Mechanisms of Ageing and Development
103 (1998) 195 – 207

Transport of glutathione conjugate in erythrocytes
from aged subjects and susceptibility to oxidative
stress following inhibition of the glutathione
S-conjugate pump
8
I: lhan Onaran a,*, Ahmet Ozaydin a, Mustafa Gultepe b,
¨
Gonul Sultuybek a
¨ ¨
a

Di6ision of Biomedical Sciences, Cerrahpasa Medical Faculty, Istanbul Uni6ersity, Istanbul, Turkey
¸
b
GATA, Military Educational Hospital, Department of Biochemistry, Istanbul, Turkey
Received 9 September 1997; received in revised form 20 February 1998; accepted 9 March 1998

Abstract
The aim of the present study was to investigate the effect of donor aging on the
glutathione conjugate transport in erythrocytes and whether it plays a role in the resistance
to oxidative stress of the erythrocytes of aging subjects. In our comparative study on intact
erythrocytes of healthy aging and young adults, in which 2,4-dinitrophenyl-S-glutathione
(DNP-SG) was used as model glutathione S-conjugate, we found that the efﬂux of DNP-SG
remained unchanged in the aged subjects. This result suggests that the detoxiﬁcation function
is maintained against the chemical stress employed in erythrocytes of aging subjects. In the
assay conditions used, which were optimized to obtain maximal inhibition of glutathione
S-conjugate transport, our results also indicated that the susceptibility of erythrocytes to in
vitro lipid peroxidation generated by cumene hydroperoxide was enhanced by pretreatment
with DNP-SG inhibitors in both age groups. However, the difference in susceptibility was
not a function of aging. Further, the results suggested that inhibition of glutathione
S-conjugate pump may impair cellular protection of the erythrocytes against oxidative
damage. © 1998 Elsevier Science Ireland Ltd. All rights reserved.

* Corresponding author. Present address: Ortaklar Cd. Butan Sk., Hyzal Apt. No:2 D:3, Mecidiyekoy
¨
¨
80290, I: stanbul, Turkey. Fax: +90 212 5299433.
0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0047-6374(98)00042-6

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Keywords: Aging; Erythrocyte; Glutathione S-conjugate; Glutathione; Transport; Oxidative
stress

1. Introduction
Transport of glutathione S-conjugates is an important element of xenobiotic
detoxiﬁcation. Glutathione is enzymatically conjugated to many electrophilic compounds in a reaction catalyzed by glutathione S-transferases (GST). The glutathione S-conjugates formed in these reactions are afterwards exported out of cells
by a speciﬁc adenosine 5%-triphosphate (ATP)-dependent glutathione S-conjugate
pump (for review see Zimniak and Awasthi, 1993). The active transport systems for
glutathione S-conjugates have been described for a number of human tissues and
this system has also been identiﬁed in human erythrocyte membranes (Awasthi et
al., 1983; Ishikawa and Sies, 1984; La Belle et al., 1986; Kunst et al., 1989). The
glutathione S-conjugate pump also plays a signiﬁcant role in cellular defence under
oxidative stress conditions since it is able to transport glutathione conjugates of
lipid peroxidation products and extrudes oxidized glutathione (GSSG) (Ishikawa,
1989; Grune et al., 1991; Akerboom et al., 1992). Erythrocytes are a good model
system for the explanation of such processes and the molecular nature of the defect
in the transport of glutathione conjugates since their lack of k-glutamyltranspeptidase precludes further metabolizing of formed conjugates. Although the transport
system exhibited a broad substrate speciﬁcity towards different types of glutathione
S-conjugate (Ishikawa, 1989), the compound most widely used in such studies is
2,4-dinitrophenyl-S-glutathione (DNP-SG) formed from 1-chloro-2,4-dinitrobenzene (CDNB) in a reaction catalyzed by GST.
Different detoxiﬁcation mechanisms such as glutathione, GST, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase but not glutathione
S-conjugate transport have been tested for aging process (Stohs et al., 1984;
Jozwiak and Jasnowska, 1985; Stohs and Lawson, 1986; Al-Turk et al., 1987;
Farooqui et al., 1987; Foldes et al., 1988; Matsubara and Machado, 1991; Picot et
al., 1992). Results from a number of studies have suggested that a glutathione-deﬁciency state is a general phenomenon in aging cells, since aging-speciﬁc decrease in
glutathione levels was observed in all tissues including erythrocytes (Stohs et al.,
1984; Stohs and Lawson, 1986; Al-Turk et al., 1987; Farooqui et al., 1987;
Matsubara and Machado, 1991). Furthermore, different results showing decreased
or unchanged GST activity in elderly subjects when compared with young subjects
were also reported (Stohs et al., 1984; Al-Turk et al., 1987; Picot et al., 1992). On
the other hand, results of our previous study indicated that the susceptibility of
intact erythrocytes to in vitro oxidative stress by cumene hydroperoxide (CumOOH) in aging subjects is not much greater than young subjects (Onaran et al.,
1997). The above observations prompted us to study how the transport of glutathione conjugate in erythrocytes, generated through the reactions of glutathione
and GST, is affected by the aging process. We also wished to examine whether

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197

glutathione S-conjugate transport has any effect on the resistance to oxidative
damage of the intact erythrocytes of aging subjects.

2. Materials and methods

2.1. Subjects
Subjects were young (aged 18 – 31) or elderly (aged 67–85) normal volunteers. All
subjects were non-smokers and had normal blood counts, normal blood levels of
urea, glucose, creatinine, albumin, alkaline phosphatase, lactate dehydrogenase and
bilirubin, and no history of hematologic abnormality, recent infectious disease or
signiﬁcant medical illness. The participants were instructed not to take aspirin-like
drugs for 2 weeks prior to blood sampling.

2.2. Reagents
All reagents were of analytical quality whenever possible, obtained mainly from
Sigma (St. Louis, MO) and Merck (Darmstadt, Germany).

2.3. Preparation of erythrocytes
Fresh heparinized fasting blood sample was centrifuged at 1500× g for 10 min.
Erythrocytes were separated from plasma and buffy coat and then washed three
times with 10 vol of phosphate buffered saline (PBS, pH 7.4). Erythrocytes
containing chronologically young population rich with reticulocytes were removed
by discontinuous Percoll density gradient essentially as described by Rennie et al.
(1979). The gradient was built up in two layers containing Percoll with speciﬁc
density values between 1.100 and 1.124 g/ml. At the end of centrifugation while the
cells were collected at the interface of the Percoll above 1.124 g/ml, enriched
fraction of reticulocytes above low density were removed. The erythrocytes were
then washed twice with PBS.
Hemoglobin (Hb) concentrations in the samples were determined with Drabkin’s
reagent as described by Beutler (1984). Reticulocyte count in the fraction was
performed on glass slides after staining with 0.1% brilliant cresyl blue saline. The
number of reticulocytes in the high density cell fractions studied did not show
signiﬁcant differences between elderly and young control groups.

2.4. Reaction systems
For inhibition of glutathione conjugate transport, erythrocytes were incubated at
hematocrit 10% in PBS (with 8 mmol/l glucose) containing one of the following
reagents (15 min at 37°C with continuous shaking at 120 cycles/min): 5–30 mmol/l
sodium ﬂuoride; 0.025 – 0.2 mmol/l sodium o-vanadate; diluted 1/4000–1/1200
Tween 80 (polyoxyethylenesorbitan-monooleate). Controls were incubated with

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PBS alone. To check whether the glutathione conjugate transport was inhibited in
erythrocyte suspensions the export of DNP-SG was measured. Hemolysis was also
checked in samples and it was always below 1–2%. After preincubation with
inhibitors, lipid peroxidation was induced by addition of freshly prepared CumOOH (ﬁnal concentration, 0.1 mmol/l). Immediately after CumOOH addition,
the tubes were sealed and incubated at 37°C with continuous shaking at 120
cycles/min. In parallel, control samples were incubated under the same conditions
but without CumOOH. After 15 min, cell suspensions were centrifuged at 7000× g
for 1 min to sediment the erythrocytes. Both pellet and supernatant were used for
malondialdehyde (MDA) determination.

2.5. Measurement of the transport of glutathione S-conjugate
Transport of DNP-SG was measured according to the procedure of Board
(1981). Washed erythrocytes were resuspended in PBS and incubated with 1 mmol/l
CDNB for 15 min at 37°C to form DNP-SG. Then the cells were washed of excess
CDNB at 4°C and suspended at a hematocrit of 20% in the PBS (pH 7.4)
containing 1 mmol/l MgCl2 and 8 mmol/l glucose. The cell suspensions were then
incubated at 37°C and the export of DNP-SG was quantiﬁed by withdrawal of
aliquots of the cell suspensions after 1, 2, 3, and 4 h. After centrifugation, DNP-SG
was detected in the supernatant by spectrophotometric determination at 340 nm
using an mmol/l extinction coefﬁcient of 9.6.

2.6. Malondialdehyde (MDA) measurement
The concentration of MDA in the samples was measured with ﬂuorometric
determination improved by synchronous ﬂuorescence (Conti et al., 1991; Onaran et
al., 1997). For erythrocytes, 0.2 ml of packed cells were suspended in 0.8 ml PBS,
0.025 ml of 4% (w/v) butylated hydroxytoluene and 0.050 ml of 0.025 mmol/l
disodium-ethylenediaminetetraacetic acid, and 0.5 ml of 30% trichloroacetic acid
was then added. Tubes were vortexed and allowed to stand in ice for 2 h. After
centrifugation, assay conditions were the same in both the supernatant from this
step and the supernatant fraction from the erythrocyte suspension (Onaran et al.,
1997). The level of MDA was expressed as total mmol MDA/g Hb.

2.7. Chemiluminescence assay
Chemiluminescence measurements were carried out in a TriCarb 1500 liquid
scintillation analyzer in the single-photon count mode (Videla et al., 1984). Brieﬂy,
erythrocytes were resuspended in 3 ml of PBS containing 8 mmol/l glucose and
preincubated with indicated concentrations of ﬂuoride, vanadate or Tween 80 for
15 min at 37°C. The erythrocytes were transferred to scintillation vials. After the
addition of CumOOH (ﬁnal concentration, 0.1 mmol/l) the chemiluminescence
measurements were taken every 10 min for 60 min. The zero time is the time of
CumOOH addition.

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199

2.8. Glutathione content and glutathione S-transferase (GST) acti6ity
The concentration of glutathione was determined by the method of Tietze (1969).
Total GST activity with CDNB as substrate was measured according to Habig et
al. (1974).

2.9. Statistical analysis
All tests were performed in duplicate or triplicate samples, and the results were
expressed as means9 S.D. Statistical difference was determined by Student’s t-test,
with signiﬁcance deﬁned as P B0.05.

3. Results
The aging group (n =18) and young controls (n = 18) used in this study showed
no difference in their erythrocyte GST activity, 2.19 0.8 and 2.1 9 0.6 mmol of
CDNB conjugated/min per g Hb, respectively (P\ 0.05). However, we found
statistically signiﬁcant lower glutathione levels in older group. The mean9 S.D.
values of glutathione were 5.24 90.69 and 6.5990.71 mmol/g Hb for the elderly
and controls, respectively (P B 0.05).
On incubation with CDNB at 37°C, glutathione is completely depleted in
erythrocytes of the two age groups. Under these experimental conditions, the rate
of DNP-SG efﬂux of the erythrocytes from aging subjects during 4 h did not differ
from those of the young controls (P\ 0.05). The rate of transport of DNP-SG in
both age groups was linear for a period of at least 4 h (Table 1).
In order to ﬁnd out whether glutathione S-conjugate transport has any effect on
the resistance to oxidative stress of the erythrocytes of aging subjects, transport of
erythrocytes was inhibited by incubation with various inhibitors, including sodium
ﬂuoride, vanadate or Tween 80. The transport process was, more markedly,
inhibited as a function of the concentration of these inhibitors. Despite the
differences in inhibition proﬁles of the transport rate of DNP-SG elicited by these
agents, the inhibition kinetics of erythrocytes from aged subjects were not different
Table 1
Transport of 2,4-dinitrophenyl-S-glutathione in erythrocytes from aging and young subjects
Time (h)

Erythrocyte efﬂux (nmol DNP-SG/ml erythrocytes)
Aging group (n=18)

1
2
3
4

Young group (n =18)

4109 77
6859 117
9719 128
11749 165

390 942
674971
933 9114
1118 9157

All data expressed as mean9S.D.

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Fig. 1. The inhibition kinetics of DNP-SG transport elicited by various inhibitors and effects of
increasing concentrations of inhibitors on CumOOH-induced MDA formation in erythrocytes. Suspensions of erythrocytes from aged (n= 5) or young (n =5) subjects were treated with increasing concentrations of Tween 80 (A), ﬂuoride (B) or vanadate (C) as described in Section 2. After centrifugation the
supernatants were assayed for DNP-SG transport. MDA levels of erythrocytes treated with 0.1 mmol/l
CumOOH after pretreatment with inhibitors were also determined. Percent inhibition was expressed as
the percentage decrease of DNP-SG efﬂux from erythrocytes. The values are means of duplicate analyses
from 10 experiments.

Fig. 2. Effects of various inhibitors of the glutathione conjugate pump on CumOOH-induced lipid peroxidation in erythrocytes from healthy aging (n =18)
and young (n= 18) adults. DNP-SG inhibitors, Tween 80 (1/1200), ﬂuoride (30 mmol/l) or o-vanadate (0.2 mmol/l) were added before the erythrocyte
suspensions were incubated with 0.1 mmol/l CumOOH for 15 min at 37°C. MDA values shown are means with standard deviations.

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201

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Fig. 3.

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203

from that of young control subjects. When the erythrocytes were exposed to 0.1
mmol/l CumOOH after preincubation with inhibitors, increasing relative concentration of inhibitors resulted in an enhanced MDA formation. The production of
MDA elicited by CumOOH corresponded generally to the degree of inhibition in
DNP-SG transport caused by different inhibitors. As shown in Fig. 1, signiﬁcant
differences in MDA formation as a function of the concentrations of inhibitors
were not observed in erythrocytes of aged subjects in comparison to that of young
subjects. Therefore, the concentrations corresponding to 30 mmol/l ﬂuoride, 0.2
mmol/l vanadate and 1/1200 Tween 80 which were effectively able to inhibit the
DNP-SG transport were selected for further experiments. In these experiments,
MDA levels of erythrocytes treated with inhibitors of DNP-SG transport were not
signiﬁcantly different from the levels of erythrocytes treated with CumOOH alone
(Fig. 2). MDA levels also remained unchanged within each group when compared
to basal conditions (data not shown). However, MDA levels of erythrocytes treated
with CumOOH after preincubation with inhibitors were signiﬁcantly different from
those treated with CumOOH without the inhibitors (PB 0.05) (about 1.6–2-fold
increase over controls with CumOOH alone). Also in this case, no differences in
MDA overproduction were observed between the aging and the young groups, i.e.,
the ratios between values of the samples treated with CumOOH alone and values of
the samples treated with CumOOH after pretreatment with inhibitors were statistically unchanged (data not shown). The MDA production in erythrocytes which
were kept under oxidative stress was very similar for Tween 80 and vanadate.
Inhibition by ﬂuoride caused slightly less MDA production than other inhibitors
(Fig. 2).
Chemiluminescence intensity of erythrocytes treated with inhibitors of DNP-SG
transport were not signiﬁcantly different from the intensity of erythrocytes treated
with CumOOH alone or from those in their basal conditions (Fig. 3A); during 60
min, light output in erythrocytes remained constant. Addition of 0.1 mmol/l
CumOOH to erythrocytes pretreated with inhibitors enhances chemiluminescence
signal in erythrocytes of both age groups. However, no differences in spectral
distributions of chemiluminescence were observed between the aging and the young
groups (Fig. 3B), i.e., the ratios of increase in chemiluminescence between treated
and control (erythrocytes treated with CumOOH alone) values were statistically
unchanged (data not shown). In addition, in the erythrocytes treated with Tween 80
or vanadate, onset and maximal chemiluminescence were slightly higher than those
of ﬂuoride.
Fig. 3. (See left) Effects of the addition of CumOOH to the reaction medium following preincubation
with various inhibitors of glutathione conjugate pump on the changes in chemiluminescence. (A)
Chemiluminescence response of erythrocytes treated with 0.1 mmol/l CumOOH following the inhibition
of DNP-SG transport of erythrocytes from aging group. (B) Comparison of the chemiluminescence
response in erythrocytes from aging and young adults. DNP-SG transport inhibitors, Tween 80, ﬂuoride
or o-vanadate were added to erythrocyte suspensions before mixing with 0.1 mmol/l CumOOH and
chemiluminescence was recorded at indicated time. Data represent the mean of 16 determinations in the
elderly group and 15 determinations in the young group.

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4. Discussion
In the present study, we employed human erythrocytes to investigate the effect of
aging on the glutathione S-conjugate transport by determining the DNP-SG
transport. We used erythrocytes other than the chronologically young population
rich with reticulocytes, the reasons for which were explained in our previous article
(Onaran et al., 1997). In experimental conditions, in which erythrocyte glutathione
is completely depleted by incubating the erythrocytes with CDNB, we did not
observed a signiﬁcant difference in the efﬂux of DNP-SG in erythrocytes from aged
subjects when compared with young subjects. The result indicates that the detoxiﬁcation function is maintained against the chemical stress employed in their
erythyrocytes.
It is known that the cells transport the glutathione S-conjugates outward through
ATP-dependent efﬂux process. The transport of glutathione S-conjugate in erythrocytes was ﬁrst reported by Board (1981) and it was shown that the addition of
CDNB to erythrocytes results in the efﬂux of DNP-SG with rapid irreversible
depletion of glutathione. The transport of this conjugate is inhibited by the
depletion of intracellular ATP or by known organic anion transport inhibitors such
as vanadate, an inhibitor of P type ATPases (La Belle et al., 1986), and ﬂuoride
(interaction with the transporter molecule versus cellular energy depletion)
(Awasthi et al., 1983). We also observed pronounced inhibition of DNP-SG
transport by these inhibitors.
Although the effects of aging on different detoxiﬁcation mechanisms such as
glutathione and GST in erythrocytes have been reported (Stohs et al., 1984;
Jozwiak and Jasnowska, 1985; Stohs and Lawson, 1986; Al-Turk et al., 1987;
Foldes et al., 1988; Matsubara and Machado, 1991; Picot et al., 1992), to our
knowledge no data on the effect of donor age on transport of glutathione
conjugates of membrane is available. It has been shown that there is an age-related
decrease in glutathione content of the erythrocytes (Stohs et al., 1984; Al-Turk et
al., 1987; Matsubara and Machado, 1991). However, some evidence pointed out
that glutathione status, physical health and longevity of life were closely interrelated
(Foldes et al., 1988; Calvin et al., 1992). Furthermore contradictory results showing
decreased or unchanged GST activity in erythrocytes with donor aging were also
reported (Stohs et al., 1984; Al-Turk et al., 1987; Picot et al., 1992). In addition, it
has been shown that changes in glutathione concentration in mammalian cells have
various effects, inﬂuencing cellular radiosensitivity and the cytotoxicity of several
chemotherapeutic agents (Bump et al., 1982; Li and Kaminskas, 1984; Fernandes
and Cotter, 1994). In the aging group that provided erythrocytes for our study,
glutathione levels were lower for about 20% and GST activities remained unchanged. The present results thus reveal that cellular glutathione status of the aging
subject has no adverse effect on glutathione S-conjugate transport and the present
amount of glutathione in their erythrocytes might be sufﬁcient to detoxify the
employed chemical stress. However, we do not know how the transport of
glutathione S-conjugate is affected by different levels of intracellular glutathione.

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It has been previously reported that transport systems for glutathione S-conjugates play signiﬁcant roles in the biotransformation of toxic products arising from
oxidative stress induced lipid peroxidation (Ishikawa et al., 1986; Grune et al., 1991;
Akerboom et al., 1992). On the other hand, results of our previous study indicated
that the intact erythrocytes from aged subjects were not more susceptible to
oxidative stress than those of young subjects (Onaran et al., 1997). In the second
part of our study we therefore decided to determine how glutathione S-conjugate
transport plays a role in the resistance to oxidative stress of the erythrocytes of the
aged subjects.
To investigate this, DNP-SG transport was inhibited by various agents, i.e.,
ﬂuoride, vanadate or Tween 80. These agents were used for inhibitory action in the
studied system as their diverse effects on glutathione conjugate pump have been
explained previously (Akerboom et al., 1992; Pulaski and Bartosz, 1995). In
preliminary studies we observed that under experimental conditions employed the
concentrations of the inhibitors did not affect the glutathione and MDA contents,
chemiluminescence formation, rate of hemolysis and GST activities in the individual’s erythrocytes. The erythrocytes were then treated with 0.1 mmol/l CumOOH in
order to induce oxidative stress and then MDA levels, a common end product of
oxidative damage, were measured to asses whether the inhibition of glutathione
transport is subjected to increased oxidative damage in erythrocytes. Analyses of
the effects of inhibitors on DNP-SG transport as a function of the inhibitor
concentrations under the in vitro standard oxidative stress conditions, indicate that
increasing the relative concentrations of inhibitors to erythrocytes resulted in an
enhanced MDA formation and that enhancement of sensitivity caused by each of
the concentrations used was unchanged as a function of aging. In addition, the
degree of the DNP-SG transport inhibition for each of inhibitors used was very
similar in young and aged subjects. Because of these results it was decided that
further experiments could be performed in the inhibition conditions which were
able effectively to prevent the conjugate transport, in order to obtain an insight into
the effects of glutathione S-conjugate transport on the resistance to oxidative stress.
At this stage of the study the extent of oxidative damage was evaluated by
measuring the chemiluminescence formation as well as MDA formation. The
present study reports that following stimulation by CumOOH after the inhibition of
DNP-SG, peroxidation- dependent changes of MDA and chemiluminescence formation took place at the same level in aging and young adults. This indicates that
the erythrocytes from the elderly are equally capable of withstanding the oxidative
stress following the inhibition of glutathione conjugate pump and oxidative effect of
lipid peroxidation is not much greater in aging individuals with diminished glutathione levels but normal GST activity than young adults. Furthermore, these
results also indicate that the erythrocytes with inhibited DNP-SG transport were
shown to be more susceptible to oxidative stress generated by CumOOH than those
which were not inhibited. We concluded, therefore, that this result indicates that
inhibition of glutathione conjugate transport may impair cellular protection to
oxidant stress. Although we are at present not able to explain the mechanisms
governing these changes, several possible explanations of the effects observed may

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be considered. As inhibitory effects of glutathione S-conjugates on GSTs as well as
glutathione reductase (Ishikawa et al., 1986; Ishikawa, 1989) and the transport of
GSSG in various tissues including erythrocytes (Bilzer et al., 1984; Akerboom et al.,
1991) have been reported (Akerboom et al., 1982, 1991; Bilzer et al., 1984; Ishikawa
et al., 1986; Ishikawa, 1989), accumulation of the S-conjugates resulting from
radical induced lipid peroxidation in the cell may be crucial for detectable oxidative
damage. It is known that GST and glutathione reductase reduce organic hydroperoxides such as CumOOH (Prohaska and Ganther, 1977; Awasthi et al., 1980), and
it was also shown that the transport of GSSG is an important process for the cell
to avoid highly oxidative stress (Adams et al., 1983). Even though such an effect is
not known, inhibition of glutathione conjugate transport may also directly compromise the oxidant suppression of the cells by inhibiting key enzymes. However,
further investigation is obviously necessary to understand the function of glutathione S-conjugate transport responsible for the protection from oxidative
damage.
In conclusion, this study indicates that the transport of DNP-SG in erythrocytes
of healthy elderly with low glutathione is unaffected and their erythrocytes are not
more susceptible to oxidative stress than those of young subjects after in vitro
pretreatment with inhibitors which inhibit glutathione conjugate pump. Our ﬁndings also suggest that the ability of the erythrocytes to withstand oxidative damage
may be diminished with inhibition of glutathione conjugate transport.

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