(Received for publication, September 11, 1995; and in revised form, February 7, 1996)
From the
A role for ascorbate-derived electrons in protection against
oxidative damage to membrane lipids was investigated in resealed human
erythrocyte ghosts. Incubation of resealed ghosts with the
membrane-impermeant oxidant ferricyanide doubled the ghost membrane
concentration of F-isoprostanes, a sensitive marker of
lipid peroxidation. Incorporation of ascorbate into ghosts during
resealing largely prevented F
-isoprostane formation due to
extravesicular ferricyanide. This protection was associated with a
rapid transmembrane oxidation of intravesicular ascorbate by
extravesicular ferricyanide. Transmembrane electron transfer, which was
measured indirectly as ascorbate-dependent ferricyanide reduction,
correlated with the content of
-tocopherol in the ghost membrane
in several respects. First, ascorbate resealed within ghosts protected
against ferricyanide-induced oxidation of endogenous
-tocopherol
in the ghost membrane. Second, when exogenous
-tocopherol was
incorporated into the ghost membrane during the resealing step,
subsequent ferricyanide reduction was enhanced. Last, incubation of
intact erythrocytes with soybean phospholipid liposomes, followed by
resealed ghost preparation, caused a proportional decrease in both the
membrane content of
-tocopherol and in ferricyanide reduction.
Incorporation of exogenous
-tocopherol during resealing of ghosts
prepared from liposome-treated cells completely restored the
ferricyanide-reducing capacity of the ghosts. These results suggest
that the transmembrane transfer of ascorbate-derived electrons in
erythrocyte ghosts is dependent in part on
-tocopherol and that
such transfer may help to protect the erythrocyte membrane against
oxidant stress originating outside the cell.
Erythrocytes possess redundant and overlapping mechanisms for
protection of their cytoplasmic contents against oxidative damage,
including catalase(1) , superoxide dismutase(2) , and
low molecular weight antioxidants such as GSH and
ascorbate(3) . Defenses against oxidant damage in the plasma
membrane are poorly understood but relate primarily to prevention and
reversal of peroxidation of unsaturated fatty acids in the lipid
bilayer(4) . Both lipophilic and cytoplasmic factors are
thought to protect the erythrocyte membrane against lipid peroxidation.
Foremost is -tocopherol(4, 5) , but
ubiquinol-10(6, 7) , membrane protein
sulfhydryls(8) , and a GSH-dependent phospholipid
hydroperoxidase (9, 10) are also likely to contribute.
Although ascorbic acid does not directly retard peroxidation of
membrane lipids(11) , it may do so indirectly by reducing the
tocopheroxyl free radical in the lipid
bilayer(12, 13) .
Beyond their ability to use
intracellular reducing potential to protect the cell membrane from
oxidant stress, erythrocytes have long been known to reduce
extracellular oxidants such as
ferricyanide(14, 15, 16) ,
methemoglobin(17) , and ferricytochrome c(18) . This export of electrons across the cell
membrane has been attributed to activity of a transmembrane
NADH:(acceptor) oxidoreductase (E.C.
1.6.99.3)(19, 20) . There are probably several such
activities in erythrocyte membranes(20) . For example, a
transmembrane enzyme has been purified (21) that can be
differentiated from the prominent inward facing cytochrome b reductase (22) by the presence of
carbohydrate, lower substrate affinities, and lack of a flavin as a
cofactor. The transmembrane enzyme has also been implicated in the
export of electrons derived from
ascorbate(15, 16, 23) . A physiologic
function of this enzyme has not been established. One possibility is
that in reticulocytes it provides electrons for reduction of
Fe
on transferrin(24) ; the resulting
Fe
is then released and can be taken up by the
cell(25) . It has also been suggested that such an enzyme may
use electrons from intracellular NADH to reduce lipid hydroperoxides in
the membrane(26) . Ascorbate might help to protect the lipid
bilayer, either by donating electrons to such an enzyme or by recycling
the lipophilic antioxidant
-tocopherol.
The goals of the
present study were to evaluate the mechanism of ascorbate-dependent
trans- or intramembrane electron transfer in human erythrocytes and to
determine whether such transfer protects against membrane lipid
peroxidation. To minimize the effects of endogenous cytoplasmic
antioxidants and to ensure study of transbilayer transfer, resealed
erythrocyte ghosts containing ascorbate were used. The results show
that ascorbate resealed within ghosts protects against
ferricyanide-induced loss of endogenous -tocopherol and against
peroxidation of membrane lipids. The protective effect of
intravesicular ascorbate involves a transmembrane transfer of
ascorbate-derived electrons, the extent of which depends on the
membrane content of
-tocopherol.
To estimate the relative amounts of ascorbate and its
oxidation products, ghosts were resealed in the presence of 400
µM of unlabeled ascorbate, 0.05 µCi of
[C]ascorbate, and 0.2 µCi of L-[
H]glucose. Sample preparation and
HPLC analysis were carried out as described in the previous paragraph,
except that the postdetector eluate was collected in 1-ml volumes with
a fraction collector. Aliquots of these fractions (0.5 ml) were added
to 5 ml of liquid scintillation fluid and counted under dual-label
conditions with efficiency correction. Both
[
C]DHA and L-[
H]glucose eluted in the column void
volume, [
C]ascorbate eluted at 5-6 min,
and 2,3-[
C]diketo-1-gulonic acid eluted at
8-9 min. A 5-min incubation at 37 °C of
[
C]ascorbate (20 nmol) with ascorbate oxidase (2
units/ml) was used to generate [
C]DHA.
To
assess the effects of the resealing process on ascorbate added during
resealing, ghosts were resealed in the presence of 400 µM unlabeled ascorbate and [C]ascorbate, and
ultrafiltrates of the ghost contents and hemolysate were analyzed by
HPLC with scintillation counting of the column eluates as outlined
above. At the end of the resealing period, the ghosts typically
retained 200-300 nmol of ascorbate/ml of intravesicular ghost
volume. After the hour of resealing at 37 °C the ascorbate
concentration in the hemolysate outside the ghosts had decreased to
130-150 µM. The decrease in ascorbate both inside
the ghosts and in the hemolysate was due largely to its conversion to
[
C]DHA, which was present in equal
concentrations inside and outside the ghosts at the end of resealing
(data not shown). Variable amounts of
2,3-[
C]diketo-1-gulonic acid were also observed.
To induce an oxidant stress on the erythrocyte membrane,
resealed ghosts that had been depleted of cytosolic contents were
incubated for 15 min with 1 mM ferricyanide at 37 °C.
Ferricyanide has long been known not to penetrate the intact
erythrocyte membrane(38) . Similarly, the agent also failed to
oxidize the residual hemoglobin present in resealed ghosts (results not
shown), indicating that any effects observed are restricted to the
outer surface of the membrane. However, ferricyanide is a relatively
mild oxidant, and it did not increase the ghost membrane content of
either thiobarbituric-reactive substances or lipid hydroperoxides
(results not shown). Therefore, F-isoprostanes were
measured to provide a more sensitive measure of membrane lipid
peroxidation in the ghosts. F
-isoprostanes are derived from
free radical-mediated oxidation of arachidonic acid and have been shown
to provide specific and extremely sensitive estimates of lipid
peroxidation, both in vivo(39) and in
vitro(40) . In contrast to lipid hydroperoxides,
F
-isoprostanes are chemically stable end products that can
remain esterified in tissues or be released by the actions of
phospholipases(36) . Ghosts resealed in the presence or absence
of ascorbate had low levels of F
-isoprostanes, and
ferricyanide treatment more than doubled F
-isoprostane
formation (Table 1). In ghosts resealed to contain ascorbate, the
membrane content of F
-isoprostanes was halved. These
results show that ferricyanide does oxidize a small amount of
arachidonic acid that is presumably located in the outer membrane
bilayer and that intravesicular ascorbate protects against this
oxidation. The findings also indicate the enhanced sensitivity of the
F
-isoprostane assay compared with other measures of lipid
peroxidation. Although the improved sensitivity of this assay is due in
part to the ease of oxidation of arachidonic acid, previous results
suggest that this level of oxidation would not be detected by measuring
changes in the membrane content of arachidonic acid(41) .
Possible mechanisms for ascorbate-dependent protection of the ghost
membrane against ferricyanide-induced lipid peroxidation were
investigated. It is unlikely that the observed protection was due
simply to consumption of most of the oxidant by intravesicular
ascorbate, since in these incubations ferricyanide was present in at
least a 17-fold molar excess over ascorbate. Whereas ferricyanide
apparently does not cross the ghost membrane, ascorbate may efflux from
the ghosts and consume ferricyanide directly in the medium. To assess
the latter, time-dependent efflux of radiolabel from ghosts that had
been loaded with [C]ascorbate was measured, with
the results shown in Fig. 1. In untreated ghosts, the rate of
efflux of the carbon-14 label was similar to that observed for L-[
H]glucose, which was used as a marker
for nonspecific diffusion. In two additional experiments, after 15 min
of incubation at 37 °C, 90% of the carbon-14 radiolabel remaining
in the ghosts was ascorbate, as determined by analysis of carbon-14
distribution in the HPLC eluates with correction for zero time values.
On the other hand, only [
C]DHA and
2,3-[
C]diketo-1-gulonic acid were found outside
the ghosts at this time; neither ascorbate nor
[
C]ascorbate was detected in the buffer. This
suggests that the observed efflux of radiolabel from the ghosts
occurred as [
C]DHA and not as
[
C]ascorbate. Similar conclusions have been
reached for intact cells(42, 43, 44) .
However, in intact cells the net efflux of
[
C]ascorbate is slower than observed in these
ghosts (42, 44) . This difference is most likely due
to the limited ability of the ghosts to recycle DHA to ascorbate
compared with intact cells, so that any DHA generated rapidly exits on
the glucose transporter(42, 43) .
Figure 1:
Efflux of
radiolabel from [C]ascorbate-loaded resealed
erythrocyte ghosts. Erythrocyte ghosts were resealed in the presence of
400 µM unlabeled ascorbate and 0.05 µCi of
[
C]ascorbate as well as 0.2 µCi of tracer L-[
H]glucose. Following the usual
centrifugation washes, the ghosts were incubated at 37 °C in PBS
containing 5 mMD-glucose in the absence (circles) or presence of 1 mM ferricyanide (squares). At the indicated times the efflux of radiolabel
from the ghosts was measured as described under ``Experimental
Procedures.'' The solid symbols show the efflux of
carbon-14, and the open symbols show the efflux of tritium.
Data from two experiments are shown ± S.E. The solid lines are monoexponential fits to the data, with r > 0.997.
The dashed horizontal line shows the residual fraction of
radiolabel that would be present in the ghosts at
equilibrium.
When ghosts
resealed with
[C]ascorbate/L-[
H]glucose
were incubated with ferricyanide, there was rapid release of about 70%
of the carbon-14, with no effect on the efflux of L-[
H]glucose (Fig. 1). Under
similar conditions, 1 mM ferricyanide also decreased the
measured intravesicular ascorbate content to less than 5% of control
concentrations within 15 min. HPLC analysis showed that following
ferricyanide treatment, 90% of the radioactivity inside the ghosts was
2,3-[
C]diketo-1-gulonic acid, and the remainder
was [
C]dehydroascorbate and
[
C]ascorbate. No
[
C]ascorbate was detected outside the ghosts.
These results suggest that extravesicular ferricyanide oxidizes
intravesicular ascorbate to DHA, which can then either escape the ghost
on the glucose transporter (42, 43, 44) or
undergo irreversible degradation to 2,3-diketo-1-gulonic
acid(45) . The latter, having undergone ring opening and
containing a negative charge, is trapped within the ghosts and accounts
for the residual carbon-14 radioactivity observed in the efflux
experiment of Fig. 1.
The separation of ferricyanide and
ascorbate across the ghost membrane implies a transmembrane transfer of
ascorbate-derived electrons, which could be estimated as the extent of
ferricyanide reduction outside the ghosts. Ferricyanide reduction
occurred rapidly over the same time course as loss of radiolabel from
ferricyanide-treated ghosts and was largely complete following 15 min
of incubation (results not shown). Several features of this
ascorbate-to-ferricyanide electron transfer are shown in Table 2.
Ghosts resealed to contain ascorbate were from 3-5-fold more
effective in reducing extravesicular ferricyanide than ghosts not
containing ascorbate. When ghosts were incubated with 100 µM DHA, a small but significant increase in ferricyanide reduction
was also observed. No increase in intravesicular ascorbate content was
detected by direct assay, but after 5 min of incubation with 400
µM [C]DHA under the conditions of Table 2, 90% of the carbon-14 present in ghosts was
[
C]ascorbate. This suggests that the mechanism
for the DHA-induced increase in ferricyanide reduction involved uptake
of DHA, intravesicular reduction of DHA to ascorbate, and transmembrane
reduction of extravesicular ferricyanide. Although glucose was required
for the small DHA effect, omission of glucose did not affect the much
larger extent of ferricyanide reduction in ghosts resealed to contain
ascorbate.
The addition of ascorbate oxidase to the ghosts several minutes before ferricyanide was added did not affect ferricyanide reduction (Table 2). In control studies not shown, the addition of 400 µM ascorbate with ascorbate oxidase under the conditions of Table 2decreased the expected amount of ferricyanide reduction by about two-thirds, indicating the ability of the enzyme to compete with ferricyanide for extravesicular ascorbate. The failure to see an ascorbate oxidase-induced decrease in ferricyanide reduction in ghosts containing ascorbate confirms the observations shown in Fig. 1, which indicated little direct efflux of ascorbate across the ghost membranes during the experiment. This further suggests a transmembrane transfer of ascorbate-derived electrons in the reduction of ferricyanide, rather than ferricyanide-induced efflux of ascorbate, followed by direct oxidation by ferricyanide outside the ghosts.
Ghosts resealed in the presence of 4 mM GSH showed a doubling in ferricyanide reduction (Table 2). GSH and ascorbate together had an additive effect on the extent of ferricyanide reduction. A role for GSH in recycling ascorbate was investigated by measuring intravesicular ascorbate concentrations just before the addition of ferricyanide. No ascorbate was detected in ghosts resealed with GSH alone, but in ghosts resealed with both 400 µM ascorbate and 4 mM GSH, ascorbate depletion was almost completely prevented (data not shown). This suggests that in ghosts resealed with GSH and ascorbate, much of the enhanced ability to reduce ferricyanide was due to increased intravesicular ascorbate concentrations. Direct participation of GSH in the transmembrane electron transfer process cannot be ruled out, however.
A common mechanism was sought to account for both transfer
of ascorbate-derived electrons and ascorbate-dependent protection of
the outer bilayer leaflet from peroxidation in response to ferricyanide
treatment. It has previously been observed that ascorbate alone does
not directly transfer reducing equivalents to membrane lipids or
hydroperoxides(11) . A more likely possibility is that
-tocopherol might mediate the effect. It is well established that
-tocopherol can intercept free radicals in lipid bilayers (4) and that ascorbate can recycle
-tocopherol from its
free radical in cell membranes(46, 47) . As shown in Fig. 2, increasing amounts of intravesicular ascorbate were
found to preserve endogenous
-tocopherol in resealed ghosts over a
range of extravesicular ferricyanide concentrations. This protection
was partial at a 400 µM loading concentration of ascorbate
and more complete at a loading concentration of 2 mM, with an
associated lag phase. The ascorbate-dependent protection of endogenous
-tocopherol against oxidation by ferricyanide fits with the
hypothesis that
-tocopherol is the proximal electron donor to
ferricyanide and that ascorbate in turn recycles
-tocopherol.
Figure 2:
Protection against loss of endogenous
-tocopherol by intravesicular ascorbate in resealed erythrocyte
ghosts. Ghosts were resealed in the absence (closed circles)
or presence of 400 µM ascorbate (closed squares)
or 2 mM ascorbate (closed triangles) and incubated
for 15 min at 37 °C with 5 mMD-glucose and the
indicated concentration of ferricyanide. The membrane content of
-tocopherol was determined and is expressed as a percentage of an
untreated control. Data are from three to six experiments ± S.E.
The difference between control and both sets of ascorbate-supplemented
ghosts was significant at the p < 0.01 level across the
three ferricyanide doses.
It is also possible that -tocopherol contributes to the
transmembrane movement of electrons from ascorbate to ferricyanide. It
has been shown that
-tocopherol can directly transfer
ascorbate-derived electrons to ferricyanide across artificial liposomal
bilayers(48) , although transbilayer movement of
-tocopherol is slow relative to quinones with short hydrophobic
tails(49) . To determine whether ascorbate-dependent
ferricyanide reduction requires
-tocopherol, two types of
experiment were carried out. In the first,
-tocopherol was added
to the ghost membranes during the resealing step. Only about 10% of
added
-tocopherol was recovered in the cell lysate before
resealing of the ghosts (see Table 3legend), possibly because of
oxidation or failure to stay in solution. However, 7-8% of the
total available
-tocopherol was found in the ghosts, resulting in
a 10-fold increase in the
-tocopherol content (Table 3). The
presence of exogenous
-tocopherol in the ghost membranes tripled
ferricyanide reduction in ghosts not containing ascorbate and doubled
it in ascorbate-containing ghosts (Table 3). It should be noted
that the incubation time of ghosts with ferricyanide was 5 min in these
experiments rather than 15 min as in the experiments shown in Table 2and Fig. 2. A shorter incubation time was used to
ensure that ascorbate depletion was not limiting for ferricyanide
reduction. Although
-tocopherol intercalates into the membrane
bilayer and can bind specifically to the erythrocyte
surface(50) , based on the measured content of
-tocopherol
in membranes not treated with ferricyanide (Table 3), simple
direct oxidation of
-tocopherol could have accounted for only a
small portion of the observed increase in ferricyanide reduction.
In
the second type of experiment designed to test for a role of
-tocopherol in facilitating the transfer of ascorbate-derived
electrons across the ghost membrane, ferricyanide reduction was
measured in ghosts depleted of endogenous
-tocopherol. Depletion
of endogenous
-tocopherol was accomplished by incubating intact
erythrocytes with soybean lecithin liposomes for 2 h followed by
centrifugation washes to remove the liposomes. As shown by the first pair of bars in Fig. 3, this treatment
decreased both the endogenous
-tocopherol content and the
ferricyanide-reducing capacity of subsequently prepared
ascorbate-containing resealed ghosts by 40-50%. A similar
decrease compared with control was observed when ascorbate was omitted
during the resealing step (results not shown). Whether
-tocopherol
was transferred from erythrocytes to the liposomes (51, 52) or oxidized by the liposomes (53) is
controversial. Although lipid peroxidation was not detected in either
liposomes or ghosts following incubation of intact cells with liposomes
in the present studies, the failure to detect quantitative transfer
supports an oxidative loss. If depletion of
-tocopherol by
liposome treatment in intact cells is the cause of the decrease in
ferricyanide reduction in resealed ghosts, then adding back
-tocopherol during the resealing phase should reverse the effect.
As shown by the second pair of bars in Fig. 3,
ghosts exposed to
-tocopherol and ascorbate during resealing had
the expected increases in measured
-tocopherol content and
ferricyanide-reducing capacity (compare to results shown in Table 3). The addition of both
-tocopherol and ascorbate
during resealing of ghosts derived from liposome-treated cells
increased the
-tocopherol content and the capacity to reduce
ferricyanide (third pair of bars in Fig. 3).
Although the
-tocopherol content of such ghosts did not attain the
level observed in
-tocopherol-treated ghosts prepared from cells
not exposed to liposomes, the ferricyanide-reducing capacity was fully
restored (compare the second and third pairs of bars in Fig. 3). Thus, both
-tocopherol depletion
and repletion correlate with ferricyanide reduction in these ghosts,
which supports the possibility that endogenous
-tocopherol might
play a role in mediating the transmembrane transfer of
ascorbate-derived electrons.
Figure 3:
Parallel changes in -tocopherol
content and ferricyanide reduction in ghosts from liposome-treated
erythrocytes with and without
-tocopherol treatment. Liposome
treatment,
-tocopherol addition, and incubation conditions are
described under ``Experimental Procedures.'' Shown are the
relative increases in
-tocopherol content (open bars) and
ferricyanide reduction (hatched bars) observed in ghosts
prepared from cells that had been treated with liposomes but not
resealed in the presence of exogenous
-tocopherol (first pair of bars), in ghosts from control cells that were treated
with
-tocopherol during resealing (second pair of bars), and in ghosts that were both prepared from
liposome-treated cells and treated with
-tocopherol during
resealing (third pair of bars). The data are from
four experiments ± S.E. and are expressed relative to responses
observed in ghosts not exposed to either liposomes or
-tocopherol.
All ghosts were resealed in the presence of 400 µM ascorbate. An asterisk indicates p < 0.05
compared with both other treatments.
Ferricyanide reduction by erythrocytes
has usually been attributed to a transmembrane oxidoreductase that uses
intracellular NADH as the electron donor(19, 20) .
Ascorbate has also been implicated as an electron donor for such an
enzyme(15, 16) , and recent results from this
laboratory indicate that ascorbate may be the preferred substrate (23) . The present results further suggest that
-tocopherol, either directly or in concert with an enzyme, may
also be involved in the electron transfer as well as in protection of
membrane lipids against oxidation by external ferricyanide.