Taurine-conjugated Bile Salts Research Articles

Systemic ASBT inactivation protects against liver damage in obstructive cholestasis in mice

Background & AimsNon-absorbable inhibitors of the apical sodium-dependent bile acid transporter (ASBT; also called ileal bile acid transporter [IBAT]) are recently approved or in clinical development for multiple cholestatic liver disorders and lead to a reduction in pruritus and (markers for) liver injury. Unfortunately, non-absorbable ASBT inhibitors (ASBTi) can induce diarrhoea or may be ineffective if cholestasis is extensive and largely precludes intestinal excretion of bile acids. Systemically acting ASBTi that divert bile salts towards renal excretion may alleviate these issues.MethodsBile duct ligation (BDL) was performed in ASBT-deficient (ASBT knockout [KO]) mice as a model for chronic systemic ASBT inhibition in obstructive cholestasis. Co-infusion of radiolabelled taurocholate and inulin was used to quantify renal bile salt excretion after BDL. In a second (wild-type) mouse model, a combination of obeticholic acid (OCA) and intestine-restricted ASBT inhibition was used to lower the bile salt pool size before BDL.ResultsAfter BDL, ASBT KO mice had reduced plasma bilirubin and alkaline phosphatase compared with wild-type mice with BDL and showed a marked reduction in liver necrotic areas at histopathological analysis, suggesting decreased BDL-induced liver damage. Furthermore, ASBT KO mice had reduced bile salt pool size, lower plasma taurine-conjugated polyhydroxylated bile salt, and increased urinary bile salt excretion. Pretreatment with OCA + ASBTi in wild-type mice reduced the pool size and greatly improved liver injury markers and liver histology.ConclusionsA reduced bile salt pool at the onset of cholestasis effectively lowers cholestatic liver injury in mice. Systemic ASBT inhibition may be valuable as treatment for cholestatic liver disease by lowering the pool size and increasing renal bile salt output even under conditions of minimal faecal bile salt secretion.Lay summaryNovel treatment approaches against cholestatic liver disease (resulting in reduced or blocked flow of bile) involve non-absorbable inhibitors of the bile acid transport protein ASBT, but these are not always effective and/or can cause unwanted side effects. In this study, we demonstrate that systemic inhibition/inactivation of ASBT protects mice against developing severe cholestatic liver injury after bile duct ligation, by reducing bile salt pool size and increasing renal bile salt excretion.

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.

Identification of Bile Salt Hydrolase and Bile Salt Resistance in a Probiotic Bacterium Lactobacillus gasseri JCM1131T.

Lactobacillus gasseri is one of the most likely probiotic candidates among many Lactobacillus species. Although bile salt resistance has been defined as an important criterion for selection of probiotic candidates since it allows probiotic bacteria to survive in the gut, both its capability and its related enzyme, bile salt hydrolase (BSH), in L. gasseri is still largely unknown. Here, we report that the well-known probiotic bacterium L. gasseri JCM1131T possesses BSH activity and bile salt resistance capability. Indeed, this strain apparently showed BSH activity on the plate assay and highly tolerated the primary bile salts and even taurine-conjugated secondary bile salt. We further isolated a putative BSH enzyme (LagBSH) from strain JCM1131T and characterized the enzymatic function. The purified LagBSH protein exhibited quite high deconjugation activity for taurocholic acid and taurochenodeoxycholic acid. The lagBSH gene was constitutively expressed in strain JCM1131T, suggesting that LagBSH likely contributes to bile salt resistance of the strain and may be associated with survival capability of strain JCM1131T within the human intestine by bile detoxification. Thus, this study first demonstrated the bile salt resistance and its responsible enzyme (BSH) activity in strain JCM1131T, which further supports the importance of the typical lactic acid bacterium as probiotics.

Characterization of a recombinant bile salt hydrolase (BSH) from Bifidobacterium bifidum for its glycine-conjugated bile salts specificity

Deconjugation of bile salts in vivo not only promotes the cholesterol metabolism of the host, but also provides cellular carbon, nitrogen and sulphur for some probiotics. In this study, the gene of BSH from Bifidobacterium bifidum (ATCC 29521) was amplified and expressed using glutathione S-transferase (GST) Gene Fusion System. The enzyme was purified in a mild conditions without denaturation and renaturation, and the molecular mass was estimated to be 35 kDa. The optimum pH for the enzyme catalysis was 5.5. The BSH enzyme was stable from 37 to 57 °C. In addition, this enzyme was completely inhibited by Cu2+, while many other metal ions only gave moderate inhibition. Hydrolysis efficiency of BSH was quantified in two ways: ninhydrin colouration method and high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD). The hydrolysis process was clearly observed through real-time picture monitoring. The activity of BSH on glycine-conjugated bile salts was better than on taurine-conjugated bile salts. The proportion of hydrolysis for both sodium glycochenodeoxycholate (GCDCA) and sodium glycoursodeoxycholate (GUDCA) were more than 90% in 60 min. However, the proportion of hydrolysis for sodium taurochenodeoxycholate (TCDCA) and sodium tauroursodeoxycholate (TUDCA) were only 68% and 43% at the same condition. The study of this paper provides a better choice for hydrolysis of glycine-conjugated bile salts. Highlights The specificity of BSH was high for glycine-conjugated bile salts. Hydrolysis process of bile acid was obtained through real-time picture monitoring. Hydrolysis efficiency of BSH was quantified in two ways: ninhydrin colouration method and HPLC-ELSD.

Critical F129 and L138 in loop III of bile salt hydrolase (BSH) in Lactobacillus plantarum B14 are essential for the catalytic activity and substrate specificity

ABSTRACTBile salt hydrolase (BSH) is a gut-bacterial enzyme that influences human health by altering the host fat digestion and cellular energy generation. BSH is essential for deconjugation of the glycine or taurine-conjugated bile salts in the small intestine of humans. Therefore, BSH may be a key microbiome target for the designing of new measures to control some diseases in humans. BSHs, a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, exhibit higher variation in substrate specificity. The phenylalanine-129 (F129) and leucine-138 (L138) in loop III of BSH, thought to be responsible for substrate specificity, are partially conserved in this superfamily. In this study, the aromatic-hydrophobic F129 and aliphatic-hydrophobic L138 of C-terminally His-tagged BSH from Lactobacillus plantarum B14 (LbBSH) was substituted for aliphatic-hydrophobic isoleucine (I) and negatively charged polar glutamate (E) amino acid, respectively, by site-directed mutagenesis and characterized using an Escherichia coli BLR(DE3) expression system. Although both mutations resulted in an assembled and stable recombinant BSHs (rBSHs), they altered the catalytic activity and substrate specificity of rBSH. This is the first experimental finding which confirmed that F129 and L138 were critical amino acids for the catalytic activity and substrate specificity turnover of BSH.

Molecular cloning, expression and characterization of bile salt hydrolase from Lactobacillus rhamnosus E9 strain

ABSTRACTBile salt hydrolase (BSH) enzyme, commonly found in probiotic bacteria of gut origin, catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts allowing for colonization of the bacteria in the gut and contributing to a decrease in levels of cholesterol. However an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid can have harmful side-effects. The aim of this study was to characterize the activity of BSH enzymes from Lactobacillus rhamnosus E9, a popular probiotic strain. The bsh gene was cloned, expressed, purified and characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activities and substrate specificities of the recombinant BSH (rBSH) enzyme were examined using six different bile acids. Nucleotide sequence analysis results indicated that the bsh of E9 contained an open reading frame (ORF) of 1014 and nucleotides encoding a 338-amino acid protein with a molecular weight of 37 kDa. Five catalytically important amino acids and the amino acid motifs located around the active site were highly conserved. The rBSH showed a slight preference towards glycine-conjugated to tauro-conjugated bile salts. This confirms that it is a safe strain for probiotics and its preference for glycine-conjugated bile salts should be further investigated.

Functional role of oppA encoding an oligopeptide-binding protein from Lactobacillus salivarius Ren in bile tolerance.

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5% oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.

Self-assembly of aqueous bilirubin ditaurate, a natural conjugated bile pigment, to contraposing enantiomeric dimers and M(-) and P(+) tetramers and their selective hydrophilic disaggregation by monomers and micelles of bile salts.

The solution behavior of bilirubin ditaurate (BDT), the first naturally occurring conjugated bile pigment to be physically and chemically characterized, was assessed in aqueous solution and in monomeric and micellar solutions of common taurine-conjugated bile salts (BS). Analytical ultracentrifugation revealed that BDT self-associates in monomer-dimer equilibria between 1 and 500 μM, forming limiting tetramers at low millimolar concentrations. Self-association was enthalpically driven with ΔG values of ≈5 kcal/mol, suggesting strong hydrophobic interactions. Added NaCl and decreases in temperature shifted the oligomerization to lower BDT concentrations. On the basis of circular dichroism spectra and the limiting size of the self-aggregates, we infer that the tetramers are composed of 2P(+) and 2M(-) enantiomeric BDT pairs in "ridge-tile" conformations interacting in a "double-bookend" structure. With added monomeric BS, blue shifts in the UV-vis spectra and tight isosbestic points revealed that BDT/BS heterodimers form, followed by BDT "decorating" BS micelles mostly via hydrophilic interactions. Conformational enantiomerism, fluorescence intensities, and anisotropy, as well as resistance of the hybrid particles to disaggregation in 6 M urea, suggested that two or three hydrogen-bonding sites bound BDT monomers to the hydroxyl groups of BS, possibly via pyrrole-π-orbital-OH interactions. BDT stabilized these interactions by enveloping the BS in its "ridge-tile" pincers with variable strain that maximized van der Waals interactions. Possibly because the BDT molecule becomes highly strained with BS subtending a 7β-hydroxyl group, BDT became totally resistant to oxidation in air. This work predicts that, because of BS dissolution of the BDT self-aggregates, BS/bilirubin hybrid particles, which are stabilized hydrophilically, are likely to be the dominant mode of transport for all conjugated bilirubins in bile.

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

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

Su1252 Gastric Juice Composition and Acid Suppression in Pediatric Gastroesophageal Reflux Disease

INTRODUCTION: Gastric acid suppression is justified to prevent severe gastro esophageal reflux (GER) disease related complications. However, it does not reduce the total amount and proximal extent of GER in the esophagus and non-acid components are able to induce (extra-esophageal) GER symptoms as well. No data on the composition of gastric juice (GJ) in children using gastric acid suppression exists. We therefore aimed to assess whether the composition of gastric juice in children using proton pump inhibitors (PPIs) differs, compared to that of their controls. METHODS: Infants and children (0-18 years) on proton pump inhibitors (PPIs) for at least six weeks and a control group not using anti reflux medication, were included. GJ was obtained through an existing nasogastric or a percutaneous endoscopic gastrostomy tube/ Mic-key gastrostomy. In the collected GJ (5 ml), pH, pepsin activity, bile salts and endotoxin (LPS) levels were determined. Pepsin was measured using a fluorometric assay using 4-Methyl-Coumaryl-7-Amide (MCA) substrate with/without pepstatin. Concentrations of deconjugated and taurine/glycine-conjugated bile salts were assessed by reversephase HPLC. Levels of LPS were determined using the spectrophotometric Limulus Amebocyte Lysate assay. RESULTS: GJ was analyzed from 16 children with (median: 3.8 yrs, range: 17.6 years) and 16 children (4.0 yrs, range: 16.0) without PPI therapy. Median duration of PPI treatment was 24 weeks (range: 514 weeks). Gastric pH was 5.0 (range: 5.0) and 1.0 (range: 4.5) in the PPI and control group respectively (p <0.001). Pepsin, unconjugated bile salts, and endotoxin were not significantly different in the two groups. Total taurine conjugated bile salts, and specifically taurocholate, was significantly higher in the PPI group (p=0.01 and p=0.005). pH and concentration of deconjugated bile and conjugated bile salts were significantly associated (p=0.006 and p=0.02). Endotoxin and bile salts were not significantly associated. CONCLUSION: Taurine conjugated bile acids are significantly higher in children chronically using PPIs compared to controls. Moreover acidity of gastric pH correlated negatively with deconjugated and conjugated bile salts. These findings imply that GJ under chronic proton pump inhibition contains nonacid components potentially harmful to esophageal mucosa and bronchial tissue.

Su1253 Abnormal Dynamics of Endogenous Acylated Ghrelin Mediates Decreases in Gastric Phase III-Like Contraction, Leading to Delayed Gastric Emptying in Urocortin1-Induced Stress Rats

INTRODUCTION: Gastric acid suppression is justified to prevent severe gastro esophageal reflux (GER) disease related complications. However, it does not reduce the total amount and proximal extent of GER in the esophagus and non-acid components are able to induce (extra-esophageal) GER symptoms as well. No data on the composition of gastric juice (GJ) in children using gastric acid suppression exists. We therefore aimed to assess whether the composition of gastric juice in children using proton pump inhibitors (PPIs) differs, compared to that of their controls. METHODS: Infants and children (0-18 years) on proton pump inhibitors (PPIs) for at least six weeks and a control group not using anti reflux medication, were included. GJ was obtained through an existing nasogastric or a percutaneous endoscopic gastrostomy tube/ Mic-key gastrostomy. In the collected GJ (5 ml), pH, pepsin activity, bile salts and endotoxin (LPS) levels were determined. Pepsin was measured using a fluorometric assay using 4-Methyl-Coumaryl-7-Amide (MCA) substrate with/without pepstatin. Concentrations of deconjugated and taurine/glycine-conjugated bile salts were assessed by reversephase HPLC. Levels of LPS were determined using the spectrophotometric Limulus Amebocyte Lysate assay. RESULTS: GJ was analyzed from 16 children with (median: 3.8 yrs, range: 17.6 years) and 16 children (4.0 yrs, range: 16.0) without PPI therapy. Median duration of PPI treatment was 24 weeks (range: 514 weeks). Gastric pH was 5.0 (range: 5.0) and 1.0 (range: 4.5) in the PPI and control group respectively (p <0.001). Pepsin, unconjugated bile salts, and endotoxin were not significantly different in the two groups. Total taurine conjugated bile salts, and specifically taurocholate, was significantly higher in the PPI group (p=0.01 and p=0.005). pH and concentration of deconjugated bile and conjugated bile salts were significantly associated (p=0.006 and p=0.02). Endotoxin and bile salts were not significantly associated. CONCLUSION: Taurine conjugated bile acids are significantly higher in children chronically using PPIs compared to controls. Moreover acidity of gastric pH correlated negatively with deconjugated and conjugated bile salts. These findings imply that GJ under chronic proton pump inhibition contains nonacid components potentially harmful to esophageal mucosa and bronchial tissue.

Sensitivity and specificity of the olfactory epithelia of two elasmobranch species to bile salts

Odor detection in vertebrates occurs when odorants enter the nose and bind to molecular olfactory receptors on the cilia or microvilli of olfactory receptor neurons (ORNs). Several vertebrate groups possess multiple, morphologically distinct types of ORNs. In teleost fishes, these different ORN types detect specific classes of biologically relevant odorants, such as amino acids, nucleotides and bile salts. For example, bile salts are reported to be detected exclusively by ciliated ORNs. The olfactory epithelium of elasmobranch fishes (sharks, rays and skates) is comprised of microvillous and crypt ORNs, but lacks ciliated ORNs; thus, it was questioned whether the olfactory system of this group of fishes is capable of detecting bile salts. The present investigation clearly indicates that the olfactory system of representative shark and stingray species does detect and respond to bile salts. Additionally, these species detect glycine-conjugated, taurine-conjugated and non-conjugated bile salts, as do teleosts. These elasmobranchs are less sensitive to the tested bile salts than reported for both agnathans and teleosts, but this may be due to the particular bile salts selected in this study, as elasmobranch-produced bile salts are commercially unavailable. Cross-adaptation experiments indicate further that the responses to bile salts are independent of those to amino acids, a major class of odorant molecules for all tested fishes.

Continuous flow synthesis and scale-up of glycine- and taurine-conjugated bile salts

A multi-gram scale protocol for the N-acyl amidation of bile acids with glycine and taurine has been successfully developed under continuous flow processing conditions. Selecting ursodeoxycholic acid (UDCA) as the model compound and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as the condensing agent, a modular mesoreactor assisted flow set-up was employed to significantly speed up the optimization of the reaction conditions and the flow scale-up synthesis. The results in terms of yield, in line purification, analysis, and implemented flow set-up for the reaction optimization and large scale production are reported and discussed.

All 4 Bile Salt Hydrolase Proteins Are Responsible for the Hydrolysis Activity in Lactobacillus plantarum ST‐III

In vertebrates, bile salt hydrolysis plays an essential role in fat metabolism. Bile salts are synthesized in the liver. And in the small intestine glycine and taurine are de-conjugated from bile salts by the enzyme bile salt hydrolase (BSH) from intestinal microbes. However, the mechanism of bile salt hydrolysis in Lactobacillus plantarum is still ambiguous. Four predicted bile salt hydrolase (bsh) genes from L. plantarum ST-III were cloned into Escherichia coli. The function of these genes was explored by overexpression. All 4 proteins that were studied showed activity against glycine- or taurine-conjugated bile salts. Substrate preference was also observed in BSH proteins, especially for the enzyme BSH1, which had high hydrolysis activity for glycodeoxycholic acid. These results suggest that all 4 bile salt hydrolases may be responsible for the bile salt hydrolysis activity in L. plantarum ST-III. Hypercholesterolemia is considered one of the major risk factors for coronary heart disease. More interest has focused on intestinal microbes because of their role in the decrease of serum cholesterol. BSH proteins play an important role in the reduction of cholesterol. This paper adds to a better understanding of BSH proteins of intestinal microbes. It gives a great hint that probiotics can be used to solve hypercholesterolemia one day.

694 A BILIARY BICARBONATE UMBRELLA PROTECTS HUMAN CHOLANGIOCYTES AND CHOLANGIOCARCINOMA CELLS AGAINST BILE ACID TOXICITY

Human cholangiocytes are physiologically exposed to millimolar concentrations of glycine-conjugated hydrophobic bile salts, which are toxic to other cell types including hepatocytes already in the micromolar range after cell entry. Apolar, protonated bile acids rather than polar, deprotonated bile salts may passively enter cholangiocytes. pKa and biliary pH determine bile acid protonation status in bile. We hypothesize that biliary HCO−3 secretion in humans serves to maintain an alkaline pH near the apical surface of cholangiocytes and fosters deprotonation of apolar bile acids to polar bile salts. This ‘biliary HCO−3-umbrella’ might be a key protective mechanism of human cholangiocytes against glycineconjugated bile acids (Hepatology 2010; 52: 1489). Our aim was to test if toxicity of unconjugated, glycine-conjugated (pKa 4–5) and taurine-conjugated (pKa < 2) bile salts in cholangiocytes is (i) pH-dependent, and (ii) antagonized by anion exchanger 2 (AE2) activity. Methods: Two human cholangiocarcinomaand one human immortalized cholangiocyte cell line were exposed to glycine-, taurine-, and unconjugated chenodeoxycholate (CDC), from 0.1mM to 2.0mM at pH8.0, 7.4, 7.1, 6.7 and 6.4 and after knockdown of AE2 by shRNA. After four hours, cell viability and apoptosis were determined by WST-1 and caspase-3/-7 assays, respectively. Results: In cholangiocarcinoma cells and non-transformed immortalized cholangiocytes, CDCand GCDC-, but not TCDCinduced cholangiocyte toxicity was pH-dependent and increased gradually when pH was lowered from 7.4 to 7.1, 6.7 or 6.4. E.g., in non-transformed cells, at pH 7.4 no bile salt-toxicity was observed while at pH 6.4 0.5mM and 1mM CDC decreased viability by 80.8±5.3 and 83.5±9.7% (p < 0.01, n = 5) and increased caspase-3/-7 activity 9and 27-fold, respectively (p < 0.001, n = 5). Acidification alone had no effect. AE2 knockdown led to 3-fold and 2-fold apoptosis induced by 0.75mM CDC or 2mM GCDC at pH 7.4 (p < 0.01, n = 6). When increasing pH to 8.0 during bile salt exposure, AE2 knockdown cells were rescued from bile salt toxicity. Conclusion: Our data support the protective function of a biliary HCO−3-umbrella against bile acid-induced cholangiocyte injury. AE2 may serve as a key element of this HCO−3-umbrella. Genetic and acquired functional defects of elements of the biliary HCO−3umbrella may contribute to development and progression of cholangiopathies.

Metabolic bone disease in lion cubs at the London Zoo in 1889: the original animal model of rickets

In 1889 Dr. John Bland-Sutton, a prominent London surgeon, was consulted about fatal rickets in over 20 successive litters of lion cubs born at the London Zoo. He evaluated the diet and found the cause of rickets to be nutritional in origin. He recommended that goat meat with crushed bones and cod-liver oil be added to the lean horsemeat diet of the cubs and their mothers. Rickets were reversed, the cubs survived, and subsequent litters thrived. Thirty years later, in classic controlled studies conducted in puppies and young rats, the definitive role of calcium, phosphate and vitamin D in prevention and therapy of rickets was elucidated. Further studies led to identifying the structural features of vitamin D.Although the Bland-Sutton diet provided calcium and phosphate from bones and vitamins A and D from cod-liver oil, some other benefits of this diet were not recognized. Taurine-conjugated bile salts, necessary for intestinal absorption of fat-soluble vitamins, were provided in the oil cold-pressed from cod liver. Unlike canine and rodent species, felines are unable to synthesize taurine, yet conjugate bile acids exclusively with taurine; hence, it must be provided in the diet. The now famous Bland-Sutton “experiment of nature,” fatal rickets in lion cubs, was cured by addition of minerals and vitamin D. Taurine-conjugated bile salts undoubtedly permitted absorption of vitamins A and D, thus preventing the occurrence of metabolic bone disease and rickets.

Olfactory sensitivity to bile fluid and bile salts in the European eel (Anguilla anguilla), goldfish (Carassius auratus) and Mozambique tilapia (Oreochromis mossambicus) suggests a `broad range' sensitivity not confined to those produced by conspecifics alone

Teleosts have high olfactory sensitivity to bile salts. To assess whether this phenomenon is involved in intra-specific chemical communication alone, or is part of a more ;broad range' sensitivity to bile salts produced by heterospecifics, we investigated possible differences in the odour of bile between the sexes and among different species - the eel (Anguilla anguilla), goldfish (Carassius auratus) and Mozambique tilapia (Oreochromis mossambicus) - using the electro-olfactogram (EOG). We also identified the main bile constituents by liquid chromatography and mass spectrometry. There were marked differences in olfactory response of the eel to thin-layer chromatography fractions of bile from both sexes, and mature and immature conspecifics. Smaller differences were seen in the potency of fractions of bile from male and female goldfish and tilapia. Eels, goldfish and tilapia demonstrated similar olfactory sensitivity to bile from a range of different species, with no apparent correlation between the olfactory potency of bile and a phylogenetic closeness and/or similarity of diet of the donor to the receiver. The three species were able to detect odorants in thin-layer chromatography fractions of heterospecific bile even in the absence of activity in conspecific bile. Eels, goldfish and tilapia responded to both sulphated C(27) bile salts (5beta-scymnol-sulphate and 5alpha-cyprinol sulphate) and to taurine-conjugated C(24) bile salts (taurochenodeoxycholic acid, taurolithocholic acid and taurocholic acid), irrespective of whether these bile salts were present in conspecific bile. Together, these results suggest that teleosts have a broad-range olfactory sensitivity to bile salts, with potential roles in both intra-specific chemical communication and in inter-specific interactions.

Diversity of bile salt hydrolase activities in different lactobacilli toward human bile salts

This study was conducted to evaluate the diversity of bile salt hydrolase (BSH) activities in eight species of lactobacilli. BSH activities were quantified based on the amount of taurine or glycine liberated from six main human bile sodium salts [glycocholic, glycodeoxycholic, glycochenodeoxycholic, taurocholic (TC), taurochenodeoxycholic, taurodeoxycholic] and a mixture of bile salts that resembled human bile. The eight species differed in their BSH activities. Specifically, Lactobacillus helveticus, Lactobacillus fermentum and Lactobacillus gallinarum had the ability to deconjugate taurine-conjugated bile salts, but not glycine-conjugated bile salts, which suggested that microbial BSHs recognize bile salts on both the cholate steroid nucleus and the amino acid moiety. Of the eight species evaluated, Lactobacillus acidophilus strains exhibited the highest specific BSH activity toward human bile salts, with the exception of TC. In addition, the L. acidophilus specific BSH activity toward glycine-conjugated bile salts was ten times higher than that toward taurine-conjugated bile salts. Moreover, the specific BSH activity of Lactobacillus plantarum did not vary significantly toward different bile salts, and Lactobacillus gasseri Am1 exhibited higher specific BSH activity toward TC than other lactobacilli. A comparison of bsh genes indicated that the LA-bshA, LA-bshB, LG-bsh and LP-bsh1 genes that encode the BSH enzymes are highly homologous (higher than 45%), while the LP-bsh2, LP-bsh3 and LP-bsh4 genes, which might not encode BSH enzymes, had lower similarity (lower than 26.3%).

Rickets in Lion Cubs at the London Zoo in 1889: Some New Insights

In 1889, when Dr John Bland-Sutton, a prominent surgeon in London, England, was consulted concerning fatal rickets in more than 20 successive litters of lion cubs at the London Zoo, he evaluated the role of diet relative to the development of rickets. He prescribed goat meat and bones and cod-liver oil to be added to the lean horse-meat diet of the cubs and their mothers. Rickets reversed, the cubs survived, and litters were reared successfully. In classic controlled studies conducted in puppies and young rats 3 decades later, the crucial role of calcium, phosphate, and vitamin D in both prevention and therapy of rickets was elucidated. Later studies led to the identification of the structural features of vitamin D. Although the Bland-Sutton interventional diet obviously provides calcium and phosphate from bones and vitamin D from cod-liver oil, other benefits of this diet were not initially recognized. Chewing bones promotes tooth and gum health and removes bacteria-laden tartar. Cod-liver oil also contains vitamin A, which is essential for the prevention of infection and for epithelial cell health. Taurine-conjugated bile salts are also necessary for the intestinal absorption of fat-soluble vitamins, including A and D. Moreover, unlike dogs and rats, all feline species are unable to synthesize taurine yet can only conjugate bile acids with taurine. This sulfur-containing beta-amino acid must be provided in the carnivorous diet of a large cat. Taurine-conjugated bile salts were provided in the oil cold-pressed from cod liver. The now famous Bland-Sutton "experiment of nature," namely, fatal rickets in lion cubs, was cured by the addition of minerals and vitamin D. However, gum health and the presence of taurine-conjugated bile salts undoubtedly permitted absorption of vitamin A and D, the latter promoting the cure of rickets.

Processing of Bile Salt Odor Information by Single Olfactory Bulb Neurons in the Channel Catfish

A chemotopic map of biologically relevant odorants (that include amino acids, bile salts, and nucleotides) exists in the olfactory bulb (OB) of channel catfish, Ictalurus punctatus. Neurons processing bile salt odorant information lie medially within this OB map; however, information as to how single neurons process bile salt odorant information is lacking. In the present report, recordings were obtained from 51 OB neurons from 30 channel catfish to determine the excitatory molecular receptive range (EMRR) of bile salt responsive neurons. All recordings were performed in vivo within the medial portions of the OB using extracellular electrophysiological techniques. Excitatory thresholds to bile salts typically ranged between 0.1 and 10 muM. The bile salt specificity of OB neurons were divided into three groups: neurons excited by taurine-conjugated bile salts only (group T), neurons excited by nonconjugated bile salts only (group N), and neurons excited by at least one member of each of the three classes of bile salts tested (group G). In addition to the conjugating group at C24 of the side-chain, OB neurons discriminated bile salts by the molecular features present at three other carbon positions (C3, C7, and C12) along the steroid backbone. These data suggest that OB neurons are selectively excited by combinations of molecular features found on the side-chain and along the steroid nucleus of bile salt molecules.