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PubMed1979

12alpha-Hydroxysteroid dehydrogenase from Clostridium group P strain C48-50 ATCC No. 29733: partial purification and characterization.

The growth of Clostridium group P strain C48-50 [an anaerobe that contains 12alpha-hydroxysteroid dehydrogenase (12alpha-HSDH) in the absence of other dehydrogenases active upon bile salts] is greatly enhanced by the addition of 2.0% d-fructose or d-glucose to the growth medium. Other sugars were less effective. The production of NADP-dependent 12alpha-HSDH paralleled the growth of the organism which was optimal at 72 hr. Growth (and enzyme production) were suppressed by the addition of bile salt to the medium; the order of suppression was deoxycholate > chenodeoxycholate >> cholate; 1 mM of either of the dihydroxy-bile salts inhibited 96% of the growth and 100% of the enzyme production. Kinetic studies on cell-free preparations of 12alpha-HSDH revealed a pH optimum of 7.8 with greater linearity of NADP evolution with time occurring only at more alkaline pH values (9-10). Lineweaver-Burke plots revealed Michaelis constant (K(m)) values in the range of 3-5 x 10(-4) M for deoxycholate and its glycine and taurine conjugates, while higher values were found for cholate and conjugates (K(m) value for taurocholate was 3 x 10(-3) M). Although there was no activity with NAD, 12alpha-HSDH was shown to bind onto both NAD- and NADP-Sepharose columns, with stronger binding on the latter. The enzyme was purified 20-fold by NAD-Sepharose chromatography. The molecular weight was estimated at 100,000 by Sephadex G-200 and a series of molecular weight markers. Substrate specificity studies showed that a variety of bile salts containing 12alpha-OH groups reacted; notably, the 3alpha-sulfates of cholate and deoxycholate were nonsubstrates.-Macdonald, I. A., J. F. Jellett and D. E. Mahony. 12alpha-Hydroxysteroid dehydrogenase from Clostridium Group P strain C48-50 #29733: partial purification and characterization.

Binding Sites↗

PubMed1983

Effects of bile acids and their glycine conjugates on gamma-glutamyl transpeptidase.

Glycine and taurine conjugates of bile acids modulate gamma-glutamyl transpeptidase by interacting with the cysteinylglycine binding site (acceptor site) of the enzyme. These compounds stimulate hydrolysis of glutamine and S-methylglutathione and the rate of the inactivation of the enzyme by the gamma-glutamyl site-directed reagent, AT-125 (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Transpeptidation between S-methylglutathione and methionine was inhibited by these compounds. These effects resemble those caused by hippurate; the glycine derivatives of bile acids, however, exhibit a much greater affinity for transpeptidase than hippurate. Cholate, as shown previously for benzoate, also seems to bind to a portion of the acceptor site as indicated by its effects on S-methylglutathione utilization and AT-125-dependent inactivation of the enzyme. The Kd values for cholate and benzoate are, however, at least one order of magnitude larger than those for their respective glycine derivatives. The acceptor site-directed modulators increase the affinity of the enzyme for AT-125 and kinetic and binding studies show that binding of gamma-glutamyl site-directed reagents increases the affinity of the enzyme for cholate. These results thus indicate cooperative interactions between the gamma-glutamyl donor and acceptor binding domains of the transpeptidase active center.

Animals↗

PubMed1980

Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced cation channel regulation.

We report the purification of acetylcholine receptors with active agonist-regulated cation channels from Torpedo californica electric organ tissue by five methods. In one method, previously used by others, contaminating proteins were removed from partially purified membranes by alkaline extraction, preserving membrane integrity throughout the procedure. In the other four methods, acetylcholine receptors were purified after solubalization with sodium cholate. The continual presence of soybean lipid in mixed micelles with cholate was required to prevent irreversible inactivation of the cation channel. Solubilized receptors were purified by affinity chromatography using either Naga naja siamensis toxin III or concanavalin A coupled to agarose. Sucrose gradient centrifugation was also used to purify solubilized receptors. The best method combined affinity chromatography on toxin-agarose and concanavalin A agarose. Receptors purified by all five methods were incorporated into soybean lipid vesicles by the cholate dialysis technique. The agonist-regulated cation channels of the receptors were equally active after reconstitution, independent of the method used for purification. All reconstituted vesicle preparations were similar in preferential orientation of acetylcholine receptor toward the external surface, dose-response to carbamylcholine, carbamylcholine-induced desensitization, and carbamycholine-induced influx of 22Na+ per mol of receptor. Carbamylcholine-induced 22Na+ influx/receptor was greater after reconstitution than in native vesicles. This was because, in native vesicles, carbamylcholine-induced 22Na+ influx was limited by equilibration of the internal volume of the vesicles with the external 22Na+ concentration, whereas in reconstituted vesicles 22Na+ influx was limited by desensitization of the receptor molecule. We demonstrate that only one of the two toxin binding sites on the receptor monomer, the one which can be affinity alkylated with 4-(N-maleimido)benzyltrimethylammonium, controls the carbamylcholine-induced opening of the cation channel.

Acetylcholine↗

PubMed1978

Effect of Na on bile acid uptake by isolated rat hepatocytes. Evidence for a heterogeneous system.

The effect of Na on cholate and taurocholate uptake was studied in isolated rat hepatocytes. Uptake of both bile acids can be divided into three components: a) a non-saturable component independent of Na, b) a Na-independent saturable and c) a Na-dependent saturable component. The two saturable components were inhibited by KCN, oligomycin and various other bile acids, which suggests carrier-mediated, energy-requiring processes. The relationship between Na concentration and the Na-dependent uptake rate was sigmoidal. The maximal uptake rate, rather than the Km, was affected by Na. These results suggest that more than one Na (probably two) are cosubstrates in the transport of one bile acid molecule and Na exerts a stimulatory effect on the translocation of the bile acid-carrier complex across the membranes. The Na-dependent uptake was inhibited by ouabain, suggesting that this uptake process is linked to the activity of membrane bound (Na-K)ATPase. Inhibition and competition studies suggest that cholate and taurocholate share a common Na-independent transport system while there are at least two Na-dependent transport systems for taurocholate, one of which is shared by cholate.

Animals↗

PubMed1982

[Lipase localization in Serratia marcescens cells].

The localization of lipase was studied in the cells of Serratia marcescens 345. When the cells were treated with sodium cholate, the enzyme bound to the cellular structures was released in the solution. The supernatant obtained after the cells were treated with sodium cholate contained no glucose-6-phosphate dehydrogenase, a well known endocellular enzyme; therefore, the cytoplasmic membrane was not damaged by the treatment. Ultrathin sections of the intact cells and the cells treated with sodium cholate were examined using electron microscopy, and no disorders were detected in the cytoplasmic and outer membranes. It appears therefore that the lipase of S. marcescens 345 is located mainly on the exterior surface of the outer membrane.

Cell Fractionation↗

PubMed1981

Bile acids modify alkaline phosphatase induction and bile secretion pressure after bile duct obstruction in the rat.

Bile acids induce synthesis of alkaline phosphatase by cultured hepatocytes. To test whether bile acids account for this enzyme's elevation during cholestasis, we developed an experimental model in which the content of the bile acid pool is controlled at the beginning of cholestasis. After depleting the bile acid pool by external biliary drainage, we obstructed bile flow in four groups of rats and then replenished the pool in three groups with taurocholate, taurochenodeoxycholate, or tauroursodeoxycholate, respectively, and replaced no bile acid in the fourth. Hepatic bile acid concentrations were elevated in all obstructed groups; however, the levels were higher in the groups that received bile acid replacement. Cholate was not metabolized, but both chenodeoxycholate and ursodeoxycholate were transformed to beta-muricholate. Although hepatic alkaline phosphatase activity rose in all obstructed animals, the levels achieved were higher in each group treated with bile acids than in the group that was not. Increases in plasma activities of alkaline phosphatase and 5'-nucleotidase occurred only after treatment with cholate or chenodeoxycholate. Bile secretion pressure was higher after ursodeoxycholate or no bile acid treatment but was lower after cholate or chenodeoxycholate replacement. Thus, all bile acids tested induced hepatic alkaline phosphatase to some degree, but only certain ones--those which reduced bile secretion pressure--released the enzyme into plasma. We conclude that, in the rat, the hepatic response to acute cholestasis is influenced by the composition of the intrahepatic bile acid pool and that various bile acids have significantly different effects on this response.

Alkaline Phosphatase↗

PubMed1980

[Serum bile acids in acute hepatitis. Clinical observations].

Diagnostic and prognostic significance of total bile acids concentrations has been evaluated in 26 patients with acute viral hepatitis. The levels of fasting and post-prandial blood cholates have been compared with the values found in normal subjects, in 20 patients with extrahepatic pathology and in 10 cases of non-viral icteric diseases. A remarkable rise in serum bile acids concentration has been showed in acute stage of hepatitis; the level of cholates is significantly higher in cholangitic jaundice, but difference is not found in respect of patients with extrahepatic cholestasis. The improvement of disease decreases the serum bile acids concentration; the decrease going on at the same rate of serum transaminases lowering but preceding the serum bilirubin one. No difference was found between fasting and post-prandial blood cholates. In conclusion, serum bile acids concentration has no diagnostic value in hepatitis, nevertheless it seems an useful test for monitoring the course of the hepatitic disease.

Adult↗

PubMed1981

Adenylate cyclase coupling proteins are not essential for agonist-specific desensitization of lymphoma cells.

Epinephrine or prostaglandin E1 pretreatment of intact S-49 lymphoma cells specifically desensitized adenylate cyclase activity. Cholate extracts of membranes from these desensitized cells showed no difference from controls in their reconstitution of hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant (cyc-) of the lymphoma cell line. Adenylate cyclase in the cyc- variant was also specifically desensitized by epinephrine in prostaglandin E1. The desensitization was assessed by reconstituting the cyc- adenylate cyclase with cholate extracts of the wild type lymphoma cells. Since the cyc- has been shown to lack the coupling or G/F proteins of the adenylate cyclase complex, and these proteins are restored by cholate extracts from wild type cells, we conclude that the G/F proteins are not altered or necessary for specific desensitization of the lymphoma cell lines.

Adenylyl Cyclases↗

PubMed1995

Hepatobiliary transport of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors conjugated with bile acids.

To obtain prodrugs with affinity to liver parenchymal cells, the hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors HR 780 and lovastatin (syn. mevinolin) were conjugated with the bile acids cholic acid, taurocholic acid, and glycocholic acid. Hepatic uptake and biliary excretion of the coupled drugs were investigated and compared with the noncoupled drugs. Studies were performed with livers of normal Wistar rats, and TR-/GT- Wistar rats with deficient drug excretion. The experiments showed that the parent drug HR 780 was slowly excreted into bile. In contrast, the excretion of the bile acid-conjugated HR 780 derivatives S 3554 (conjugated with cholate), S 3898 (conjugated with glycocholate), and S 4193 (conjugated with taurocholate) was rapid and very efficient in both groups of rat strains. The bile acid-conjugated HMG-CoA reductase inhibitors showed a 10 to 20 times higher affinity for the uptake systems of bile acids than the noncoupled parent drug compounds, and even higher affinities than the bile acids themselves. The cholate conjugate of HR 780 (compound S 3554) was shown to be a noncompetitive inhibitor of taurocholate uptake and a competitive inhibitor of sodium-independent cholate uptake (Ki = 1 mumol/L). Uptake of radiolabeled S 3554 into isolated rat hepatocytes was observed to be rapid, cell specific, saturable, energy dependent, and carrier mediated. However, the carrier for S 3554 uptake was found not to be the cloned Na(+)-dependent taurocholate cotransporting polypeptide Ntcp. Expression of this carrier cRNA in Xenopus laevis oocytes did not stimulate S 3554 uptake.

ATP-Binding Cassette Transporters↗

PubMed1979

Solubilization and reconstitution of dopamine-sensitive adenylate cyclase from bovine caudate nucleus.

Dopamine-sensitive adenylate cyclase was reconstituted from the cholate-soluble components of caudate nucleus homogenate. Biological function was restored by precipitating the components from cholate and phospholipid with ammonium sulfate, dialyzing the resuspended precipitate, and activating the particulate complex with phospholipid in the assay. The reconstituted adenylate cyclase was stimulated 3- to 4-fold by dopamine and 8- to 12-fold by guanyl-5'-yl imidodiphosphate. The catecholamine stimulation was specific for dopamine and required the addition of GTP. The cholate-soluble component(s) of the basal adenylate cyclase were separated from the component(s) that conferred dopamine sensitivity by gel filtration chromatography. Dopamine-sensitive adenylate cyclase was also reconstituted from digitonin-soluble components. These were resolved into two fractions by DEAE-cellulose chromatography: one fraction contained adenylate cyclase, but both fractions were required for reconstitution of dopamine-sensitive adenylate cyclase.

Adenylyl Cyclases↗

PubMed1995

Histopathology of cholestasis.

Cholestasis may be extrahepatic or intrahepatic in origin. The block in bile secretion may be complete or incomplete to variable extent. Complete cholestasis occurs in case of primary parenchymal disease (intrahepatic cholestasis) or total obstruction of extrahepatic bile ducts (extrahepatic cholestasis). Incomplete block in bile secretion is due to incomplete obstruction of intra- or extrahepatic bile ducts (intra- or extrahepatic cholestasis or both). Histologically, it is useful to distinguish between bilirubionstasis and cholate-stasis. Complete secretory block causes as early changers: bilirubinostasis (in hepatocytes, canaliculi and Kupffer cells) in acinar zone 3, and "ductular reaction" in acinar zone 1. The latter refers to an increase in periportal ductular profiles, associated with neutrophil infiltration. With longer duration of cholestasis, further lesions ensue: feathery degeneration of hepatocytes due to retention of detergent bile acids, cholestatic liver cell rosettes representing a shift from hepatocellular to biliary differentiation, xanthomatous cells reflecting hyperlipidemia, cholate stasis in acinar zone 1 due to overload of membrane-damaging bile acids, eventually paraportal bile infarcts, and progressive ductular reaction. The latter may be due to multiplication of pre-existing ductules, to metaplasia of periportal hepatocytes, or to activation of progenitor cells. It is invariably associated with periductular fibrosis: the pacemaker for increasing matrix deposition, resulting in biliary fibrosis and eventually in true biliary cirrhosis. Incomplete cholestasis (e.g. PBC, PSC) is characterized by absence of bilirubinostasis during long periods of time, whereas the afore mentioned features of chronic cholestasis do appear. Hence follows that the most reliable markers of chronic incomplete cholestasis are cholate stasis, cholestatic rosettes and ductular reaction. Bilirubinostasis is only a late and often ominous sign.

Adult↗

PubMed1997

Activity and stability of a Rhizomucor miehei lipase in hydrophobic media.

The effects of detergents and organic solvents on a commercial lipase (Lipozyme) from Rhizomucor miehei were investigated. It was shown that the detergent sodium cholate is possibly an activator of the enzyme, increasing lipase activity 2.5 times (250% of the control) when the enzyme was preincubated with 7 mM cholate. Lipozyme activity was over twice as high (230% of the control) in the presence of 80 mM Tween 80 or 90 mM Tween 20 (polyoxyethylenesorbitan monolaurate), apparently through an additional emulsifying action on the substrate. Preincubation with Tween 80 (polyoxyethylenesorbitan mono-oleate) did not affect enzyme activity. In contrast, lipase activity was completely inhibited in the presence of an 8.9 mM concentration of another non-ionic detergent, Brij 58, whereas with a 16.4 mM concentration of the cationic detergent cetyltrimethylammonium bromide (CTAB), enzyme activity was reduced by 80%. Preincubation of Lipozyme with the same concentrations of Brij 58 [poly(oxyethylene)20 cetyl ether] and CTAB promoted total inactivation of the enzyme. Organic solvents had different effects on lipase activity and stability. Of the tested solvents, hexane was least deleterious to lipase activity and did not alter enzyme stability on preincubation. These results suggest that Lipozyme can be used in esterification reactions with hexane as solvent or in hydrolysis reactions with Tween 20 or Tween 80 as emulsifying agents, after pretreatment with sodium cholate.

Culture Media↗

PubMed1976

Deoxycholate metabolism in alcoholic cirrhosis.

Deoxycholate is often absent in bile of patients with alcoholic cirrhosis. The purpose of this study was to define the mechanism for this abnormality in bile acid metabolism in alcoholic cirrhosis. Excretion and hepatic metabolism of exogenous [14C]deoxycholate were determined, quantitative and qualitative analyses of fecal bile acids were performed, and ability of fecal bacteria to metabolize cholate to deoxycholate in vitro was measured both in cirrhotic patients and in controls. There was no evidence for deoxycholate malabsorption or rehydroxylation. In cirrhotic patients without biliary deoxycholate, both in vivo levels of deoxycholate and lithocholate in feces and in vitro fecal bacterial conversion of cholate to deoxycholate were significantly decreased as compared to controls. The marked decrease in 7 alpha-dehydroxylase activity of fecal bacteria of some patients with alcoholic cirrhosis results in impaired conversion of cholate to deoxycholate and explains the lack of biliary deoxycholate in these patients.

Adult↗

PubMed1976

[The non-surgical treatment of postoperatively retained hepato-choledochal calculi].

Postoperatively retained stones in the extrahepatic bile ducts can be chemically dissolved or removed mechanically. One method of chemically dissolving stones is sodium cholate perfusion through the T-tube. In the light of one personal case of successful treatment with sodium cholate, this relatively little-used method is described and recommended as a useful alternative to reoperation. Before reoperation for residual cholesterol gallstones in the extrahepatic bile ducts, dissolving of stones with sodium cholate should at least be attempted.

Aged↗

PubMed2006

Novel O-palmitoylscleroglucan-coated liposomes as drug carriers: development, characterization and interaction with leuprolide.

Polysaccharide-coated liposomes have been studied for their potential use for peptide drug delivery by the oral route because they are able to minimize the disruptive influences on peptide drugs of gastrointestinal fluids. The aim of this work was to synthesize and characterize a modified polysaccharide, O-palmitoylscleroglucan (PSCG), and to coat unilamellar liposomes for oral delivery of peptide drugs. To better evaluate the coating efficiency of PSCG, also scleroglucan (SCG)-coated liposomes were prepared. We studied the surface modification of liposomes and the SCG- and PSCG-coated liposomes were characterized in terms of size, shape, zeta potential, influence of polymer coating on bilayer fluidity, stability in serum, in simulated gastric and intestinal fluids and against sodium cholate and pancreatin. Leuprolide, a synthetic superpotent agonist of luteinizing hormone releasing hormone (LHRH) receptor, was chosen as a model peptide drug. After polymer coating the vesicle dimensions increased and the zeta potential shifted to less negative values. These results indicate that both SCG- and PSCG-coated liposomes surface and DSC results showed that PSCG was anchored on the liposomal surface. The stability of coated-liposomes in SGF, sodium cholate solution and pancreatin solution was increased. From this preliminary in vitro studies, it seems that PSCG-coated liposomes could be considered as a potential carrier for oral administration.

Animals↗

PubMed2006

Rapid-onset intranasal delivery of metoclopramide hydrochloride Part II: Safety of various absorption enhancers and pharmacokinetic evaluation.

In the present study, several nasal absorption enhancers, used in metoclopramide hydrochloride (MCP HCl) nasal solutions, have been screened for their possible damaging effect in the in vitro human erythrocytes lysis experiment. Moreover, the in vivo leaching of biological markers from the rat nasal epithelium was used as a quantitative assessment for possible nasal mucosal irritation whereby the extent of release of total protein and lactate dehydrogenase (LDH) in the nasal lavage fluid was determined. Results showed that insignificant hemolysis from normal saline (P<0.05) occurred with the enhancer protamine sulphate while poly-l-arginine and sodium cholate demonstrated very low (<15%) hemolysis and caused insignificant protein and LDH release from the rat nasal mucosa. Conversely, sodium deoxycholate and chitosan polymers (either of low or high molecular weight) showed high (>60%) hemolysis in vitro and the release of the biological markers in vivo was significantly higher (P<0.05) than the control solution (no enhancer). A significant correlation (P<0.05) existed between the enhancement effect of MCP HCl nasal absorption and the amounts of protein (r=0.85) and LDH (r=0.88). Furthermore, the pharmacokinetics of MCP HCl was determined after intravenous (IV), per-oral and intranasal administration of 10mg drug dose in rabbits. The application of a nasal spray (NS) solution containing 0.5% sodium cholate resulted in a significant improvement (P<0.05) in both the rate and extent of absorption of MCP HCl where the T(max) achieved was 23.3min as compared to 50min in case of the oral solution while the area under the serum concentration-time curve (AUC(0-infinity)) were 506.1, 434.9 and 278.7microg/mlmin for IV, NS and oral solutions, respectively. These values corresponded to absolute bioavailabilities of 87.21 and 55.61% for the NS and oral solutions, respectively. It could thus be concluded that NS of MCP HCl represents a viable approach to achieving rapid and high systemic drug absorption during the emergency treatment of severe emesis.

Absorption↗

PubMed2006

Enhancing effect of surfactants on fexofenadine.HCl transport across the human nasal epithelial cell monolayer.

The effect of various surfactants (sodium cholate, sodium taurocholate, Tween 80 and Poloxamer F68) on enhancing the transepithelial permeability of fexofenadine.HCl was evaluated in a human nasal epithelial cell monolayer model. The cytotoxicity of the surfactants on the human nasal epithelial cells was evaluated by the MTT assay. A dose-dependent reduction of cell viability was observed at higher than critical micelle concentration (CMC) of the surfactants, and the IC50 of non-ionic surfactants (Tween 80 and Poloxamer F68) was higher than that of ionic surfactants (sodium cholate and sodium taurocholate). The TEER values significantly decreased after 2 h incubation with the ionic surfactants, but were recovered after 24 h in the fresh culture media. Ionic surfactants significantly increased the transepithelial permeability (P(app)) of fexofenadine.HCl compared to the non-ionic surfactants. The reduction of TEER values upon exposing the cell monolayer to the surfactants for 2 h correlated well with the P(app) of fexofenadine.HCl, which suggests that the permeation-enhancing mechanism of the ionic surfactants is by altering the tight junction property of the paracellular pathway. F-actin staining showed that the effect of ionic surfactants on the tight junction is temporary and reversible, which is consistent with the TEER value recovery within 24 h. These results imply that ionic surfactants are potentially useful permeation enhancers for nasal delivery of hydrophilic compounds, such as fexofenadine.HCl. This study also indicated the usefulness of the human nasal epithelial cell monolayer model not only for evaluating the in vitro nasal drug transport but also for studying the mechanism and toxicity of enhancers.

Cell Membrane Permeability↗

PubMed1999

Enzymatic reactions in liposomes using the detergent-induced liposome loading method.

Microcompartmentalization is a crucial step in the origin of life. More than 30 years ago, Oparin et al. proposed models based on biochemical reactions taking place in so-called coacervates. Their intention was to develop systems with which semipermeable microcompartments could be established. In the present work we follow their intuition, but we use well-characterized bilayer structures instead of the poorly characterized coacervates. Liposomes from phospholipids can be used as microreactors but they exhibit only a modest permeability and, therefore, chemical reactions occurring inside these structures are depleted after a relatively short period. Here it is shown that even highly stable liposomes from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be used as semipermeable microreactors when treated with sodium cholate. Using this kind of mixed liposomes, we describe a biochemical reaction occurring inside the liposomes while the same reaction is prevented in the external medium. In addition, we show that this cholate-induced permeability of POPC bilayers can even be used to load macromolecules such as enzymes from the outside.

Cholates↗