Sulfated Bile Acids Research Articles

Sulfated Bile Acids in Serum as Potential Biomarkers of Disease Severity and Mortality in COVID-19.

The fight against coronavirus disease 2019 (COVID-19) continues. Since the pandemic's onset, several biomarkers have been proposed to assess the diagnosis and prognosis of this disease. This research aimed to identify potential disease severity biomarkers in serum samples of patients with COVID-19 during the disease course. Data were collected using untargeted and targeted mass spectrometry methods. The results were interpreted by performing univariate and multivariate analyses. Important metabolite classes were identified by qualitative untargeted metabolomics in 15 serum samples from survivors of COVID-19. Quantitative targeted metabolomics on a larger patient cohort including 15 non-survivors confirmed serum 3-sulfate bile acids (i.e. GLCA-3S) were significantly increased in non-survivors compared to survivors during the early disease stage (p-value < 0.0001). Notably, it was associated with a higher risk of mortality (odds ratio of 26). A principal component analysis showed the ability to discriminate between survivors and non-survivors using the BA concentrations. Furthermore, increased BA-S is highly correlated with known parameters altered in severe clinical conditions.

Expanding Role of Endogenous Biomarkers for Assessment of Transporter Activity in Drug Development: Current Applications and Future Horizon.

The evaluation of transporter-mediated drug-drug interactions (DDIs) during drug development and post-approval contributes to benefit-risk assessment and helps formulate clinical management strategies. The use of endogenous biomarkers, which are substrates of clinically relevant uptake and efflux transporters, to assess the transporter inhibitory potential of a drug has received widespread attention. Endogenous biomarkers, such as coproporphyrin (CP) I and III, have increased mechanistic understanding of complex DDIs. Other endogenous biomarkers are under evaluation, including, but not limited to, sulfated bile acids and 4-pyridoxic acid (PDA). The role of endogenous biomarkers has expanded beyond facilitating assessment of transporter-mediated DDIs and they have also been used to understand alterations in transporter activity in the setting of organ dysfunction and various disease states. We envision that endogenous biomarker-informed approaches will not only help to formulate a prudent and informed DDI assessment strategy but also facilitate quantitative predictions of changes in drug exposures in specific populations.

Sulfated bile acid is a host-derived ligand for MAIT cells.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin-based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue-derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function.

Integrated post-acquisition data processing strategy for rapid steroid sulfate characterization in Toad gall-bladder

LC–MS serves as a workhorse for chemical profile characterization of Chinese medicinal materials (CMMs) attributing to the ability of measuring fruitful MS/MS spectral information. However, it is laborious to extract the information belonging to the compounds-of-interest from the massive data matrixes even employing those well-defined post-acquisition data processing strategies. Here, efforts were devoted to propose an integrated strategy allowing rapid chemical homologs-focused data filtering through integrating the fit-for-purpose existing strategies, such as molecular weight imprinting (MWI), diagnostic fragment ion filtering (DFIF), neutral loss filtering (NLF), and isotope pattern filtering (IPF). Homologs-focused chemical characterization of a precious CMM namely Toad gall-bladder (Chinese name: Chandan) that is rich of diverse effective steroid sulfates, particularly bufogenin sulfates, bile acid sulfates and bilichol sulfates, was employed as a proof-of-concept. Recombinant human SULT2A1-catalyzed in vitro metabolism was undertaken to generate eight bufogenin sulfates to facilitate summarizing MS/MS spectral behaviors. After in-house data library construction and MS1 and MS2 spectral acquisition, data filtering was conducted as follows: 1) MWI and IPF was utilized in combination to capture deprotonated molecular ions and the 34S isotopic ions for the sulfates of those reported steroids; 2) m/z 79.9568 (SO3−·) and 96.9596 (HSO4−) were applied to DFIF; and 3) SO3 (79.9568 Da) served as the feature to achieve NLF. Those captured MS/MS information subsequently participated in tentatively structural annotation through applying those empirical mass fragmentation rules. As a result, 71 compounds including 7 bufogenin sulfates, 17 bile acid sulfates, 13 bilichol sulfates and a C-23 steroid sulfate were detected from Toad gall-bladder and thereof, 39 ones received plausible identities assignment. Above all, the steroid sulfates in Toad gall-bladder were profiled in depth, and more importantly, the proposed strategy should be a meaningful option for, but not limited to, submetabolome characterization in CMMs.

Sulfation of chondroitin and bile acids converges to antagonize Wnt/β-catenin signaling and inhibit APC deficiency-induced gut tumorigenesis

Sulfation is a crucial and prevalent conjugation reaction involved in cellular processes and mammalian physiology. 3′-Phosphoadenosine 5′-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the primary enzyme to generate the universal sulfonate donor PAPS. The involvement of PAPSS2-mediated sulfation in adenomatous polyposis coli (APC) mutation-promoted colonic carcinogenesis has not been reported. Here, we showed that the expression of PAPSS2 was decreased in human colon tumors along with cancer stages, and the lower expression of PAPSS2 was correlated with poor prognosis in advanced colon cancer. Gut epithelial-specific heterozygous Apc deficient and Papss2-knockout (ApcΔgut-HetPapss2Δgut) mice were created, and the phenotypes were compared to the spontaneous intestinal tumorigenesis of ApcΔgut-Het mice. ApcΔgut-HetPapss2Δgut mice were more sensitive to gut tumorigenesis, which was mechanistically accounted for by the activation of Wnt/β-catenin signaling pathway due to the suppression of chondroitin sulfation and inhibition of the farnesoid X receptor (FXR)-transducin-like enhancer of split 3 (TLE3) gene regulatory axis. Chondroitin sulfate supplementation in ApcΔgut-HetPapss2Δgut mice alleviated intestinal tumorigenesis. In summary, we have uncovered the protective role of PAPSS2-mediated chondroitin sulfation and bile acids–FXR–TLE3 activation in the prevention of gut carcinogenesis via the antagonization of Wnt/β-catenin signaling. Chondroitin sulfate may be explored as a therapeutic agent for Papss2 deficiency-associated colonic carcinogenesis.

Analysis of the Bile Acid Composition in a Fibroblast Growth Factor 19-Expressing Liver-Humanized Mouse Model and Its Use for CYP3A4-Mediated Drug-Drug Interaction Studies.

Numerous biomedical applications have been described for liver-humanized mouse models, such as in drug metabolism or drug-drug interaction (DDI) studies. However, the strong enlargement of the bile acid (BA) pool due to lack of recognition of murine intestine-derived fibroblast growth factor-15 by human hepatocytes and a resulting upregulation in the rate-controlling enzyme for BA synthesis, cytochrome P450 (CYP) 7A1, may pose a challenge in interpreting the results obtained from such mice. To address this challenge, the human fibroblast growth factor-19 (FGF19) gene was inserted into the Fah-/- , Rag2-/- , Il2rg-/- NOD (FRGN) mouse model, allowing repopulation with human hepatocytes capable of responding to FGF19. While a decrease in CYP7A1 expression in human hepatocytes from humanized FRGN19 mice (huFRGN19) and a concomitant reduction in BA production was previously shown, a detailed analysis of the BA pool in these animals has not been elucidated. Furthermore, there are sparse data on the use of this model to assess potential clinical DDI. In the present work, the change in BA composition in huFRGN19 compared with huFRGN control animals was systematically evaluated, and the ability of the model to recapitulate a clinically described CYP3A4-mediated DDI was assessed. In addition to a massive reduction in the total amount of BA, FGF19 expression in huFRGN19 mice resulted in significant changes in the profile of various primary, secondary, and sulfated BAs in serum and feces. Moreover, as observed clinically, administration of the pregnane X receptor agonist rifampicin reduced the oral exposure of the CYP3A4 substrate triazolam. SIGNIFICANCE STATEMENT: Transgenic expression of FGF19 normalizes the unphysiologically high level of bile acids in a chimeric liver-humanized mouse model and leads to massive changes in bile acid composition. These adaptations could overcome one of the potential impediments in the use of these mouse models for drug-drug interaction studies.

Fast profiling of primary, secondary, conjugated, and sulfated bile acids in human urine and murine feces samples.

Bile acids (BAs) are a complex class of metabolites that have been described as specific biomarkers of gut microbiota activity. The development of analytical methods allowing the quantification of an ample spectrum of BAs in different biological matrices is needed to enable a wider implementation of BAs as complementary measures in studies investigating the functional role of the gut microbiota. This work presents results from the validation of a targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 28 BAs and six sulfated BAs, covering primary, secondary, and conjugated BAs. The analysis of 73 urine and 20 feces samples was used to test the applicability of the method. Concentrations of BAs in human urine and murine feces were reported, ranging from 0.5 to 50nmol/g creatinine and from 0.012 to 332nmol/g, respectively. Seventy-nine percent of BAs present in human urine samples corresponded to secondary conjugated BAs, while 69% of BAs present in murine feces corresponded to primary conjugated BAs. Glycocholic acid sulfate (GCA-S) was the most abundant BA in human urine samples, while taurolithocholic acid was the lowest concentrated compound detected. In murine feces, the most abundant BAs were α-murocholic, deoxycholic, dehydrocholic, and β-murocholic acids, while GCA-S was the lowest concentrated BA. The presented method is a non-invasive approach for the simultaneous assessment of BAs and sulfated BAs in urine and feces samples, and the results will serve as a knowledge base for future translational studies focusing on the role of the microbiota in health.

Characterization of Bile Acid Sulfate Conjugates as Substrates of Human Organic Anion Transporting Polypeptides.

Drug interactions involving the inhibition of hepatic organic anion transporting polypeptides (OATPs) 1B1 and OATP1B3 are considered important. Therefore, we sought to study various sulfated bile acids (BA-S) as potential clinical OATP1B1/3 biomarkers. It was determined that BA-S [e.g., glycochenodeoxycholic acid 3-O-sulfate (GCDCA-S) and glycodeoxycholic acid 3-O-sulfate (GDCA-S)] are substrates of OATP1B1, OATP1B3, and sodium-dependent taurocholic acid cotransporting polypeptide (NTCP) transfected into human embryonic kidney 293 cells, with minimal uptake evident for other solute carriers (SLCs) like OATP2B1, organic anion transporter 2, and organic cation transporter 1. It was also shown that BA-S uptake by plated human hepatocytes (PHH) was inhibited (≥96%) by a pan-SLC inhibitor (rifamycin SV), and there was greater inhibition (≥77% versus ≤12%) with rifampicin (OATP1B1/3-selective inhibitor) than a hepatitis B virus myristoylated-preS1 peptide (NTCP-selective inhibitor). Estrone 3-sulfate was also used as an OATP1B1-selective inhibitor. In this instance, greater inhibition was observed with GDCA-S (76%) than GCDCA-S (52%). The study was expanded to encompass the measurement of GCDCA-S and GDCA-S in plasma of SLCO1B1 genotyped subjects. The geometric mean GDCA-S concentration was 2.6-fold (90% confidence interval 1.6, 4.3; P = 2.1 × 10-4) and 1.3-fold (1.1, 1.7; P = 0.001) higher in individuals homozygous and heterozygous for the SLCO1B1 c.521T > C loss-of-function allele, respectively. For GCDCA-S, no significant difference was noted [1.2-fold (0.8, 1.7; P = 0.384) and 0.9-fold (0.8, 1.1; P = 0.190), respectively]. This supported the in vitro data indicating that GDCA-S is a more OATP1B1-selective substrate (versus GCDCA-S). It is concluded that GCDCA-S and GDCA-S are viable plasma-based OATP1B1/3 biomarkers, but they are both less OATP1B1-selective when compared to their corresponding 3-O-glucuronides (GCDCA-3G and GDCA-3G). Additional studies are needed to determine their utility versus more established biomarkers, such as coproporphyrin I, for assessing inhibitors with different OATP1B1 (versus OATP1B3) inhibition signatures.

The effects of sulfated secondary bile acids on intestinal barrier function and immune response in an inflammatory in vitro human intestinal model

Dysbiosis-related perturbations in bile acid (BA) metabolism were observed in inflammatory bowel disease (IBD) patients, which was characterized by increased levels of sulfated BAs at the expense of secondary BAs. However, the exact effects of sulfated BAs on the etiology of IBD are not investigated yet. Therefore, we aimed to investigate the effects of sulfated deoxycholic acid (DCA), sulfated lithocholic acid (LCA) and their unsulfated forms on intestinal barrier function and immune response. To this end, we first established a novel in vitro human intestinal model to mimic chronic intestinal inflammation as seen during IBD. This model consisted of a co-culture of Caco-2 and HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation to represent the mucus layer. A pro-inflammatory environment was created by combining the co-culture with LPS-activated dendritic cells (DCs) in the basolateral compartment. The presence of activated DCs caused a decrease in transepithelial electrical resistance (TEER), which was slightly restored by LCA and sulfated DCA. The expression of genes related to intestinal epithelial integrity and the mucus layer were slightly, but not significantly increased. These results imply that sulfated BAs have a minor effect on intestinal barrier function in Caco-2 and HT29-MTX-E12 cells. When exposed directly to DCs, our results point towards anti-inflammatory effects of secondary BAs, but to a minor extent for sulfated secondary BAs. Future research should focus on the importance of proper transformation of BAs by bacterial enzymes and the potential involvement of BA dysmetabolism in IBD progression.

P066 Lower serum concentration of sulfated bile acids is associated with Primary Sclerosing Cholangitis Inflammatory Bowel Disease comorbidity and advanced liver fibrosis

Abstract Background Primary sclerosing cholangitis associated Inflammatory bowel disease (PSC-IBD) carries significant morbidity risk. Alterations in bile acid profiles has been shown to modulate chronic inflammation in IBD, but data in PSC-IBD comorbidity is scarce. We aimed to assess associations of serum and stool bile acids (BA) with PSC-IBD phenotype versus ulcerative colitis (UC) and the degree of liver fibrosis in PSC-IBD patients Methods In this cross-sectional study, bile acids were profiled in 52 PSC-IBD and 50 UC subjects. Quantitation of bile acids was performed by reversed phase ultrahigh performance liquid chromatography/multiple reaction monitoring mass spectrometry with negative ion detection (TMIC, Alberta, CA). After data filtering and normalization, 71 serum and 76 stool BA were available for analysis. Logistic regression was used to assess associations between BA levels and disease phenotype (PSC-IBD versus UC). Among the PSC-IBD group, linear regression was used to assess associations of fibrosis degree (severe (F4) versus no/mild (F0-3) fibrosis, measured by FibroScan®) with BA profile. Age, sex and ursodeoxycholic acid treatment were included as co-variates in all models and alkaline phosphatase (ALP) was added as co-variate for the liver fibrosis analysis. False discovery rate&amp;lt;0.05 was considered significant Results PSC-IBD and UC group had similar age and sex distribution. In the multivariable logistic regression, 26 serum BA were significantly associated with PSC-IBD (Figure 1). Decreased concentrations of taurine/glycine-primary BA [odds ratio(OR)=0.97, 95% confidence interval (CI) 0.95–0.99], taurine/glycine-secondary BA [OR=0.98, 95%CI 0.97–0.99] and sulfated-BA [OR=0.96, 95%CI 0.95–0.98], and increased concentrations of conjugated-BA [OR=1.02, 95%CI 1.01–1.03] and glucuronidated-BA [OR=1.06, 95%CI 1.02–1.1] were associated with PSC-IBD phenotype (q&amp;lt;0.05). Decreased sulfated-BA concentrations remained associated with PSC-IBD [OR=0.96, 95%CI 0.94–0.99, q=0.06] even among PSC-IBD subjects with normal ALP&amp;lt;2XULN (n=67). In contrast, stool BA analysis did not show significant associations with PSC-IBD versus UC. Additionally, in the PSC-IBD group, F4 fibrosis degree compared to F0-3 was associated with lower serum concentrations of taurine/glycine- and sulfated-BA, and higher serum concentrations of conjugated-BA (q&amp;lt;0.05) Conclusion Decreased concentration of sulfated-BA was associated with PSC-IBD phenotype with and without cholestasis compared to UC group. Lower sulfated-BA concentration was also associated with severe degree of liver fibrosis among PSC-IBD subjects. This inverse relationship between sulfated-BA levels and PSC-IBD and the degree of fibrosis should be further explored in future mechanistic studies

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS.

Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) "Guideline on bioanalytical method validation". To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

The role of fecal sulfur metabolome in inflammatory bowel diseases

Sulfur metabolism and sulfur-containing metabolites play an important role in the human digestive system, and sulfur compounds and pathways are associated with inflammatory bowel diseases (IBD). In fact, cysteine metabolism results in the production of taurine and sulfate, and gut microbes catabolize them into hydrogen sulfide, a signaling molecule with various biological functions. Besides metabolites originating from sulfur metabolism, several other sulfur-containing metabolites of different classes were detected in human feces, consisting of non-volatile and volatile compounds. Sulfated steroids and bile acids such as taurine-conjugated bile acids are the major classes along with sulfur amino acids and sulfur-containing peptides. Indeed, sulfur-containing metabolites were described in stool samples from healthy subjects, patients suffering from colorectal cancer or IBD. In metabolomics-driven studies, around 50 known sulfur-containing metabolites were linked to IBD. Taurine, taurocholic acid, taurochenodeoxycholic acid, methionine, methanethiol and hydrogen sulfide were regularly reported in IBD studies, and most of them were elevated in stool samples from IBD patients. We summarized from this review that there is strong interplay between perturbed gut microbiota in IBD, and the consistently higher abundance of sulfur-containing metabolites, which potentially represent substrates for sulfidogenic bacteria such as Bilophila or Escherichia and promote their growth. These bacteria might shift their metabolism towards the degradation of taurine and cysteine and therefore to a higher hydrogen sulfide production.

Longitudinal Profiles of Dietary and Microbial Metabolites in Formula- and Breastfed Infants.

The early-life metabolome of the intestinal tract is dynamically influenced by colonization of gut microbiota which in turn is affected by nutrition, i.e. breast milk or formula. A detailed examination of fecal metabolites was performed to investigate the effect of probiotics in formula compared to control formula and breast milk within the first months of life in healthy neonates. A broad metabolomics approach was conceptualized to describe fecal polar and semi-polar metabolites affected by feeding type within the first year of life. Fecal metabolomes were clearly distinct between formula- and breastfed infants, mainly originating from diet and microbial metabolism. Unsaturated fatty acids and human milk oligosaccharides were increased in breastfed, whereas Maillard products were found in feces of formula-fed children. Altered microbial metabolism was represented by bile acids and aromatic amino acid metabolites. Elevated levels of sulfated bile acids were detected in stool samples of breastfed infants, whereas secondary bile acids were increased in formula-fed infants. Microbial co-metabolism was supported by significant correlation between chenodeoxycholic or lithocholic acid and members of Clostridia. Fecal metabolites showed strong inter- and intra-individual behavior with features uniquely present in certain infants and at specific time points. Nevertheless, metabolite profiles converged at the end of the first year, coinciding with solid food introduction.

The crystal structure of mouse SULT2A8 reveals the mechanism of 7α-hydroxyl, bile acid sulfation

Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3′-phosphoadenosine-5′-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.

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.

Intestinal Sulfation Is Essential to Protect Against Colitis and Colonic Carcinogenesis

Sulfation is a conjugation reaction essential for numerous biochemical and cellular functions in mammals. The 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 2 (PAPSS2) is the key enzyme to generate PAPS, which is the universal sulfonate donor for all sulfation reactions. The goal of this study was to determine whether and how PAPSS2 plays a role in colitis and colonic carcinogenesis. Tissue arrays of human colon cancer specimens, gene expression data, and clinical features of cancer patients were analyzed. Intestinal-specific Papss2 knockout mice (Papss2ΔIE) were created and subjected to dextran sodium sulfate-induced colitis and colonic carcinogenesis induced by a combined treatment of azoxymethane and dextran sodium sulfate or azoxymethane alone. The expression of PAPSS2 is decreased in the colon cancers of mice and humans. The lower expression of PAPSS2 in colon cancer patients is correlated with worse survival. Papss2ΔIE mice showed heightened sensitivity to colitis and colon cancer by damaging the intestinal mucosal barrier, increasing intestinal permeability and bacteria infiltration, and worsening the intestinal tumor microenvironment. Mechanistically, the Papss2ΔIE mice exhibited reduced intestinal sulfomucin content. Metabolomic analyses revealed the accumulation of bile acids, including the Farnesoid X receptor antagonist bile acid tauro-β-muricholic acid, and deficiency in the formation of bile acid sulfates in the colon of Papss2ΔIE mice. We have uncovered an important role of PAPSS2-mediated sulfation in colitis and colonic carcinogenesis. Intestinal sulfation may represent a potential diagnostic marker and PAPSS2 may serve as a potential therapeutic target for inflammatory bowel disease and colon cancer.

Bariatric surgery stirs symbionts to counteract diabesity by CA(7)Sting a liver-generated bile acid into the mix

Obesity and diabetes are major health challenges for the 21st century. Effective treatments affect nutrient signaling along the gut microbiome-bile acid axis. In this issue of Cell, Chaudhari etal. demonstrate that a gut microbial secondary bile acid stimulates hepatic synthesis of a sulfated bile acid with anti-diabetic properties.

Hypothyroidism Increases Cholesterol Gallstone Prevalence in Mice by Elevated Hydrophobicity of Primary Bile Acids.

Background: Thyroid hormone (TH) deficiency has been associated with increased cholesterol gallstone prevalence. Hypothyroidism impacts hepatic lipid homeostasis, biliary secretion, gallbladder motility, and gallstone (LITH) gene expression, all potential factors contributing to cholesterol gallstone disease (CGD). However, how TH deficiency may lead to gallstone formation is still poorly understood. Therefore, we performed molecular studies in a CGD mouse model under lithogenic conditions and modulation of TH status. Methods: Male, three-month-old C57BL/6 mice were randomly divided into a control (euthyroid) group, a hypothyroid (hypo) group, a gallstone (litho) group, and a gallstone+hypothyroid (litho+hypo) group and were treated for 2, 4, and 6 weeks (n = 8/treatment period). Gallstone prevalence, biliary composition and cholesterol crystals, hepatic expression of genes participating in cholesterol, bile acid (BA), and phosphatidylcholine synthesis (Hmgcr, Cyp7a1, Pcyt1a), and canalicular transport (Abcg5, Bsep, Abcb4) were investigated. Results: Increased cholesterol gallstone prevalence was observed in hypothyroid mice under lithogenic diet after 4 and 6 weeks of treatment (4 weeks: 25% vs. 0%; 6 weeks: 75% vs. 37.5%). Interestingly, neither the composition of the three main biliary components, cholesterol, BAs, and phosphatidylcholine, nor the hepatic expression of genes involved in synthesis and transport could explain the differences in cholesterol gallstone formation in the mice. However, TH deficiency resulted in significantly increased hydrophobicity of primary BAs in bile. Furthermore, downregulation of hepatic sulfonation enzymes Papss2 and Sult2a8 as well as diminished biliary BA sulfate concentrations in mice were observed under hypothyroid conditions all contributing to a lithogenic biliary milieu as evidenced by microscopic cholesterol crystals and macroscopic gallstone formation. Conclusions: We describe a novel pathogenic link between TH deficiency and CGD and suggest that the increased hydrophobic character of biliary BAs due to the diminished expression of hepatic detoxification enzymes promotes cholesterol crystal precipitation and enhances cholesterol gallstone formation in the bile of hypothyroid mice.

The Effects of Sulfated Secondary Bile Acids on Intestinal Barrier Function and Immune Response in an Inflammatory &lt;i&gt; in vitro&lt;/i&gt; Human Intestinal Model

Dysbiosis-related perturbations in bile acid (BA) metabolism were observed in inflammatory bowel disease (IBD) patients, which was characterized by increased levels of sulfated BAs at the expense of secondary BAs. However, the exact effects of sulfated BAs on the etiology of IBD are not investigated yet. Therefore, we aimed to investigate the effects of sulfated deoxycholic acid (DCA), sulfated lithocholic acid (LCA) and their unsulfated forms on intestinal barrier function and immune response. To this end, we first established a novel in vitro human intestinal model to mimic chronic intestinal inflammation as seen during IBD. This model consisted of a co-culture of Caco-2 and HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation to represent the mucus layer. A pro-inflammatory environment was created by combining the co-culture with LPS-activated dendritic cells (DCs) in the basolateral compartment. The presence of activated DCs caused a decrease in transepithelial electrical resistance (TEER), which was slightly restored by LCA and sulfated DCA. The expression of genes related to intestinal epithelial integrity and the mucus layer were slightly, but not significantly increased. These results imply that sulfated BAs have a minor effect on intestinal barrier function in Caco-2 and HT29-MTX-E12 cells. When exposed directly to DCs, our results point towards anti-inflammatory effects of secondary BAs, but not sulfated secondary BAs. These findings emphasize the importance of proper transformation of BAs by bacterial enzymes and the potential involvement of BA dysmetabolism in IBD progression.

Making the Diagnosis of Bile Acid Diarrhea.

Bile acid diarrhea is a frequent, treatable cause of functional diarrhea but is difficult to diagnose when the nuclear medicine seleno-taurohomocholic acid test is unavailable. An alternative approach is testing blood for the bile acid precursor, 7α-OH-4-cholesten-3-one, which is raised with increased bile acid synthesis. A recent article has defined measurements that have high negative and positive predictive values, further exploring how they can be improved by incorporating measures such as age, stool number, fibroblast growth factor 19, or plasma sulfated bile acids. Other articles have looked at the percentage of fecal primary bile acids. Together, they promise better use of diagnostic biomarkers for this condition.