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Mechanisms
for
the
Oxidation
of Reduced
Stimulated
By Arthur
We
have
have
reported
an
ble
irreversible
sulfhydryls
ent
study.
release
one
of these
in the
supernatant.
to
enzyme.
However,
oxidize
HO2
these
since
hydroxyl
heme
factors.
radical,
change
and
rate
S
EVERAL
ported
concentration
stimulation
of
or
the
The
neither
scavengers,
the
to
since
nism
or
or
nor
benzoic
has
acid,
recently
LABORATORIES
have
recently
rethe reduced
soluble
sulfhydryl
(RSS)
of human
granulocytes
falls following
by opsonized
zymosan
particles
or phorbol
This
consumption
of
glutathione
(GSH),
on myeloperoxidase
or heme,
the reaction
is inhibited
by azide.23
The mechafor the consumption
of the endogenous
RSS by
polymorphonuclear
studies
cells
appears
(PMNs)
similar
observed
to that
reported
in
our
recently
by
Tsan et al. for the degradation
of methionine.6
Those
investigators
found
that stimulated
PMNs
generate
a
reactive
oxygen
species
with
the capacity
to oxidize
methionine
to a sulfoxide.6
They hypothesized
that the
compound
mediating
the reaction
may be either singlet
oxygen
oxygen
or hypochlorous
species
appears
tion
of
methionine
inhibitors.9
proteins
related
acid.6
This
to be involved
reactive
inactiva-
n-formyl
methionyl
chemotactic
peptide,7
enkephalin,8
and
serum
alpha
protease
In all cases,
the methionine
group
of these
becomes
oxidized.
The
factor
may
also be
to the cytotoxicity
target
same
in the
of PMNs
against
and
a variety
of
cells.’#{176}”
hypochlorous
with
ROS
Our
the
Supported
Society,
Submitted
Address
University
OH
Ohio
Central
Presented
hal
of
by NC1
Heart
Ohio
in part
CA
1978
November
(Clin
1. 1982;
requests
OSUHospital.
by Grune
Ohio
State
and
by grants
is a Senior
from
Investigator
Meeting
410
ofthe
Res 26:356,
accepted
July
Reticuloendothe-
1978.
studies,
L. Sagone.
Jr..
that
stimulated
reactive
oxygen
State
Columbus,
with
The
corn-
H2O2
and
and
compound
the
is either
derived
capacity
of GSH
for
this
from
to
react
tripeptide
injury.
to elucidate
further
of RSS
by
proved
difficult
because
intact
cell system.”2
For
the possibility
that
consume
extracellular
of
this
stimulated
GSH.
We
granulocytes
species
generate
that
amounts
ofGSH.
One compound
demonstrated
in the supernatants
one
oxidize
large
is stable enough
of granulocyte
to be
sus-
pensions
after
stimulation
by zymosan.
Once
formed,
the same species,
in addition
to its capacity
to consume
GSH,
is able to oxidize
iodide
to iodine,
fix halogen
to
proteins,
and react with methionine.
Our observations
indicate
that
the ROS
reacting
with GSH
is probably
identical
to that which
reacts
with
methionine.
Likewise,
our results
suggest
that
cellular
GSH
may be important
in the protection
of
intracellular
enzymes
damage
by this ROS.
and
MATERIALS
Cell
cell
AND
membranes
against
METHODS
Preparation
blood
blood)
dextran
was
from
collected
healthy
sedimentation.
platelets,
removed,
(SBSS),
and
layered
remove
platelets
fuged
for
35 mm
and
SBSS
and
over
containing
100 mg/dl
than
95%-99%
Cells
were
counted
then
glucose.
The
electronically
using
button
shock
were
washed
lysis
with
saline
contained
FN
to
to the
1 % mononuclear
a model
and
solution
centri-
Hypotonic
preparation
than
1 hr
was
phosphate-buffered
final
less
by
mixture
down
cells.
Cells
Dulbecco’s
and
salt
mixture
aspirated
red
erythrocytes.
granulocytes
leukocytes
Ficoll-Hypaque
This
cc
isolated
at 37#{176}C
for
balanced
cells.”2
a few
in
were
incubated
in Seligman’s
g and
remaining
cc of 5% solution/lO
containing
a standard
and
resuspended
was
plasma,
mononuclear
at 800
(0.3
Leukocytes
blood
The
mixed
granulocytes
the
in EDTA
volunteers.
Whole
was
eliminated
Ohio
on
designed
have
react
protein.
role
The
halogen
granulocytes
in the consumption
evaluated
might
found
to
oxidant
involved
reason,
we
granulocytes
more
a
fix
GSH,
production.
The
granulocytes,
nature
of the
containing
18. 1983.
W. 10th Avenue-N1022,
Inc.
abstr).
or
stimulated
the complex
or
its
against
previous
r
an additional
protection
in 0.1 vol of 4% dextran.
32321
A.L.S.
to Dr. Arthur
& Stratton.
0006-4971/84/6301-0012$01.00/0
Oncology.
OH.
Association.
at the Annual
December
reprint
Grant
Association.
Heart
and
Columbus,
43210.
© 1984
96
Hematology
ofMedicine.
in part
the Central
ofthe
Division
College
(HOCI)
suggests
stimulated
dependent
for
as a chloramine.
the mechanism
whole
From
acid
this
Venous
University
appears
could
GSH.
and
a
and
methionine
to oxidize
fix
by
H202
degrades
iodide
that
and
system
on
that
that
to oxidize
oxidize
therefore,
such
found
capacity
a sulfoxide
dependent
ROS
indicate
the
myeloperoxidase
HOCI,
We
data
with
to
is
same
capacity
Our
a ROS
oxidized
that
the
the
to proteins.
in cellular
that
be dependent
with
has
pound.
superoxide
and
also
be
species
production.
methionine,
with
dismutase
Methionine
its
react
release
oxidants
reacts
is not
for
ROS
to
oxygen
directly
a heme-containing
ROS
myristate
acetate,
(PMA).’5
RSS,
which
includes
reduced
appears
heme
One
stable
superoxide
reaction.
pres-
be detected
compound
mannitol
the
to
and Earl N. Metz
demonstrated
reactive
with
(GSH).
enough
of these
formed,
been
(ROS)
glutathione
formation
once
In the
M. Sue O’Dorisio,
solu-
granulocytes
species
is stable
Husney,
granulocytes
reduced
stimulated
oxygen
reduced
The
require
without
the
reactive
human
stimulation.
that
compounds
appears
hydroxyl
zymosan
more
to
more
GSH
that
endogenous
in their
demonstrate
or
capacity
the
previously
by
Granulocytes
Jr., Rose Marie
fall
following
we
L. Sagone,
Gluthathione
Coulter
more
cells.
Coun-
ter.
Blood,
Vol. 63. No.
1 (January),
1984:
pp. 96-104
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MECHANISMS
FOR
THE
OXIDATION
Studies
to Evaluate
Zymosan-Stimulated
OF
GSH
BY
97
PMNs
similar
the Effect
of
PMNs
on Extracellular
conditions
as a control. The solution was then incubated
1 5 mm, and the GSH remaining
was determined.
additional
GSH
In some
quantity
of 15-20
x
Dulbecco’s
phosphate-buffered
A
placed
in
1 2-ml
magnetic
were
stir bars.
Louis,
MO)
One
fresh
part
was
was
opsonized
added
bated
at 37#{176}C
for
10 mm
final
Chicago,
terminated
IL)
in a water
incubations
with
suspension
removed.
of approximately
they
by adding
solutions
per
at
ofsuspension.’3
12.5
been
were
of oxygen
stoppered
addition
and
was
then
of opsonized
determined,
gassed
were
was
vortexed
for
zymosan
particles,
suspensions
air or nitrogen
as previously
before
the
reported.’
In the
tants
ofPMN
second
series
the
capacity
of zymosan-stimulated
granulocyte
was studied.
For most experiments,
GSH
suspension
(5
zymosan
zymosan
ratio
x
cells/mi)
106
was
in
of the
with
constant
at
5 mg/20
x
l0
cells.
The
and
in which
tors
were
studied,
the
agent
for
particles.
then
ml
the
solution
10 mm
suspensions
mixed,
precipitating
We
tors
to
DTNB
also
on the
the
was
then
incubated
addition
of the
ofGSH,
0.05
for
solution.’3
The
remaining
evaluated
the
activity
supernatant.
GSH
effect
of scavengers
In
these
experiments,
for
I 5 mm
at 37#{176}C
with
the
An
aliquot
of the
supernat.nt
the
the
agent
was
of
species
by
The
reason
for
of granulocytes
the same
amount
this
particles,
we
in the
of stable
oxidant.
Produced
From
by Stimulated
also
incubated
The
was
addition
under
of
to GSSG.
The
glutathione
as
of GSSG
in the buffer
by
we eval-
supernatants
reductase,
previously
Supernatant
and
the
described.’
system
The
was
used
as a
From
established
that
to Oxidize
granulocytes,
described
by Saltzman
for the
supernatants
from
Iodide
following
experiments
involving
to a tube
GSH.
stimu-
Na2HPO4-KH2PO4
was
read
developed
was
unstable
was
at
read
given
produced
352
granulocyte
I
The
upon
mixing,
since
decreased
with
with
for
of 12 produced
iO
cm’.6
ability
i07
ml)
a Beckman
(see Table
PMNs
had
from
incubated
supernatants
0.5
.tCi
Corp.,
Arlington
OD
the nanomoles
of
of 2.64
and
suspensions
zymosan
incubated
in a total
(20
IL),
was
x I 06
Aliquots
volume
activity
x
Clark.’5
for 20 mm.
(specific
12
The
to protein
of Klebanoff
Heights,
that
The
The
an E352
granulocyte
iodide
(OD)
color
time.
to fix iodine
with
were
‘25I-Na
density
the
was
in 0.1
ml of supernatant.
assuming
method
ml
spectrophotometer.
0.6
supernatants
obtained
optical
4) indicate
using
of the
been
DB
calculated
of neutrophil
were
of the
gradually
was
by a modification
that
0.6
KI solution
solution.
and
suspensions
supernatant,
neutral
in the results
from
each
2.4 ml of a 1 25%
immediately
nm
amount
tested
micrudeter-
20 mm of incubation
at 37#{176}C
was identical to that used in
Of
containing
produced
M’
iodometric
zymosan-stimulated
(13-20
x 106/ml) were obtained
after
with continuous
stirring. The procedure
of 0.8
1 3-1 7 mCi/
40 nmole
KI,
2 mM
mM CaC12, and 3% human
serum.
After
incubation
for 20
mm at 37#{176}C,
the iodination
reaction
was stopped by addition
of 0.1
ml of 0.1 M sodium
metabisulfite
in 0.15 M Na-KPO4
buffer, pH
7.3, and 0.3 ml 30% trichioroacetic
acid (TCA).
Samples
were
centrifuged
at 1,000 g for 10 mm. Pellets were washed
3 times with 9
ml 10% TCA and counted
in a Beckman
Gamma
7000 scintillation
MgCl2,
supernatant
oxidized
degraded
of organic
oxidants
and ozone using neutral
buffered
iodide.’4 In this method, the oxidation
ofiodide
to iodine is
with the development
of increased
absorption
at 352 nm.
gig, Amersham
inhibi-
it was
was
PMNs,
Granulocytes
to Protein
studies
procedure
ml containing
directly
GSH
zymosan-stimulated
determined
ofthe
initial
(0.6
them
the
Our
the
bath.
that
from
lation with zymosan,
released
a factor into the supernatant
that
could degrade GSH. The supernatants
from these suspensions
were
then studied for their capacity
to oxidize iodide and fix halogen to
protein. The method used to quantitate
the oxidation
of iodide was
to I
metabolic
before
Capacity
added
was determined
by adding
The
cell/mi)
0. 1 mM),
GSH
Zymosan-Stimulated
and Fix Halogen
was
cc of metaphosphoric
and
activity
zymosan
number
NADPH,
of 50 nmole
Supernatants
zymosan
above.
of the supernatants
of
GSH
in a 37#{176}C
water
0.75
as described
cc was
GSH
I 5 mm
shunt
oxygen
control.
The
with
to degrade
by adding
incubated
GSH.
to the
terminated
assay
GSH
preincubated
concentration
regeneration
inhibi-
was
solution
(final
and
acid
the
2 mM
with
values
were
or metabolic
suspension
of the 1-ml supernatant
Ofa
reaction
of scavengers
at 37#{176}C
prior
supernatant
The
using
effects
granulocyte
The capacity
determined.
of
the
the
that
M
without
stirred
constantly.
The reaction
was terminated
by placing
the
suspensions
in an ice bath. The suspensions
were then immediately
centrifuged
in a Sorvall RC-2B at 4#{176}C
for 20 mm at 27,700 g. In
experiments
the possibility
transferred
particles
for I 5 mm in a 37#{176}C
water bath. The amount
of
added to these suspensions
was adjusted
so that the final
was
the
demonstrated
obtained
supplemented
the
superna-
suspensions
to degrade
1.4 ml of the granulocyte
incubated
lot.’2
the Product
Generated
experiments
were
mination
potassium
associated
Suspensions
of experiments,
the
the less potent
to generate
uated
the
Studies
to Determine
Whether
or Not the
GSH-Reactive
Compound
Could
be Demonstrated
the Supernatant
our
regeneration
acid precipitating
granulocyte
either
to
With
to increase
in order
of reactive
PMNs
Once
of buffer
reactions
solution
the
with
it necessary
the supernatant
30 sec. permitted
to stand for 5 mm, and then centrifuged
for 10 mm
at 27,700 g. The amount
of GSH
remaining
in the supernatant
fluid
was determined
using the DTNB assay for quantitating
GSH.’3 In
some experiments,
we evaluated
the effect of several enzymes
and
metabolic
inhibitors
on the degradation
of extracellular
GSH by
zymosan-stimulated
granulocytes.
In this case, the granulocyte
suspension
was incubated
with the compound
or enzyme at 37#{176}C
for
10 mm before adding
the zymosan.
For the experiments
in which the
importance
according
zymosan
monophosphate
generation
Studies
to Determine
GSH by the Compound
preincu-
amount
37#{176}C.The
The
varies
is not clear.
particles
ofopsonized
x 106 PMNs)
had
0.75 cc of metaphosphoric
milliliter
the hexose
the
lot of zymosan
the capacity
3
was
The zymosan
(5 mg/20
a different
that
of 50
incubated
An identical
bath
cells
as follows.
The
noted
to augment
difference
was added
to the control
samples.
Samples
were incubated
for
intervals
up to 1 hr with constant
stirring
(Magna
4, Cole Palmer
Inst.,
these
serum
was
we used
have
to stimulate
autologous
at a concentration
We
and
found
after
stirring.
a
experiments,
PMNs
Chemical,
suspension
with
particles
(Sigma
supernatant
suspensions
were
Teflon
particles
at a concentration
to granulocyte
8-mm
(71-F-8086).
in
suspension
zymosan
glucose,
I ml
the suspensions
at 37#{176}C
for 30 mm.
and most ofthe
in
(GSH)
saline
zymosan
in buffer
The
in fresh
serum
with
containing
glutathione
in normal
of the
resuspended
mg/mi.
opsonized
autologous
then centrifuged
(DPBS)
of experiments,
reduced
suspended
suspended
tubes
1 mM. Zymosan
were
was
mg/mi.
parts
with
of about
Zymosan
saline
centrifuge
In the first series
supplemented
concentration
St.
Nalgene
granulocytes,
106
an
counter.
protein.
1
The
results
were
expressed
as
picograms
of
251 fixed
to
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
SAGONE
98
Reagents
tion,
Superoxide
tase
(type
azide,
catalase
NADPH
Sigma
Sharp,
(St.
and
(Fair
Corp
FMC
sodium
Lawn,
Sigma.
NJ).
lot
tabolism
the
oxidative
were
done
Fisher
was
with
from
found
oxidative
The
effect
was
determined
and
ZymoWe
of the
meusing
assays
for
described
from
laboratory.’2
Statistical
were
independent
analyzed
samples.
according
Results
to
the
are expressed
test
for
as mean
dependent
±
SD
or
or
±
SE.
granulocyte
suspen-
with
opsonized
zymosan
(data
not
shown),
under
anaerobic
condiof GSH
by more than
indicating
a requirement
for
oxygen
in this reaction.
We next studied
the effect of
several
scavengers
on the consumption
of GSH
(see
Table
1 The hydroxyl
scavenger,
mannitol,
had no
protective
effect.
Similarly,
superoxide
dismutase
was
).
not protective.
Catalase
(25 g/m1)
provided
protection
(10%).
The failure
of catalase
more protection
was unexpected.
Therefore,
were
catalase
catalase
done
using
a higher
minimal
to provide
additional
concentration
of
(200
ig/ml).
This
higher
concentration
of
provided
increased
but incomplete
production
(20%-30%).
tive.
Azide
((104M)
ofOpsonized
Zymosan
Extracellular
GSH
Granulocyte
enough
GSH
suspensions
so that
the
on the Recovery
in Granulocyte
found
in the
of
Suspensions
were
supplemented
final concentration
supernatant
was 1 .2 mM. The amount
system
(1,000-1,200
nmole)
exceeds
our experiments
most of the GSH,
The Capacity
ofthe
Against
Oxidation
Our
factor
RESULTS
amount
in the
incubated
was also
partially
protec-
Analysis
Data
Effect
consumed
were
experiments
of zymosan
standard
as previously
the
obtained
lot 80-F-0478.
oxidation
from
by Beutier.’6
stimulation
species,
obtained
to confirm
were
of granulocytes
oxygen
from
from
titrated
lot 80-F-0478.
of ‘4C- 1-glucose
of reactive
obtained
7l-F-8086,
less
than
was
solution
was
90%
Merck,
cyanide
described
and
metabolism
stimulation
the detection
H202
Na
solution
were obtained
from
rapidly
which
particles.
Incubation
of PMN
tions reduced
the consumption
sodium
methionine,
obtained
was
sions,
reduc-
C40),
III,
Phenol
A 30%
produced
of granulocytes
PA).
a method
experiments
7I-F-8086
was
and
80-F-0478
Cat.
type
and zymosan
MO)
The
using
lots
Most
that
Point,
NJ).
(Buffalo,
particles,
U/mg,
Mannitol
Louis,
glutathione
salt
benzoate,
(West
concentration,
H2O2
san
U/mg),
(36,000
MO).
(Saint
Scientific
1, 2,900
tetrasodium
Louis,
Dohme
Mallinckrodt
our
beefliver
IV),
glutathione,
from
the
(type
GSSG,
reduced
on
dismutase
ET AL.
number
of GSH
in the
by 25 times
the
of granulocytes
(40 nmole).
when added
with
in the
As indicated
in a I .2-mM
used
in
in Fig.
concentra-
1,
results,
degrading
than
H202,
suspensions
GSH
against
Catalase
by H202
discussed
GSH
since
the amount
of catalase
seemed
more
than
adequate
oxidation
by H2O2. Further,
oxidation
by
Table
H2O2
in the
1 . Recovery
of GSH
(25 j.tg/mI)
(200
Stimulated
mannitol
Values
ig/ml)
30
60
MInutes
Fig. 1 .
Consumption
of supernatant
GSH by PMNs
during
interaction
with
opsonized
zymosan
particles.
The values
are
expressed
as a percent
of the GSH recovered
in PMN suspensions
at each time following
the ingestion
of zymosan
particles
cornpared
to the value recovered
in suspensions
incubated
without
zymosan.
For 6 paired
experiments.
the recovery
of GSH from
unstimulated
suspensions
after 60 mm was 1 .207 nmole
± 41 (SD)
compared
to 53 nmoles
± 35 (SD)
for zymosan-stimulated
ones.
same
the
1.200
PMN/ml)
lot
of
±
1 06
<0.1
3
353
±
59
<0.05
3
63
±
23
NS
3
79
±
19
NS
3
344±40
(80-F-0478).
nanomoles
nmole
There
of GSH
of GSH
incubated
of GSH
added
in Dulbecco’s
zymosan
recovery
buffer
particles
was
from
recovered
no effect
buffer
supplemented
was
unstimulated
in the
by all stimulated
unstimulated
heat-inactivated
a different
one
(p
<
catalase
lot of zymosan
used
for
without
suspensions
used.
particles
after
the
The
or scavengers
cells.
effect
The
The
on the
or solutions
p value
with
given
additives
to
The consumption
higher
was
were
with
from
experiments
suspensions
experiments
(7 1-F-8O86)
mm
glucose.
these
is significantly
protective
60
of granulocytes
with
all
of enzymes
granulocyte
0.0 1 ). No
was
<0.05
to a 1-mI suspension
compares
the mean of the stimulated
PMN suspensions
the stimulated
PMN suspension
without
the additive.
of GSH
-
185
(40 mM)
indicate
(2 x i0
25
28
+
approximately
20
±
p Value
4
azide(104M)
15
17
+
Stimulated
10
7 1
SD
±
±
+
SOD(1Oig/ml)
5
1 .234
+
catalase
msan
Mean Va’ue
Number
and
Suspensions
4
Stimulated
S.E.
Unstimulated
with
+
catalase
C
From
4
Stimulated
C
(Dulbecco’s
Granulocyte
of Suspension
Stimulated
I Ii
medium
Zymosan-Stimulated
Stimulated
I
U,
0
added
to
to protect
it was diffi-
cult to explain
the protective
effect of azide if the GSH
were
simply
reacting
with
H2O2.
To test
this,
we
studied
the capacity
of catalase
to protect
GSH against
Type
E
GSH
above,
suggested
that
the
might
be a compound
other
Unstimulated
S
to Protect
similar
than
the
found
when
repeated
with
results.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
MECHANISMS
FOR
THE
OXIDATION
OF
GSH
BY
99
PMNs
glucose)
used for our experiments.
A 0.1-mM
GSH
solution
was incubated
for 1 5 mm at 37#{176}C
with final
concentrations
of H202 ranging
from 0. 1 to 10 mM. A
0. 1-mM
concentration
of H202 decreased
the
tration
ofGSH
to 0.07, and a 0.3-mMconcentration
H202 to 0.026
mM. Concentrations
of H202,
from
0.5 to 10 mM,
the highest
(200 g/ml)
of GSH
against
of H2O2.
superoxide
protect
oxidized
all the GSH.
concentrations
could completely
used
for
protect
oxidation
by the
Other
scavengers,
such
dismutase
(SOD),
GSH
against
oxidation
10 mM
Effect
consumption
ofGSH
natants
were
then
x
iO
in
92
±
6.2
-
35
±
14
-
4
88
±
9
4
25
±
12
NS
Phenol(104M)
3
90±
10
<0.01
Benzoic
3
33
±
13
NS
2
34
±
12
3
53±4
3
90
(25
acid (20 mM)
Mannitol
mannitol,
did not
(40 mM)
Cyanide(104M)
Methionine
of
was
zymosan
1 ml of
time (Fig.
1). The superfrom
these
suspensions
produced
enough
approximately
then
used
to
factor
produced
Effect
a zymosan-stimulated
a concentration
of the compound
ofMetabolic
Inhibitors
Incubation
In
this
series
then
The
determined
results
are
benzoate,
prevent
protective.
5
mm
to
of experiments,
and
Scavengers
on the
GSH
oxidize
the
effect
of
of several
formation
of the factor
was evaluated.
suspensions
were supplemented
with
The activity
of the supernatant
was
after the initial
incubation
indicated
in Table
2. The
mannitol,
and
the formation
Cyanide
and
as before.
scavengers,
superoxide
dismutase,
did not
of the factor.
Catalase
was
phenol
also provided
a protec-
tive effect.
Since
methionine
has been shown
to react
with
a ROS
generated
by PMNs,
studied
the protective
effect of this compound
hundred
with
There
The
p value
zymosan
was
the
the additives.
required
recently
we also
(Table
value
without
from
to generate
of experiments,
the
CN
this
same
provided
mg/S
106
(see
to
the
with
GSH
1 ml of the
values
stimulated
additives.
The
Materials
react
added
mean
lot
experiments.
to
was
The
1 5 mm.
are given
mean
in
with
The
recovery
of GSH
98 to 1 10, with
a mean
value of
compared
With
x
these
in the recovery
The experiments
(7 1-F-8086).
all
compares
ranged
additives
(1 .25
for 1 5 mm. The same
at 37 #{176}C
for an additional
determined.
no difference
with
of zymosan
series
then
(5 x 10
the granulocytes
of GSH
incubated
was
<0.02
suspension
of the supernatant
nanomoles
to the stimulated
buffer
without
was
SD.
±
additives
from
recovered
and the solution
remaining
nanomoles
from
then
for
used
NS
1
±
zymosan
of the additive
was
of GSH
to the
were repeated
controls
using a different
lot,
an increased
amount
of the
enhanced
in unstimulated
unstimulated
number
stable
protection
lot
of PMNs
oxidant.
In this
(67%-75%).
2). This amino
acid protected
GSH
against
oxidation
by the factor.
This could be demonstrated
with concentrations
of methionine
also impaired
the
shown).
Experiments
as low as 0. 1 mM. Serum
(3%)
recovery
of the factor
(data
not
to Determine
From
GSH
Once
we
by Stimulated
observed
supernatants
prepared
ulocyte
suspensions,
Added
Activity
GSH
opsonized
and the capacity
One
102.
granulocyte
susx 106 cells/ml
in 1 5
were
supernatants
50-80
nmole
of GSH.
This system
was
further
characterize
the nature
of the
by granulocytes
(see Table
2).
to the Granulocyte
the Supernatants
agents
on the
The granulocyte
several
agents.
of
(8O-F-0478)
supernatant
The
with
or absence
<0.01
<0.1
1 .4 ml of a anulocyte
incubated
in the presence
and Methods),
all of the GSH.
The experiments
were then repeated
using
lower concentrations
of granulocytes
in the mitial incubations.
We found
that
1 ml of supernatant
from
with
experiments,
supernatants
in
mM)
(0.2
For these
and
their
capacity
to consume
GSH
studied.
The
supernatants
were
incubated
for 1 5 mm with
100
nmole
of GSH
(final
concentration
0. 1 mM).
There
was a factor
present
in the supernatant
that consumed
obtained
pension
big/mI)
(10 zg/ml)
SOD
1 5 mm with and without
5
particles.
In our prior experithat
there
was significant
by this
recovered
p Value
4
Catalase
concentration
cells
to
10
by H202.
(2
Nanomoles
GSH Recovered
Number
Stimulated
tested.
for
(Added
Supernatants
Unstimulated
ranging
Studies
to Determine
Whether
or Not a Factor
Reacting
With GSH
Could
be Detected
in
Supernatants
of Granulocyte
Suspensions
suspensions
Inhibitors
of Zymosan-Stimulated
of
PMN)
Granulocyte
and Metabolic
on the Activity
Granulocyte
PMN/ml)
buffer)
were incubated
mg ofopsonized
zymosan
ments,
we demonstrated
of Scavengers
Suspensions)
Type of
Supernatant
experiments
degradation
as benzoate,
and
phenol,
2.
concen-
Catalase
our
the
Table
Granulocyte
the Product
Generated
Granulocytes
that
GSH
was
degraded
by
from zymosan-stimulated
we evaluated
the possibility
the
granthat
the GSH
was oxidized
to GSSG.
Therefore,
we regenerated
any GSSG
produced
from
GSH
during
the
reaction
by further
incubating
the supernatants
for 35
mm at 37#{176}C
with glutathione
reductase,
NADPH,
and
then measuring
the GSH.
In 3 experiments,
we were
able to regenerate
more than 80% of the GSH
that had
been
degraded
by the activated
supernatant.
This
observation
indicates
that
most
of the
GSH
is oxidized
during
the reaction
with the factor(s)
in the granulocyte supernatant.
Similarly,
we were able to regenerate
more than 80% of the GSH
in supernatants
collected
from incubations
in which
the GSH
had been added
directly
ogenous
to the
GSH).
stimulated
granulocyte
suspension
(cx-
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
100
Effect
ofMetabolic
Inhibitors
Directly
to the Supernatant
Factor
with
the
Similarly,
reaction.
100 nmole
the
agents
for
azide
and
The amount
added
to prevent
1 5 mm.
Following
catalase
of GSH
this,
also failed
to
recovered
from
to supernatants
the
formation
that
of the
the interaction
of zymosan
with
scavenging
a substrate
required
pound
or by inhibition
reaction.
The failure
GSH
that
when
the
added
had
compound
supernatants
reacting
factor
been
during
the PMNs,
to produce
of an enzyme
of catalase
and
to the
with
GSH
either
by
the com-
essential
for the
azide
to protect
also
indicates
appears
compound
Characteristics
ofthe
Additional
studies
to be due
directly.
in the
superna-
to its capacity
to react
Factor
were
done
to characterize
the
factor.
We found
that the activity
was destroyed
by
boiling
for 5 mm. This also occurred
if the supernatant
was heated
at 56#{176}C
for 30 mm. The factor
remained
active when the supernatants
were kept at 4#{176}C
for 2 hr,
but gradually
lost activity
when
incubated
at 37#{176}C.
Also,
the activity
of the factor
was
destroyed
by
acidifying
the supernatants
with metaphosphoric
acid.
In some experiments,
the supernatants
were incubated
with
10 mM H2O2 for 15 mm at 37#{176}Cand supplemented
with catalase
before
adding
the GSH.
This did
not alter
the reactivity
of the factor.
It could
not be
extracted
to decrease
with ether
or chloroform.
Trypsin
the activity
of the factor,
but
appeared
to be nonspecific.
heat-denatured
enzyme
0.05
From
Activity
of
on the GSH
Zymosan-Stimulated
AL.
Granulocyte
Nanomoles
GSH Oxidized
59
mM
29
0.1mM
For this
22
appeared
the effect
The factor
was removed
by
as well
as the
unheated
10
experiment,
supernatants
and zymosan-stimulated
were
1 5 mm at 37
for
0
ml of the solution
and
the
tion
the
in the
nmole.
results
enzyme.
indicate
from
The
ofthe
1 experiment
The values
The GSH following
unstimulated
typical
incuba-
cells
to Oxidize
of a factor
was
99
of 3 done.
trypsin
to oxidize
with the
unable
to
since freezing,
even
a loss of activity.
Supernatant
to 1
1 5 mm.
(NEM)-treated
with
identification
added
an additional
the ability
of the factor
that the factor
was reacting
of the protein.
We were
store the supernatants,
was also associated
Capacity
the
Aliquots
the methionine
was then determined.
N-Ethylmaleimide
did not decrease
GSH,
suggesting
sulfhydryl
group
unstimulated
of GSH were
incubated
of GSH oxidized.
obtained
from
(5 x 1 06/mI).
and without
100 nmole
of the solutions
supernatants
The
this,
with
supernatant
nanomoles
net
collected
suspensions
then incubated
C. Following
The GSH concentration
indicate
were
granulocyte
of the supernatants
cytes
with
methionine,
tant cannot
be H202,
does not require
myeloperoxidase, and is unlikely
to be singlet
oxygen.
In contrast
to
the other
agents
studied,
methionine
was protective
when added
to the supernatant
again
in relatively
low
concentrations
(Table
3). Thus,
the protective
effect of
methionine
with the
of Methionine
0
then
preincubated
with catalase
(25
zg/ml)
was 23 ± 7
(SD).
The value for azide
(104M)
was 29 ± 9 (SD).
The
corresponding
value
for the control
for these
experiments
(stimulated
supernatants
without
additives) was 28 ± 1 1 . Therefore,
both azide and catalase
appear
Effect
Obtained
0.5mM
the capacity
of the supernatant
to consume
GSH
was
again
determined.
As expected,
SOD
and hydroxyl
radical
scavengers
did not provide
protection
(data
not
shown).
alter the
The
Concentration
of Methionine
was designed
to
of action
of these
with the factor
or a
In order
to do this,
were recovered
from zymosan-stimulated
suspensions.
The supernatant
was
incubated
3.
Supernatants
ET
Suspensions
Our
next
series
of experiments
determine
whether
the mechanism
agents
was due to a direct
reaction
capacity
to prevent
its production.
supernatants
granulocyte
Table
and Scavengers
Added
on the GSH Oxidizing
SAGONE
at
-
70#{176}C,
Iodide
released
from
the capacity
to react
with both
and the apparent
requirement
granuloGSH
and
for myelo-
peroxide
(MPO)
activity,
raised
a possible
relationship
of this compound
to the one generated
by stimulated
PMNs
that
causes
fixation
of halogen.’5
For this
reason,
two types of experiments
were done.
First,
we
determined
whether
or not the supernatants
had the
capacity
to oxidize
iodide
to iodine.
Second,
we determined
whether
halogen
The
to protein.
supernatants
pensions
after
or
not
the
supernatants
obtained
stimulation
from
with
factor
that was able to oxidize
4). The amount
of 12 produced
present
in 1 ml of supernatant
was
would
granulocyte
zymosan
fix
sus-
contained
a
iodide
to iodine
(Table
from 1 by the oxidant
derived
from I 0 PMNs
35
nmole.
The
factor
involved
in the reaction
to be similar
to the one reacting
with GSH.
It
required
H2O2 but not superoxide
for its production.
When
GSH
or methionine
was added
to the granuloappeared
cyte
not
suspension
be detected
during
in the
stimulation,
supernatant.
the factor
Further,
could
if the
supernatant
was incubated
with GSH,
no oxidation
of
iodide could be demonstrated.
Once formed,
the factor
released
by stimulated
PMNs
into the supernatant
did
not require
H202
or myeloperoxidase
activity,
since
preincubation
azide
did
not
of the
impair
supernatant
the reaction
with
(data
catalase
and
not shown).
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
MECHANISMS
Table
FOR THE
OXIDATION
Capacity
of Zymosan-Stimulated
4.
Supernatant
OF GSH
to Oxidize
Type of
Supernatant
101
BY PMNs
No. of
Experiments
Stimulated
10
22.8
3
6.0
Stimulated
+ 0.2
Stimulated
For these
2.8
3
22.0
±
2.6
3
10.0
±
4.1
bated
GSH before
1 .0 mM
GSH
2
1 .3
±
0.7
of I
granulocyte
were
supernatants
to oxidize
the nanomoles
supernatant.
The
(SD) per
supernatants.
OD
to 12 quantitated
of 12 produced
suspensions
without
supernatants
also
(Table
catalase
reaction.
the
text).
1 per
PMNs
than
GSH.
suspensions,
The
by 0.6
was
PMNs
with
(±
values
1O PMNs
0.O7/1O
±
lower
experiments.
supernatants
stimulated
are all significantly
since
the
from
and 0.20
for
In some
enzymes.
from
(see
for the unstimulated
1O PMNs
unstimulated
with
recovered
were
I
The values
to protein
suspensions.
supplemented
from
for
and
addition
serum
GSH,
with
the corresponding
value
the additive.
contained
a factor
The
5).
factor
that
did
could
not
added
to the incubation
However,
the factor
fix
not
be
by GSH.
a factor
or
factors
that
granulocytes,
folzymosan
particles,
are
reduced
glutathione.
Granulocytes
large
amounts
of these
compounds,
from our
degraded
greater
vations).
studies
that the
by optimally
there
was
suspensions
no protective
to
oxidize
amount
of GSH
that can
stimulated
granulocytes
than
100 nmole/
1 06 cells
In our initial
experiments,
the granulocyte
able
appear
to release
and we estimate
with
effect
(unpublished
obserwe supplemented
GSH.
In this
of superoxide
system,
dismutase
4.68
0.066
1 , the supernatants
precipitate
of the
( 1 mM)
incubated
was
under
supernatants
(25
similar
were
we
then
supernatants
and catalase
incubated
.tg/ml),
incubated
for 20
for
respectively.
A second
with
1251
that
GSH
2 and
3,
with
GSH
aliquot
was
as a control.
The
KI, and serum
of ‘25I into the TCA precipitate
found
in
1 5 mm
the additive
without
could
#{176}
The cpm
In experiments
preincubated
C
mm at 37
and KI (see text).
determined.
were
conditions
then
4.17
were
3% serum,
iodide,
for 20 mm,
quantitated.
be
degraded
by
high
concentrations
of hydrogen
peroxide
(greater
than 0.3 mM).
However,
catalase
was able to prevent
the oxidation
of GSH
by a 10 mM concentration
of
hydrogen
peroxide.
that a compound,
be
is
0.068
and
iCi of “I-Na
others,’7
results
indicate
that human
stimulation
by opsonized
release
TCA
0.037
before
of l
and the incorporation
DISCUSSION
Our
lowing
the
aliquots
require
did
could
0.5
0.029
15mm
catalase
In experiment
with
7.42
incu-
serum
for
0.034
of 1 and
addition
and
6.91
incu-
batedfor
stimulated
catalase,
0.029
1 5 mm before
Supernatant
±
From
Cells
addition
Supernatant
SD)
ml of
Supernatants
Stimulated
3
bated
0.0 1 5
Fixed
I25
incu-
and serum
and
(B)
S
From
Cells
15 mm with
Experiment
(A)
to
to Protein
and
Supernatants
0.63
collected
Unstimulated
Suspensions
incu-
of l
0.42
were
Halogen
serum
±
supernatants
From
Granulocyte
1 5 mm before
±
The
capacity
Supernatants
bated
2.4
methionine.
removed
2
3.7
suspensions
stimulated
1
Experiment
addition
experiments,
methionine
Experiment
2
granulocyte
I
±
(B)
and zymosan-stimulated
H202,
impair
8.6
2
+
The
±
GSH
Stimulated
Supernatants
Pico’am
mM
+ 0.5
of the
Supernatants
Unstimulated
mM
methionine
0.008
0.81
+ 1 mM
Stimulated
indicate
±
(A)
methionine
then
1.6
+ SOD
(10sg/mI)
Capacity
Fix
+ catalase
Stimulated
The
Nanomoles
2
per iO Cells
10
(25 ag/mI)
5.
and Zymosan-Stimulated
(1
Unstimulated
Stimulated
Table
Granulocyte
Iodine
These
observations
suggest
to us
or compounds,
in addition
to H2O2,
was released
with GSH.
by granulocytes
In this regard,
be involved.
The
reaction
produced
tion
capacity
suggests
that one
by myeloperoxidase.
of the
reaction
could
with the capacity
multiple
mechanisms
of azide
to inhibit
mechanism
However,
not
be
to react
may
part
of the
involves
a ROS
a sizable
por-
inhibited
by
azide.
or the hydroxyl
Catalase,
when
concentrations,
scavengers,
mannitol
and benzoate.
added
to the system
in relatively
large
did provide
some
partial
protection.
This suggests
that GSH
is also reacting
with a species
that has yet to be identified.
One possibility
is that the
oxidant
is a lipid
peroxide,
since
the formation
of
Azide,
which
is
the activity
of
singlet
oxygen,
consumption
of
known
to have the capacity
to inhibit
heme
enzymes
as well as scavenge
also
provided
protection.
Since
the
GSH
by stimulated
PMNs
could have
been
reaction
leukotriene
by PMNs
is now known
to involve
GSH.
Therefore,
the possibility
that GSH
is reacting
with a
lipid peroxide
requires
additional
study.
The complex
nature
of the intact
cell system
made it
difficult
to clarify
the reaction
involving
GSH.
We
therefore
evaluated
the possibility
that
one of the
factors
released
by granulocytes
might
be stable
enough
to be detected
in the supernatants
of these cells
studies
capacity
in the
a
direct
to evaluate
of hydrogen
buffer
used
with
H202,
we
did
further
this possibility.
We studied
the
peroxide
to directly
oxidize
GSH
for our experiments.
Similar
to
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
102
SAGONE
after
incubation
with zymosan.
case,
although
the amount
detected
represents
a small
oxidizing
scavengers
during
This proved
to be the
of the stable
oxidant
fraction
of the GSH-
capacity
in the cell
and agents
to the
stimulation
with
system.
The
granulocyte
zymosan
addition
suspensions
suggests
that
of
the
compound
required
hydrogen
peroxide
and heme,
or
heme-containing
enzyme,
for its production.
This was
indicated
by the protection
afforded
by catalase
and
cyanide.
The factor
does not seem to require
superoxide or hydroxyl
radical,
since SOD,
benzoic
acid, and
mannitol
did not impair
the production
of the factor.
In
contrast
to the inhibitory
effects
of azide and catalase
on the oxidation
of GSH
when added
to the granulocyte suspension,
neither
of the agents
prevented
the
reaction
of the factor
with GSH
when
added
to the
supernatant
indicates
production
that
after the granulocytes
were removed.
This
H202 and heme may be required
for the
of the compound
the reaction
of the factor
excludes
the possibility
Further,
this
exclude
Tsan
experimental
but
are not participants
with GSH.
This
that
the compound
result
also
singlet
oxygen
as the ROS.
has recently
demonstrated
oxidized
to a sulfoxide
would
that
by a compound
in
observation
is H202.
seem
to
is
is gener-
ated by zymosan-stimulated
granulocytes.6
This compound,
similar
to the factor
we have
measured,
is
dependent
on hydrogen
peroxide
and
heme
for its
production,
but not superoxide
or hydroxyl
radical.6
Tsan
suggested
Therefore,
interact
supernatants.
methionine
the
we
with
was
ROS
might
Our
experiments
able to react
factor
that oxidized
GSH.
This
with relatively
low concentrations
suggests
that the
with methionine,
ROS
may
GSH.
Hypochlorous
oxidizing
factor.
Other
and Tsan
are
They observed,
be hypochlorous
studied
the ability
the factor
present
described
be the
be oxidized
directly
of H202. However,
lower concentration
demonstrated
directly
with
the
to
that
same
could be demonstrated
of methionine.
This
by Tsan, which
same
one reacting
acid
is one candidate
recent
observations
also consistent
similar
to our
acid.
of methionine
in our granulocyte
with
results,
reacts
with
for the
by Turkall
this possibility.’7
that GSH
could
by relatively
high concentrations
GSH
could be oxidized
by a much
of H202 in the presence
of myelo-
peroxidase
and halogens.
The reaction
appears
to be
mediated
by hypochlorous
acid. These observations
are
directly
revelant
to ours and establish
several
points.
First,
GSH
can react
nonenzymatically
with hypochlorous
acid. Second,
the rate constant
for the reaction
of GSH
with hypochlorous
acid appears
to be higher
than
for the reaction
of GSH
explain
the apparent
preferential
the hypochlorous
acid released
with H202. This
reaction
ofGSH
by PMNs
rather
H2O2,
as we observed
in our
experiments.
regard,
the factor
reported
by Tsan
that
methionine
and the one reported
by Carp9
yates
serum
alpha-I
protease
inhibitors
with
dryl
the methionine
group
and possibly
other
sulfhygroup
of this enzyme
are probably
the same.
Recently,
to bacteria
relatively
Thomas
react
stable
These
oxidants,
react
with the
regard,
Thomas
dryls,
suggesting
in
In this
reacts
with
that mactiby reacting
the
bacteria
et al. reported
rapidly
oxidants,
HOC1
added
as well as HOC1,
have the capacity
to
sulfhydryl
groups
of bacteria.
In this
used DTNB
to measure
the sulfhythat the major
sulfhydryl
consumed
was
GSH.
Further,
Thomas
that the biologic
half-life
of HOC1
its reactivity.
This
consideration
stable
oxidant
detected
tions may not be HOCI.
zymosan-
that
with amino
acids to produce
known
as chloramines.’8”9
under
Weiss
suggested
is short
suggests
because
that
our experimental
et al. demonstrated
or PMA-stimulated
granulocytes
of
the
condithat
could
chlo-
rinate
an exogenous
amine
(taurine)
by a myeloperoxidase-dependent
process
(azide
inhibitable)
to form
methionine
that
with
ET AL.
would
with
than
chloramine.2#{176} This
compound
could
be detected
a
by its
capacity
to oxidize
the thiocompound,
5-thio-2-nitrobenzoic
acid (TNB),
to DTNB
and oxidize
I to 12.
Further,
the supernatants
from stimulated
granulocyte
were found
to have some capacity
to oxidize
TNB
in
the
absence
stable
studies.
of taurine,
suggesting
oxidant
similar
to the
The amount
of oxidant
the
presence
of a
one observed
generated
from
in our
zymo-
san-stimulated
PMNs
reported
by Weiss
is similar
to
that observed
in our experiments.
Therefore,
the observations
of Thomas,
et al. and Weiss
et al. suggest
that
the relatively
stable
be a chloramine(s).
with this possibility.
with
failure
natant
HOC1
but
not
oxidant
noted in our studies
could
One of our results
is consistent
H2O2 is known
to react
rapidly
with
of H202 to remove
is more consistent
chloramine.
the
with
Therefore,
oxidant
from
a chloramine
the
the superthan with
HOC1.
On the other
hand,
Thomas
has shown
that
monochloramine
(NH2C1)
can be extracted
by chloroform.
The oxidant
demonstrated
in our studies
could
not be extracted
by chloroform
or ether.
Therefore,
if
the oxidant
is a chloramine,
it would
be most
higher
molecular
weight
or more polar.’8”9
A similarity
of the apparent
mechanism
production
of the compound
that reacts
with
likely
of
for the
GSH
to
that reported
for the oxidation
of halogens
by Klebanoff et al.’5 prompted
us to do additional
experiments
on the capacities
of the factor
to react
with halogen.
Our observations
factor
that has
indicate
the capacity
that the supernatant
to oxidize
iodide
and fix iodide
to protein.
The capacity
tant to oxidize
I to 12 was somewhat
observed
for GSH,
but in a similar
has a
to iodine
of the supernalower than that
range.
The reac-
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MECHANISMS
tions
FOR THE
103
BY PMNs
However,
H202
is clearly
required
for the
of the factor,
since granulocyte
suspensions
species
strates
may be derived
as a consequence
with
failed
catalase
to produce
during
this
reaction
factor.
Also,
Whether
with
the
and GSH
removed
the
that was related
to this oxidation
reaction.
vides evidence
that the compound
reacting
compound
This prowith both
methionine
halogen
could
inorganic
hypochlorous
We are
of the
hampered
However,
tant
and
be
mediated
organic
currently
by
one
compounds
attempting
in
of
to identify
the
to
with
a chloramine-relatively
reactivity
with
1,
thiocompound.
We have
recently
oxidized
reaction
by
that
radicals.2’
is
also
reported
stable,
and
reported
zymosan-stimulated
appears
to be
the
ability
that
One
reaction
resistant
to
a
granulocytes
mediated
by
acid
is
in
hydroxyl
a
Of note is that the oxidation
of benzoic
acid
azide
inhibitable,
similar
to the
reaction
here for GSH.
However,
benzoic
acid in high
concentrations
factor
did
related
not
to the
prevent
the
consumption
formation
of the
of GSH
for
granulocyte
to oxidize
benzoic
considerations
reactive
oxygen
from H2O2 or equivalent
subof a heme-related
cofactor.
reactions
are
mediated
or different
study.
by one
ones
is not
enzyme
clear
and
supernatants
of these
cells and which
is present
in
amounts
sufficient
to oxidize
several
times the endogenous GSH content
of the cells from which
it is derived.
Presumably,
this compound
is released
by PMN
at
sites
of inflammation
and may
mediate
damage
to
tissues
nature
factor
further.
Our
experiments
have
been
by instability
of the oxidant
during
storage.
the characteristics
of the factor
seem consis-
H202,
these
These
of multiple
In summary,
our
experiments
demonstrate
that
stimulated
granulocytes
release
a reactive
oxygen
species that
is stable
enough
to be harvested
in the
several
addition
the production
(myeloperoxidase)
requires
additional
addi-
GSH
and methionine
is also capable
of reacting
with
halogen.
However,
this does not aid in the definite
identification
of the compound,
since the reaction
with
the
compound.
that
zymosan
of
by the same
suggest
supplemented
tion
mediated
since
the
added
activity.
production
OF GSH
be attributed
to hydrogen
peroxide,
to the supernatant
did not eliminate
cannot
catalase
OXIDATION
or remove
length
of
and
have
ceased
its enzymes
important
of GSH
time
after
the
to function.
feature
of our observations
with this factor
may provide
is that the
a quanti-
tative
measure
of the amount
of this compound
that is
produced
by granulocytes.
Our experiments
also mdicate that the rate constants
for the reaction
of methionine and GSH
with the factor
are similar.
This observation
indicates
that GSH
may be important
in the
cellular
groups
protection
of other
observations
of methionine
amino
acids
and
indicate
protection
of GSH
and
the factor
from the supernatants
obtained
from granulocytes
after
zymosan
stimulation.
Thus,
it appears
that the oxidation
of benzoic
acid and GSH
are not
a considerable
of cells
to protect
other
thione
an additional
role
against
oxidant
the cell against
organic
peroxides
peroxidase
experiments
directly
with
and the
proteins.
has
provide
oxidants
sulfhydryl
Thus,
our
for GSH
injury.
The
hydrogen
as a cofactor
been
well
in the
capacity
peroxide
for
gluta-
can
react
documented.
evidence
that
GSH
other than peroxides.
Our
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1984 63: 96-104
Mechanisms for the oxidation of reduced gluthathione by stimulated
granulocytes
AL Jr Sagone, RM Husney, MS O'Dorisio and EN Metz
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