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Antibacterial action of bile acids against Helicobacter pylori and changes in its ultrastructural morphology: effect of unconjugated dihydroxy bile acid.

Although it has been shown that bile acids possess antibacterial activity against Helicobacter pylori, few reports on their activity have been published. We determined the minimum inhibitory concentration at 72 h of various unconjugated and conjugated bile acids against laboratory standard strains and clinical isolates of H. pylori, and studied morphologic changes of H. pylori under the scanning electron microscope during treatment with deoxycholic acid and ursodeoxycholic acid at the minimum inhibitory concentrations for 24 h. We found that only the unconjugated form of dihydroxy bile acid has antibacterial activity. The minimum inhibitory concentration of deoxycholic acid is 200-400 microg/ml, that of chenodeoxycholic acid is similar to that of deoxycholic acid, and that of ursodeoxycholic acid is 400-800 microg/ml. The morphology of H. pylori changed from its primary rodlike shape to a spherical shape with blebs on the cell surface, and was further degraded to an irregularly condensed mass, following an increase in bile acid. This morphologic change in H. pylori was different from the change to a spherical shape caused by amoxicillin. On the basis of these results, it seems that unconjugated dihydroxy bile acid might be a candidate drug for eradication of H. pylori, but further investigations of the clinical usefulness of bile acid for this purpose should be done.

Bile Acids and Salts↗

Effect of phospholipase C on cholesterol solubilization in model bile. A concanavalin A-binding nucleation-promoting factor from human gallbladder bile.

Human bile contains a phospholipase C activity. To examine its pathophysiological importance, the effect of phospholipase C on the dynamics of lipid solubilization and nucleation (cholesterol crystal formation) were investigated in model bile. Phospholipase C from gallbladder bile from patients with gallstones was partially purified by competitively eluting from a concanavalin A (con A)-Sepharose (Sigma, St. Louis, MO) column and incubating with Pronase (Calbiochem, Behring Diagnostics, La Jolla, CA). Phospholipase C activity was resistant to Pronase digestion. When this fraction (concentrated to half the original volume) was mixed with model bile (1:1, vol/vol), a transfer of cholesterol and phospholipid from the micellar to the vesicular phase and an accelerate nucleation time were found concomitant with phospholipid hydrolysis. These effects were prevented by inhibiting the phospholipase C activity with ethylenediaminetetraacetic acid. To confirm that the results were caused by phospholipase C activity and not some other nucleation-promoting factor within the biliary con A preparation, model bile was incubated with bacterial phospholipase C. An identical cascade of events to that found with the partially purified biliary enzyme was observed. Further purification of the con A-bound proteins on DEAE-Sephadex (Pharmacia, Uppsala, Sweden) did not resolve any separate nucleation-promoting activity to that associated with phospholipase C activity. In conclusion, this study has identified phospholipase C as a/the con A nucleation-promoting activity in human gallbladder bile and has characterized a possible molecular mechanism by which cholesterol nucleation is stimulated by this fraction.

Bile↗

Metabolism of bile alcohols in the perfused rabbit liver - C26 bile alcohols.

The metabolism of a C26 bile alcohol (I, 24-nor-5beta-cho-lestane-3alpha, 7alpha,25-triol) was studied in the isolated perfused rabbit liver. The new bile alcohol and bile acid metabolites secreted into the bile were isolated and identified by a combination of TLC, GLC and GLC-MS. The following bile alcohols were found: II, 24-nor-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol, III, 24-nor-5beta-cholestane-3alpha,7alpha,12alpha,25,26-pentol; IV, 24-nor-5beta-cholest-23-ene-3alpha,7alpha,12alpha-triol; and V, 24-nor-5beta-cholest-23-ene-3alpha,7alpha-diol. In the bile acid fraction, 24-nor-cholic acid and 3alpha,7alpha,12alpha-trihydroxy-24-nor-5beta-cholest-23-en-26-oic acid were present. The perfused nor-triol was not resistant to 12alpha-hydroxylation.

Animals↗

Preparation of [3beta-3H] labeled bile acids and bile alcohols.

[3beta-3H]-bile acids and bile alcohols may be useful for metabolic studies in man and animals because the 3-position is invulnerable to bacterial attack. A number of tritium labeled bile acids and bile alcohols were prepared by selective oxidation of the hydroxyl group at carbon-3 followed by reduction with NaBT4. In each case, the bile acids and bile alcohols epimeric at carbon-3 were resolved by analytical and preparative thin-layer chromatography and characterized by gasliquid chromatography. The average yield was 60--65% and specific activities of the final products were in the range of 7.4 x 10(7) dpm/mg.

Bile Acids and Salts↗

Effect of the adaptation to high bile salts concentrations on glycosidic activity, survival at low PH and cross-resistance to bile salts in Bifidobacterium.

Six derivatives with increased resistance to ox gall (MIC: > or = 1% w/v) and one derivative resistant to sodium cholate (MIC: 0.8% w/v) were obtained from more sensitive original Bifidobacterium strains. These microorganisms, and two additional cholate resistant derivatives obtained in a previous study (Int. J. Food Microbiol. 82 (2003) 191), were partially characterised in this study. Acquisition of resistance against a given bile salt, also conferred cross-resistance to other bile salts, and promoted an increase in the survival of these microorganisms at low pH. Bile resistance levels of derivatives were dependent on the external pH so that the resistance was lower at neutral pH values than in acidic environments. In addition, the acquisition of bile resistance induced changes on glycoside-hydrolysing activities of derivatives obtained from five out of eight original strains, with certain activities such as beta-glucosidase showing more than tenfold increases in some of these microorganisms. These data suggest that the exposure to high bile salts concentrations may have induced a synergic response on Bifidobacterium for the adaptation to the conditions of the gastrointestinal tract. This could have improved the survival at low pH in these microorganisms, the resistance to high bile salts concentrations, and the assimilation of non-digestible carbohydrates by the enhancement of some glycoside-hydrolysing activities.

Adaptation, Physiological↗

Bile acids of patients with renal failure (possibility of bile acid secretion in the distal tubule).

Bile acids of patients with renal failure and of healthy subjects were analyzed by capillary gas chromatography after group separation. The amount of bile acids in the total dialysate (150 L) of the patients was smaller than that in the 24-h urine of healthy subjects. Polar bile acid sulfates constituted 17.3% and 30.9% of the total bile acids in serum and urine of healthy subjects, respectively, 26.0% in predialysis serum of patients, and only 11.3% in dialysate of patients. The amount of bile acid sulfates in the hemodialysate converted during a 24-h period dialysis, was still smaller than that in 24-h urine of healthy subjects. We propose that the distal tubule secretes bile acid sulfates.

Bile Acids and Salts↗

Enzymic measurement of primary bile acids and the primary bile acid ratio in serum with the IL-Multistat III Fluorescence Light-Scattering Centrifugal Analyzer.

Enzymic fluorimetric methods are described for the determination of primary bile acids and of chenodeoxycholic acid (CDC) and cholic acid (C) in serum. Bile acids are extracted from 0.3 mL of serum in a simple 5-min step with use of Sep-Pak C cartridges. Total primary bile acids are measured by an equilibrium technique after reaction with beta-NAD in the presence of 7 alpha-hydroxysteroid dehydrogenase. Chenodeoxycholic acid (and its conjugates) is measured by a reaction-rate technique employing the same reaction as above but under different experimental conditions. A small contribution of cholic acid (and its conjugates) to the reaction rate is eliminated by simple calculations. Cholic acid is calculated by difference of the two determinations. In both assays NADH fluorescence is measured with the Multistat centrifugal analyzer. Absolute recovery of bile acids from serum was about 87%. Day-to-day standard deviations for CDC and C were 1.6 and 2.0 mumol/L at serum concentrations of 22.1 and 24.1 mumol/L respectively. Comparison data with a cholylglycine RIA procedure gave the following correlation coefficients (x = RIA, y = proposed method): r = 0.980 (RIA vs total primary bile acids), r = 0.918 (RIA vs CDC) and r = 0.989 (RIA vs C). The methods described appear more practical for use on a routine basis than methods in the literature for the calculation of the primary bile acid ratio.

Adult↗

Randomised trial of laparoscopic exploration of common bile duct versus postoperative endoscopic retrograde cholangiography for common bile duct stones.

BACKGROUND: The management of stones in the common bile duct in the laparoscopic era is controversial. The three major options are preoperative endoscopic retrograde cholangiography (ERCP), laparoscopic exploration of the common bile duct (LECBD), or postoperative ERCP. METHODS: Between August, 1995, and August, 1997, 471 laparoscopic cholecystectomies were done in our department. In 427 (91%), satisfactory peroperative cholangiography was obtained. In 80 (17%) of these cases there were stones in the common bile duct, 40 patients were randomised to LECBD and 40 to postoperative ERCP. If LECBD failed, patients had either open exploration of the common bile duct or postoperative ERCP. If one postoperative ERCP failed, the procedure was repeated until the common bile duct was cleared of stones or an endoprosthesis was placed to prevent stone impaction. The primary endpoints were duct-clearance rates, morbidity, operating time, and hospital stay. Analyses were by intention to treat. FINDINGS: Age and sex distribution of patients was similar in the randomised groups. Duct clearance after the first intervention was 75% in both groups. By the end of treatment, duct clearance was 100% in the laparoscopic group compared with 93% in the ERCP group. Duration of treatment was a median of 90 min (range 25-310) in the laparoscopic group (including ERCPs for failed LECBD) compared with 105 min (range 60-255) in the postoperative ERCP group (p = 0.1, 95% CI for difference -5 to 40). Hospital stay was a median of 1 day (range 1-26) in the laparoscopic group compared with 3.5 days (range 1-11) in the ERCP group (p = 0.0001, 95% CI 1-2). INTERPRETATION: LECBD is as effective as ERCP in clearing the common bile duct of stones. There is a non-significant trend to shorter time in the operating theatre and a significantly shorter hospital stay in patients treated by LECBD.

Cholangiopancreatography, Endoscopic Retrograde↗

Effects of hydrophobic and hydrophilic bile salt mixtures on cholesterol crystallization in model biles.

UNLABELLED: The hydrophilic bile salt ursodeoxycholate is frequently used to dissolve cholesterol gallstones. We have now quantitated crystallization as a function of bile salt hydrophobicity, phospholipid content, cholesterol saturation and total lipid concentration (TLCo). METHODS: Crystallization in supersaturated model biles with low phospholipid contents (left two-phase-micelles and crystal-containing-zone) was assessed during 21 days by microscopy and chemical measurement of crystal mass. For model biles with higher phospholipid contents (central three-phase-micelles, vesicles and crystal-containing-zone), lipid distribution into various phases was determined by combined ultracentrifugation-filtration-dialysis methodology (Biochim. Biophys. Acta 1532 (2001) 15-27). RESULTS: In the left two-phase zone, crystal numbers and masses were highest in case of more hydrophilic bile salt composition (TUDC 100%>TC/TUDC 70%/30%>TC 100%>TC/TDC 70%/30%>TDC 100%) and decreased with increasing phospholipid contents, lower TLCo and lower cholesterol saturation index (CSI). In contrast, in the presence of vesicles (three-phase zone), crystallization decreased at increasing bile salt hydrophilicity, with concomitant increased vesicular cholesterol solubilization. CONCLUSIONS: Presence of vesicular phases is a prerequisite for inhibition of cholesterol crystallization by tauroursodeoxycholate.

Bile Acids and Salts↗

Pathways of cholesterol crystallization in model bile and native bile.

Hypersecretion of hepatic cholesterol, chronic supersaturation of bile with cholesterol and rapid precipitation of cholesterol crystals in the gallbladder from cholesterol-enriched vesicles represent the primum movens in cholesterol gallstone formation. Physical-chemical factors and pathways leading to cholesterol crystallization can be investigated in artificial model biles and ex vivo in fresh human bile. Depending on modulatory factors (i.e., lipid concentration, bile salt or phospholipid species, humidity, mucins, etc.), cholesterol can precipitate in several forms (i.e., monohydrate, anhydrous) and habits (i.e., plate-like, needle-like, intermediate arcs, filaments, tubules, spirals). Careful analysis of biliary cholesterol crystals includes biochemical analysis of precipitated crystals, polarizing quantitative light microscopy, and turbidimetric methods. In this paper, recent concepts on cholesterol crystallization in artificial model biles as well as in human bile will be reviewed.

Bile↗

Nasal membrane and intracellular protein and enzyme release by bile salts and bile salt-fatty acid mixed micelles: correlation with facilitated drug transport.

The effects of four bile salts, one fusidate derivative, and one mixed micellar formulation of bile salt-fatty acid combination on the nasal mucosal protein and enzyme release have been investigated in rats using an in situ nasal perfusion technique. Deoxycholate (NaDC) was found to possess the maximum protein solubilizing activity, followed by taurodihydrofusidate (STDHF), cholate, glycocholate (NaGC), and taurocholate (NaTC) in a descending order. The difference in protein solubilization of NaDC and NaGC was further characterized by the release of 5'-nucleotidase (5'-ND), a membrane-bound enzyme, and lactate dehydrogenase (LDH), an intracellular enzyme, in the perfusate. While both NaDC and NaGC caused comparable 5'-ND release from nasal membrane, intracellular LDH release was significantly higher with NaDC. The greater protein and LDH solubilizing effects of NaDC corresponded well with its faster rate of disappearance from the nasal perfusate. Therefore, the dihydroxy bile salt NaDC tends to cause intracellular damage and cell lysis, whereas the trihydroxy bile salt NaGC appears to produce primarily mucosal membrane perturbations. Linoleic acid in the form of soluble mixed micelles with glycocholate caused a further increase in nasal protein release. However, the rate and extent of nasal membrane protein release by the mixed micelles composed of 15 mM glycocholate and 5 mM linoleic acid were significantly lower than those caused by either deoxyholate or STDHF at the same concentrations. Nasal absorption of acyclovir, a non-absorbable hydrophilic model antiviral agent, was found to be enhanced in the presence of conjugated trihydroxy bile salts and bile salt-fatty acid mixed micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗

Identification of bile acids in the serum and urine in cholestasis. Evidence for 6alpha-hydroxylation of bile acids in man.

In this qualitative study of the pattern of bile acid excretion in cholestasis, methods are described for the isolation of bile acids from large volumes of urine and plasma. The bile acids were subjected to a group separation and identified by combined gas chromatography-mass spectrometry. The techniques were developed to allow identification of the minor components of the bile acid mixture. Four bile acids that have not previously been described in human urine and plasma were detected, namely 3beta, 7alpha-dihydroxy-5beta-cholan-24-oic acid, 3alpha, 6alpha-dihydroxy-5beta-cholan-24-oic acid (hyodeoxycholic acid), 3alpha, 6alpha, 7alpha-trihydroxy-5beta-cholan-24-oic acid (hyocholic acid) and 3alpha, 7beta, 12alpha-trihydroxy-5beta-cholan-24-oic acid. In addition three C27 steroids were found; 26-hydroxycholesterol and a trihydroxy cholestane, probably 5 beta-cholestane-3alpha, 7alpha, 26-triol were found in the sulphate fraction of plasma and urine. In the plasma sample, a sulphate conjugate of 24-hydroxycholesterol was found. The presence of these compounds probably reflects the existence of further pathways for bile acid metabolism. It is not yet known whether this is a consequence of the cholestasis or whether they are also present in normal man, at much lower concentrations.

Bile Acids and Salts↗

The isolation and further characterization of the bilirubin tetrapyrroles in bile-containing human duodenal juice and dog gall-bladder bile.

Bilirubin and its conjugates were extracted from either dog gall-bladder bile or bile-containing human duodenal juice into chloroform containing 10mm-tetraheptylammonium chloride. The intact bilirubin tetrapyrroles were then separated by t.l.c. Structural elucidation was made after coupling of the individual pigments with diazonium salts. Four azopigments were detected: azopigment alpha(o) or dipyrrolic azobilirubin; azopigment delta or dipyrrolic azobilirubin monoglucuronide; azopigment alpha(3) or dipyrrolic azobilirubin monoglucoside; and, from dog gall-bladder bile, azopigment alpha(2). The last conjugate required further verification of its structure. After methanolysis, it was shown by combined g.l.c.-mass spectrometry to contain xylose in a 1:1 molar ratio with the azopigments of bilirubin. Human bile contained 86% bilirubin diglucuronide, 7% bilirubin monoglucuronide monoglucoside diester, 4% bilirubin monoglucuronide and 3% bilirubin. Dog gall-bladder bile had a considerably different composition; it contained 47% bilirubin diglucuronide, 40% bilirubin monoglucuronide monoglucoside diester, 8% bilirubin monoglucuronide, 4% bilirubin diglucoside, 1-2% bilirubin and traces of conjugates containing xylose. The total bilirubin content and proportions of the conjugates did not change in bile that was frozen and stored at -20 degrees C under N(2), whereas in the chloroform/tetraheptylammonium chloride extract, similarly stored, total pigment was slowly lost and the diglucuronide conjugate converted into the monoglucuronide.

Animals↗

Bile leakage and resultant bile peritonitis during or after diagnostic laparoscopy: an unpredictable event.

Bile peritonitis secondary to bile leakage is a rare complication of laparoscopy-guided liver biopsy. We report two cases of bile peritonitis occurring, respectively, during and 6 days after liver biopsy in patients with an initial diagnosis of intrahepatic cholestasis associated with chronic pancreatitis. In both cases, bile leakage was first managed by compression with a palpating probe and, at onset or worsening of symptoms, by suture during laparotomy. However, while the first patient progressed uneventfully during the postoperative period, the second died 13 days after laparotomy. In the latter patient, bile leakage was associated with bleeding from the biopsy site with consequently diminished peritoneal irritation and delayed onset of symptoms. In this patient, pancreatic carcinoma, of which evidence was found at autopsy, may also have contributed to the fatal outcome. In conclusion, bile leakage and secondary peritonitis may also occur in the absence of dilation of the biliary tree and may be fatal, especially if not recognized and treated early.

Adult↗

The maximum capacity of the bovine liver to excrete manganese in bile, and the effects of a manganese load on the rate of excretion of copper, iron and zinc in bile.

1. The maximum capacity of the bovine liver to excrete manganese in bile was determined in three Friesian-cross steers surgically prepared to allow bile flow-rate to be measured and samples collected. Plasma Mn concentrations were increased by infusing manganese chloride solutions into a jugular vein and the biliary excretion rates of Mn, copper, zinc and iron were measured. 2. The maximum capacity of the liver to excrete Mn in bile was exceeded at an infusion rate of approximately 4000 microgram Mn/min, and at this rate there was a significant decrease in the concentration of Cu, Fe and Zn in bile. The maximum concentration (mean +/- SE) of Mn in bile was 193 +/- 19 microgram/ml, and the maximum excretion rate (mean +/- SE) was 1210 +/- 130 microgram/min for three animals. There was no reduction in bile flow or evidence of live damage as a result of the infusions.

Animals↗

Long-term course in collagenous colitis and the impact of bile acid malabsorption and bile acid sequestrants on histopathology and clinical features.

BACKGROUND: Bile acid malabsorption is common in collagenous colitis, although long-term follow-up data on the impact of bile acids are limited. The aim was to study whether bile acid malabsorption is a permanent finding, with an impact on histopathology and clinical features in collagenous colitis. METHODS: The objective was to reinvestigate 27 patients with collagenous colitis > or = 3 years after index investigation. The clinical course was evaluated by means of an interview, a review of the hospital records and registration of symptoms over a period of 7 days. The patients were invited to undergo a repeat colonoscopy and 75SeHCAT measurement. Initial and follow-up data and 75SeHCAT values from 29 controls were compared. RESULTS: The median follow-up time was 4.2 (range 3-5.3) years. Twenty-two patients underwent a repeat 75SeHCAT test, 23 patients a colonoscopy and in 25 patients the clinical course could be evaluated. The 75SeHCAT values were abnormal in 32% at follow-up versus 44% at index, and the median retention value was 19% (range 2-69) versus 12% (range 0.5-41) (P = 0.024) although lower than in the control groups figure of 38% (range 8-91) (P < 0.005). Histopathology had improved independently of bile acid malabsorption, gender, smoking and autoimmune disease at follow-up. Four were normalized. Patients on bile acid binders had no significant change of histopathology. Four patients had recovered, seven displayed an intermittent course and 14 had continuous diarrhoea. CONCLUSIONS: Collagenous colitis and bile acid malabsorption seem to be associated yet independent disorders. The histopathology improves during the long-term course although only a few patients resolve.

Bile Acids and Salts↗

Dietary psyllium increases fecal bile acid excretion, total steroid excretion and bile acid biosynthesis in rats.

Psyllium, a source of dietary fiber rich in soluble components results in lower serum cholesterol concentration in several species. Suggested mechanisms for the hypocholesterolemic effect include a greater excretion of fecal bile acids and total steroids, and up-regulation of bile acid biosynthesis. The activity of cholesterol 7alpha-hydroxylase (7alphaOHase), the rate limiting enzyme in bile acid biosynthesis, is higher in rats fed 5% psyllium. Whether this higher activity corresponds to an increase in mRNA levels has not been determined. Four groups of 10 rats were fed a semipurified diet containing 5% cellulose (CEL; control), 5% cellulose plus 1% cholic acid (CCA), 5% cellulose plus 2% cholestyramine (CHY) or 5% psyllium hydrocolloid (PSY) for 3 wk. Liver cholesterol concentration, fecal bile acid and total steroid excretion, 7alphaOHase activity and 7alphaOHase mRNA levels were measured. Liver cholesterol content in rats fed CCA was significantly higher than in all other groups. Rats fed CHY and PSY had significantly lower liver cholesterol content than those fed CEL. Total fecal steroid and bile acid excretions were significantly greater in rats fed CCA, CHY and PSY than in those fed CEL. Activities and mRNA levels of 7alphaOHase in rats fed CHY and PSY were significantly higher than in rats fed CEL or CCA. These data indicate that feeding psyllium to rats increases fecal bile acid and total steroid excretion as well as 7alphaOHase activity and 7alphaOHase mRNA levels.

Animals↗

Dietary psyllium increases expression of ileal apical sodium-dependent bile acid transporter mRNA coordinately with dose-responsive changes in bile acid metabolism in rats.

Psyllium (PSY), a type of dietary fiber containing mainly soluble components, has been shown to decrease serum cholesterol concentrations in several species; however, mechanisms involved are not clearly defined. Four groups of 10 rats were fed semipurified diets containing 10% dietary fiber from cellulose and/or PSY for 21 d. Increasing levels of PSY were fed (0,3.33, 6.67 and 10% PSY) with the remaining 10% made up with cellulose. Liver cholesterol, cholesterol 7alpha-hydroxylase (CYP7A) activity and mRNA, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) mRNA, ileal apical sodium-dependent bile acid transporter (ASBT) mRNA, fecal bile acids and total steroids, and intestinal bile acid content were measured. All variables responded in a dose-dependent manner to PSY in the diet. Total liver cholesterol content was significantly reduced in all groups fed PSY compared to cellulose-fed controls [138(a), 105(b), 105(b) and 93(c) micromol (SEM = 4.2) for 0, 3.33, 6.67 and 10% PSY, respectively]. Activity of CYP7A was significantly greater in all groups fed PSY compared to the cellulose-fed controls [6.36(c), 16.92(b), 15.28(b) and 20.37(a) pmol x min(-1) x mg protein(-1) (SEM = 3.19) for 0, 3.33, 6.67 and 10% PSY, respectively]. These differences in CYP7A activity were similar to differences in CYP7A, HMGR and ASBT mRNA levels. Fecal bile acid and total steroid excretion as well as total intestinal bile acids were significantly greater in rats fed PSY-containing diets compared to 0% PSY-fed rats. These results suggest that the reduction in liver cholesterol involves modulating the size and composition of the bile acid pool via regulation of ileal ASBT, CYP7A and HMGR mRNA levels.

Animals↗