Oxygen free radicals in acute pancreatits of the rat

Abstract

This study aimed to assess the role of oxygen free radicals in acute pancreatitis. Acute pancreatitis was induced in rats by infusion of the CCK-analogue cerulein (5 micrograms/kg per hour) for 30 minutes, 3.5 hours, and 12 hours. After the infusion, serum enzymes and conjugated tissue dienes and malondialdehyde were measured and tissue samples were subjected to electron and light microscopy. Electron microscopy after 30 minutes showed moderate intracellular alterations. After 3.5 hours of cerulein infusion interstitial oedema and intravascular margination of granulocytes in the pancreatic gland were seen. After 12 hours histological evaluation showed pronounced zymogen degranulation, extensive tissue necrosis, and migration of granulocytes into the tissue. Amylase and lipase activities increased 15 and 35-fold respectively during this time. After 30 minutes of cerulein infusion conjugated dienes and malondialdehyde increased, they reached their peak after 3.5 hours and decreased to normal values after 12 hours. Treatment with superoxide dismutase (100,000 U/kg/hour) and catalase (400,000 U/kg/hour) either before or after the start of the cerulein infusion prevented lipid peroxidation and reduced zymogen degranulation and tissue necrosis. Tissue oedema and inflammatory response, however, were not affected in any of the treated rats. Oxygen free radicals are instrumental in the development of acute pancreatitis. Even after its onset, scavenger treatment reduced the tissue damage normally observed.

Full text

Gut,
1990,
31,
1138-1143
LIVER,
BILIARY,
AND
PANCREAS
Oxygen
free
radicals
in
acute
pancreatitis
of
the
rat
M
H
Schoenberg,
M
Buchler,
M
Gaspar,
A
Stinner,
M
Younes,
I
Melzner,
B
Bultmann,
H
G
Beger
Abstract
This
study
aimed
to
assess
the
role
of
oxygen
free
radicals
in
acute
pancreatitis.
Acute
pan-
creatitis
was
induced
in
rats
by
infusion
of
the
CCK-analogue
cerulein
(5
tg/kg
per
hour)
for
30
minutes,
3*5
hours,
and
12
hours.
After
the
infusion,
serum
enzymes
and
conjugated
tissue
dienes
and
malondialdehyde
were
measured
and
tissue
samples
were
subjected
to
electron
and
light
microscopy.
Electron
microscopy
after
30
minutes
showed
moderate
intracellular
alterations.
After
3*5
hours
of
cerulein
infusion
interstitial
oedema
and
intra-
vascular
margination
of
granulocytes
in
the
pancreatic
gland
were
seen.
After
12
hours
histological
evaluation
showed
pronounced
zymogen
degranulation,
extensive
tissue
necrosis,
and
migration
of
granulocytes
into
the
tissue.
Amylase
and
lipase
activities
in-
creased
15
and
35-fold
respectively
during
this
time.
After
30
minutes
of
cerulein
infusion
conjugated
dienes
and
malondialdehyde
in-
creased,
they
reached
their
peak
after
3
5
hours
and
decreased
to
normal
values
after
12
hours.
Treatment
with
superoxide
dismutase
(100
000
U/kg/hour)
and
catalase
(400
000
U/kg/
hour)
either
before
or
after
the
start
of
the
cerulein
infusion
prevented
lipid
peroxidation
and
reduced
zymogen
degranulation
and
tissue
necrosis.
Tissue
oedema
and
inflammatory
response,
however,
were
not
affected
in
any
of
the
treated
rats.
Oxygen
free
radicals
are
instrumental
in
the
development
of
acute
pan-
creatitis.
Even
after
its
onset,
scavenger
treat-
ment
reduced
the
tissue
damage
normally
observed.
Departments
of
General
Surgery
and
Pathology,
University
of
Ulm,
West
Germany
M
H
Schoenberg
M
Buchler
M
Gaspar
A
Stinner
I
Melzner
B
Bultmann
H
G
Beger
Department
of
Toxicology,
University
of
Liibeck,
West
Germany
M
Younes
Correspondence
to:
Dr
H
G
Beger,
Department
of
General
Surgery,
University
of
Ulm,
Steinhovelstr
9,
7900
Ulm,
West
Germany
Accepted
for
publication
20
November
1989
Acute
pancreatitis
in
humans
leads
to
severe
acinar
cell
damage,
extensive
interstitial,
partly
haemorrhagic
oedema,
and
migration
of
neutro-
philic
granulocytes
into
the
damaged
pancreatic
gland.'
2
It
has
been
shown
that
oxygen
free
radicals
play
an
important
role
in
the
development
of
inflammation
in
other
diseases.3
Because
of
this
there
have
been
various
studies
on
the
role
of
oxygen
free
radicals
in
the
pathogenesis
of
acute
pancreatitis.
Sanfey
et
al
caused
acute
pan-
creatitis
in
an
ex
vivo
perfused
canine
pancreas
by
oleic
acid
infusion,
partial
duct
obstruction,
and
a
two
hour
period
of
ischaemia.
These
stimuli
simulated
an
acute
pancreatitis
in
man
due
to
ischaemia/reperfusion
injury,
transient
outflow
obstruction,
and
alcohol-induced
hyper-
lipaemia.
The
authors
showed
that
enhanced
generation
of
oxygen
radicals
added
consider-
ably
to
the
damages
observed
in
all
forms
of
pancreatitis.4'
Guice
et
al
showed
in
an
in
vivo
cerulein-induced
pancreatitis
model
that
pretreatment
with
superoxide
dismutase
and
catalase
reduced
pancreatic
damage
normally
observed.6
These
results
were
recently
con-
firmed
by
Wisner
et
al.
In
the
same
experimental
model
these
authors
were
able
to
show
that
pretreatment
with
polyethylene
glycol-linked
superoxide
dismutase
minimised
tissue
oedema,
histological
damage,
and
the
increase
in
a-
amylase
in
the
serum.7
We
used
the
same
experimental
model
of
cerulein-induced
pancreatitis
to
monitor
the
generation
of
oxygen
radicals
and
its
relation
to
the
development
of
acute
pancreatitis.
To
assess
the
onset
of
free
radical
generation,
we
measured
conjugated
dienes
and
malondialdehyde,
which
are
both
indirect
parameters
for
the
formation
of
lipid
hydroperoxides,
at
various
times
during
the
development
of
acute
pancreatitis.
We
then
compared
them
with
the
extent
of
tissue
damage.
In
addition,
we
examined
the
effects
of
treatment
with
the
oxygen
radical
scavengers
superoxide
dismutase
and
catalase,
given
before
and
after
the
onset
of
acute
pancreatitis.
Material
and
methods
EXPERIMENTAL
PROCEDURE
Fifty
four
male
one
strain
WKY
rats
(weight
ranging
from
300-350
g)
were
given
inhalation
anaesthesia
with
halothane,
and
a
heparin
rinsed
polyethylene
catheter
was
placed
in
the
jugular
vein.
The
animals
were
allowed
to
recover
for
12
to
14
hours
before
being
given
an
infusion
of
cerulein.
The
catheters
were
then
flushed
with
0
3
ml
normal
saline
solution
in
order
to
confirm
patency
before
the
start
of
the
infusion.
There-
after,
the
cerulein
(synthetic
cerulein
provided
by
Deutsche
Farmitalia,
Freiburg,
West
Germany)
was
dissolved
in
isotonic
saline
solution
and
infused
continuously
by
means
of
a
Havard
pump
via
the
implanted
catheter.
A
standard
dose
of
cerulein,
5
[tg/kg
per
hour,
was
given
at
an
infusion
rate
of
0
6
ml/hour
for
periods
ranging
from
30
minutes
to
12
hours.
During
infusion
the
rats
had
free
access
to
pelleted
food
and
water.
After
the
infusion
blood
was
sampled
for
plasma
amylase
and
lipase
measurements
and
pancreatic
tissue
samples
were
excised
for
light
and
electron
microscopic
1138

Oxygen
free
radicals
in
acute
pancreatitis
of
the
rat
examination
and
for
determination
of
conjugated
tissue
dienes
and
malondialdehyde
concentra-
tions.
CONTROL
GROUPS
Untreated
rats
were
divided
into
four
groups
of
10
animals
each.
Control
rats
were
prepared
in
the
same
way
but
received
an
isotonic
saline
solution
for
12
hours
instead
of
cerulein.
The
other
three
groups
were
infused
continuously
with
cerulein
(dosage
see
above)
for
30
minutes,
3
5
hours,
and
12
hours,
respectively.
TREATED
GROUPS
Fourteen
rats
were
divided
into
two
groups
and
received
a
continuous
superoxide
dismutase
in-
fusion
(bovine
superoxide
dismutase
by
Grunen-
thal
Co,
Aachen,
West
Germany)
at
a
dosage
of
100
000
U/kg
per
hour.
In
addition,
the
animals
received
during
the
first
three
hours
a
bolus
injection
of
400
000
U
catalase
(bovine
catalase
provided
by
Boehringer
Co,
Mannheim,
West
Germany),
starting
with
the
onset
of
the
super-
oxide
dismutase
infusion.
The
treatment
started
one
hour
before
the
cerulein
infusion
(dosage:
5
,tg/kg
per
hour)
and
paralleled
this
for
3
-
5
hours
(n=7)
or
12
hours
(n=7).
In
another
set
of
experiments,
seven
rats
were
treated
with
superoxide
dismutase
and
catalase
using
the
same
dosage,
preparation,
and
method,
but
the
treatment
started
one
hour
after
the
beginning
of
the
cerulein
infusion.
After
one
hour
the
cerulein
infusion
was
paral-
leled
by
the
continuous
superoxide
dismutase
treatment
for
11
hours.
In
addition,
catalase
was
given
as
an
intravenous
bolus
for
the
first
three
hours
after
the
beginning
of
the
superoxide
dismutase
infusion.
The
cerulein
and
superoxide
dismutase/catalase
infusions
were
given
through
the
same
jugular
catheter,
since
it
had
been
shown
previously,
and
reaffirmed
before
the
experiments,
that
neither
superoxide
dismutase
nor
catalase
affected
the
toxic
properties
of
cerulein
in
vitro.0
MEASUREMENTS
Alpha
amylase
and
lipase
activities
in
the
plasma
were
determined
by
a
standard
clinical
method
for
automated
analysis
(amylase
PNP,
lipase
automated
analysis,
Boehringer,
Mannheim,
Germany).
The
tissue
concentrations
of
con-
jugated
dienes
and
malondialdehyde
were
measured
by
the
methods
of
Buege
and
Aust8
as
well
as
according
to
Freeman
and
Crapo,9
and
were
corrected
for
the
protein
content
of
the
tissue.
The
protein
concentration
of
the
pan-
creatic
tissue
was
determined
by
the
method
of
Lowry
et
al.
'°
LIGHT
MICROSCOPY
Tissue
samples
removed
from
the
caput,
corpus,
and
cauda
of
the
pancreas
were
fixed
in
a
5%
formalin-picrin
acid
mixture
for
24
hours,
were
paraffin-embedded,
and
then
stained
with
haematoxylin
and
eosin.
The
light
microscopical
slides
were
examined
and
graded
by
the
patholo-
gist,
who
was
unaware
of
the
previous
treatment
(blind
observer
design).
GRADING
AND
STAGING
SCALE
Oedema
was
graded
0-3:
grade
0=no
tissue
oedema;
grade
1
=interlobular
oedema;
grade
2=moderate
interlobular
and
intra-acinar
oedema;
and
grade
3=severe
interlobular
and
intra-acinar
oedema.
NEUTROPHILIC
INFLAMMATORY
RESPONSE
Neutrophilic
inflammatory
response
was
staged
1-3:
stage
1
=intravascular
margination
of
granulocytes
in
capillaries
and
postcapillary
venules;
stage
2=granulocytes
present
in
the
perivascular
tissue;
and
stage
3
=
diffuse
infiltra-
tion
of
the
entire
pancreatic
gland.
CELL
DAMAGE
The
extent
of
zymogen
degranulation
and
cell
necrosis
was
assessed
planimetrically
by
counting
the
degranulated
and
necrotic
cells
in
approximately
100
acinar
lobuli
per
sample
(at
least
four
histological
sections/sample).
The
fre-
quency
of
degranulation
and
cell
necrosis
in
the
lobuli
is
given
as
a
percentage.
ELECTRON
MICROSCOPY
Immediately
after
the
death
of
cerulein
infused
rats,
1
mm3
of
their
pancreatic
tissue
was
removed
and
incubated
in
3%
glutaraldehyde
in
phosphate
buffer
(pH=7
3).
After
10
hours
the
tissue
was
rinsed
extensively
in
buffer
and
post-
fixed
by
incubation
with
OS04
(1%
in
phosphate
buffer).
After
fixation,
the
samples
were
washed
in
Veronal
acetate
buffer
and
dehydrated
in
a
graded
series
of
alcohols.
Thereafter
the
samples
were
embedded
in
Epon
812.
Sections
were
cut
with
a
diamond
knife
on
an
LKB
ultramicro-
tome,
stained
with
5%
uranylacetate
and
lead
citrate,
and
were
examined
with
a
Philips
EM
10
A
electron
microscope.
Sixty
acinar
cells/tissue
sample
(one
tissue
sample/animal;
five
animals/cerulein-treated
group)
were
evaluated
for
the
content
and
distri-
bution
of
zymogen
granules,
intracytoplasmatic
vacuole
formation,
and
damage
to
the
organelles
and
membranes
within
the
acinar
cells.
The
results
are
given
as
the
mean
(SD)
percentage
of
cells
showing
abnormal
findings
in
each
experi-
mental
group.
STATISTICAL
METHODS
The
data
are
expressed
as
mean
(SD)
values.
The
data
were
evaluated
statistically
by
the
Institute
of
Biomathematics
and
Statistics,
University
of
Ulm.
Differences
in
conjugated
dienes,
malon-
dialdehyde,
a
amylase,
and
lipase
in
the
plasma
were
tested
pairwise
for
statistical
significance
according
to
the
Mann-Whitney
test
and
were
re-
evaluated
by
the
Student's
t
test.
In
addition
the
histological
data
were
evaluated
for
statistical
significance
using
the
Student's
t
test
for
inde-
pendent
means.
In
all
instances
p
values
of
<0
05
were
considered
to
be
significant.
1139

Schoenberg,
Buchler,
Gaspar,
Stinner,
Younes,
Melzner,
Bultmann,
Beger
Histological
grading,
assessment
of
cell
necrosis,
and
zymogen
degranulation
at
various
stages
of
acute
pancreatitis
Zymogen
Cell
necrosis
degranulation
(%)
(%)
(mean
Groups
No
Oedema
Neutrophils
(mean
(SD))
(SD))
Control
10
0
0
0
0
30
Minutes
after
cerulein
10
0
0
0
0
3
5
Hours
after
cerulein
10
1-2
1
20-8
(9
7)
12-3
(4
5)
(range:
10-30)
(range:
5-20)
12Hoursaftercerulein
10
3
3
84-2(7-4)
60-0(7
1)
(range:
75-95)
(range:
50-70)
SOD/CAT:
I
hour
before
7
33
39-3
(10-6)*
21-0
(6-2)*
(range:
30-60)
(range:
12-30)
1
hourafter
7
3
3
50-7(11-7)*
21-4(10-7)*
(range:
35-70)
(range:
10-40)
Effects
of
superoxide
dismutase/catalase
treatment.
*p<0-001
compared
with
the
corresponding
value
in
untreated
rats
receiving
12
hours'
cerulein
infusion.
Results
UNTREATED
RATS
Light
microscopy
of
the
pancreatic
glands
of
the
rats
which
had
received
a
30
minute
infusion
of
cerulein
showed
that
these
seemed
undamaged.
The
first
lesions
become
apparent
after
3
5
hours
and
affected
the
entire
gland.
They
were
characterised
by
a
moderate
interstitial
oedema,
intravascular
margination
of
granulocytes,
the
beginning
of
zymogen
degranulation,
and
patchy
acinar
cell
necrosis
(see
Table).
These
lesions
became
worse
after
12
hours
of
continuous
cerulein
infusion.
The
tissue
was
severely
oedematous
and
showed
extensive
zymogen
degranulation
and
cell
necrosis
as
well
as
a
pronounced
migration
and
accumulation
of
polymorphonuclear
leucocytes
into
the
tissue.
Even
after
30
minutes
of
cerulein
infusion
electron
microscopic
examination
showed
the
formation
of
small
(in
54
(1
1)%)
and
large
(27
(11)%)
intracytoplasmic
vacuoles
and
a
reduc-
tion
of
zymogen
granules
in
46
(27)%
of
all
acinar
cells.
Most
impressive
was
the
finding
of
swelling
of
the
mitochondria
in
60
(15)%
of
the
cells,
which
in
14
(9)%
led
to
the
disruption
of
the
cristae
and
the
matrix
(see
Figs
1
and
2).
After
3*5
hours
the
damage
had
progressed
further,
leading
to
large
vacuoles
in
57
(12)%
of
the
cells
that
indicated
autophagic
processes
in
the
exocrine
cell.
Moreover,
the
tight
junctions
in
55
(12)%
of
the
cells
began
to
loosen
as
an
early
sign
of
cell
membrane
damage.
After
12
hours'
infusion
the
vacuoles
were
seen
to
occupy
a
large
part
of
the
acinar
cells
in
91
(9)%,
the
oedema
within
the
connective
tissue
had
worsened,
and
polymorphonuclear
leucocytes,
as
well
as
a
few
mononuclear
cells,
had
accumulated
in
the
interstitial
space.
In
contrast
with
the
early
changes
seen
by
electron
microscopy
after
30
minutes,
plasma
values
of
both
a
amylase
and
lipase
remained
at
control
values.
After
3*5
hours
we
measured
a
significant
accretion
of
these
enzymes,
and
after
12
hours
their
serum
concentrations
had
increased
more
than
15
and
35
times
respect-
ively.
Interestingly,
the
interindividual
compari-
son
showed
no
positive
correlation
between
the
amount
of
cell
and
tissue
damage
and
the
serum
enzyme
activities
(see
Figs
3
and
4).
As
well
as
the
early
electron
microscopical
damage
of
the
pancreatic
gland
after
30
minutes
of
cerulein
infusion,
the
tissue
concentrations
of
both
conjugated
dienes
and
malondialdehyde
were
raised.
After
3
5
hours,
the
conjugated
dienes
increased
progressively
from
2-3
(0
2)
to
4
0
(0
5)
.tmol/mg
protein
and
malondialdehyde
from
15-7
(2
5)
to
48
(12)
nmol/mg
protein
(see
Figs
5
and
6).
After
12
hours,
at
which
time
the
histological
signs
of
acute
pancreatitis
were
seen,
both
conju-
gated
dienes
and
malondialdehyde
returned
to
their
control
values.
Figure
1:
Electron
microscopical
picture
after
30
minutes
of
cerulein
infusion
(original
magnification
xSOOO).
1140

Oxygen
free
radicals
in
acute
pancreatitis
of
the
rat
Figure
2:
Enlargement
of
the
electron
microscopical
picture
(see
Fig
1).
Arrows
show
a
swollen
mitochondria
with
disrupted
cristae
(original
magnification
x
11
000).
TREATED
RATS
Superoxide
dismutase
and
catalase
treatment
before
infusion
prevented
the
increase
in
conju-
gated
dienes
in
the
pancreatic
tissue
normally
observed
after
3-5
hours.
At
the
same
time
the
tissue
malondialdehyde
concentrations
were
only
slightly
raised.
They
did
not,
however,
differ
significantly
from
the
control
values.
After
12
hours
both
conjugated
dienes
and
malondial-
dehyde
concentrations
were
the
same
as
the
normal
range
in
the
untreated
rats
(see
Figs
5
and
6).
Histological
evaluation
of
the
pancreatic
tissue
after
3-5
hours
of
cerulein
infusion
in
untreated
and
pretreated
rats
showed
that
there
were
no
significant
differences
between
the
groups.
After
12
hours
of
cerulein
infusion,
however,
histo-
logical
examination
showed
that
superoxide
dismutase
and
catalase
treatment,
given
one
hour
before
cerulein,
reduced
zymogen
degranu-
lation
and
acinar
cell
necrosis.
Only
21
(6
2)%
of
the
acinar
cells
were
severely
damaged
or
necrotic
compared
with
60
(7)%
in
the
untreated
group
(p<00001).
At
the
same
time,
zymogen
degranulation
was
39-2
(10-6)%,
significantly
lower
than
in
the
untreated
rats
(84-2
(7-3)%)
5004
E
400
Es1
300
E
Cs
co
200-
E
Q-100-
(p<OOOO1).
Pretreatment
with
superoxide
dis-
mutase and
catalase,
however,
had
no
influence
on
the
development
of
tissue
oedema
or
the
stage
of
inflammatory
response
(see
Table).
More-
over,
in
spite
of
the
differences
in
zymogen
de-
granulation,
the
enzyme
activities
did
not
differ
significantly
from
the
values
after
3
5
and
12
hours
in
the
untreated
group
(see
Figs
3
and
4).
Histological
findings
in
animals
treated
with
superoxide
dismutase
one
hour
after
beginning
cerulein
infusion
showed
almost
identical
oedema
formation
and
the
same
stage
of
inflam-
matory
response
as
those
in
untreated
rats.
The
extent
of
cellular
necrosis
was
21-4
(10-6)%,
significantly
less
(p<0-0001)
than
in
untreated
rats
(see
Table).
Zymogen
degranulation,
al-
though
less
than
in
untreated
rats,
was
still
more
pronounced
than
in
the
pretreated
group.
At
the
same
time,
amylase
and
lipase
activities
were
lower
in
the
treated
animals
but
because
of
the
considerable
variation
the
difference
did
not
reach
statistical
significance
(see
Figs
3
and
4).
As
in
the
other
experimental
groups,
the
lipid
peroxidation
in
animals
treated
one
hour
after
cerulein
infusion
was
the
same
as
control
values
(see
Figs
5
and
6).
3°c0
min
Hour
"O
mrin
Hour
Figure
3:
The
changes
of
the
pancreatic
amylase
in
the
plasma
Figure
4:
The
changes
of
the
pancreatic
lipase
in
the
during
the
course
of
pancreatitis
with
and
without
scavenger
development
of
acute
pancreatitis
and
the
effects
of
treatment
treatment
(mean
(SD)).
SOD=superoxide
dismutase.
with
superoxide
dismutase
(SOD)
and
catalase
(mean
(SD)).
I
1
141

Schoenberg,
Buchler,
Gaspar,
Stinner,
Younes,
Melzner,
Bultmann,
Beger
Untreated
rats
-
-
-
SOD/catalase
treated
rats
*
£
SOD/catalase
treated
rats
I
h
after
cerulein
I
/~
~~~-------
1
0)
.eH
min
Figure
5:
The
changes
in
the
tissue
concentrations
of
conjugated
dienes
in
pancreatic
tissue
during
the
development
of
the
disease
in
untreated
and
superoxide
dismutase
(SOD)
and
catalase
treated
rats.
*Significance
p<002
level
(mean
(SD)).
Discussion
Oxygen
radicals
react
preferentially
with
phos-
pholipids
of
cell
membranes.
This
direct
reaction
induces
lipid
peroxidative
chain
reactions
within
the
membrane,
disintegration
of
the
cell
membrane,
and,
finally,
irreversible
cell
damage."
Indirectly,
oxygen
radicals
generate
a
chemotactic
factor
which
leads
to
an
accumula-
tion
and
margination
of
polymorphonuclear
leucocytes
in
the
damaged
tissue.
12
Because
of
their
high
reactivity,
oxygen
radicals
are
difficult
to
measure
directly.
Their
involvement
in
various
pathological
mechanisms
has
therefore
to
be
proved
by
circumstantial
evidence.
One
possibility
is
to
measure
the
amount
of
lipid
peroxidation
in
the
tissue
and
compare
it
with
the
development
of
tissue
injury.
Conjugated
dienes
or
malondialdehyde,
or
both
are
by-
or
fragmentation
products
of
lipid
peroxi-
dation'3
14
and
we
measured
these
to
assess
the
onset
and
further
development
of
oxygen
radical
generation
in
our
model
of
acute
pancreatitis.
After
30
minutes
of
cerulein
infusion,
the
tissue
concentrations
of
conjugated
diene
and
malondialdehyde
had
already
increased
slightly.
Electron
microscopy
showed
vacuole
formation,
the
beginning
of
zymogen
degranulation,
and
mitochondrial
changes.
Conjugated
dienes
and
malondialdehyde
reached
their
highest
tissue
concentrations
3
5
hours
after
the
start
of
cerulein
infusion,
and
at
the
same
time
incomplete
disassembly
of
tight
junctions
and
moderate
inflammatory
reaction
of
the
pancreatic
gland
was
observed.
After
12
hours
of
cerulein
infusion,
there
was
severe
tissue
oedema,
cell
necrosis,
and
a
mainly
neutrophilic
infiltration
of
the
pancreatic
gland.
At
the
same
time
the
serum
enzyme
activities
increased
significantly.
The
tissue
values
of
conjugated
dienes
and
malondialdehyde,
how-
ever,
returned
to
normal.
This
is
probably
due
to
the
extensive
cell
necrosis
or
further
fragmenta-
tion
of
these
labile
products,
or
both.
The
increase
in
these
parameters
at
an
early
stage
of
acute
pancreatitis
suggests
that
free
radicals
induce
enhanced
formation
of
lipid
peroxides
in
the
tissue,
before
severe
tissue
damage
can
be
seen
on
electron-
and
light
microscopy.
The
individual
increases
in
con-
jugated
dienes
and
malondialdehyde
as
well
as
in
a
amylase
and
lipase
in
the
plasma,
however,
did
not
correlate
with
the
development
of
histo-
+
~~~~~Untreated
rats
-
SOD
catalase
treated
rats
4
/
SOD
catalase
treated
rats
m
~~~~~~~~~~~~~1
h
after
cerutein
'103
E
0
Hour
Figure
6:
The
development
of
malondialdehyde
in
the
pancreatic
tissue
at
various
times
after
cerulein
infusion.
*Significance
p<005
(mean
(SD)).
SOD=superoxide
dismutase.
logical
damage
and
the
degree
of
inflammation.
Another
indirect
way
of
evaluating
the
role
of
oxygen
radicals
is
to
scavenge
them
by
the
highly
specific
antioxidant
enzymes
superoxide
dis-
mutase
and
catalase.
In
our
model
the
super-
oxide
dismutase/catalase
treatment
given
before
and
one
hour
after
cerulein
infusion
prevented
an
increase
in
conjugated
dienes
and
malondial-
dehyde
and
reduced
significantly
the
extent
of
zymogen
degranulation
and
acinar
cell
necrosis.
Tissue
oedema
and
neutrophilic
inflammatory
response,
however,
remained
as
severe
as
in
the
untreated
animals.
These
results
suggest
that
once
free
radicals
and
their
derivatives
are
generated
at
an
early
stage
of
disease,
followed
by
direct
or
indirect
membrane
damage
to
the
acinar
cells,
the
later
development
of
tissue
oedema
and
inflammation
seems
to
be
indepen-
dent
of
the
enhanced
oxygen
radical
generation.
This
may
be
explained
by
the
further
release
of
chemotactic
stimuli
(leucotrienes,
complement
activation)
generated
from
the
remaining
necrotic
tissue.
These
results
partly
confirm
those
in
the
study
by
Sanfey
et
al,
in
which
an
ex
vivo
perfused
canine
pancreas
preparation
was
used
and
an
acute
pancreatitis
was
induced
by
ischaemia,
free
fatty
acid
infusion,
or
partial
pancreatic
duct
occlusion.
Pretreatment
with
superoxide
dis-
mutase
and
catalase
significantly
reduced
the
pancreatic
tissue
oedema
and
hyperamylasaemia
normally
observed.4
Wisner
et
al
confirmed
these
results.
In
an
acute
pancreatitis
model
induced
by
a
three
hour
cerulein
infusion,
treatment
with
polyethylene
glycol-linked
superoxide
dismu-
tase
(PEG-SOD)
reduced
tissue
oedema,
serum
amylase
activity,
and
vacuolisation
of
the
acinar
cells.
As
in
our
study,
treatment
with
PEG-SOD
did
not
reduce
the
degree
of
inflammation.7
In
contrast,
Rutledge
et
al
showed
that
in
diet-
induced
haemorrhagic
pancreatitis
in
mice,
oxygen
radicals
were
apparently
of
minor
importance.
Treatment
with
various
scavengers
reduced
the
peripancreatic
oedema.
All
the
other
features
that
characterise
this
model
of
pancrea-
titis
were
unchanged.
The
development
of
the
inflammatory
response
with
and
without
scavenger
treatment
was
not
assessed
in
this
study.'5
In
all
these
studies,
however,
antioxidant
treatment
was
given
before
the
induction
of
acute
pancreatitis.
Guice
et
al6
used
superoxide
dismutase
and
catalase
treatment
before
and
1142

Oxygenfree
radicals
in
acute
pancreatitis
ofthe
rat
1143
after
a 12
hour
cerulein
infusion
and
compared
the
histological
outcome
and
the
recovery
of
the
pancreatic
gland.
In
their
study
a
single
post-
injury
injection
of
superoxide
dismutase
and
catalase
was
as
effective
as
a
continuous
infusion
of
these
scavengers
given
at
the
same
time
as
cerulein.
In
their
model,
however,
the
pancreas
was
examined
12
hours
after
the
end
of
cerulein
infusion,
that
is
at
a
time
when
the
pancreas
is
beginning
to
recover
from
the
cerulein
infusion.
16
Nevertheless
the
study
of
Guice
et
al
and
our
own
results
show
that
even
after
the
initial
onset
of
free
radical
generation
and
subsequent
damage,
treatment
with
free
radical
scavengers
continues
to
be
beneficial.
This
reflects
the
clinical
situation
more
closely
where
the
patient
is
admitted
to
the
hospital
after
suffering
from
the
first
symptoms
of
acute
pancreatitis.
Moreover,
in
patients
suffering
from
pancrea-
titis,
oxygen
radicals
seem
to
be
important
in
the
development
of
the
disease.
Braganza
et
al
and
Schoenberg
et
al
found
increased
amounts
of
lipid
peroxidation
products,
possibly
derived
from
free
radical
oxidation,
in
the
bile
and
tissue
of
patients
suffering
from
acute
or
recurrent
pancreatitis,
or
both.
7
8
In
addition,
Braganza
et
al
administered
acetylcystein,
a
well
known
radical
scavenger,
to
a
patient
suffering
from
multiple
organ
failure
due
to
severe
pancreatitis.
Within
72
hours
of
the
treatment
the
patient's
lung
and
renal
function
improved
consider-
ably.'9
Thus,
it
seems
reasonable
to
assume
that
oxygen
free
radicals
are
generated
in
acute
pancreatitis
in
man
and
add
to
the
damage
observed.20
Consequently,
treatment
with
free
radical
scavengers
could
possibly
interrupt
these
damaging
pathomechanisms
and
the
develop-
ment
of
complications,
and
improve
the
final
outcome
in
the
disease.
This
study
was
supported
by
grants
from
the
Deutsche
Forschungsgemeinschaft,
Bad
Godesberg
(Scho
309-2).
1
Adler
G,
Kern
HF.
Fine
structure
and
biochemical
studies
in
human
acute
pancreatitis.
In:
Gyr
KE,
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MV,
Sarles
H,
eds.
Pancreatitis.
Amsterdam:
Excerpta
Medica,
1984:
37-
42.
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Aho
HJ,
Nevalainen
TJ,
Havia
VT,
Heinonen
RJ.
Human
acute
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Acta
Pathol
Microbiol
Immunol
Scand
(A)
1982;
90:
367-73.
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GB.
The
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oxygen
free
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in
human
disease
processes.
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1983;
94:
407-11.
4
Sanfey
H,
Bulkley
GB,
Cameron
JL.
The
role
of
oxygen-
derived
free
radicals
in
the
pathogenesis
of
acute
pancreati-
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Ann
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1984;
200:
405-13.
5
Sanfey
H,
Bulkley
GB,
Cameron
JL.
The
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of
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the
source
and
role
of
oxygen-derived
free
radicals
in
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Ann
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1985;
201:
633-9.
6
Guice
KS,
Miller
DE,
Oldham
KT,
Townsend
CM,
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and
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a
poss-
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in
established
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1986;
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Wisner
J,
Green
D,
Ferrell
L,
Renner
I.
Evidence
for
a
role
of
oxygen
derived
free
radicals
in
the
pathogenesis
of
caerulein
induced
acute
pancreatitis
in
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Gut
1988;
29:
1516-23.
8
Buege
JA,
Aust
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Microsomal
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Methods
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1978;
52:
302-10.
9
Freeman
BA,
Crapo
JD.
Free
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and
tissue
injury.
Lab
Invest
1982;
47:
412-26.
10
Lowry
OH,
Rosenbrough
NI,
Farr
AL,
Randell
RI.
Protein
measurements
with
the
folinphenol
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J
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Chem
1951;
193:
265-75.
11
Slater
TF.
Free
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mechanism
in
tissue
injury.
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1984;
222:
1-15.
12
Petrone
WF,
English
DK,
Wong
K,
McCord
JM.
Free
radicals
and
inflammation:
superoxide-dependent
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of
a
neutrophil
chemotactic
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in
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Proc
Natl
Acad
Sci
1980;
77:
1159-63.
13
Floyd
RA,
Zaleska
MM.
Involvement
of
activated
oxygen
species
in
membrane
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possible
mechanisms
and
biological
consequences.
In:
Bors
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ed.
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in
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York:
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14
Younes
M,
Schoenberg
MH,
Fredholm
BB,
Haglund
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tissue
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following
regional
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and
reperfusion
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Res
Exp
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1984;
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15
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PL,
Saluia
AL,
Powers
RE,
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in
mice.
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1987;
93:
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16
Adler
G,
Hupp
T,
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382:
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17
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JM,
Wickens
DL,
Cawood
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Dormandy
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Lipid
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in
bile
of
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with
pancreatic
disease.
Lancet
1983;
ii:
375-8.
18
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MH,
Buchler
M,
Beger
HG.
Lipid
peroxidation
products
in
the
pancreatic
tissue
of
patients
with
acute
pancreatitis.
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1988;
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JM,
Holmes
AM,
Morton
AR,
Stalley
L,
Ku
R,
Kishen
R.
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to
treat
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of
pancreati-
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Lancet
1986;
i:
914-5.
20
Braganza
JM,
Rinderknecht
H.
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and
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1988;
94:
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