated that glyco-/tauro-conjugated primary BAs were commonly increased in nonobese and obese NASH, while unconjugated primary BAs increased only in nonobese NASH. These characteristic primary BA level changes were maintained even after stratification according to diabetes status and were replicated in the independent validation cohort. Compared to nonobese NAFL patients, nonobese NASH patients exhibited upregulated hepatic expression of CYP8B1. BA metabolism is dysregulated as the histological severity of NAFLD worsens, independent of obesity and diabetes status; dysregulation is more prominent in nonobese NAFLD patients. Metabolome-driven omics approach provides new insight into our understanding of altered BA metabolism associated with individual phenotypes of NAFLD.

Understanding the physiological functions of the host xenobiotic-sensing nuclear receptors PXR and CAR on the gut microbiome using genetically modified mice

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

Liver Bile Acid Changes in Mouse Models of Alzheimer's Disease.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive cognitive impairment. It is hypothesized to develop due to the dysfunction of two major proteins, amyloid-β (Aβ) and microtubule-associated protein, tau. Evidence supports the involvement of cholesterol changes in both the generation and deposition of Aβ. This study was performed to better understand the role of liver cholesterol and bile acid metabolism in the pathophysiology of AD. We used male and female wild-type control (C57BL/6J) mice to compare to two well-characterized amyloidosis models of AD, APP/PS1, and AppNL-G-F. Both conjugated and unconjugated primary and secondary bile acids were quantified using UPLC-MS/MS from livers of control and AD mice. We also measured cholesterol and its metabolites and identified changes in levels of proteins associated with bile acid synthesis and signaling. We observed sex differences in liver cholesterol levels accompanied by differences in levels of synthesis intermediates and conjugated and unconjugated liver primary bile acids in both APP/PS1 and AppNL-G-F mice when compared to controls. Our data revealed fundamental deficiencies in cholesterol metabolism and bile acid synthesis in the livers of two different AD mouse lines. These findings strengthen the involvement of liver metabolism in the pathophysiology of AD.

Impact on Bile Acid Concentrations by Alveolar Echinococcosis and Treatment with Albendazole in Mice.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis is a chronic, progressive liver disease widely distributed in the Northern Hemisphere. The main treatment options include surgical interventions and chemotherapy with benzimidazole albendazole (ABZ). To improve the current diagnosis and therapy of AE, further investigations into parasite–host interactions are needed. This study used liquid chromatography–tandem mass spectrometry (LC-MS/MS) to assess serum and liver tissue bile acid profiles in the i.p. chronic E. multilocularis-infected mouse model and evaluated the effects of the anthelmintic drug ABZ. Additionally, hepatic mRNA and protein expression of enzymes and transporters regulating bile acid concentrations were analyzed. AE significantly decreased unconjugated bile acids in serum and liver tissue. Taurine-conjugated bile salts were unchanged or increased in the serum and unchanged or decreased in the liver. Ratios of unconjugated to taurine-conjugated metabolites are proposed as useful serum markers of AE. The expression of the bile acid synthesis enzymes cytochrome P450 (CYP) 7A1 and aldo-keto reductase (AKR) 1D1 tended to decrease or were decreased in mice with AE, along with decreased expression of the bile acid transporters Na+/taurocholate cotransporting polypeptide (NTCP) and bile salt efflux pump (BSEP). Importantly, treatment with ABZ partially or completely reversed the effects induced by E. multilocularis infection. ABZ itself had no effect on the bile acid profiles and the expression of relevant enzymes and transporters. Further research is needed to uncover the exact mechanism of the AE-induced changes in bile acid homeostasis and to test whether serum bile acids and ratios thereof can serve as biomarkers of AE and for monitoring therapeutic efficacy.

Hyperoside attenuates non-alcoholic fatty liver disease in rats via cholesterol metabolism and bile acid metabolism

IntroductionNon-alcoholic fatty liver disease (NAFLD) results from increased hepatic total cholesterol (TC) and total triglyceride (TG) accumulation. In our previous study, we found that rats treated with hyperoside became resistant to hepatic lipid accumulation. ObjectivesThe present study aims to investigate the possible mechanisms responsible for the inhibitory effects of hyperoside on the lipid accumulation in the liver tissues of the NAFLD rats. MethodsLabel-free proteomics and metabolomics targeting at bile acid (BA) metabolism were applied to disclose the mechanisms for hyperoside reducing hepatic lipid accumulation among the NAFLD rats. ResultsIn response to hyperoside treatment, several proteins related to the fatty acid degradation pathway, cholesterol metabolism pathway, and bile secretion pathway were altered, including ECI1, Acnat2, ApoE, and BSEP, etc. The expression of nuclear receptors (NRs), including farnesoid X receptor (FXR) and liver X receptor α (LXRα), were increased in hyperoside-treated rats’ liver tissue, accompanied by decreased protein expression of catalyzing enzymes in the hepatic de novo lipogenesis and increased protein level of enzymes in the classical and alternative BA synthetic pathway. Liver conjugated BAs were less toxic and more hydrophilic than unconjugated BAs. The BA-targeted metabolomics suggest that hyperoside could decrease the levels of liver unconjugated BAs and increase the levels of liver conjugated BAs. ConclusionsTaken together, the results suggest that hyperoside could improve the condition of NAFLD by regulating the cholesterol metabolism as well as BAs metabolism and excretion. These findings contribute to understanding the mechanisms by which hyperoside lowers the cholesterol and triglyceride in NAFLD rats.

Development of an Efficient and Sensitive Chemical Derivatization-Based LC-MS/MS Method for Quantifying Gut Microbiota-Derived Metabolites in Human Plasma and Its Application in Studying Cardiovascular Disease.

Recently, the gut microbiota has been found to be associated with many diseases, such as inflammatory bowel disease, depression, Parkinson's disease, cancer, metabolic syndrome, and cardiovascular disease (CVD). Among various gut microbiota-derived metabolites (GMs), short-chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) metabolites are the most frequently discussed metabolites. LC-MS/MS shows advantages in quantifying the levels of metabolites with good sensitivity and selectivity; however, the poor ionization efficiency and polar characteristics of SCFAs make their analysis challenging, especially when analyzing plasma samples with low SCFA concentrations. Moreover, without characteristic fragment ions for unconjugated BAs and different detection ion modes for TRP metabolites and BAs, GM analysis is complex and time-consuming. To overcome these problems, we developed a derivatization method combined with LC-MS/MS to enhance the sensitivity and LC retention of GMs. Through derivatization with 3-nitrophenylhydrazine (3-NPH), 7 SCFAs, 9 bile acids, and 6 tryptophan metabolites can be simultaneously analyzed via separation within 14 min on a reversed-phase C18 column. For accurate quantification, 13C6-3NPH-labeled standards were used as one-to-one internal standards. This derivatization approach was optimized and then validated. We further applied this method to investigate the targeted GM profile in patients with CVD. The results showed a significant reduction in plasma butyrate levels in CVD patients compared with healthy controls, suggesting its potentially protective role in CVD. In summary, this work provides a sensitive and effective LC-MS/MS method for simultaneously quantifying gut microbiota-related metabolites in human plasma, which could benefit various future gut microbiota-related studies.

Abstract P131: Changes In Bile Acid Subtypes And Improvements In Adiposity And Energy Metabolism In Response To Weight-loss Diets

Introduction: Bile acids (BAs) are synthesized in the liver from cholesterol, and gut microbiota transform the host-derived primary BAs into secondary BAs in the intestine. Emerging evidence suggests that different types of circulating BAs may play pivotal roles in regulating energy metabolism and body adiposity. Hypothesis: We comprehensively examined whether changes in different BA subtypes after consuming weight-loss diets were associated with improvements in energy metabolism and body adiposity among patients with obesity. Methods: This study included 551 overweight and obese adults who participated in a 2-year weight-loss dietary intervention, the POUNDS Lost trial. Blood levels of 14 types of BAs (primary and secondary unconjugated BAs and their taurine-/glycine-conjugates) were measured at baseline and 6 months after the intervention; changes in BAs from baseline to 6 months after the intervention were calculated. We evaluated changes in resting energy expenditure, weight, and waist circumference. Also, body composition was assessed by the dual-energy X-ray absorptiometry (DEXA) scans, and fat distribution was assessed by computed tomography (CT) scans. Results: At baseline, higher primary and secondary BAs were related to greater degrees of adiposity and energy expenditure. At 6 months after the intervention, greater decreases in primary BAs (cholic acid [CA] and chenodeoxycholic acid) and secondary BAs (deoxycholic acid [DCA] and lithocholic acid [LCA]) and their conjugated subtypes (except for glycolithocholic acid) were significantly associated with more decreases in weight and waist circumference at 6 months after the intervention (P values after controlling for the multiple testing, P FDR &lt;0.05). Greater reductions in the primary BAs (both unconjugated and conjugated types) and secondary BAs (DCA and its taurine-/glycine-conjugated forms) were also significantly ( P FDR &lt;0.05) associated with more decreases in resting energy expenditure at 6 months. We found that reductions in two BA subtypes, glycocholic acid (GCA) and glycoursodeoxycholic acid (GUDCA), were consistently and significantly associated with improvements in energy metabolism, general and central adiposity, as well as body fat composition and visceral adipose tissue mass. Further, the initial (6-month) changes in several primary and secondary BAs (including GCA and GUDCA) were significantly predictive of the long-term successful weight loss (weight loss of more than 5% loss from the initial weight) at 2 years. Conclusions: Weight-loss diet-induced changes in circulating various BAs may be involved in improving general and central adiposity and energy metabolism. Changes in specific BA subtypes would be potential targets for improving regional adiposity and achieving successful weight-loss among patients with obesity.

A comprehensive time course and correlation analysis of indomethacin-induced inflammation, bile acid alterations and dysbiosis in the rat small intestine

It has been proposed that changes in microbiota due to nonsteroidal anti-inflammatory drugs (NSAIDs) alter the composition of bile, and elevation of hydrophobic secondary bile acids contributes to small intestinal damage. However, little is known about the effect of NSAIDs on small intestinal bile acids, and whether bile alterations correlate with mucosal injury and dysbiosis. Here we determined the ileal bile acid metabolome and microbiota 24, 48 and 72 h after indomethacin treatment, and their correlation with each other and with tissue damage in rats. In parallel with the development of inflammation, indomethacin increased the ileal proportion of glycine and taurine conjugated bile acids, but not bile hydrophobicity. Firmicutes decreased with time, whereas Gammaproteobacteria increased first, but declined later and were partially replaced by Bilophila, Bacteroides and Fusobacterium. Mucosal injury correlated negatively with unconjugated bile acids and Gram-positive bacteria, and positively with taurine conjugates and some Gram-negative taxa. Strong positive correlation was found between Lactobacillaceae, Ruminococcaceae, Clostridiaceae and unconjugated bile acids. Indomethacin-induced dysbiosis was not likely due to direct antibacterial effects or alterations in luminal pH. Here we provide the first detailed characterization of indomethacin-induced time-dependent alterations in small intestinal bile acid composition, and their associations with mucosal injury and dysbiosis. Our results suggest that increased bile hydrophobicity is not likely to contribute to indomethacin-induced small intestinal damage.

Fitness for purpose of stabilized stool samples for bile acid metabolite analyses

Biobanks and cohort studies are increasingly utilizing chemical stabilizers to collect and store stool samples for downstream DNA-based microbiome analyses. While stabilizers permit ambient-temperature collection and storage of samples for gut microbiome studies, the use of the same sample type for downstream metabolomics assays has not been explored. Microbiome-metabolomics analysis of fecal samples is increasingly getting attention to further elucidate the mechanisms by which the gut microbiota influences the host. In this study, we evaluated fitness-for-purpose of OMNIgene-GUT-collected stool samples for downstream metabolomics assays in the scope of fecal bile acids (BA) quantification. Biocrates Bile Acids Kit was used for the quantification of BA from eight healthy donors’ samples collected in (1) OMNIgene-GUT kit and (2) snap frozen in −80 °C in duplicates. A highly selective reversed phase LC–MS/MS analysis method in negative ion multiple reaction monitoring (MRM) detection mode was applied to determine the BA concentrations in each sample.Total fecal BA levels were detectable in OMNIgene-GUT-collected samples (range: 29.9–903.7 pmol/mg). Paired t-test confirmed that there was a significant difference in the total BAs between the OMNIgene-GUT and snap frozen samples (p < 0.05). Extractions from snap frozen samples resulted in higher concentrations of total BAs (range: 243.7–1136.2 pmol/mg). Qualitative differences between individual donors’ BA profiles were detectable using the two sample collection methods. No significant difference was found in the relative concentrations of primary (CA, CDCA) or secondary (DCA, LCA, UDCA) unconjugated BAs to the total BA concentrations in OMNIgene-GUT-collected samples as compared with the snap frozen samples (Wilcoxon-Mann–Whitney test, p > 0.05). Passing-Bablok method comparison and correlation analyis showed a high degree of correlation in the relative concentrations of CA, CDCA, DCA and LCA between OMNIgene-GUT and snap frozen samples. For these four bile acids, the two methods are comparable at an acceptability bias of 30%. We conclude that the OMNIgene-GUT-collected stool samples are fit-for-purpose for downstream fecal bile acids analysis.

Weakly Acidic Bile Is a Risk Factor for Hypopharyngeal Carcinogenesis Evidenced by DNA Damage, Antiapoptotic Function, and Premalignant Dysplastic Lesions In Vivo.

Simple SummaryThe etiologic role of biliary reflux in hypopharyngeal cancer is supported by clinical data. Although, reflux episodes often occur at pH 4.0, they can also occur at weakly acidic pH (5.5–6.0). The carcinogenic effect of bile at strongly acidic pH (pH 3.0) was recently documented in vivo. Here, we provide novel in vivo evidence that a weakly acidic pH of 5.5, similarly to a strongly acidic pH of 3.0, increases the risk of bile-related hypopharyngeal neoplasia. We document that chronic exposure of hypopharyngeal mucosa to bile at pH 5.5 promotes premalignant lesions with DNA damage, NF-κB activation, and deregulated mRNA and miRNA phenotypes, including Bcl-2 and miR-451a. The oncogenic effects of bile over a wider pH range suggests that antacid therapy may be insufficient to fully modify the effects of a bile induced oncogenic effect.Background: There is recent in vivo discovery documenting the carcinogenic effect of bile at strongly acidic pH 3.0 in hypopharynx, while in vitro data demonstrate that weakly acidic bile (pH 5.5) has a similar oncogenic effect. Because esophageal refluxate often occurs at pH > 4.0, here we aim to determine whether weakly acidic bile is also carcinogenic in vivo. Methods: Using 32 wild-type mice C57B16J, we performed topical application of conjugated primary bile acids with or without unconjugated secondary bile acid, deoxycholic acid (DCA), at pH 5.5 and controls, to hypopharyngeal mucosa (HM) twice per day, for 15 weeks. Results: Chronic exposure of HM to weakly acidic bile, promotes premalignant lesions with microinvasion, preceded by significant DNA/RNA oxidative damage, γH2AX (double strand breaks), NF-κB and p53 expression, overexpression of Bcl-2, and elevated Tnf and Il6 mRNAs, compared to controls. Weakly acidic bile, without DCA, upregulates the “oncomirs”, miR-21 and miR-155. The presence of DCA promotes Egfr, Wnt5a, and Rela overexpression, and a significant downregulation of “tumor suppressor” miR-451a. Conclusion: Weakly acidic pH increases the risk of bile-related hypopharyngeal neoplasia. The oncogenic properties of biliary esophageal reflux on the epithelium of the upper aerodigestive tract may not be fully modified when antacid therapy is applied. We believe that due to bile content, alternative therapeutic strategies using specific inhibitors of relevant molecular pathways or receptors may be considered in patients with refractory GERD.

The effects of different plant extracts on bile salt hydrolase activity of Lactobacillus strains isolated from the gastrointestinal tract of poultry

The bile salt hydrolysis (BSH) enzyme weakens fat metabolism through bile salt deconjugation and reduces poultry performance, in order to cope with the antibacterial properties of the bile. Therefore, reducing the activity of this enzyme through the use of feed additives is probably a promising alternative to antibiotics for improving poultry performance. Plant extracts have long been used as feed additives for promoting poultry growth. In the current experiment, five Lactobacillus strains including Lactobacillus animalis, Lactobacillus acidophillus, Lactobacillus gallinarum, Lactobacillus lactis, and Lactobacillus returi were obtained from the poultry hindgut and were used as the probiotic application. A plate test and two-step enzymatic reaction method were used for deconjugation activity determination of the Lactobacillus strains. Further, four plant extracts (i.e., the aerial parts of Rosemary (Rosmarinus officinalis), Roselle calyx (Hibiscus sabdariffa), Berberis vulgaris root, and Green tea) were examined in terms of BSH enzyme inhibitors using the cell-free extracts as the potential antibiotic alternative. Furthermore, the gallbladders of the broilers were freshly collected from the poultry slaughterhouses, and their contents were extracted. The results showed that all Lactobacillus strains could hydrolyze the taurocholate acid (TCA) and chicken bile salt mixture (CBSM) to unconjugated bile acid. Moreover, ethanolic extracts of B. vulgaris root and Green tea relatively reduced the activity of the BSH enzyme that could potentially be investigated as an appropriate alternative in poultry feed in vivo. In conclusion, all five Lactobacillus strains were resistant to bile salts (i.e. TCA and CBSM) by BSH activity, and the addition of Green tea and B. vulgaris root extracts to the bacterial medium demonstrated inhibitory effects against the BSH enzyme.

Bile acids during pregnancy: Trimester variations and associations with glucose homeostasis.

Background and aimsBile acids are known to contribute to hepatic glucose and lipid metabolism regulation. Although glucose homeostasis sustains well‐characterized modifications during uncomplicated pregnancies, changes in bile acids concentrations and relative proportions throughout pregnancy remain unknown. Furthermore, literature shows strong associations between bile acids profiles and glucose homeostasis under normal metabolic conditions. We seek, first, to characterize bile acids' metabolic changes across trimesters and, second, to evaluate associations between changes in bile acids and glucose homeostasis indexes in the first and second trimesters.MethodsA total of 78 women were recruited and followed at each trimester of pregnancy. Fasting serum samples were collected once per trimester in which quantitative measurement of 30 different bile acids' molecules were performed using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Glucose homeostasis indexes were measured in the first and second trimesters, after a 12‐hour fast and following a 75 g oral glucose tolerance test.ResultsTotal bile acids increased from the first trimester to late pregnancy, along with the cholic acid: chenodeoxycholic acid and conjugated: unconjugated bile acids ratios. Changes in bile acids were positively associated with elevated peripheral and hepatic insulin resistance indexes, as well as with trimestral changes in these indexes.ConclusionOur findings suggest that modifications occurring in bile acids' profiles during normal pregnancy are associated with changes in glucose homeostasis. Further research is needed to examine the nature of those associations and the possible outcome of bile acids changes on pathological glucose homeostasis alterations during pregnancy.

STARD1 promotes NASH-driven HCC by sustaining the generation of bile acids through the alternative mitochondrial pathway

Besides their physiological role in bile formation and fat digestion, bile acids (BAs) synthesised from cholesterol in hepatocytes act as signalling molecules that modulate hepatocellular carcinoma (HCC). Trafficking of cholesterol to mitochondria through steroidogenic acute regulatory protein 1 (STARD1) is the rate-limiting step in the alternative pathway of BA generation, the physiological relevance of which is not well understood. Moreover, the specific contribution of the STARD1-dependent BA synthesis pathway to HCC has not been previously explored. STARD1 expression was analyzed in a cohort of human non-alcoholic steatohepatitis (NASH)-derived HCC specimens. Experimental NASH-driven HCC models included MUP-uPA mice fed a high-fat high-cholesterol (HFHC) diet and diethylnitrosamine (DEN) treatment in wild-type (WT) mice fed a HFHC diet. Molecular species of BAs and oxysterols were analyzed by mass spectrometry. Effects of NASH-derived BA profiles were investigated in tumour-initiated stem-like cells (TICs) and primary mouse hepatocytes (PMHs). Patients with NASH-associated HCC exhibited increased hepatic expression of STARD1 and an enhanced BA pool. Using NASH-driven HCC models, STARD1 overexpression in WT mice increased liver tumour multiplicity, whereas hepatocyte-specific STARD1 deletion (Stard1ΔHep) in WT or MUP-uPA mice reduced tumour burden. These findings mirrored the levels of unconjugated primary BAs, β-muricholic acid and cholic acid, and their tauroconjugates in STARD1-overexpressing and Stard1ΔHep mice. Incubation of TICs or PMHs with a mix of BAs mimicking this profile stimulated expression of genes involved in pluripotency, stemness and inflammation. The study reveals a previously unrecognised role of STARD1 in HCC pathogenesis, wherein it promotes the synthesis of primary BAs through the mitochondrial pathway, the products of which act in TICs to stimulate self-renewal, stemness and inflammation. Effective therapy for hepatocellular carcinoma (HCC) is limited because of our incomplete understanding of its pathogenesis. The contribution of the alternative pathway of bile acid (BA) synthesis to HCC development is unknown. We uncover a key role for steroidogenic acute regulatory protein 1 (STARD1) in non-alcoholic steatohepatitis-driven HCC, wherein it stimulates the generation of BAs in the mitochondrial acidic pathway, the products of which stimulate hepatocyte pluripotency and self-renewal, as well as inflammation.

Changes in fasting bile acid profiles after Roux-en-Y gastric bypass and sleeve gastrectomy.

Bile acid is an essential factor that plays a role in metabolic regulation, but how bile acid is regulated after Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) remains unclear. This meta-analysis aimed to investigate changes in the levels of fasting bile acids following RYGB and SG. A systematic literature search of the PubMed, EMBASE, Cochrane Library and Web of Science databases through July 2020 was performed in accordance with the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) guidelines and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. The concentrations of bile acids were evaluated. Thirteen studies with 289 patients were included. Our results showed that patients who underwent RYGB had increased levels of fasting total bile acids, primary bile acids, secondary bile acids, conjugated bile acids, and unconjugated bile acids, but no significant differences in all these bile acid levels were observed in patients who underwent SG. Furthermore, 12a-hydroxylated bile acid levels and the 12a-hydroxylated/non-12a-hydroxylated bile acid ratio also increased following RYGB. In this study, we found that fasting bile acid levels, especially 12a-hydroxylated bile acids levels, were increased after RYGB. However, no differences in fasting bile acid levels were observed following SG.

Metabolic Profiling of Bile Acids in the Urine of Patients with Alcohol-Associated Liver Disease.

Bile acids (BAs) play important functions in the development of alcohol‐associated liver disease (ALD). In the current study, urine BA concentrations in 38 patients with well‐described alcohol‐associated hepatitis (AH) as characterized by Model for End‐Stage Liver Disease (MELD), 8 patients with alcohol‐use disorder (AUD), and 19 healthy controls (HCs) were analyzed using liquid chromatography–mass spectrometry. Forty‐three BAs were identified, and 22 BAs had significant changes in their abundance levels in patients with AH. The potential associations of clinical data were compared to candidate BAs in this pilot proof‐of‐concept study. MELD score showed positive correlations with several conjugated BAs and negative correlations with certain unconjugated BAs; taurine‐conjugated chenodeoxycholic acid (CDCA) and MELD score showed the highest association. Cholic acid, CDCA, and apocholic acid had nonsignificant abundance changes in patients with nonsevere ALD compared to HCs but were significantly increased in those with severe AH. Receiver operating characteristic analysis showed that the differences in these three compounds were sufficiently large to distinguish severe AH from nonsevere ALD. Notably, the abundance levels of primary BAs were significantly increased while most of the secondary BAs were markedly decreased in AH compared to AUD. Most importantly, the amount of total BAs and the ratio of primary to secondary BAs increased while the ratio of unconjugated to conjugated BAs decreased as disease severity increased. Conclusion: Abundance changes of specific BAs are closely correlated with the severity of AH in this pilot study. Urine BAs (individually or as a group) could be potential noninvasive laboratory biomarkers for detecting early stage ALD and may have prognostic value in AH morbidity.