Gut1992;33:
1113-1117
Inhibition
of
human
gall
bladder
mucus
synthesis
in
patients
undergoing
cholecystectomy
M
Rhodes,
A
Allen,
R
H
Dowling,
G
Murphy,
T
W
J
Lennard
Abstract
Hypersection
of
gall
bladder
mucus
is
associated
with
gall
stone
formation
in
animal
models.'
Aspirin
inhibits
both
mucus
synthesis
and
secretion,
prevents
gail
stone
formation
in
animals2
and
reduces
gall
stone
recurrence
in
man
after
dissolution
therapy.3
Mucus
bio-
synthesis
in
human
gall
bladder
mucosal
explants
is
inhibited
by
aspirin
in
vitro.4
We
have
studied
the
effects
of
aspirin
in
vivo.
Fifty
five
patients
with
functioning
gail
bladder
and
stones
have
been
randomised,
27
to
group
1
(aspirin
EC
300
mg
once
daily
for
seven
days
before
cholecystectomy)
and
28
to
group
2
(controls).
Gall
bladder
bile
composition
was
analysed
and
mucus
synthesis
rates
measured
using
3H-glucosamine
incorporation
into
mucosal
explants
cultured
for
24
hours.4
Patient
age,
sex,
and
gall
bladder
histology
were
similar
in
both
groups.
There
were
no
dif-
ferences
in
stone
composition,
gall
bladder
bile
calcium
concentration,
cholesterol
saturation
and
cholesterol
nucleation
time.
The
mean
3H-glucosamine
incorporation
in
aspirin
treated
patients
was
1347
fmol/g
wet
weight
as
compared
with
2008
fmol/g
wet
weight
in
controls
(95%
confidence
interval
222-1100,
p<0
005,
unpaired
t
test).
This
reduction
in
biosynthesis
was
associated
with
gall
bladder
bile
mucus
concentrations
of
7.6
mg/ml
in
patients
and
7.1
mg/ml
in
controls
(ns).
Treatment
with
aspirin
led
to
a
significant
reduction
in
mucus
biosynthesis
by
the
gall
bladder
mucosa.
This
action
is
consistent
with
a
role
for
aspirin
in
the
prevention
of
gall
stones.
(Gut
1992;
33:
1113-1117)
Departments
of
Surgery
and
Physiological
Sciences,
New
Medical
School,
Framlington
Place,
Newcastle
upon
Tyne
M
Rhodes
A
Allen
T
W
J
Lennard
Gastroenterology
Unit,
Guy's
Campus,
Division
of
Medicine,
UMDS
of
Guy's
and
St
Thomas'
Hospitals,
London
R
H
Dowling
G
Murphy
Correspondence
to:
Mr
M
Rhodes,
FRCS,
Department
of
General
Surgery,
Frenchay
Hospital,
Frenchay,
Bristol.
Accepted
for
publication
30
December
1991
The
advent
of
laparoscopic
cholecystectomy
is
making
a
dramatic
impact
on
the
management
of
symptomatic
patients
with
gall
bladder
stones.
Despite
this,
many
patients
are
still
being
treated
with
alternative
approaches
to
cholecystectomy
(both
open
abdominal
and
laparoscopic)
in
which
the
stones
are
removed
or
dissolved,
but
the
gall
bladder
is
left
in
situ.
These
include
oral
bile
acid
treatment,5-'
contact
gall
stone
dissolution
with
solvents
such
as
methylterbutylether,8
extra-
corporeal
shock
wave
lithotripsy
(ESWL)
+/-
adjuvant
oral
bile
acids9"'
and
percutaneous
cholecystolithotomy.'2
'3
A
major
disadvantage
of
all
these
approaches
is
that
approximately
50%
of
the
patients
develop
recurrent
stones.">'
As
yet,
there
is
no
reliable
way
of
preventing
this
recurrence
but
studies
of
the
pathogenesis
of
recurrent
stones
are
important
as
they
may
provide
clues
about
primary
gall
stone
formation.
At
least
three
factors
contribute
to
the
development
of
both
primary
and
recurrent
cholesterol
stones:
(i)
supersaturated
bile
in
which
the
amount
of
cholesterol
present
exceeds
the
limits
of
thermodynamic
equilibrium,'920
(ii)
stasis
associated
with
gall
bladder
motor
dys-
function2'
22
and
(iii)
abnormal
nucleation
of
cholesterol
crystals
from
the
supersaturated
bile
because
of
an
excess
of
promoters
or
a
deficiency
of
inhibitors
of
crystallisation,
or
both.23
Several
lines
of
evidence
suggest
that
excess
mucus
glycoprotein
synthesis
by
the
gall
bladder
mucosa,
and
secretion
into
the
gall
bladder
lumen
may
play
an
important
role
in
the
nucleation
and
trapping
or
immobilising
of
cholesterol
crystals.2425
First,
in
prairie
dogs
fed
a
lithogenic
diet,
increased
mucus
glycoprotein
synthesis
and
secretion
by
the
gall
bladder
mucosa
antedate
the
appearance
of
crystals
and
stones.'
Aspirin
treatment,
however,
prevents
this
excess
mucus
glycoprotein
production
and
the
development
of
gall
stones
in
this
animal
model.2
Second,
during
weight
reduction,
obese
patients
are
at
risk
of
developing
microcrystals,
microstones
and
gall
stones
but
Broomfield
and
colleagues26
showed
that
aspirin
reduced
this
risk.
Third,
in
a
retrospective
survey
of
patients
in
the
British-Belgian
post
dissolution
trial,
Hood
et
all
found
that
regular
non-steroidal
anti
inflammatory
drug
(NSAID)
ingestion
prevented
gall
stone
recurrence.
The
aim
of
the
present
investigation,
therefore,
was
to
extend
these
preliminary
observations
by
studying,
prospectively,
the
effect
of
preopera-
tive
oral
enteric
coated
aspirin
treatment
on
the
composition
of
gall
bladder
bile
and
on
the
rate
of
mucus
glycoprotein
synthesis
by
explanted
gall
bladder
mucosa
from
gall
stone
patients
scheduled
for
elective
open
cholecystectomy.
Methods
PATIENTS
Patients
awaiting
elective
open
cholecystectomy
for
gall
stones
were
recruited
from
three
hospitals:
the
Royal
Victoria
Infirmary
and
the
Freeman
Hospital
in
Newcastle
upon
Tyne
and
Ashington
Hospital
in
Northumberland.
Patients
with
a
non-functioning
gall
bladder,
previous
peptic
ulceration,
or
hypersensitivity
to
aspirin
were
excluded
from
the
trial.
In
addition,
patients
already
taking
long
term
non-steroidal
antiinflammatory
agents
were
excluded.
Seventy
three
patients
were
randomised
into
two
groups,
37
to
group
1
(aspirin
treated)
and
36
to
group
2
(untreated
controls).
Patients
in
group
1
were
given
300
mg
of
enteric
coated
aspirin
(aspirin
EC)
once
daily
for
seven
days
before
1113
Rhodes,
Allen,
Dowling,
Murphy,
Lennard
cholecystectomy
and
a
further
single
600
mg
dose
two
hours
before
surgery.
Controls
received
no
NSAIDs
during
the
week
before
surgery.
The
choice
of
aspirin
dose
was
based
on
the
known
safety
and
efficacy
of
300
mg
aspirin
EC/
day
while
the
decision
to
treat
the
patients
for
seven
days
before
cholecystectomy
was
made
to
improve
compliance
and
facilitate
administration
of
the
trial.
ETHICAL
CONSIDERATIONS
Informed
consent
was
obtained
from
each
patient
and
ethical
approval
for
the
study
was
granted
by
both
the
Newcastle
and
Northumberland
Ethical
Committees.
LABORATORY
METHODS
MUCUS
GLYCOPROTEIN
BIOSYNTHESIS
IN
MUCOSAL
EXPLANTS
Mucosal
explants
of
freshly
excised
gall
bladder
were
grown
for
24
hours
in
tissue
culture.27
Glucosamine
hydrochloride,
D-[6-3H(N)]-,
with
a
specific
activity
of
1110
0
GBq/mmol,
was
used
to
label
macromolecules.
3H-glucosamine
was
used
at
a
concentration
of
74
KBq/ml
in
the
tissue
culture
medium.
Total
3H-glucosamine
incorporation
into
mucin
was
measured
after
removal
of
protein
and
low
molecular
weight
radioactive
material
by
papain
digestion
(72
hours)
and
exhaustive
dialysis
against
distilled
water
(144
hours).
Papain
digestion
is
an
effec-
tive
method
for
isolation
of
mucus
glycoprotein.
After
digestion,
dialysis
removes
protein
and
non-glycosylated
regions
of
protein
core,
leaving
behind
non-dialysable,
mucus
glycoprotein.28
Glucosamine
incorporation
into
the
glyco-
protein
component
of
mucin
was
confirmed
by
fractionation
on
a
caesium
chloride
density
gradient29
and
by
gel
filtration
on
sepharose
4B.
All
radioactive
mucin
fractionated
in
a
peak
coincident
with
purified,
papain
digested
human
gall
bladder
mucin.
Gall
bladder
mucosa,
killed
in
liquid
nitrogen
was
used
as
a
control
to
check
for
non-specific
interactions
between
the
tissue
and
3H-glucosamine.
MUCUS
GLYCOPROTEIN
CONTENT
OF
GALL
BLADDER
BILE
Quantitation
of
mucus
glycoprotein
was
undertaken
using
a
modified
'Slotblot'
tech-
nique.30
After
blotting
samples
of
bile
onto
nitrocellulose
membranes,
mucus
glycoprotein
was
visualised
using
the
periodic
acid/Schiff
reaction.3'
Colour
yeild
was
then
measured
using
a
scanning
densitometer
(Shimadzu,
Japan).
Results
were
compared
with
those
obtained
by
measuring
glycoprotein
after
purification
of
the
mucin
by
the
traditional
method
of
fractionation
on
a
caesium
chloride
density
gradient
and
showed
close
correlation.
CHOLESTEROL
The
cholesterol
concentration
in
bile
was
measured
using
the
enzymatic
method
first
described
by
Roda32
and
modified
by
Bolton.33
Briefly,
this
involved
diluting
samples
of
gall
bladder
bile
obtained
by
needle
puncture
at
laparotomy
1
in
10
with
isopropanol
and
incubating
the
diluted
sample
with
three
enzymes.
Sequential
treatment
of
bile
with
catalase,
cholesterol
esterase
and
cholesterol
oxidase
(Sigma,
UK)
produces
cholestenone
and
hydrogen
peroxidase.
The
hydrogen
peroxide
is
then
used
as
an
oxidising
agent
in
a
colorimetric
assay.
The
cholesterol
content
of
gall
stones
was
measured
by
crushing
the
dried
stones
with
a
pestle
and
mortar,
extracting
the
cholesterol
in
isopropanol
and
quantifying
it
as
described
above.
PHOSPHOLIPID
Phospholipid
was
assayed
using
an
enzymatic
method
first
described
by
Takayama34
and
modified
by
Qureshi
et
al.35
Dual
enzymatic
digestion
of
bile
with
phospholipase
and
choline
oxidase
(Wako
Chemical
Company,
Eastleigh,
UK)
leads
to
the
release
of
hydrogen
peroxide
which
is
used
as
the
oxidant
in
a
colorimetric
assay.
BILE
ACIDS
The
total
bile
acid
content
of
gall
bladder
bile
was
measured
using
an
enzymatic
method
in
which
3-a-hydroxy-steroid
dehydrogenase
(Sigma
UK)
is
used
to
oxidise
bile
acids
allowing
reduction
of
nicotinamide
adenine
dinucleotide
to
nicotinamide
adenine
dinucleotide
hydride
(personal
communication).
CHOLESTEROL
SATURATION
The
cholesterol
saturation
was
determined
by
calculating
the
actual
percentage
of
cholesterol
in
bile
and
dividing
it
by
the
theoretical
maximal
cholesterol
solubility.36
CALCIUM
The
total
calcium
of
gall
bladder
bile
was
measured
using
atomic
absorption
spectro-
photometry.37
CHOLESTEROL
NUCLEATION
TIME
Nucleation
time
was
measured
in
the
so
called
isotropic
'micellar'
phase
obtained
by
centri-
fugation
of
bile
for
one
hour
at
200
000
g.
After
centrifugation
samples
were
examined
for
the
presence
of
cholesterol
crystals
then
incubated
at
TABLE
I
Ten
patients
in
the
aspirin
treated
group
were
withdrawn
from
the
study
as
compared
with
eight
in
the
control
group.
Reasons
for
withdrawal
are
indicted
together
with
the
numbers
of
patients
in
each
category
Patients
withdrawn
from
trial
Group
-
I
Group
-
2
Aspirin
Control
7
Women-
3
mucocele
3
Women-
2
fibrosis
2
fibrosis
1
oedema
1
adenocarcinoma
5
Men-
2
mucocele
1
aspirin
withdrawn
1
no
bile
3
Men
-
3
aspirin
withdrawn
1
oedema
1
infected
1114
Inhibition
of
human
gall
bladder
mucus
synthesis
in
patients
undergoing
cholecystectomy
TABLE
II
Age,
sex,
symptom
duration
and
histology
in
the
two
groups.
The
severity
of
cholecystitis
was
graded
independently
by
a
pathologist
Patient
age,
sex,
symptoms
and
histology
Group
-
I
Aspinin
Group
-
2
Control
n
27
28
Age
(yr)
54.4
(range
21-84)
52.0
(range
23-74)
F:M
21:6
26:2
Symptoms
(yr)
2-7
(SD
2.7)
2.9
(SD
2.5)
Histology
normal
-
3
normal
-
2
mild
chronic
mild
chronic
cholecystitis-
14
cholecystitis
-
13
moderate
chronic
moderate
chronic
cholecystitis-
7
cholecystitis-
5
37°C
under
nitrogen.
Further
aliquots
of
this
isotropic
phase
were
removed
daily
and
examined
with
polarizing
light
microscopy
(Leitz,
Wetzlar)
to
look
for
cholesterol
crystals.38
STATISTICAL
ANALYSIS
Data
were
compared
visually
using
histograms
and
statistically
using
the
Francia
Shapiro
W'
test
to
test
for
normality.
Where
data
were
found
to
be
normally
distributed,
the
unpaired
t
test
was
used
to
compare
the
two
patient
groups.
For
those
data
which
were
not
normally
distributed,
log
transformations
was
utilised
to
obtain
a
normal
distribution
and
the
unpaired
t
test
was
then
used
to
compare
the
two
groups.
Results
Of
73
patients
randomised,
18
were
withdrawn
from
the
study
(Table
I).
Patients
who
had
fibrotic
oedematous
or
acutely
infected
gall
bladders
were
withdrawn
from
the
study
because
it
was
not
possible
to
conduct
explant
culture.
Of
the
55
patients
who
were
suitable
for
further
study,
27
were
randomised
to
the
aspirin
treated
group
and
28
to
the
control
group.
The
male
to
female
ratio,
mean
age
and
mean
duration
of
symptoms
were
similar
in
the
two
groups
(Table
II).
Gall
bladder
histology
was
also
comparable
in
both
groups.
MUCUS
GYLCOPROTEIN
SYNTHESIS
BY
GALL
BLADDER
MUCOSA
Gall
bladder
mucus
biosynthesis,
as
measured
by
3H-glucosamine
incorporation,
was
significantly
less
in
aspirin
treated
patients
than
in
the
untreated
controls.
Gall
bladder
explants
from
controls
incorporated
a
mean
of
2008
fmol/g
wet
weight
(SD
892)
of
3H-glucosamine
as
compared
with
1347
fmol/g
wet
weight
(SD
722)
in
aspirin
treated
patients
(p<0005,
95%
confidence
interval
for
the
difference
222
to
1100,
unpaired
t
test),
(Figure).
Incorporation
of
3H-glucosamine
TABLE
III
Gall
bladder
bile
composition
and
gall
stone
composition
in
the
two
groups
Gall
bladder
bile
and
gall
stone
composition
Aspirin
Control
n=27 n=28
t
test
Mucus
glycoprotein
(mg/ml)
76
(SD
77)
7-1
(SD
5.4)
ns
Nucleation
time
(days)
6
(SD
2.4)
5-2
(SD
1-9)
ns
Cholesterol
(mmol/l)
15-3
(SD
7.9)
13
5
(SD
8)
ns
Phospholipids
(mmol/l)
36
1
(SD
19.1)
33.2
(SD
17
1)
ns
Bile
acids
(mmol/l)
103
(SD
50-1)
102
(SD
49
2)
ns
Saturation
(%
cholesterol)
145
(SD
39)
160
(SD
82)
ns
Total
calcium
(mmol)
6-9
(SD
3.3)
5-4
(SD
2.9)
ns
Stone
composition
(%
cholesterol)
66
(SD
34)
75
(30)
ns
into
mucosal
explants
linear
over
24
hours.
Controls
containing
liquid
nitrogen
killed tissue
incorporated
a
maximum
of
3
fmol/g
wet
weight
of
3H-glucosamine.
GALL
BLADDER
BILE
COMPOSITION
Both
gall
bladder
bile
mucus
concentration
and
cholesterol
nucleation
time
were
similar
in
the
two
groups
(Table
III).
Cholesterol
saturation,
calcium
concentration
and
stone
composition
were
also
similar
in
the
two
groups
(Table
III).
SIDE
EFFECTS
AND
COMPLICATIONS
There
was no
postoperative
morbidity
in
the
control
group.
Two
patients
in
the
aspirin
treated
group
had
complications.
One
had
evidence
of
a
prolonged
clotting
time
postoperatively
and
her
haemoglobin
fell
from
12.5
g/dl
to
10
g/dl
on
the
first
postoperative
day.
No
transfusion
was
needed
and
the
patient
showed
no
clinical
signs
of
blood
loss.
The
second
had
a
small
melaena
after
two
days
of
aspirin
treatment
and
he
was
therefore
withdrawn
from
the
study.
Discussion
This
study
shows
that
low
dose
aspirin
significantly
inhibits
mucus
synthesis
in
the
human
gall
bladder
in
vivo.
Our
earlier
work4
has
shown
that
aspirin
will
inhibit
human
gall
bladder
mucus
glycoprotein
biosynthesis
in
vitro
and
this
present
study
shows
that
this
inhibition
occurs
in
vivo.
This
is
not,
however,
accompanied
by
significant
changes
in
soluble
mucin
content
in
gall
bladder
bile
or
gall
bladder
bile
composition.
This
may
be
as
a
result
of
short
term
treatment
(seven
days),
or
because
of
the
low
dose
of
aspirin
chosen
for
this
study.
What
is
clear
is
that
the
mucus
biosynthetic
machinery
of
the
gall
bladder
mucosa
has
been
reduced
by
short
term
aspirin
treatment.
Over
a
longer
period
of
time
this
would
be
expected
to
establish
a
decreased
level
of
mucus
production
by
the
gall
bladder
and
thus
diminish
its
potential
as
a
nucleating
agent.
Mucus
has
been
shown
to
be
a
nucleating
agent
in
vitro39`
and
in
vivo
increased
mucus
glycoprotein
synthesis
and
secretion
antedates
gall
stone
formation
in
the
rabbit4'
and
the
prairie
dog.'
Aspirin
inhibits
mucus
synthesis
and
secretion
and
prevents
gall
stones
in
the
prairie
dog.7
Our
results
here
show
that
in
man
aspirin
has
similar
actions
on
gall
bladder
mucus
glycoprotein
biosynthesis.
Stone
reccurrence
is
a
major
problem
with
all
forms
of
gall
stone
treatment
which
leave
the
gall
bladder
in
situ.
Ruppin
et
al'6
followed
54
patients
successfully
treated
with
oral
bile
acids
for
a
mean
of
23
months
and
found
that
25
(46%)
suffered
recurrent
stones.
O'Donnell
et
al'7
found
a
similar
recurrence
rate
in their
study
of
40
patients,
50%
of
whom
had
a
recurrence
within
five
years.
In
a
longer
term
study,
Villanova
et
al
'4
found
a
61%
gall
stone
recurrence
rate
at
11
years
in
86
patients.
Gall
stone
recurrence
has
also
been
studied
in
patients
after
percutaneous
cholecystolithotomy
and
shock
wave
lithotripsy
although
the
length
of
follow
up
is
much
shorter
than
in
post
dissolution
studies.
Eight
months
11
15
1116
Rhodes,
Allen,
Dowling,
Murphy,
Lennard
4000
(D
0
E
3000
0~~~0
0
L._
o
S*
°
2000
**
°
1000
-
0
-
*
Aspirin
Control
(n
=
27)
(n
=
28)
Patients
Figure:
Mucus
glycoprotein
biosynthesis
as
measured
by
3H-glucosamine
incorporation
over
24
hours
and
treatment
with
papain
for
72
hours
followed
by
exhaustive
dialysis
(144
hours).
Each
patient
is
represented
by
a
dot
and
the
mean
of
each
group
by
a
horizontal
line.
Mean
incorporation
in
aspirin
treated
patients
with
1347
fmollg
wet
weight
(SD
722)
as
compared
with
2008
(SD
892)
in
controls;
p<0
005,
unpaired
t
test,
95%
confidence
intervalfor
the
difference
222-1
100.
follow
up
in
53
patients
who
underwent
success-
ful
percutaneous
removal
of
their
gall
stones
revealed
recurrent
stones
in
five'8
whilst
the
same
duration of
follow
up
in
49
patients
who
under-
went
shock
wave
lithotripsy
also
showed
five
to
have
recurrent
stones.
II
It
has
been
suggested
that
recurrent
stones
are
amenable
to
repeat
dissolution
therapy42
but
experience
with
this
approach
is
limited.
One
possible
way
of
preventing
gall
stone
recurrence
is
the
use
of
long
term,
low
dose
aspirin.
There
is
extensive
evidence
that
aspirin
prevents
gall
stone
formation
in
the
prairie
dog
through
its
inhibition
of
mucus
synthesis
and
secretion.2
Broomfield
et
al26
studied
the
effects
of
aspirin
(1200
mg/day)
and
urosodeoxycholic
acid
(1300
mg/day)
on
duodenal
bile
composition
and
on
microcrystal,
microstone
and
gall
stone
for-
mation
in
53
patients
dieting
to
lose
weight.
Patients
taking
aspirin
or
ursodeoxycholic
acid
had
significantly
lower
bile
glycoprotein
concentrations
when
compared
with
the
placebo
group
and
both
had
a
reduced
incidence
of
gall
stone,
microstone
and
cholesterol
crystal
formation.
Hood
et
al3
found
that
gall
stone
recurrence
after
dissolution
was
significantly
less
in
patients
taking
long
term
non-steroidal
anti
inflammatory
agents,
a
result
consistent
with
the
work
of
Broomfield.
Our
study
has
shown
inhibition
of
gall
bladder
mucin
biosynthesis
using
aspirin
at
a
dose
of
300
mg/day.
At
this
dose
we
have
failed
to
show
significant
prolongation
of
cholesterol
nucleation
time.
Longterm
treatment
with
aspirin
at
a
dose
of
300
mg/day
has
been
shown
to
be
safe
in
asymptomatic
subjects
who
took
part
in
The
Physicians
Health
Study43
and
the
UK
TIA
study.44
If
future
studies
show
prolongation
of
cholesterol
nucleation
time
as
a
consequence
of
longterm
treatment
with
aspirin
at
a
dose
between
300
mg/day
and
1200
mg/day,
there
might
be
a
role
for
the
drug
in
the
prevention
of
gall
stones
among
high
risk
groups.
This
work
was
funded
by
the
Newcastle
Health
Authority
Scientific
and
Research
Committee
and
the
University
of
Newcastle
upon
Tyne
William
Edmund
Harker
Bequest.
Our
thanks
to
several
surgeons
in
the
Northern
Region
who
provided
patients
for
the
study
and
in
particular
Mr
A
Gunn
who
helped
with
the
administration
of
the
trial
in
Northumberland.
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SP,
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JT,
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MC.
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the
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Clin
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1981;
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SP,
Carey
MC,
LaMont
JT.
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1981;
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K,
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1990;
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1986;
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10
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M,
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1988;
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N,
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A,
Jazrawi
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Heaton
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655-8.
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Donald
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MC.
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the
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20
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RT,
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M,
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M,
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K.
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D,
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in
the
pathogenesis
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Gastroenterology
1979;
77:
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22
Forgacs
IC,
Maisey
MN,
Murphy
GM,
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RH.
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of
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and
ursodeoxycholic
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on
gall-
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Gastroenterology
1984;
87:
299-307.
23
Doty
JE,
Pitt
HA,
Kuckenbecker
SL,
et
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the
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Hale
W,
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ML,
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B.
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PH,
Chopra
R,
Sheinbaum
RC,
et
al.
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the
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Engly
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