Conjugated Bile Salts Research Articles

PI3 kinase mediates bile salt induced proliferation in a telomerase-immortalized Barrett’s cell line

Abstract Introduction: Bile exposure is implicated in the malignant progression of Barrett's esophagus. Our previous studies demonstrated that bile salt exposure in a Barrett's adenocarcinoma cell line induces proliferation by activating the PI3 kinase/Akt pathway. However, it is not clear that these findings in cancer cells are applicable to the non-neoplastic cells of Barrett's esophagus. Therefore, we studied the effects of bile on PI3 kinase activation and proliferation in a novel, telomerase-immortalized, non-neoplastic Barrett's cell line. Methods: A human, telomerase-immortalized Barrett's cell line (BAR D2847 hTERT lox) was exposed to the conjugated bile salt glycochenodeoxycholic acid (GCDA) at neutral pH. Proliferation was measured by the MTT incorporation assay and Coulter counter. PI3 kinase activity was inferred from western blots for phosphorylated (active form) and total Akt, the major down-stream effector of PI3 kinase. LY294002, a PI3 kinase inhibitor, was used to block the PI3 kinase/Akt pathway. Results: A 5-minute exposure to the clinically relevant concentration of 200uM GCDA produced a 50% cell number increase (P Conclusions: Conjugated bile salts stimulate proliferation in a non-neoplastic Barrett's cell line in a PI3 kinase dependent fashion. We propose that bile exposure acts as a growth signal in the early stages of non-neoplastic Barrett's, thereby promoting progression to adenocarcinoma.

An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts.

F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions. A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception was traW. A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.

Bile salt exposure causes phosphatidyl-inositol-3-kinase–mediated proliferation in a Barrett's adenocarcinoma cell line

BackgroundThe mechanisms by which gastroesophageal reflux promotes malignant progression in Barrett's esophagus are poorly understood. The phosphatidylinositol-3-kinase (PI3 kinase)/Akt pathway regulates proliferation and apoptosis. We hypothesized that the PI3 kinase/Akt pathway mediates the pro-proliferative and antiapoptotic effects of bile. MethodsThe Barrett's adenocarcinoma cell line, SEG-1, was exposed to the conjugated bile salt, glycochenodeoxycholic acid (GCDA). Cell number was measured by the MTT incorporation assay and by Coulter counter. PI3 kinase/Akt activity was inferred from Western blots of phosphorylated and total Akt. Proliferation and apoptosis were determined by BrdU incorporation and cell death ELISA. ResultsA dose-dependent cell number increase was seen with a 20-minute exposure to GCDA. On Western blot, 200 μmol/L GCDA caused a 3-fold increase in Akt phosphorylation within 20 minutes, which was inhibited by 90% with the addition of PI3 kinase inhibitor, LY294002. LY294002 produced dose-dependent inhibition of GCDA-induced cell number increases. 200 μmol/L GCDA decreased apoptosis by 25%. Addition of LY294002 did not completely inhibit the antiapoptotic effect of bile. ConclusionsBile salts activate the PI3 kinase/Akt signaling pathway and stimulate cell growth in SEG-1. The majority of this PI3 kinase–mediated effect is secondary to increases in proliferation rather than to decreases in apoptosis.

Sigma(B)-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor sigma(B) contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific sigma(B)-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative sigma(B)-dependent genes based on the presence of a predicted sigma(B)-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm sigma(B)-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes deltasigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes deltasigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0.5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of sigma(B) activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA-gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0.5 M NaCl or 0.5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific sigma(B)-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.

Binding of Bile Salts by Soluble Fibers and Its Effect on Deconjugation of Glycocholate by Lactobacillus acidophilus and Lactobacillus casei.

ABSTRACT: Binding of sodium cholate, sodium taurocholate, and sodium glycocholate by guar gum, soluble oat fiber, xanthan gum, and inulin was studied. All soluble fibers were able to bind sodium cholate, sodium taurocholate, and sodium glycocholate from a mixture of the three, but when tested individually, the fibers bound little or no sodium cholate. In general, the fibers bound higher concentrations of conjugated bile salts than they did free bile salts. Soluble oat fiber, which bound as much or more sodium cholate and sodium glycocholate as did the other 3 fibers, was tested for its effect on deconjugation of glycocholate by Lactobacillus acidophilus and Lactobacillus casei. The presence of the fiber significantly increased (P < 0.05) deconjugation by all cultures.

Effects of microflora status, dietary bile salts and guar gum on lipid digestibility, intestinal bile salts, and histomorphology in broiler chickens

The aim of the present study was to reexamine the effects of intestinal viscosity, microflora, and bile salts, and their interactions in order to clarify the mechanisms that explain the effect of intestinal viscosity on lipid digestibility, especially those that could involve microflora. Effects of intestinal viscosity, microflora status, and dietary bile salts on lipid digestibility, intestinal bile salts, and intestinal histomorphology were tested in a 2 x 2 x 2 factorial experiment. The effect of microflora status was examined by comparing conventional chickens to chickens with limited microflora obtained by rearing birds in sterilized conditions. Viscosity and dietary bile salts were tested with guar gum (0 or 0.5% in diets) and sodium taurocholate (0 or 0.3% in diets), respectively. Guar gum was autoclaved and added to the gamma-irradiated diets by mixing inside the sterile isolators. The intestinal concentration of lactic acid and cecal concentration of short-chain fatty acids were both very low in birds with limited microflora compared to conventional birds (P = 0.0001). Chickens with limited microflora had higher gain:feed ratios (P = 0.002), higher fecal lipid digestibility (P = 0.0001), more intestinal conjugated bile salts (P = 0.0001), less intestinal unconjugated bile salts (P = 0.0001), and their gastrointestinal compartments were smaller (P < 0.05) than those of conventional chickens. Addition of bile salts in diets increased the lipid digestibility (P = 0.0001) with a more pronounced effect in conventional birds (P = 0.0001) and in birds fed on guar gum diets (P = 0.002). Feeding the guar gum diets increased the intestinal supernatant viscosity (P = 0.0001) for both microflora status. Guar gum addition increased (P < 0.05) the lactic acid concentration in the small intestine of conventional chickens. Guar gum decreased the fecal lipid digestibility (P = 0.0001) and the intestinal conjugated bile salts (P = 0.0001) for both microflora status. However, the magnitude of lipid digestibility decrease due to guar gum was lower (P = 0.05) in birds with limited microflora than in conventional birds, and the negative effect of guar gum on intestinal conjugated bile salts was more pronounced (P = 0.02) in birds with limited microflora. Bile salt addition reduced the negative effect of guar gum on lipid digestibility (P = 0.02) for both microflora status. The mean lipid digestibilities were negatively correlated (P = 0.0001) with the ratio [Ln(viscosity)/total bile salt] measured in intestinal digesta. Except for gizzard, size of digestive compartments and villus heights increased (P < 0.05) with feed efficiency and digestibility decreased. In conclusion, the results provided evidences that the negative effect of guar gum on lipid digestibility was mainly mediated by its direct effect on intestinal bile salt concentration and efficiency. The small interaction between guar gum and microflora status effects on lipid digestibility had to be accounted for by the low basal level of intestinal bile salts in conventional chickens.

CbsT2 from Lactobacillus johnsonii 100-100 Is a Transport Protein of the Major Facilitator Superfamily That Facilitates Bile Acid Antiport

We previously identified two conjugated bile acid transporters, CbsT1 and CbsT2, in Lactobacillus johnsonii 100-100 and Lactobacillus acidophilus KS-13 that are gene duplicates encoded in tandem with a conjugated bile salt hydrolase (BSH) [Elkins and Savage, J. Bacteriol. 180:4344–4349, 1998; Elkins et al., Microbiology 147: 3403–3412, 2001]. CbsT2 from 100–100 was shown to increase taurocholic acid (TCA) uptake in Escherichia coli; however, higher levels were achieved when an extracellular factor (EF) from 100–100 was present in the assay medium (spent medium from 100–100, pH 4.2). We continued this study here to determine the role of EF in this transport system. Kinetic studies revealed that the previously observed CbsT2- and EF-mediated TCA accumulation is rapid (<15 s) but not saturable, suggesting that EF is limiting. In addition, uptake of TCA by E. coli expressing CbsT2 was insensitive to ionophores, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, and thus, is independent of the proton motive force. Since BSH converts [24-¹⁴C]TCA to [24-¹⁴C]cholic acid (CA), we measured net radiolabel uptake in E. coli cells expressing transporter(s) and BSH. Interestingly, such cells accumulated less ¹⁴C radiolabel (by approximately half) than cells expressing CbsT2 alone. These data can be explained if CA diffuses out of E. coli through the transporter(s). We, therefore, added exogenous, unlabeled CA to EF-spent media, which under our assay conditions, performed similarly to EF+ culture supernatant in TCA and CA uptake assays. Thus, unlabeled CA (a protonated, neutral lipophile) can partition directly into E. coli cells especially at low pH. These findings were validated in uptake assays with [³H]TCA, which yields [³H]taurine (a hydrophilic moiety) upon hydrolysis by the BSH. Amounts of cell-associated ³H radiolabel remained similar in cells expressing CbsT2 and BSH versus cells expressing only CbsT2, both of which were higher than in cells expressing BSH alone. Our data support a hypothesis that these transporters, which comprise a new subfamily of the major facilitator superfamily, facilitate antiport of TCA and CA.

Characterization of cholylglycine hydrolase from a bile-adapted strain of Xanthomonas maltophilia and its application for quantitative hydrolysis of conjugated bile salts.

Purified bile salt hydrolase from bile-adapted Xanthomonas maltophilia displays Michaelis-Menten kinetics on cholylglycine and cholyltaurine and hydrolyzes bile salts also in crude bovine bile. The protein is a dimer and is resistant to proteinases and to heating at 55 to 60 degrees C for up to 60 min, in agreement with calorimetric data.

Bile salt transporters.

Bile salts are the major organic solutes in bile and undergo extensive enterohepatic circulation. Hepatocellular bile salt uptake is mediated predominantly by the Na(+)-taurocholate cotransport proteins Ntcp (rodents) and NTCP (humans) and by the Na(+)-independent organic anion-transporting polypeptides Oatp1, Oatp2, and Oatp4 (rodents) and OATP-C (humans). After diffusion (bound by intracellular bile salt-binding proteins) to the canalicular membrane, monoanionic bile salts are secreted into bile canaliculi by the bile salt export pump Bsep (rodents) or BSEP (humans). Both belong to the ATP-binding cassette (ABC) transporter superfamily. Dianionic conjugated bile salts are secreted into bile by the multidrug-resistance-associated proteins Mrp2/MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by non-ionic diffusion and the apical sodium-dependent bile salt transporter Asbt/ASBT, transported back into the periductular capillary plexus by Mrp3/MRP3 [and/or a truncated form of Asbt (tAsbt)], and subjected to cholehepatic shunting. The major portion of biliary bile salts is aggregated into mixed micelles and transported into the intestine, where they are reabsorbed by apical Oatp3, the apical sodium-dependent bile salt transporter (ASBT), cytosolic intestinal bile acid-binding protein (IBABP), and basolateral Mrp3/MRP3 and tAsbt. Transcriptional and posttranscriptional regulation of these enterohepatic bile salt transporters is closely related to the regulation of lipid and cholesterol homeostasis. Furthermore, defective expression and function of bile salt transporters have been recognized as important causes for various cholestatic liver diseases.

Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase

Intestinal alkaline sphingomyelinase (SMase) has physiological roles in the digestion of sphingomyelin (SM) and clinical implications in colonic carcinogenesis. In the present work, the enzyme from rat has been purified 1,589-fold with 11% recovery by elution of the intestine with bile salt, precipitation of the proteins by acetone, and several types of chromatographies. Its molecular mass was 58 kDa and optimal pH was 9 to 9.5. Under the optimal conditions, the Vmax was 930 μmol/h/mg and Km was about 1.25 mM. The enzyme could hydrolyze phosphatidylcholine at pH 7.4 in the presence of Ca2+; the rate was about 8% of that for SM. The activity against SM was dependent on bile salt. Taurine conjugated bile salts were much more effective than glycine conjugated ones, and the most effective bile salts were taurocholate and taurochenodeoxycholate. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and Triton X100 (TX100) had no stimulatory effects. Unlike neutral SMase, intestinal alkaline SMase was not Mg2+ dependent, not inhibited by EDTA, and not inhibited by glutathione. The enzyme was stable during incubation with temperatures up to 50°C and in pHs from 7 to 10. Trypsin and chymotrypsin had no effects on its activity, and 10 mM dithiothreitol reduced its activity by 25%. A specific antibody against the enzyme was developed, and Western blot showed that the enzyme was expressed in the intestine but not in other organs. In conclusion, we purified a potentially important SMase in the intestine with several properties different from neutral SMase.—Cheng, Y., Å. Nilsson, E. Tömquist, and R-D. Duan. Purification, characterization, and expression of rat intestinal alkaline sphingomyelinase.

Micellar electrokinetic chromatography of tri aza aromatic ligand compounds of iron (II): Influence of bile salt type on enantiomeric separation

Micellar electrokinetic chromatography is used with a variety of bile salt micelles to separate the enantiomers of bis(8-((pyridine-2-methylene)amino)quinoline)iron(II) hexafluorophosphate, Fe(PMAQ)2(PF6)2; bis(8-((pyridine-2-methylene)amino)lepidine iron(II) hexafluorophosphate, Fe(PMAL)2(PF6)2; and bis(1-(2-pyridinyl)ethylidine)-8-aminoquinoline iron(II) hexafluorophosphate, Fe(PEAQ)2(PF6)2. The influence of ten different bile salts on the resolution of each pair of enantiomers is investigated. Significant changes in resolution are seen depending upon the bile salt used. The dihydroxy bile salts are superior to the trihydroxy bile salts in terms of resolution, and the taurine or glycine conjugated bile salts yield better results than the unconjugated bile salts. Resolution for most enantiomers is maximized in a buffer solution containing 10-15% acetone and employing either taurochenodeoxycholic or glycochenodeoxycholic acid as the bile salt. Evidence for the separation of the corresponding Fe(III) complexes is presented.

Bile salt hydrolase activity and resistance to toxicity of conjugated bile salts are unrelated properties in lactobacilli.

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.

Characterization of the mouse bile salt export pump overexpressed in the baculovirus system.

The bile salt export pump (Bsep), a member of the ATP-binding cassette superfamily of transporters, mediates the ATP-dependent canalicular secretion of bile salts. We have cloned and expressed the mouse Bsep (mBsep) protein in Sf9 insect cells, and characterized its transport and ATPase properties. Because its deduced amino acid sequence predicts multiple phosphorylation sites for protein kinase A, protein kinase C (PKC) and Ca(2+)-calmodulin dependent kinase II, we have also tested whether mBsep undergoes phosphorylation. MBsep transports both glycine and taurine conjugated bile salts. Sf9 cell membranes that express mBsep exhibit higher basal ATPase activity than control membranes, and this is further stimulated by bile salts and inhibited by vanadate. Taurochenodeoxycholate is transported with the highest affinity and is the most potent inducer of ATPase activity. Cyclosporin A, glibenclamide and rifamycin SV, all competitive inhibitors of Bsep transport, also reduced the bile salt-stimulated ATPase activity. MBsep exists as a phospho-protein when expressed in Sf9 cells and the immunoprecipitated mBsep complex is a substrate for the catalytic subunit of PKC. When mBsep and the alpha-isoform of mouse PKC are co-expressed in Sf9 cells, a ninefold stimulation of phosphorylation occurs. This is further increased to 18-fold after activation by phorbol ester. Given that bile salts activate selected PKC isoforms in hepatocytes, including the alpha isoform, the phosphorylation of mBsep by PKCalpha may represent a point of regulation for this transporter that is mediated by its own substrate.

Germinated Barley Foodstuff Exhibits Different Adsorption Properties for Hydrophilic versus Hydrophobic Bile Acids

Background/Aims: Germinated barley foodstuff (GBF), a type of dietary fiber, exhibits therapeutic effects in ulcerative colitis (UC) patients. However, the precise mechanisms responsible for these effects are still under investigation. On the other hand, it has been suggested that bile salts in the gut lumen play an important role in the integrity of the intestinal mucosa. The aim of the present study was to investigate the ability of GBF to adsorb bile salts in vitro. Methods: The binding capacities of GBF, enzymatically digested GBF (GBF-fiber), and α-cellulose for unconjugated and conjugated bile salts were measured using Langmuir’s method. The morphology of these fibers was observed by light and fluorescence microscopy. Results: GBF adsorbed bile salts very strongly, especially hydrophobic bile salts. Even after enzymatic digestion, the GBF fiber still exhibited strong binding capacity, whereas α-cellulose exhibited very low binding capacity. Microscopically, GBF consists mainly of aleurone, a lattice-like cell wall with cytoplasm enclosed. After enzymatic digestion, the cytoplasm was also digested. Conclusion: GBF possesses a great capacity to adsorb bile salts. This may be part of the mechanism for the therapeutic effects of GBF in UC patients.

Hepatocellular transport proteins and their role in liver disease

TRANSPORT PROTEINS Basolateral transport systems Na-dependent bile salt uptake Uptake of bile salts into the liver was first characterized in experimental models such as the isolated perfused rat liver, isolated hepatocyte cultures and basolateral plasma membrane vesicles. These studies indicated that more than 80% of taurocholate uptake but less than 50% of cholate uptake into hepatocytes is sodiumdependent. Whereas unconjugated bile salts are uncharged molecules that can traverse membranes by passive nonionic diffusion, conjugation with glycine or taurine decreases their pKa values and necessitates the presence of a specific carrier protein for hepatocellular uptake. The chief uptake system for conjugated bile salts in mammalian liver was isolated by expression and molecular cloning strategies and has been called the Na + taurocholate cotransporting polypeptide (gene symbol: SLC10A1). Rat Ntcp consists of 362 amino acids with an apparent molecular mass of 51 kD and is expressed exclusively at the basolateral membrane of hepatocytes (Figure 1). Ntcp mediates sodium-dependent uptake of taurocholate and other bile salts when expressed in stably transfected COS-7, Chinese hamster ovary (CHO) and hepatoblastoma (HepG2) cells or in cRNA injected Xenopus laevis oocytes, with apparent Km values between 17-42 μmol/L. The only non-bile salt substrates that are transported by Ntcp are selected sulfated steroid conjugates such as estrone-3sulfate and dehydroepiandrosterone sulfate (DHEAS) . In human liver, NTCP represents a 349-amino acid protein. NTCP is structurally related to the intestinal bile salt transporter (IBAT), that also mediates the Nadependent uptake of bile saltsand that is expressed not only in ileum, but also in the kidney and in cholangiocytes. Na-dependent taurocholate uptake is reduced in experimental models of cholestasis such as bile duct l igat ion , endotoxinemia and par t ia l hepatectomy, is reduced in primary hepatocyte cultures and is absent in various hepatoma cell lines . These changes in hepatic Na dependent bile salt uptake correlate with expression levels of Ntcp. Thus, Ntcp mRNA and protein levels are decreased in bile duct ligation, endotoxinemia and ethinyl estradiol induced cholestasis. In patients with a diagnosis of extrahepatic biliary atresia and clinical evidence of cholestasis, NTCP mRNA levels are also decreased.

Physiologic Concentrations of Bile Salts Inhibit Rat Hepatic Alkaline Phosphatase but Not the Intestinal Isoenzyme

Objective: The effect of bile salts on alkaline phosphatase (EC 3.1.3.1) activity from Wistar rat liver, duodenum, jejunum, and serum was investigated. Design and results: For concentrations higher than 1 mM conjugated bile salts (glycocholate, glycochenodeoxycholate, taurocholate, taurodeoxycholate, and taurochenodeoxycholate) inhibited hepatic ALP but, up to concentrations of 10 mM, had no effect on intestinal ALP. Also cholate, deoxycholate, and chenodeoxycholate, within the same concentration range, did not have any effect on intestinal ALP. ALP inhibition induced by conjugated bile salts was significantly higher in serum of starved rats than in serum of fed animals, what is in good agreement with the known higher proportion of hepatic ALP and lower proportion of intestinal ALP in serum of starved rats. Conclusions: Bile salts can, thus, be used to help discriminating between tissue-nonspecific and intestinal ALP isoenzymes and identifying pathologic conditions where the relative quantities of these isoenzymes are altered in serum. Inhibition of hepatic ALP by physiologic concentrations of bile salts may bear some relation to the bile salts effects on their own enterohepatic circulation and/or biosynthesis.

Inhibiting the Activity of Calf Pregastric Lipase: Effect of Bile Salts, Lecithin, Liposomes, Phenyl Boronic Acid and Diethyl 4-Nitrophenyl Phosphate

A commercial extract from oropharyngeal tissue of calves has been used as the source of partially purified pregastric lipase. Activity of the enzyme against 4-nitrophenyldecanoate was inhibited by the conjugated bile salts taurocholate, taurodeoxycholate, and tauro- and glyco-chenodeoxycholate in their monomeric form. Although solutions of L-α-lecithin (0-0.75 mg mL -1 ) enhanced the activity of the lipase at all concentrations studied, with maximum rate enhancement (∼190%) occurring within the range (0.11-0.34) mg mL -1 , even this concentration of L-α-lecithin could not remove the inhibitory effect of the bile salts. The Michaelis-Menten parameters, V and K M , were determined for the activity of the enzyme against 4-nitrophenylacetate in the absence and presence of egg phosphatidylcholine (egg-PC) and egg-PC:cholesterol (10:1 mol/mol) liposomes. While values of V decreased slightly (and to the same extent) in the liposomal suspensions, the value of K M was decreased by 50% in the normal liposome but increased by 30% in the cholesterol impregnated liposome. The phenyl boronic acid / lipase dissociation constant was evaluated as 1.9 mM and pronounced inhibition was obtained in the presence of diethyl 4-nitrophenyl phosphate (E600). These inhibition results confirmed the presence of serine in the active site of calf pregastric lipase.

Bile salt toxicity to some bifidobacteria strains: role of conjugated bile salt hydrolase and pH.

The purpose of this work was to study some aspects of bile salt toxicity towards bifidobacteria. A strain (Bifidobacterium coryneforme ATCC 25911) was selected for its lack of conjugated bile salt hydrolase activity (CBSH-), and was used with three deconjugating strains (CBSH+), for study of their growth and viability in the presence of two dihydroxylated conjugated bile salts (tauro- and glyco-deoxycholic acids). The presence of the glycoconjugate induced a more significant growth inhibition for the four strains than the tauroconjugate. The viability of the strains was measured at several pH levels. Glycodeoxycholic acid, but not taurodeoxycholic acid, exerted a lethal effect, which increased at low pH. This phenomenon was more pronounced for the CBSH- strain. We explain some of these results using an hypothesis based on the consequence of dissociation of conjugated and deconjugated bile salts, and the value of their pKa.

Effect of Conjugated Bile Salts on Antibiotic Susceptibility of Bile Salt–Tolerant Lactobacillus and Bifidobacterium Isolates

Virtually every antibiotic may cause in vivo alterations in the number, level, and composition of the indigenous microbiotae. The degree to which the microbiotae are disturbed depends on many factors. Although bile may augment antibiotic activity, studies on the effect of bile on the antibiotic susceptibility of indigenous and exogenous probiotic microorganisms are lacking. It was against this background that the antibiotic susceptibility of 37 bile salt–tolerant Lactobacillus and 11 Bifidobacterium isolates from human and other sources was determined in the presence of 0.5% wt/wt oxgall (conjugated bile salts). Oxgall did not affect the intrinsic resistance of lactobacilli to metronidazole (5 μg), vancomycin (30 μg), and cotrimoxazole (25 μg), whereas it resulted in a complete loss of resistance to polymyxin B (300 μg) and the aminoglycosides gentamicin (10 μg), kanamycin (30 μg), and streptomycin (10 μg) for most strains studied (P < 0.001). Oxgall did not affect the intrinsic resistance of bifidobacteria to metronidazole and vancomycin, whereas polymyxin B and co-trimoxazole resistance was diminished (P < 0.05) and aminoglycoside resistance was lost (P < 0.001). Seven lactobacilli, but no bifidobacteria strain, showed unaltered intrinsic antibiotic resistance profiles in the presence of oxgall. Oxgall affected the extrinsic susceptibility of lactobacilli and bifidobacteria to penicillin G (10 μg), ampicillin (10 μg), tetracycline (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), and rifampicin (5 μg) in a source- and strain-dependent manner. Human strain–drug combinations of lactobacilli (P < 0.05) and bifidobacteria (P < 0.01) were more likely to show no change or decreased susceptibility compared with other strain-drug combinations. The antimicrobial activity spectra of polymyxin B and the aminoglycosides should not be considered limited to gram-negative bacteria but extended to include gram-positive genera of the indigenous and transiting microbiotae in the presence of conjugated bile salts. Those lactobacilli (7 of 37) that show unaltered intrinsic and diminished extrinsic antibiotic susceptibility in the presence of oxgall may possess greater upper gastrointestinal tract transit tolerance in the presence of antibiotics.

A comparison of the permeation enhancement potential of simple bile salt and mixed bile salt:fatty acid micellar systems using the CaCo-2 cell culture model

The aim of this study was to compare the permeation enhancing potential and toxicity of simple bile salt and bile salt:fatty acid mixed micellar systems using the CaCo-2 cell culture model. The effects of micellar systems of sodium cholate, (NaC), and sodium taurocholate, (NaTC), on the permeability of the hydrophilic markers, mannitol (182) and polyethylene glycols (PEGS) 900 and 4000, were assessed. Simple micelle systems of the unconjugated bile salt, NaC, caused greater enhancement of the hydrophilic markers than the conjugated bile salt, NaTC. In the case of NaC systems the enhancement was coincident with excess membrane disruption and toxicity as indicated by altered TEERs, TEMs, MTT values, and, the lack of recovery following removal of the enhancer. In contrast, the NaTC systems were less toxic, and, in the simple micelle form the likely mechanism of enhancement of the hydrophilic markers is via a transient effect on the tight junctions. Formation of mixed micellar systems with linoleic acid (LA) accentuated the toxic effects of NaC. In comparison, NaTC:LA mixed micelles showed superior permeability enhancement versus simple micelles without increasing membrane toxicity. The mechanism of enhancement of NaTC:LA appears more complex and involves a possible combination effect on both the paracellular and transcellular routes.