Lipid components of bile increase the protective effect of conjugated bile salts against antifungal drugs

Fungi and bacteria can persist in the human gall bladder. Previous studies have shown that bile protects Candida albicans in this cryptic host niche from antifungals, providing a reservoir for intestinal re-colonization after discontinuation of antifungal therapy. Bile and conjugated bile salts trap antifungals in micelles, thereby reducing their bioavailability and possibly promoting the development of drug resistance. Here we show that the protective effect of bile and conjugated bile salts is not limited to C. albicans, but also observed with other fungi. Interestingly, bile, but not conjugated bile salts conferred resistance of C. albicans against fluconazole and only bile mediated resistance of Aspergillus terreus against voriconazole. To investigate this higher potency of bile we aimed in a step-wise reconstitution of bile from conjugated bile salts. Neither addition of phospholipids nor saturated fatty acids protected from azoles. In contrast, supplementation with polyunsaturated fatty acids increased azole resistance and decreased the critical micelle concentration of conjugated bile salts to the level of bile. Therefore, polyunsaturated fatty acids are vital for mixed micelle formation with high potential to trap antifungals. As biliary infections are difficult to treat, drug efficacy in the biliary system should be tested by using reconstituted synthetic bile

The ulcerogenic effect of bile and bile acid in rats during immobilization stress

The effect of different concentrations of oxen bile and individual bile acids or their sodium salts on the gastric mucosa of rats was investigated in combination with immobilization stress. A statistically significant higher frequency of ulcers was only determined in the application of 10% oxen bile. Dosages on 10% sodium glycocholic acid demonstrated strong toxic damage with atonic dilation of the stomach and extensive mucosal bleeding

Synthesis and in vitro cholesterol dissolution by 23- and 24-phosphonobile acids

A new class of 23- and 24-phosphonobile acids have been synthesized from bile acid and their in vitro cholesterol-dissolving efficiency have been estimated. 24-Phosphonobile salts (PBSs) are slightly more efficient in solubilizing cholesterol than 23-PBSs and natural bile salts. The cholesterol solubilizing power is influenced by the structure of PBSs, and is considerably reduced with an increase in the bulk pH

Bile resistance and bile salt deconjugation activity of Bifidobacterium pseudocatenulatum G4 in a simulated colonic pH.

The ability of Bifidobacterium pseudocatenulatum G4 to survive and tolerate bile acids exposure and its bile salt hydrolase activity was investigated. The growth rate of B. pseudocatenulatum G4 (106, 108 and 1010cfu/ml) decreased in the presence of 2.0% oxgall compared to the control (without oxgall), however, the colonic concentration of bile acids (0.1%) did not show any significant effect (p<0.05) on the growth rate of this strain in three different simulated colonic pH (5.7, 6.2 and 6.8). Bile salt hydrolase activity, which is the measurement of enzyme activity responsible for bile salt deconjugation, was quantified by high pressure liquid chromatography (HPLC) assay. B. pseudocatenulatum G4 demonstrated high deconjugation rate (82 to 100%) in TPY broth supplemented with 0.25 mM and 5.0 mM of all six different types of bile acids including: taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic (GCDCA), taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA). Overall, the percentage of deconjugation activity was higher in TPY medium supplemented with 0.25 mM bile acids compared to TPY broth with 5.0 mM bile acids. Also, B. pseudocatenulatum G4 showed good tolerance to bile acids. Generally, B. pseudocatenulatumG4 deconjugated glycoconjugated bile acids in higher amount compared to tauroconjugate ones. Bifidobacterium pseudocatenulatum G4 demonstrated good tolerance of bile acids suggesting that it would be capable of surviving in the colon and deconjugating bile salts if used as a probiotic

Bile resistance and bile salt deconjugation activity of Bifidobacterium pseudocatenulatum G4 in a simulated colonic pH.

The ability of Bifidobacterium pseudocatenulatum G4 to survive and tolerate bile acids exposure and its bile salt hydrolase activity was investigated. The growth rate of B. pseudocatenulatum G4 (106, 108 and 1010cfu/ml) decreased in the presence of 2.0% oxgall compared to the control (without oxgall), however, the colonic concentration of bile acids (0.1%) did not show any significant effect (p<0.05) on the growth rate of this strain in three different simulated colonic pH (5.7, 6.2 and 6.8). Bile salt hydrolase activity, which is the measurement of enzyme activity responsible for bile salt deconjugation, was quantified by high pressure liquid chromatography (HPLC) assay. B. pseudocatenulatum G4 demonstrated high deconjugation rate (82 to 100%) in TPY broth supplemented with 0.25 mM and 5.0 mM of all six different types of bile acids including: taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic (GCDCA), taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA). Overall, the percentage of deconjugation activity was higher in TPY medium supplemented with 0.25 mM bile acids compared to TPY broth with 5.0 mM bile acids. Also, B. pseudocatenulatum G4 showed good tolerance to bile acids. Generally, B. pseudocatenulatumG4 deconjugated glycoconjugated bile acids in higher amount compared to tauroconjugate ones. Bifidobacterium pseudocatenulatum G4 demonstrated good tolerance of bile acids suggesting that it would be capable of surviving in the colon and deconjugating bile salts if used as a probiotic

Catabolism of cholesterol to bile salts by rat hepatocytes maintained in monolayers

The liver plays a central role in the metabolism of cholesterol being the major site at which lipoproteins are both assembled and degraded and the only organ where cholesterol can be degraded to bile salts. The synthesis of bile salts by the liver provides the major pathway for the removal of cholesterol from the body.The results in this thesis describe the characterisation of a rat hepatocyte monolayer system, suitable for studying the synthesis of bile salts. The utilization of the cholesterol derived from a high density lipoprotein subfraction (HDL2) for the synthesis of bile salts was also investigated.Following the isolation of a viable cell preparation, hepatocytes were maintained in monolayers for up to 24h. During this period hepatocytes were shown to maintain their viability and to synthesise and secrete bile salts, as determined by radioimmunoassay of conjugated cholic, chenodeoxycholic and ß-muricholic acids. The rate of synthesis of these bile salts by hepatocytes was increased by feeding rats cholestyramine for at least 5 days prior to the preparation of hepatocyte monolayers.Incubation of hepatocyte monolayers with rat HDL2 had no effect on the synthesis of the three bile acid conjugates measured when the cells were obtained from rats fed the pellet diet. However, when the experiment was repeated using hepatocytes obtained from rats fed cholestyramine, HDL2 was found to increase the synthesis of the bile salts measured. This is the first report that a defined lipoprotein fraction can increase the synthesis of bile salts.In an attempt to ascertain the reason for the increase in the synthesis of bile salts, hepatocytes isolated from cholestyramine- vi fed rats were incubated in the presence of HDL2 radiolabelled with either [4- 14Clcholesterol or [4- 14C]cholesteryl oleate. The degradation of the radiolabelled HDL2-cholesterol to bile salts was subsequently determined. The results indicated that the increase in the synthesis of bile salts was due to the utilization of HDL2-cholesteryl ester.Finally, the effect of HDL2 on the synthesis of cholesterol and the utilization of newly synthesised cholesterol for the synthesis of bile salts in hepatocyte monolayers was determined. The results showed that HDL2 had no effect on either cholesterol synthesis or the utilization of newly synthesised cholesterol for the synthesis of bile salts

Bile salts as olfactory and gustatory stimuli 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) and forebrain (FB) of channel catfish, Ictalurus punctatus (Chapter one). Neurons processing bile salt odorant information lie medially within these bilaterally symmetric structures; however, information as to how single neurons discriminate and process this odorant information is lacking. Chapters two and three of the dissertation identify the range of odorant bile salt molecules that excite these neurons [i.e. the excitatory molecular receptive range (EMRR)] within the bile salt chemotopic zones of the OB and FB. The results of the investigations of single bile salt responsive neurons within the OB indicate that these neurons are selectively excited by combinations of molecular features found on the side-chain and the steroid nucleus of bile salt molecules. Further, the results of the investigations of single bile salt responsive neurons within the FB indicate that their EMRRs are virtually identical to that of OB neurons suggesting that little modification of the neural olfactory quality code for these molecules occurred between the OB and the FB. Bile salts are known olfactory stimuli to teleosts, but only a single report (Yamashita et al. 2006) indicated that the taste system of a fish was sensitive to this class of stimuli. Chapter four investigates the gustatory sensitivity of the facial taste system to bile salts in the channel catfish. Bile salts were shown to be highly effective facial taste stimuli with estimated electrophysiological thresholds of approximately 10-11M-10-10M. Multiunit cross-adaptation experiments indicate that bile salts and amino acids bind to relatively independent receptor sites; however, nerve twig data and a few single fiber recordings suggest that both independent and shared neural pathways exist for the transmission of bile salt and amino acid information to the primary gustatory nucleus of the medulla. The findings of the present report aid in understanding how bile salt molecules are detected and initially processed by the olfactory and gustatory systems in catfish and further suggest that bile salt odorant information is not greatly transformed by central olfactory neurons (Chapter five)

Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition

The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it mediates the elimination of monovalent bile salts into the bile. Inhibition of BSEP is considered a susceptibility factor for drug-induced liver injury that often goes undetected during nonclinical testing. Although in vitro assays exist for screening BSEP inhibition, a reliable and specific method for confirming Bsep inhibition in vivo would be a valuable follow up to a BSEP screening strategy, helping to put a translatable context around in vitro inhibition data, incorporating processes such as metabolism, protein binding, and other exposure properties that are lacking in most in vitro BSEP models. Here, we describe studies in which methods of quantitative intravital microscopy were used to identify dose-dependent effects of two known BSEP/Bsep inhibitors, 2-[4-[4-(butylcarbamoyl)-2-[(2,4-dichlorophenyl)sulfonylamino]phenoxy]-3-methoxyphenyl]acetic acid (AMG-009) and bosentan, on hepatocellular transport of the fluorescent bile salts cholylglycyl amidofluorescein and cholyl-lysyl-fluorescein in rats. Results of these studies demonstrate that the intravital microscopy approach is capable of detecting Bsep inhibition at drug doses well below those found to increase serum bile acid levels, and also indicate that basolateral efflux transporters play a significant role in preventing cytosolic accumulation of bile acids under conditions of Bsep inhibition in rats. Studies of this kind can both improve our understanding of exposures needed to inhibit Bsep in vivo and provide unique insights into drug effects in ways that can improve our ability interpret animal studies for the prediction of human drug hepatotoxicity

A high-throughput LC-MS/MS method for the measurement of the bile acid/salt content in microbiome-derived sample sets

Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a host of microbiologically-relevant matrices. Here, we provide a detailed bioanalytical protocol that contains several modifications of a method previously described by Wegner et al. [1]. Briefly, this modified method exhibits the following advantages for the measurement of cholic acid (CA), taurocholic acid (TCA), and deoxycholic acid (DCA) in microbiome-relevant sample matrices: i) fecal sample processing has been streamlined by the elimination of lyophilization and manual homogenization steps; ii) the Sciex 6500 QTRAP hybrid triple-quadrupole/linear ion trap mass spectrometer has sufficient sensitivity to perform the measurement of bile acids/salts in negative ion mode – ammonium adducts of bile acids/salts are not required for detection; and, iii) assay throughput has been boosted by more than 5-fold by shortening the chromatographic duty cycle of a single sample injection from 45 min to 8.4 min. Recently, the method was used to perform 508 sequential injections (72 calibration standards, 52 blank-internal standard sample, and 368 MiniBioReactor Array (MBRA)-derived samples) from four separate batches over a 4-day time period

Polyamines as olfactory stimuli in goldfish

The effects of polyamines as odorants to goldfish olfactory receptors were investigated by in vivo electrophysiological recordings. Electro-olfactogram (EOG) recordings indicated that polyamines (putrescine, cadaverine and spermine) are potent olfactory stimuli for goldfish with estimated electrophysiological thresholds of 10-100nM, similar to that for L-arginine, the most stimulatory amino acid. Although thresholds were similar, the magnitude of the EOG responses to intermediate and high concentrations of polyamines dwarfed those to amino acids and single amine containing compounds (amylamine and butylamine). The EOG responses to 0.1mM putrescine, cadaverine and spermine were, respectively, 4.2x, 4.3x and 10.3x that of the standard, 0.1mM L-arginine. Electrophysiological cross-adaptation experiments indicated the independence of polyamine receptor sites from those to L-amino acids (arginine, methionine, alanine, glutamate, lysine and ornithine), bile salts (Na+ taurocholate and taurolithocholate), single amine containing compounds, and ATP. Further, cross-adaptation experiments indicated that independent receptors exist for the different polyamines tested. During continuous application of forskolin (5-20micromolar), an adenylate cyclase activator, EOG responses to bile salts were eliminated, while responses to L-amino acids, polyamines and ATP were only partially attenuated. These results suggest that polyamine odorants, in contrast to bile salt, L-amino acid and nucleotide odorants are transduced by a non-cAMP second messenger pathway. Although polyamines result in large EOG responses, olfactory receptor neurons responding excitedly to polyamines are likely few in number, because polyamine application to the olfactory mucosa failed to increase integrated multi-unit activity recorded from the sensory surfaces of olfactory lamellae. Preliminary data suggest that olfactory bulb neurons that respond excitedly to L-amino acids are inhibited by polyamines. The present results indicate polyamines are potent odorants to goldfish, distinct from L-amino acids, bile salts and nucleotides