Hydrolysis of bile acid conjugates by selected fungi

Summary Fungi which have been previously shown to hydrolyse glycocholic acid, with liberation of the free bile acid, have now been shown to be similarly capable of hydrolysing glycodeoxycholic acid. Sodium taurocholate, however, is much less susceptible and its hydrolysis has been demonstrated with only one of the selected fungi, Penicillium chrysogenum, growing in a medium containing the conjugate as the sole sulphur source. It is concluded that the nature of the amino acid moiety is important in determining the ease of hydrolysis of bile acid conjugates by whole cells of the fungi under test.     

Hydrolysis of bile acid conjugates by Clostridium bifermentans

Summary Two strains of Clostridium bifermentans have been investigated for their ability to hydrolyse bile acid conjugates under conditions suited to further transformation of the free acids liberated. In batch fermentation at 0.5 g/l substrate concentration, growing cells effected the near-quantitative hydrolysis of glycodeoxycholate, taurodeoxycholate and taurocholate within 48 h; glycocholate was 88% hydrolysed. At substrate concentration greater than 1.0 g/l however, taurine conjugates were less well hydrolysed. Further transformation of the liberated cholic acid to deoxycholic acid and/or 7-ketodeoxycholic acid was achieved, but quantitative conversion was not observed.     

Synthesis and antimicrobial evaluation of bile acid tridentate conjugates

Two series of novel bile acid tridentate conjugates with different linkers were synthesized and characterized, and their biological activities in vitro were evaluated. The procedure was straightforward and efficient to be carried out with high overall yield. The antimicrobial activity of the synthesized compounds against Saccharomyces cerevisiae, Aspergillus niger, Escherichia coli and Staphylococcus aureus was investigated in vitro. The best activity of minimal inhibitory concentrations (MICs) for 1c, 1c′, 2c and 2c′ against S. cerevisiae was up to 0.125 μg/mL.     

Hypocholesterolemic effects of fatty acid bile acid conjugates (FABACs) in mice

Fatty acid bile acid conjugates (FABACs) prevent and dissolve cholesterol gallstones and prevent diet induced fatty liver, in mice. The present studies aimed to test their hypocholesterolemic effects in mice. Gallstone susceptible (C57L/J) mice, on high fat (HFD) or regular diet (RD), were treated with the conjugate of cholic acid with arachidic acid (FABAC; Aramchol). FABAC reduced the elevated plasma cholesterol levels induced by the HFD. In C57L/J mice, FABAC reduced plasma cholesterol by 50% (p < 0.001). In mice fed HFD, hepatic cholesterol synthesis was reduced, whereas CYP7A1 activity and expression were increased by FABAC. The ratio of fecal bile acids/neutral sterols was increased, as was the total fecal sterol excretion. In conclusion, FABACs markedly reduce elevated plasma cholesterol in mice by reducing the hepatic synthesis of cholesterol, in conjunction with an increase of its catabolism and excretion from the body.     

Effect of organic anions and bile acid conjugates on biliary excretion of taurine-conjugated bile acid sulfates in the rat

Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, canalicular multispecific organic anion transporter/multidrug resistance protein 2. On the other hand, a multiplicity of canalicular organic anion transporter/multidrug resistance protein 2 has been suggested. Therefore, to examine the effect of hydrophobicity on the substrate specificity of canalicular multispecific organic anion transporter/multidrug resistance protein 2, we examined the effect of organic anions and bile acid conjugates on biliary excretion of three taurine-conjugated bile acid sulfates with different hydrophobicity, taurolithocholate-3-sulfate, taurochenodeoxycholate3-sulfate, and taurocholate-3-sulfate in rats. Biliary excretions of these bile acid conjugates were delayed in Eisai hyperbilirubinemic rats. Biliary excretion of these bile acid conjugates was inhibited by sulfobromophthalein, whereas biliary excretion and taurocholate-3-sulfate was not inhibited by phenolphthalein glucuronide. Taurolithocholate-3-sulfate and ursodeoxycholate-3-glucuronide decreased biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate, but ursodeoxycholate-3,7-disulfate did not affect biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate. These findings indicate that very hydrophilic organic anions are not good substrates of canalicular multispecific organic anion transporter/multidrug resistance protein 2.     

The use of microwave oven for the rapid hydrolysis of bile acid methyl esters.

An efficient and convenient procedure for the hydrolysis of bile acid methyl esters is described. This is achieved by the addition of aqueous lithium hydroxide in methanol/dioxane/tetrahydrofuran (or dimethylformamide) in the microwave oven. Under these conditions the formates as well as the acetate derivatives prepared under microwave irradiation conditions were also hydrolyzed, and the desired bile acids were isolated in 86-94% yield. All these reactions were completed in the microwave oven within 45-60 s.     

Chemical synthesis of 24-beta-D-galactopyranosides of bile acids: a new type of bile acid conjugates in human urine

A method is reported for the preparation of the C-24 carboxyl-linked beta-D-galactopyranosides of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids, two of which were recently identified as a novel type of the metabolites of bile acids excreted in human urine. Direct esterification (galactosidation) of the unprotected bile acids with 2,3,4,6-tetra-O-benzyl-D-galactopyranose in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent and subsequent hydrogenolysis of the resulting benzyloxy-protected bile acid 24-beta-D-galactopyranosides over 10% palladium on charcoal under atmospheric pressure afforded the title compounds. The structures of the bile acid acyl galactosides were confirmed by measuring several (1)H-(1)H and (1)H-(13)C shift correlated 2D NMR.     

Efficient extraction of bile acid conjugates with tetraheptylammonium chloride, a liquid ion exchanger

Ethyl acetate or chloroform solutions of tetraheptylammonium chloride, an oil-soluble quaternary amine, quantitatively extract polar, anionic lipids such as steroid or bile salt conjugates from aqueous solution by a process of anion exchange.     

The anionic conjugates of bilirubin and bile acids stimulate ATP hydrolysis by S-(dinitrophenyl)glutathione ATPase of human erythrocyte.

These studies demonstrate that bilirubin-ditaurate (an analog of bilirubin-diglucuronide), lithocholic acid 3-O-sulfate, and lithocholic acid 3-O-glucuronide, which are believed to be transported from liver into bile through an active transport process stimulate ATP hydrolysis by purified dinitrophenylglutathione ATPase of human erythrocytes. The Km and Vmax values of the enzyme for these substrates are similar to those for dinitrophenylglutathione indicating the transport mechanisms for bilirubin conjugates, and anionic bile acid-conjugates from hepatocytes to bile and transport of GSH-conjugates from erythrocytes may be mediated by similar mechanisms.     

Glucuronic acid conjugates

The methods of assay in body fluids of 1-β-alkyl, 1-β-phenyl and 1-β-acyl glucuronic acids (“glucuronide conjugates”) have been reviewed. Most of the 78 references cited (from the literature of the period 1990–1997) concern the glucuronide conjugates of drug metabolites, and these have been considered, for reasons of accessibility, within sections of individual drug classes such as analgesics, anti-cancer agents and opioids. Other glucuronide conjugates are considered under “miscellaneous compounds”. A few gas chromatography and capillary electrophoresis methods are described, but the major technique of assay (62 citations) is reversed-phase high-performance liquid chromatography.     

Adriamycin(hydrazone)-antibody conjugates require internalization and intracellular acid hydrolysis for antitumor activity

Summary Adriamycin hydrazone (ADM-Hzn) immunoconjugates have previously been shown to exhibit antibody-directed antitumor activity in vitro and in vivo. In this report, the biological and biochemical properties of the mAb and linker were investigated. Conjugates prepared with two antibodies 5E9 [anti-(transferrin receptor)] and G28.1 (anti-CD37), (which internalize from the surface of target cells following binding) were more cytotoxic in vitro and had greater antitumor activity against Daudi B lymphoma tumor xenografts than a non-internalizing immunoconjugate prepared with mAb 2H7 (anti-CD20). In addition, the 13-acylhydrazone bond linking the drug to the mAb was labile at pH 5 and released unmodified ADM at a rapid rate (t1/2 = 2.5 h). Immunoconjugates prepared with an oxime linkage at the C-13 position were stable to acid and were not cytotoxic. These findings suggest that internalization of ADM-Hzn immunoconjugates and release of free ADM from the mAb in acidic intracellular compartments were important steps in the mechanism of action of ADM-Hzn immunoconjugates.     

The synthesis of TNF-alpha conjugates with alendronic acid

Conjugates of recombinant human tumor necrosis factor alpha (TNFα) and alendronic acid linked through the protein sulfhydryl, carboxyl, and amino groups were obtained with crosslinking agents of different types. The conjugation reactions were conducted in solution and on a solid phase. Unlike the conjugation reactions in solution, the method involving immobilization of active components on a hydroxyapatite column was shown to result in the conjugates with a specified stoichiometry and a high degree of homogeneity. The TNFα conjugates retained the specific cytolytic activity and demonstrated the higher affinity to hydroxyapatite, an analogue of the bone mineral matrix, than TNFα.     

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).     

Separation of individual sulfated bile acid conjugates as calcium complexes using reversed-phase partition thin-layer chromatography.

A method for separating individual monosulfated primary bile acid conjugates by reversed-phase partition thin-layer chromatography on octadecyl-bonded silica gel is described. The solvent system is acetonitrile containing calcium, probably as calcium carbamate. Excellent resolution of the 3- and 7-monosulfated glycine conjugates, as well as 3- and 7-monosulfated taurine conjugates of cholic and chenodeoxycholic acids is reported. A convenient class separation of sulfated from nonsulfated primary bile acid conjugates by adsorption thin-layer chromatography on low-polarity silica gel is also described.     

Design of novel synthetic MTS conjugates of bile acids for site-directed sulfhydryl labeling of cysteine residues in bile acid binding and transporting proteins

The purpose of this study was to design bile acid-containing methanethiosulfonate (MTS) agents with appropriate physical attributes to effectively modify the cysteine residues present in the human apical sodium-dependent bile acid transporter. Four physical properties including surface area, molecular volume, ClogP, and dipole moment were calculated for each semiempirically optimized structure of MTS compounds. The specificity of the synthesized bile acid-MTS conjugate toward native cysteines and putative bile acid interacting domains of hASBT was supported by the effect of 1mM cholyl-MTS, cholylglycyl-MTS, and 3-amino-cholyl-MTS on uptake activity, that displayed a significant decrease in TCA affinity (K(T)=69.9+/-4.5, 69.01+/-6.2, and 63.24+/-0.26 microM and J(max)=35.8+/-0.3, 24.03+/-1.22, 46.49+/-5.01 pmol mg protein min(-1), respectively). These compounds prove to be effective tools in probing the structural and functional effects of cysteine residues in bile acid binding and transporting proteins.