Exp ToxicPathol 1992; 44: 339-343
Gustav Fischer Verlag Jena
Institute of Pharmacology and Toxicologyl), Friedrich Schiller University lena, Germany
Department of Pediatrics2 ), Friedrich Schiller University lena, Germany
The influence of systemic hypoxia and reoxygenation on the gluta-
thione redox system of brain, liver, lung and plasma in newborn rats
A. REUTER2) and W. KLINGER!)
With 4 figures
Received: August 29, 1991; Accepted: September 21, 1991
Address for correspondence: Dr. med.A. REUTER, Friedrich-Schiller-Universitat lena - Kinderklinik, KochstraBe 2,
D - 0 - 6900 lena, Deutschland
Keywords: Glutathione; Hypoxia; Fasting; Newborn rats; Reoxygenation; Brain, glutathione redox system; Liver, glutathione
redox system; Lung, glutathione redox system.
Summary
The concentrations of reduced (GSH) and oxidized
glutathione (glutathione disulfide, GSSG) in lung, liver, brain
and plasma of newborn rats were investigated under the condi-
tion of reversible hypoxia. Brain and lung of newborn rats seem
to be susceptible to reversible hypoxia. We found an increase in
GSSG concentration after hypoxia in these organs. This altera-
tion of the GSH-GSSG redox system was reversible within 2
hours of reoxygenation. A second increase in cerebral GSSG
concentration after 4 hours of reoxygenation was connected
with fasting during the experiment. In the liver we found a
hypoxia dependent decrease in the GSHlevel, followed by a
decrease in GSSG concentration. The increased GSSG concen-
trations in lung and brain are accompanied by an enhancement
of plasma GSSG concentration.
Introduction
Glutathione (reduced form: GSH; oxidized form:
glutathione disulfide, GSSG) is ubiquitously distributed in
most mammalian tissues. GSH is known to influence various
biological phenomena. The protection of cells from the
influence of free radicals, which cause formation of lipid
peroxides, followed by destruction of cells, is one of the
main functions of GSH (MEISTER and ANDERSON 1983;
NAGASAKO et al. 1989). YOUNES et al. (1984) showed a
close correlation between the activity of GSH peroxidase and
formation of lipid peroxides. They observed the formation of
lipid peroxides in liver after administration of
acetaminophen only in GSH deficiency. JAECKSON (1990)
found a marked increase in GSSG level in lung tissue after
hypoxia and reoxygenation. Similar effects were observed
by ADAMS et al. (1983) in plasma after administration of
diquat and t-butylhydroperoxides. Also CHANG et al. (1989)
described a reversible increase in the GSSG concentration of
10*
plasma after hypoxia. The liver may playa central role in
GSH homeostasis of the organism because it is supposed at
present to be the main GSH export organ. In general, the
plasma GSSG level is accepted as an index of oxidative
stress.
The level of cerebral GSH is age dependent. BIEN et al.
(1990) found an increase in GSH concentration in brain of
praenatal rats until birth, followed by a decrease immedia-
tely after birth and a postnatal increase in GSH concentra-
tion. KRETZSCHMAR et al. (1990) found an increase in GSSG
level parallel to an increase in lipid peroxide concentration in
brain of newborn rats under the condition of reversible
hypoxia.
We investigated the GSH status in various organs of
newborn rats under conditions of reversible hypoxia. We
were interested in the reaction of the GSH status of several
organs in this situation and in this age. In addition we
investigated the change of GSH concentrations in organs
under the condition of 5 hours of starvation corresponding to
the duration of the experiment to find out possible additional
changes after this period of starvation.
Material and methods
Animals: Newborn rats (Uje-WIST) of both sexes of our
Institute's colony bread at an age of 19 hours at maximum
were used in these experiments. The dams were raised and
'housed under controlled conventional conditions, natural
day-night cycle, temperature 22-24°C, humidity> 50 %,
free access to tap water and standard diet (pellets VTD-I,
R13; Futtermittelwerke Biesenthal).
Hypoxia and reoxygenation experiment: In the first
series of experiments the animals of the experimental groups
Exp Toxic Pathol 44 (1992) 6 339
[n = 5-6] were placed for one hour in an airtight jar venti- B
lated with nitrogen containing 9 % oxygen. Thereafter the
newborn rats were killed immediately or restored for 1, 2 or <5
4 hours under normal conditions (temperature 25-30°C, L..
1::
o
humidity > 50 %). The animals of the control group u

[n = 5-6] were killed at the beginning of the experiment.

After killing whole liver, brain, lung and plasma were ~10 , .. __ x ~ 100~
~ ~.
removed rapidly, weighed or measured and processed (see
:g 60 60
below). In the second series of experiment the animals were
removed from the dams and placed for 5 hours under normal o 2 3 5 o 2 3 5
condition.
Biochemical procedure: For the determination of GSH
<5
and GSSG lung, brain and liver tissue samples were
homogenized with 0.2 M sodium phosphate -0.005 M ~
EDTA buffer (pH 8.0) and 25 % metaphosphoric acid in ....o
u

relation 1 : 11 : 4. The homogenates were centrifuged

(12,000 g, 30 min, O°C). Supernatants were used for the
~10~ 100~

determination of GSH by ELLMAN'S method (1959) and :g 60 60

GSSG as described by HrSSIN and HILF (1976). Blood
samples pooled from 3 animals were obtained by heparinised
o 2 3 5 o 2 3 5
time (hours) time (hours)
pipettes. The samples were put into the buffer described in
the original methods without delay, centrifuged for plasma,
and the plasma GSH and GSSG levels were measured
Fig. 1. The influence of hypoxia and duration of reoxygenation
according to ADAMS et al. (1983).
(A) as well as of 5 hours of starvation (B) on the contents of
Statistics: All data are given as percentage of daily GSH and GSSG in the liver of newborn rats. Means ± SEM are
controls. The statistic significance between the sets of data given in % of daily control in the time 0 [n = 5 -6]. Asterisks
was assessed using the Mann-Whitney-test, p:::; 0.05. indicate significant differences between experimental and con-
trol groups (p:::; 0.05).
Results
A B
The effects of hypoxia and reoxygenation as well as of 5
h~ia
hours of starvation on GSH and GSSG concentration in
liver, brain, lung and plasma are shown in the figures 1-4. 0L..
x
1::
The liver showed a significant decrease in GSH concentra-
~
0

tion after 1 hour of hypoxia, followed by an increase during ....

u

reoxygenation, but the control level was not achieved after 4

hours of reoxygenation. In the fasted newborn rats without
~100
0
:x::
~ 100

hypoxic exposure the GSH level was significantly decreased

:g 60 60
after 5 hours of starvation. Hepatic GSSG levels tended to 0 2 3 5 0 2 3 5
decrease during hypoxia, followed by a further decrease
during reoxygenation, but after 4 hours of reoxygenation the
control levels were achieved. In contrast, animals without h~ia x
hypoxic exposure showed a significant decrease in GSSG
concentration after 5 hours of starvation compared to con- ....<5c:
L..

trols. 0
u x
Cerebral GSH concentrations were not changed under the x x
't5
conditions of hypoxia and reoxygenation. In fasted rats brain ~100 100 ~
GSH levels increased significantly during 1 hour of starva- l:J
V1
tion, followed by a second increase after 3 hours but begin to :g 60 60
decrease thereafter. GSSG concentrations in brain increased
0 2 3 5 0 2 3 5
during hypoxia and remained significantly increased after 1
time ( hours) time (hours)
hour of reoxygenation. After 2 hours of reoxygenation
GSSG levels returned to control level, followed by a second
Fig. 2. The influence of hypoxia and duration of reoxygenation
marked enhancement after 4 hours of reoxygenation. After 3
(A) as well as of 5 hours of starvation (B) on the contents of
and 5 hours of starvation we found a significant increase in GSH and GSSG in the brain of newborn rats. Means ± SEM are
brain GSSG concentrations. given in % of daily control in the time 0 [n = 5-6]. Asterisks
In the lung we found similar changes as in the brain. Lung indicate significant differences between experimental and con-
GSSG concentrations increased markedly after 1 hour of trol groups (p:::;O.05).

340 Exp Toxic Pathol44 (1992) 6

 A B hypoxia and remained significantly increased in comparison
to controls after 1 hour of reoxYgenation. With increasing
duration of reoxygenation we observed a continuous
decrease in GSSG concentrations in lung. The GSH levels of
the lung remained unchanged under the conditions of
hypoxia and reoxygenation. In fasted rats a small decrease in
GSSG levels was shown during the fIrst 2 hours, followed by
a return to control levels. The GSH levels were unchanged
60
during fIve hours of starvation.
0 2 3 5 o 2 3 5 In plasma we found only negligible variations of GSH
concentrations in hypoxia and reoxygenation as well as
without hypoxia. Also in fasted newborn rats without
h~Q
-0 )(
hypoxia we found no signifIcant changes in plasma GSSG
.....C concentrations, but the plasma GSSG concentration

-
L.

0
u increased continuously under the conditions of hypoxia and
C)
reoxygenation. After 4 hours of reoxygenation a small
decrease in GSSG concentration was measurable, but it was
l:)
VI still signifIcant compared to the control level.
~6 60
0 2 3 5 o 2 3 5 Discussion
ti me (hours) time (hours)
The results show the different reactions of the organs of
Fig. 3. The influence of hypoxia and duration of reoxygenation newborn rats under the condition of hypoxia and reoxygena-
(A) as well as of 5 hours of starvation (B) on the content of GSH tion. Our results are similar to the results of KRETZSCHMAR
and GSSG in the lung of newborn rats . Means ± SEM are given et aI . (1990). The decrease in hepatic GSH concentration
in % of daily control in the time 0 [n = 5-6] . Asterisks indicate under hypoxic conditions could be connected with an inhibi-
significant differences between experimental and control groups tion of the ATP dependent GSH synthesis. ATP defIciency is
(p::50.05) . caused by decreased oxidative phosphorylation during
hypoxia (VENCENT et aI . 1982). The liver formes the amino
acid cystein, as precursor of GSH synthesis, from
methionine by the cystathione pathway. S-adenosyl-
B methionine synthetase is more susceptible to ATP deficiency
as glutamylcystein synthetase and GSH synthetase (SHAN et
al. 1990). It was concluded that ATP deficiency could cause
-0 the decrease in GSH synthesis by an inhibition of the cystein
~o supply. SHAN et al. (1990) demonstrated a reduced con-
u
sumption of oxygen in isolated hepatocytes during reoxyge-
~100~ 100~ nation after anoxia in connection with diminished ATP
~
:x::: synthetase activity simultaneously with a reduced Ca2 + -level
~ 60 60 of mitochondria. The decreased ATP supply for GSH
synthesis could be responsible for the slowness of the post-
0 2 3 5 o 2 3 5
hypoxic increase in hepatic GSH concentration. The
hypoxia
,...--, decrease in GSSG concentration during reoxygenation might
-0
L.
)(
be caused by a preceding decrease in GSH level. Also
1::

-
0
u
JAESCHKE (1990) observed a diminution of GSSG concentra-
0 tion in isolated rat liver after perfusion with a hypoxic
~100 100 solution. The reduction of GSSG formation is assumed to be
l:)
VI
caused by a suppressed formation of superoxide anions
:g 60 60 during hypoxia. Hepatic GSSG formation is recognized as
marker for oxidative stress. We observed a significant
0 2 3 5 0 2 3 5 decrease in GSSG as well as GSH concentrations in the liver
time (hours) time (hours) of newborn rats after 5 hours of starvation. It could be
possible that these decreases are connected with a failure of
Fig. 4. The influence of hypoxia and duration of reoxygenation
(A) as well as of 5 hours of starvation (B) on the contents of food supply.
GSH and GSSG in the plasma of newborn rats. Means ± SEM In the lung we found a marked increase in GSSG concen-
are given in % of daily control in the time 0 [n=5-6] . tration immediately after 1 hour of hypoxia. This increase
Asterisks indicate significant differences between experimental could be a consequence of the formation of SUbstances,
and control groups (p::50.05). which are reduced by reaction with GSH. Experiments of

Exp Toxic Pathol 44 (1992) 6 341

JENKINSON et al. (1988) with isolated and ventilated lungs of
rats showed both an increased GSSG level in lung tissue and
an increased delivery of GSSG from cells into the perfusate
during reoxygenation after hypoxia. The authors suppose a
connection between the increase in GSSG concentration with
the appearance of free radicals and the formation of hydro-
genperoxide as well as of lipid hydroperoxides. Xanthine
oxidase could be responsible for the formation of superoxide
anions in posthypoxic tissues. Xanthine dehydrogenase as
precursor of xanthine oxidase is present in normal tissues.
Xanthine dehydrogenase is converted into xanthine oxidase
during acidotic stages, caused by hypoxia. Also a conversion
of ATP to monophosphate can be observed, followed by the
formation of hypoxanthine. Increased xanthine oxidase activ-
ity as well as the presence of oxygen during reoxygenation are
prerequisites for an increased formation of superoxide anions
(DENEKE and FANBURG 1989; JENKINSON et al. 1988; PICK-
FORD et al. 1990). Under conditions of starvation over 5 hours
only small changes of GSSG concentrations were found in
lungs, which should be connected with biological variations.
Cerebral GSSG concentration increased during hypoxia. The
cause of the second marked increase in GSSG concentration
of brain 4 hours after reoxygenation is not clear. We expect a
connection with a failure of food. Without hypoxia we
observed a small but significant increase in cerebral GSSG
concentrations after 3 and 5 hours of starvation. It could be
possible that this increase in GSSG concentration is amplified
by hypoxia. The cause of ip.creased GSSG concentration in
the brain of fasted rats is unknown. It might be possible that
the increase in GSSG concentration is an effect of fluid loss.
Under normal conditions glucose is the only source of energy
in the brain. The lack of exogenous glucose supply lead to a
deficiency ofNADPH, which is a cofaktor ofGSH reductase.
In this way usually formed GSSG cannot be reduced to GSH
(SHAN et al. 1990; SMITH et al. 1990; TRIBBLE and JONES
1990). We observed a significant increase in the GSH
concentration of brain after 5 hours of starvation. The cause of
this increased cerebral GSH concentration is unknown. KAN-
NAN et al. (1990) observed an uptake of labeled GSH from
blood into brain. This uptake was influenced neither by
inhibition of gamma-glutamyltranspeptidase nor by several
amino acids. A reduced uptake was only observed after
administration of unlabeled GSH. A particular carrier of GSH
was postulated which transports GSH across the blood-brain-
barrier. The synthesis of GSH inside of the brain should be
also possible because enzymes of synthesis and breakdown
were found (BIEN et al. 1990). Because of the incomplete
blood-brain-barrier and the high plasma concentration of
GSH in newborn rats as well as low activities of GSH
synthesis enzymes in the brain of newborn rats
(KRETZSCHMAR et al. unpublished results) the uptake of GSH
from blood into brain is more likely.
In plasma we found a continuous increase in GSSG
concentrations immediately after hypoxia up to 2 hours of
reoxygenation. After 4 hours of reoxygenation we established
a small decrease in GSSG level but it was still significant
compared to controls. These results correspond to observa-
tions by ADAMS et al. (1983), JAESCHKE (1990), and JENKIN-
342 Exp Toxic Pathol 44 (1992) 6
SON et al. (1988). They consider plasma concentration as a
marker for oxidative stress. When GSSG levels increase
within the cells, it is removed out of the cells resulting in an
increase in plasma GSSG concentration and, consequently, in
the maintenance of the intracellular redox potential. There
should be a strong connection between the formation of
hydroperoxides and free radicals during reoxygenation. For-
mation of lipid peroxides in hepatocytes, following the
formation of radicals, occurs only by simultaneous decrease
in GSH concentration to less than 10-15 % of normal level.
This part of cellular GSH content is supposed to correspond to
mitochondrial GSH (COMPORTI 1989; KApLOWITz et al.
1985). In contrast to these results, KRETZSCHMAR et al.
(1990) demonstrated a connection between an increase in
GSSG concentration and the lipid peroxide level of brain in
newborn rats under the condition of reversible hypoxia. The
cerebral GSH level was unchanged. The hepatic lipid perox-
ide as well as GSSG concentrations were unchanged under the
same conditions although the hepatic GSH level was dimished
to 70 % of controls. The brain of newborn rats is supposed to
be susceptible to reversible hypoxia without marked alteration
of GSH concentration.
The causal role of lipid peroxidation of cellular membranes
in oxidative cell damage is doubted. TRIBBLE et al. (1987)
reported an alteration of membrane fluidity and permeability
to various compounds and ions prior to the destruction of
special transport systems. It is assumed that the Ca2+ -ATPase
of mitochondria and the endoplasmatic reticulum play an
important role. The oxidation of sulfhydroxyl groups of this
enzyme caused its inactivation, followed by an increase in
cytosolic Ca2+ concentration. The consequence of increased
cytosolic Ca2 + concentration is the activation of several
enzymes such as phospholipase and some proteases. The
uncontrolled activation of these enzymes causes the cell
damage, finally. GSH is able to protect sulfhydryl groups of
Ca2 + - ATPase from oxidation. In this way the cells can be
protected against oxidative stress via a GSH dependent
action.
Acknowledgements: The authors wish to thank ELKE KARGE for
her technical assistance.
References
ADAMS JD, LAUTERBERG J, MITCHEL JR: Plasma glutathione
and glutathione disulfide in the rat: Regulation and response
to oxidative stress. J Pharmacol Exp Ther 1983; 227:
749-754.
BIEN E, SKORKA G, REX, H, et al. : Glutathione in developing rat
brain. Biogenic Amines 1990; 7: 278-281.
CHANG SW, STELZNER TJ, WElL JV, et al.: Hypoxia increases
plasma glutathione disulfide in rats. Lung 1989; 167:
269-276.
COMPORTI M: Glutathione depleting agents and lipid peroxida-
tion. Chern. Phys. Lipids 1989; 45: 143-169.
DENEKE SM, FANBURG BL: Regulation of cellular glutathione.
Am J Physiol 1989; 257: L163-173.
ELLMAN GL: Tissue sulfhydryl groups. Arch Biochem Biophys
1959; 82: 70-77.
HISSIN PJ, HILF R: A fluorometric method for determination of
oxidized and reduced glutathione in tissues. Anal Biochem
1976; 74: 214-226.
JAECKSON RM, VEAL CF: Effects of hypoxia and reoxidation on
lung glutathione system. Amer J Physiol 1990; 259:
H518-524.
JAESCHKE H: Glutathione disulfide as index of oxidant stress in
rat liver during hypoxia. Amer J Physiol 1990; 258:
G499-505.
JENKINSON SG, MARCUM RF, PICKARD JS, et al.: Glutathione
disulfide formation during hypoxia and reoxygenation of rat
lung. J Lab Clin Med 1988; 112: 471-480.
KANNAN R, KUHLENKAMP JF, JEANDIDIER E, et al.: Evidence
for carrier-mediated transport of glutathione across the
blood-brain-barrier in the rat. J Clin Invest 1990; 85:
2009-2013.
KAPLOWITZ N, Aw TY, OOKHTENS M: The regulation of
hepatic glutathione. Ann Rev Pharmacol Toxicol 1985; 25:
715-744.
KRETZSCHMAR M, GLOECKNER R, KLINGER W: Glutathione
levels in liver and brain of newborn rats: Investigations of
the influence of hypoxia and reoxidation of lipid peroxida-
tion. Physiol Bohemslov 1990; 39: 257-260.
MEISTER A, ANDERSON ME: Glutathione. Ann Rev Biochem
1983; 52: 711-760.
NAGASAKO Y, FUJLL S, KUNEKO T: Microsomal glutathione
dependent protection against lipidperoxidation. Arch
Biochem Biophys 1989; 2711: 82-86.
PICKFORD MA, GOWER JD, DaRE C, et al.: Lipid peroxidation
and ultrastructural changes in rat lung isografts after single
passage organ flush and 48 hour could storage with and
without one-hour reperfusion in vivo. Transplantation 1990;
144: 210-218.
SHAN X, Aw TY, JONES DP: Glutathione dependent protection
against oxidative injury. Pharmacol Tl}erap 1990; 47:
61-71.
SMITH LJ, HORCHER P, ANDERSON J, et al.: Effect of fasting on
the lung glutathione redox cycle in air- and oxygen-exposed
mice: Beneficial effects of sugar. J Lab Clin Med 1990; 116:
717-723.
TRIBBLE DL, Aw TY, JONES DP: The pathological significance
of lipid peroxidation in oxidative cell injury. Hepatology
1987; 7: 377-387.
TRIBBLE DL, JONES DP: Oxygen dependence of oxidative
stress. Rate of NADPH supply for maintaining the GSH pool
during hypoxia. Biochem Pharmacol 1990; 39: 729-736.
VENCENT MF, VAN DEN BERGHE G, HERS HG: The pathway of
adenine nucleotide catabolism and its control in isolated rat
hepatocytes subjected to hypoxia. Biochem J 1982; 202:
117-123.
YOUNES M, SIEGER LP: Interrelation between lipidperoxidation
an other hepatotoxic events. Biochem Pharmacol 1984; 33:
2001-2003.
Exp Toxic Pathol 44 (1992) 6 343