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University of ZurichZurich Open Repository and Archive
Winterthurerstr. 190
CH-8057 Zurich
http://www.zora.uzh.ch
Year: 2008
Ursodeoxycholic acid reduces lipid peroxidation and mucinsecretagogue activity in gallbladder bile of patients with
cholesterol gallstones
Jüngst, C; Sreejayan, N; Zündt, B; Müller, I; Spelsberg, F W; Hüttl, T P;Kullak-Ublick, G A; del Pozo, R; Jüngst, D; von Ritter, C
Jüngst, C; Sreejayan, N; Zündt, B; Müller, I; Spelsberg, F W; Hüttl, T P; Kullak-Ublick, G A; del Pozo, R; Jüngst,D; von Ritter, C (2008). Ursodeoxycholic acid reduces lipid peroxidation and mucin secretagogue activity ingallbladder bile of patients with cholesterol gallstones. European Journal of Clinical Investigation, 38(9):634-639.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:European Journal of Clinical Investigation 2008, 38(9):634-639.
Jüngst, C; Sreejayan, N; Zündt, B; Müller, I; Spelsberg, F W; Hüttl, T P; Kullak-Ublick, G A; del Pozo, R; Jüngst,D; von Ritter, C (2008). Ursodeoxycholic acid reduces lipid peroxidation and mucin secretagogue activity ingallbladder bile of patients with cholesterol gallstones. European Journal of Clinical Investigation, 38(9):634-639.Postprint available at:http://www.zora.uzh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch
Originally published at:European Journal of Clinical Investigation 2008, 38(9):634-639.
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Ursodeoxycholic acid reduces lipid peroxidation and mucinsecretagogue activity in gallbladder bile of patients with
cholesterol gallstones
Abstract
BACKGROUND: Recently it has been postulated that gallbladder mucin hypersecretion observed in thepathogenesis of cholesterol gallstone disease may be induced by biliary lipid peroxidation.Ursodeoxycholic acid treatment reduces mucin concentration and the formation of cholesterol crystals inthe gallbladder bile of patients with cholesterol gallstones and this effect might be mediated by adecrease of biliary lipid peroxidation. MATERIAL AND METHODS: In a double-blind,placebo-controlled trial patients with symptomatic cholesterol gallstones received eitherursodeoxycholic acid (750 mg daily) (n = 10) or placebo (n = 12) 10-12 days prior to cholecystectomy.As a marker for lipid peroxidation malondialdehyde was measured in bile together with mucinconcentration. In addition, the mucin secretagogue activity of the individual bile samples was assessedin cultured dog gallbladder epithelial cells. RESULTS: Ursodeoxycholic acid therapy resulted in asignificant reduction of lipid peroxidation in bile as determined by the biliary malondialdehydeconcentration (1.36 +/- 0.28 vs. 2.05 +/- 0.38 micromol L(-1); P < 0.005) and the malondialdehyde(micromol L(-1))/total bile acid (mmol L(-1)) ratio (0.02 +/- 0.005 vs. 0.06 +/- 0.01; P < 0.001).Furthermore, a decrease in mucin concentrations (0.7 +/- 0.3 vs. 1.3 +/- 0.5 mg mL(-1); P < 0.005) andof the mucin secretagogue activity of gallbladder bile (0.9 +/- 0.2 vs. 2.2 +/- 0.3 times control; P <0.001) was observed. CONCLUSIONS: The reduction of lipid peroxidation and mucin secretagogueactivity of gallbladder bile induced by ursodeoxycholic acid treatment may contribute to the beneficialeffects of this drug on gallbladder bile composition and symptoms in cholesterol gallstone patients.
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Ursodeoxycholic acid reduces lipid peroxidation and mucin secretagogue activity in
gallbladder bile of patients with cholesterol gallstones
C.Jüngst1 , N.Sreejayan2, B.Zündt2, I. Müller2, F.W.Spelsberg3,
T.P. Hüttl3, G.A.Kullak-Ublick1, R. del Pozo4, D.Jüngst2 and C.von Ritter5
Division of Clinical Pharmacology and Toxicology, University Hospital, Zurich, Switzerland1,
Departments of Medicine II2 and Surgery3, Klinikum Grosshadern, Ludwig-Maximilians-University,
Munich, Germany,
Universidad Catolica de la Santisima, Concepcion4, Chile,
Department of Internal Medicine, Triamed Kreisklinik, Prien5, Germany.
Corresponding author:
Dieter Jüngst, MD, Department of Medicine II, Klinikum Grosshadern, Marchioninistr. 15, 81377
Munich, Germany
Phone: +49-89-7095 2376, Fax: +49-89-7095 5374, E-Mail: [email protected]
Running head: Ursodeoxycholic acid and lipid peroxidation in bile
Total number of words in the document without abstract and references: 2575
Total number of words in abstract: 255
ACKNOWLEDGEMENTS
The authors are grateful to Birgit Eberlein and Viera Stefanek for their help in preparing the manuscript and to
Gundula Straub for the illustrations. Cultured dog gallbladder epithelial cells were kindly provided by Sum Ping
Lee, Seattle, WA, USA. The study was supported by the Else-Kröner-Fresenius Foundation, Bad Homburg,
Germany and the Deutsche Forschungsgemeinschaft (DFG Ri-584/3).
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ABSTRACT
Background. Recently it has been postulated that gallbladder mucin hypersecretion observed in the
pathogenesis of cholesterol gallstone disease may be induced by biliary lipid peroxidation.
Ursodeoxycholic acid treatment reduces mucin concentration and the formation of cholesterol crystals
in gallbladder bile of patients with cholesterol gallstones and this effect might be mediated by a
decrease of biliary lipid peroxidation.
Material and methods. In a double blind placebo controlled trial patients with symptomatic cholesterol
gallstones received either ursodeoxycholic acid (750 mg daily) (n=10) or placebo (n=12) 10-12 days
prior to cholecystectomy. As a marker for lipid peroxidation malondialdehyde was measured in bile
together with mucin concentration. In addition, the mucin secretagogue activity of the individual bile
samples was assessed in cultured dog gallbladder epithelial cells.
Results. Ursodeoxycholic acid therapy resulted in a significant reduction of lipid peroxidation in bile
as determined by the biliary malondialdehyde concentration (1.36 ± 0.28 vs. 2.05 ± 0.38 µmol/l) and
the malondialdehyde (µmol/l) / total bile acid (mmol/l) ratio (0.02 ± 0.005 vs. 0.06 ± 0.01; p<0.001).
Furthermore, a decrease in mucin concentrations (0.7 ± 0.3 vs. 1.3 ± 0.5 mg/ml; p<0.005) and of the
mucin secretagogue activity of gallbladder bile (0.9 ± 0.2 vs. 2.2 ± 0.3 times control; p<0.001) was
observed.
Conclusions. The reduction of lipid peroxidation and mucin secretagogue activity of gallbladder bile
induced by ursodeoxycholic acid treatment may contribute to the beneficial effects of this drug on
gallbladder bile composition and symptoms in cholesterol gallstone patients.
Key words: Ursodeoxycholic acid, lipid peroxidation, gallbladder bile, mucin, gallstones, cell culture
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INTRODUCTION
Mucin-type glycoproteins are abundant in bile, and mucins are found in the nuclei of both cholesterol
and pigment gallstones (1). Several studies have shown that mucin hypersecretion precedes cholesterol
crystal formation in bile and stimulates cholesterol crystal growth in supersaturated model and human
bile (2-9). Furthermore, the highly viscous mucus gel on the luminal side of gallbladder epithelium
and the soluble mucin in bile are believed to enhance the residence time of lithogenic bile and
cholesterol crystals in the gallbladder, allowing the growth of crystals and, thereby, serving as a nidus
for gallstone formation (10). Recent results support a promoting effect of products of biliary lipid
peroxidation on gallbladder mucin hypersecretion. Lipid peroxidation in bile may therefore contribute
to rapid formation of cholesterol crystals in cholesterol gallstone disease (11).
Antioxidant properties of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, have been proposed
as a potential mechanism for its therapeutic activity (12, 13). Moreover, Guarino et al. have
demonstrated recently that UDCA treatment improves gallbladder muscle contractility by decreasing
the cholesterol content in the plasma membrane and the biochemical parameters of inflammation and
lipid peroxidation in gallbladder muscle cells (14).
On this background, we have performed a double blind placebo controlled trial in patients with
symptomatic cholesterol gallstone disease to investigate the effect of UDCA treatment on lipid
peroxidation in gallbladder bile as determined by malondialdehyde an end product of lipid
peroxidation. In addition, the mucin secretagogue activity of the individual bile samples was assessed
in cultured dog gallbladder epithelial cells.
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MATERIALS AND METHODS
Materials
Vitrogen was purchased from Celtrix Laboratories (Palo Alto, CA, USA). Tissue culture plates were
from Falcon (Lincoln Park, NJ, USA). [3H]N-acetyl-D-glucosamine and Nystatin were from
Amersham Life Science (Braunschweig, Germany). Gentamycin, L-glutamine, trypsin-
ethylenediaminotetraacetic acid (10X) and HEPES buffer were obtained from Gibco (Eggenstein,
Germany). Phosphoric acid (relative density 1.69, 44.0 molL) and Entellan microscopic mounting
solution were from Merck (Darmstadt, Germany), methanol spectroscopic grade from Carl Roth
GmbH (Karlsruhe, Germany). Dulbecco´s modified Eagle medium, butylated hydroxyl toluene,
1,1,3,3-tetraethoxypropane, 2-thiobarbituric acid (TBA), potassium dihydrogen phosphate,
trichloracetic acid (TCA), phosphotungstic acid (PTA), 3,3`-diaminobenzidine tetrahydrochloride,
trypsin and ethylenediaminotetraacetic acid disodium salt were from Sigma Chemical Co. (St. Louis,
MO, USA).
Patients, study design and collection of bile
Twenty-two patients, 17 women and 5 men (age 26 to 68 years), who underwent laparoscopic surgery
because of symptomatic cholesterol gallstone disease were included in the study. Gallstones were
visualized by ultrasonogaphy and patients shown to have a loss of the gallbladder reservoir or cystic
duct obstruction were excluded from the study. All patients gave informed consent after a detailed
explanation of the procedure required for intraoperative bile collection. During laparoscopic surgery
the gallbladder was punctured and a flexible probe with side ports was inserted; bile was aspirated as
completely as possible because of the known stratification of human gallbladder bile (15). Stones were
removed, washed with distilled water, dried, weighed and ground to a powder. The cholesterol content
of the stones as measured chemically after extraction with organic solvent and expressed as percentage
of dry weight (16) was not significantly different in the 10 patients of the UDCA group (83.1 ± 8.0 %)
as compared to the 12 patients of the control group (80.6 ± 9.4 %). Five patients with gallstones
containing less than 50 % cholesterol were excluded from the study.
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No concomitant medication known to influence the composition of gallbladder bile was allowed. The
patients were kept on a regular diet but were advised to avoid fatty meals.
The study was prospective, randomized and double blind. Blinded UDCA and placebo medication was
generously provided by the Else-Kröner-Fresenius Foundation. Ursodeoxycholic acid (3 capsules of
250 mg daily before bedtime, equivalent to 8-11 mg/kg body weight) and placebo were given 10-12
days prior to cholecystectomy. There were 10 patients (eight women and two men; three patients with
solitary and seven with multiple stones) in the UDCA group and 12 patients (nine women and three
men; five patients with solitary and seven with multiple stones) in the placebo group. The study
protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in an a
priori approval by the ethical committee of the institution.
Cholesterol crystal observation time
After collection, bile samples were mixed thoroughly. For the determination of cholesterol crystal
observation time (COT) 4 ml of gallbladder bile were centrifuged for 1h at 100, 000 x g in a Beckman
L 50 ultracentrifuge (Beckman Instruments, Fullerton, CA, USA) to obtain crystalfree bile as
described by Holan et al. (17). The top 1 ml and the next 2ml interphase were removed by aspiration
and mixed carefully to avoid any stratification of multilamellar vesicles. An aliquot of this supernatant
was immediately examined microscopically to confirm the absence of cholesterol monohydrate
crystals (CMCs); the rest was placed in sterile tubes, flushed with nitrogen, sealed and incubated at
37°C. Five µl 20% sodium azide/ml bile were added to prevent bacterial growth. The supernatant was
investigated daily for the appearance of cholesterol crystals. The interval between the start of the
incubation and the first detection of a cholesterol crystal in the sample was taken as the COT and was
measured in days.
Analysis of bile composition
For the analysis of bile composition duplicate aliquots of native gallbladder bile were stored at –30°C
prior to determination. Cholesterol was determined colorimetrically with the Liebermann-Burchard
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reaction after double extraction of 1 ml methanolic bile sample with petroleum ether (18).
Phospholipids were measured as total biliary phosphate after hydrolysis at 150°C with sulphuric acid,
using the colorimetric assay of Fiske-Subbarow, and total bile salts were determined by a modified 3-
α-hydroxysteroid dehydrogenase method (19, 20). The cholesterol saturation index (CSI) of each
sample was calculated in the gallbladder bile by dividing the cholesterol molar ratio by the maximum
cholesterol solubility according to Carey and was corrected for the total lipid content of each
individual bile (21). Individual bile salts were analyzed by capillary gas-liquid chromatography with
hyodeoxy cholic acid as internal standard (22). Biliary mucin concentration was determined according
to a modification of the classical method as earlier proposed by us (23). Lipid peroxidation levels in
bile were determined by measuring the malondialdehyde-thiobarbituric acid (MDA-TBA) adduct by
the high-performance liquid chromatography (HPLC) method employing a fluorescence detector as
described previously (11,24).
Culturing of gallbladder epithelial cells
Gallbladder epithelial cells were isolated from dog gallbladders as previously described (11, 25).
Stock cultures were grown on 60-mm Petri dishes coated with 1 mL vitrogen gel (1:1 mixture of
vitrogen and media); 1 ml/30 mm dish) in Eagle´s minimum essential media supplemented with 10%
fetal calf serum, 2 mM L-glutamine, 20 mM HEPES, 0.5 mg/ml gentamycin and 50 u/mL Nystatin.
Media were changed twice a week, and the cells were maintained in a 37°C incubator with 5% CO2.
The cells were passaged when confluent (every 10-15 days) using trypsin (2.5 g/L) and
ethylenediaminotetraacetic acid (1 g/L) treatment.
Mucin secretion assay
Dog gallbladder epithelial cells were grown to confluence on 12-well tissue culture plates exposing the
apical side to the supernatant. The cells were labelled overnight (16-24 hours) with 3 µCi/well of
[3H]N-acetyl-D-glucosamine in media containing 10% fetal calf serum. After labelling, the cells were
washed with sterile phosphate-buffered saline (PBS), pH 7.4, for 30 minutes, followed by washing for
another 30 minutes with serum-free media to remove unincorporated label. The cells were then loaded
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with 0.1 mL of the bile sample (from patients) and 0.9 mL of serum free medium or 1.0 ml of serum
free medium as control. This set up was then incubated at 37°C overnight. Following incubation the
medium was harvested and centrifuged at 500 g for 10 minutes to pellet released cells. A half milliliter
of the supernatant was then mixed with 8 ml of 10% TCA, vortexed, and incubated overnight at 4°C.
After precipitation overnight, the samples were centrifuged at 1500g for 15 minutes and the resulting
protein pellet was washed twice, first with 5 ml of TCA/PTA and then with 2 ml of 90% ethanol.
Finally, the pellets were dissolved in 0.5 mL of water and counted in 10 mL of scintillation fluid.
Results were expressed as counts per minute per well. Mucin release of dog gallbladder epithelial cells
is variable depending of plating density and the number of cells per well. Therefore, we present
normalized data dividing the counts per minute in individual wells obtained after incubation with the
bile sample with the counts measured after incubation with medium alone (control). Each experiment
including the controls was performed in quadruplicate and thus a single value represents the mean
value determined in four different wells.
Statistical analysis
Results are presented as the group mean or median and standard deviation of the mean (SEM). Group
mean differences were compared by the Mann-Whitney-U-test. Individual variables were tested by a
Pearson Chi-square test. Double-sided P-values are reported. P-values of less than 0.05 were
considered statistically significant.
RESULTS
Effect of UDCA treatment on the biliary composition of individual bile acids
The composition of different bile acids in bile of the placebo and UDCA treated groups is given in
table 1. The bile samples of the placebo group were rich in hydrophobic bile acids (cholic acid,
deoxycholic acid and chenodeoxycholic acid), which constituted about 95% of the bile acid pool while
the hydrophilic bile acid UDCA amounted to only about 4% of the total bile acids. Treatment with
UDCA resulted in a significant enrichment (p < 0.001) of bile of this hydrophilic bile acid. The
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relative concentration of UDCA increased about ten fold (45% of total bile acids) and a simultaneous
reduction in the hydrophobic bile acid pool was observed.
Effect of UDCA treatment on other biliary components and cholesterol crystal observation time
Table 1 shows the already well described effects of UDCA therapy on biliary concentrations of
cholesterol, total bile acids, phospholipids, CSI and mucin: A significant (p < 0.005) reduction in the
levels of total cholesterol, mucin and CSI in bile was observed in the treated group. No significant
differences were observed in the total bile acid concentrations and biliary phospholipids following
treatment with UDCA (table 1). The COTs of the ultracentrifuged bile samples of the patients treated
with UDCA were significantly longer than those of the control groups (median ≥ 21 vs. 2 days, p <
0.0001).
Effect of UDCA-treatment on lipid peroxidation
Lipid peroxidation was assessed by HPLC-measurement of the biliary levels of malondialdehyde, the
major end product of peroxidation of lipids. To normalize for different concentrations of gallbladder
bile samples data are presented in addition as the ratio of MDA (µmol/l) / total bile acids (mmol/l) as
proposed by Alan Hofmann (San Diego, CA, USA) (figure 1). Treatment with UDCA was found to be
associated with a significant (p<0.005) decrease of MDA concentrations (1.36 ± 0.28 vs. 2.05 ± 0.38
µmol/l) and of MDA / total bile acid ratios in gallbladder bile (0.02 ± 0.005 vs 0.06 ± 0.01; p<0.001).
Effect of UDCA-treatment on bile-induced mucin secretion
The effect of different bile samples on epithelial mucin secretion was measured in cultures of dog
gallbladder epithelial cells. The average counts/min/well of the control wells was 1.1 x 103. As shown
in figure 2, bile samples of the placebo group induced a significant (p<0.001) increase in mucin
secretion (2.2 ± 0.3 times control). Incubation of cultured gallbladder epithelial cells with bile samples
from patients who had undergone UDCA treatment showed no increase in mucin secretion (0.9 ± 0.2
times control).
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DISCUSSION
Our earlier in vitro experiments have shown that lipid peroxidation may induce cholesterol crystal
formation in model bile (26). We have recently been able to show that lipid peroxidation is also
increased in vivo in gallbladder bile of patients with cholesterol gallstone disease (11). Increased levels
of hydrophobic bile acids may be responsible for lipid peroxidation in bile. This notion is supported by
our finding that replacement of hydrophobic bile acids by the hydrophilic bile acid UDCA reduces
lipid peroxidation in bile. In our earlier in vitro experiments using pure lipid model systems, we have
found that hydrophobic bile acids, in the presence of free iron ions can increase the rate of lipid
peroxidation in phosphatidylcholine liposomes and arachidonic acid by over three folds as compared
to the lipid peroxidation induced by iron ions alone (27). A close correlation between the
hydrophobicity and the pro-oxidant potential of individual bile acids was observed. We were now able
to confirm these in vitro observations by the present randomized controlled clinical trial which shows
that in cholesterol gallstone patients UDCA therapy effectively displaces hydrophobic bile acids from
the bile acid pool and causes a significant reduction in biliary lipid peroxidation.
It is well documented that hypersecretion of mucin contributes to cholesterol crystal formation in bile
(1, 10). Our previous results demonstrate that the mucin secretagogue activity of human gallbladder
epithelial cells may be increased by incubation with rapid nucleating gallbladder bile samples (11).
This effect of lithogenic bile may at least in part be caused by the different concentrations of MDA we
observed in these bile samples. The present clinical trial now shows that the UDCA induced decrease
in biliary lipid peroxidation in bile is associated with a marked decrease of the mucin secretagogue
activity of human bile. Co-incubation of bile samples of untreated patients with cultured gallbladder
epithelial cells were found to significantly increase mucin secretion by the epithelial cells. In contrast,
bile samples of the UDCA-treated patients with a lower level of lipid peroxidation failed to show such
an effect. The claim that lipid peroxidation induces mucin hypersecretion is further substantiated by
our previous in vitro experiments which demonstrate that the lipid peroxidation product MDA is a
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potent stimulator of mucin secretion by cultured dog gallbladder epithelial cells in concentrations
similar to the concentrations measured in native gallbladder bile (11).
Our results correspond to a double blind, placebo-controlled, 4-week study by Guarino et al.
comparing the effects of UDCA (8-11.2 mg/kg per day) with those of placebo in patients scheduled to
undergo cholecystectomy for symptomatic gallbladder stones (14). The authors demonstrate that the
normalization of gallbladder muscle contraction observed following UDCA-treatment was associated
not only with a reduction in plasma membrane cholesterol levels but interestingly also with a decrease
of MDA in gallbladder muscle cells (1.3 ± 0.4 vs. 2.5 ± 0.7 mmol/100 mg protein).
The therapeutic consequences of these observations are still unclear, but a long-term treatment with
UDCA in selected gallstone patients may be a future option. Indeed, a long-term study on UDCA
treatment revealed a significant decrease in the incidence of acute cholecystitis (28). In contrast, a 3-
months randomized placebo-controlled study with UDCA showed no beneficial effect on biliary pain
or complications of gallstone disease (29). The finding of Guarino et al. that UDCA treatment restores
gallbladder muscle functions together with a decrease in biochemical markers of oxidative stress and
inflammation (14) and the observation that the beneficial effects of UDCA appears to be at least in
part independent of gallstone dissolution (28) supports the notion that UDCA mediates an antioxidant
and anti-inflammatory effect on the gallbladder wall which, in turn, reduces lithogenicity of bile.
In conclusion, our clinical trial shows that UDCA treatment reduces biliary lipid peroxidation. In
addition, mucin concentrations in bile are reduced by UDCA and this effect is mediated by a
decreased mucin secretagogue activity of gallbladder bile. These novel properties of UDCA may
contribute to its already known beneficial effects on gallbladder bile composition and symptoms in
patients with cholesterol gallstone disease (30-32).
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Table 1: Effect of UDCA treatment on the biliary composition of individual bile acids, biliary lipids,
CSI, mucin (mean ± SEM) and crystal observation time (COT, median and range) in gallbladder bile
of patients with cholesterol stones.
Placebo
(n = 12)
UDCA
(n = 10)
Cholic acid % 37.8 ± 2.7a 20.8 ± 3.1
Deoxycholic acid % 14.0 ± 0.6 8.8 ± 0.3
Chenodeoxycholic acid % 43.2 ± 1.1a 24.9 ± 0.6
Ursodeoxycholic acid % 4.0 ± 0.1a 44.9 ± 2.5
Phospholipids (mmol/l) 35.4 ± 3.6 38.9 ± 2.4
Total bile acids (mmol/l) 87.5 ± 3.8 95.6 ± 6.8
Cholesterol (mmol/l) 18.8 ± 1.1b 10.1 ± 0.5
CSI 1.42 ± 0.16b 0.86 ± 0.08
Mucin (mg/ml) 1.3 ± 0.5b 0.7 ± 0.3
COT (days) 2 (1 ≥ 21)a 21 (3 ≥ 21)
Page 18
16
0
0,02
0,04
0,06
0,08
0,1M
alon
dial
dehy
de (µ
mol
/l) /
Tota
l Bile
Aci
ds (m
mol
/l) R
atio
Placebo UDCA
p < 0.001
Figure 1. Effect of UDCA therapy on biliary lipid peroxidation. Lipid peroxidation in bile
was determined by measuring malondialdehyde (MDA) using the thiobarbituric acid (TBA)
method. To normalize for different concentration of gallbladder bile samples MDA
concentrations are given as the ratio of MDA (µmol/l) / total bile acids (mmol/l). MDA /
biliary total bile acid ratios were significantly lower in the UDCA treated group as compared
to the placebo-control group (p < 0.001).
Page 19
17
0
1
2
3M
ucin
Sec
retio
n/C
ontr
ol
Placebo UDCA
p < 0.001
Figure 2. Effect of gallbladder bile on mucin secretion of dog gallbladder epithelial cells. The
average mucin-secretion under control conditions (medium alone) was 1.1 x 103
counts/minute/well. Mucin secretagogue activity of bile samples from the placebo group was
markedly higher than that observed with the bile samples of the ursodeoxycholic acid-treated
group (2.2 ± 0.3 vs. 0.9 ± 0.2 times control; p<0.001 ).
