(Received for publication, April 4, 1995; and in revised form, July 31, 1995)
From the
We performed a detailed kinetic analysis of the uptake of
dehydroascorbic acid by HL-60 cells under experimental conditions that
enabled the differentiation of dehydroascorbic acid transport from the
intracellular reduction/accumulation of ascorbic acid. Immunoblotting
and immunolocalization experiments identified GLUT1 as the main glucose
transporter expressed in the HL-60 cells. Kinetic analysis allowed the
identification of a single functional activity involved in the
transport of dehydroascorbic acid in the HL-60 cells. Transport was
inhibited in a competitive manner by both
3-O-methyl-D-glucose and 2-deoxy-D-glucose.
In turn, dehydroascorbic acid competitively inhibited the transport of
both sugars. A second functional component identified in experiments
measuring the accumulation of ascorbic acid appears to be associated
with the intracellular reduction of dehydroascorbic acid to ascorbic
acid and is not directly involved in the transport of dehydroascorbic
acid via GLUT1. Transport of dehydroascorbic acid by HL-60 cells was
independent of the presence of external Na, whereas
the intracellular accumulation of ascorbic acid was found to be a
Na
-sensitive process. Thus, the transport of
dehydroascorbic acid via glucose transporters is a
Na
-independent process which is kinetically and
biologically separable from the reduction of dehydroascorbic acid to
ascorbic acid and its subsequent intracellular accumulation.
Vitamin C is fundamental to human physiology(1, 2, 3) . Since humans cannot synthesize vitamin C, it must be provided exogenously in the diet and transported intracellularly(4, 5) . Vitamin C is present in human blood at an average concentration of about 50-100 µM, and at least 95% is in the reduced form (ascorbic acid) with the remaining 5% in the oxidized form (dehydroascorbic acid)(5) . The observation that cells and tissues accumulate characteristic intracellular concentrations of the reduced form of vitamin C, both in vivo and in vitro, suggests that the transport and cellular accumulation of vitamin C is a highly regulated process(5) .
Data accumulated over a number of years pointed to the participation of glucose transporters in the cellular transport of vitamin C(6, 7, 8, 9, 10, 11, 12) , although functional evidence for the existence of additional transporters is also available(13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) . We recently demonstrated directly, by expression in Xenopus laevis oocytes, that the glucose transporters GLUT1, GLUT2, and GLUT4 are efficient transporters of dehydroascorbic acid(25) . We extended these studies to show that glucose transporters are also the main pathway mediating the transport of dehydroascorbic acid in normal human neutrophils and HL-60 myeloid leukemia cells(25, 26) . There has been controversy regarding the issue of the identity of the form of vitamin C, reduced or oxidized, transported by human neutrophils. Freshly isolated human neutrophils contain millimolar concentrations of reduced ascorbic acid, and in vitro they accumulate high concentrations of ascorbic acid when incubated with ascorbic or dehydroascorbic acid(5, 27) . Current evidence indicates, however, that dehydroascorbic acid, and not ascorbic acid, is preferentially transported into cells(9, 18, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36) . We, and others, have shown that under the incubation conditions used by most laboratories, ascorbic acid undergoes oxidation to dehydroascorbic acid which is then transported intracellularly(25, 26, 37, 38) . The transported dehydroascorbic acid is reduced back to ascorbic acid, providing the mechanism for its cellular accumulation. The oxidation of ascorbic acid to dehydroascorbic acid appears to be part of the mechanism by which cells of the host defense system, such as neutrophils, increase their uptake of dehydroascorbic acid when activated by physiological stimuli.
Kinetic analysis of the uptake of ascorbic acid in human neutrophils has revealed the presence of two functional activities with different affinities for ascorbate, an observation that was interpreted as suggesting the existence of at least two separate transport systems involved in the cellular uptake of ascorbic acid(27) . We detected two functional activities, one with high affinity and one with low affinity, involved in the uptake of dehydroascorbic acid in X. laevis oocytes expressing GLUT1 (25) and in human neutrophils and HL-60 cells(25, 26) . The X. laevis expression experiments suggested the association of both functional activities with the expression of a single transport protein.
The studies mentioned above were performed under conditions leading to the accumulation of ascorbic acid in cells incubated with dehydroascorbic acid. Uptake under these conditions is a function of both transport and intracellular trapping. No clear distinction between transport of dehydroascorbic acid and accumulation of ascorbic acid has been presented, and, therefore, it has been impossible to assign the functional activities detected to the steps of transport or accumulation. An understanding of the mechanisms that regulate the cellular content of ascorbic acid requires the identification of the events involved in the transport of dehydroascorbic acid as well as in the cellular accumulation of ascorbic acid. We studied this issue by analyzing the accumulation of ascorbic acid in HL-60 cells under experimental conditions that allowed us to dissociate the transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid. We identified GLUT1 as the glucose transporter expressed in HL-60 cells, characterized it functionally, established the conditions to measure transport as distinct from accumulation, and applied these conditions to measure the transport of dehydroascorbic acid. We now provide evidence indicating that only one functional system, corresponding to the glucose transporter GLUT1, is directly involved in the facilitated transport of dehydroascorbic acid by HL-60 cells. A second functional activity apparently associated with the trapping and accumulation of the reduced form of ascorbic acid is not directly involved in the transport of dehydroascorbic acid via the glucose transporters.
Figure 1: Expression of GLUT1 in HL-60 cells. A, for immunoblotting, equal amounts of protein were loaded on each lane of a sodium dodecyl sulfate-containing polyacrylamide gel, electrophoresed, transferred to Immobilion, reacted with the different anti-GLUT antibodies, and visualized using horseradish peroxidase anti-rabbit IgG and enhanced chemiluminescence. B and C, for immunofluorescence, cytospin preparations were reacted with preimmune serum (panel B) or anti-GLUT1 antibodies (panel C), and visualized using fluorescein-coupled secondary antibodies. No reactivity was observed with anti-GLUT2, -3, -4, or -5 antibodies.
Figure 2:
Kinetics of the uptake of methylglucose by
HL-60 cells and the effect of competition with different sugars. A, time course of the uptake of 1 mM methylglucose. B, dose-response of the transport of methylglucose using 30-s
uptake assays. C, double-reciprocal plot of the substrate
dependence for methylglucose transport. D, semi-log plot of
the concentration dependence for inhibition of methylglucose transport
in HL-60 cells by different sugars. Measurements were performed at 1
mM methylglucose using 30-s uptake assays. DOG,
deoxyglucose; OMG, methylglucose; -MethylG,
-methyl-D-glucopyranoside.
Dose-response experiments examining uptake at 30 s indicated that
transport of methylglucose approached saturation at millimolar
concentrations of methylglucose (Fig. 2B), with an
apparent K for transport of 8.5 mM (Fig. 2C). Uptake was decreased in the presence of
deoxyglucose, a typical substrate of the facilitative glucose
transporters(40) . Deoxyglucose, at 3 mM, inhibited by
50% (IC
= 3 mM) the transport of
methylglucose by the HL-60 cells (Fig. 2D). Maltose, a
disaccharide that binds to the glucose transporters but is not
transported(40) , inhibited the uptake of methylglucose with an
IC
of approximately 25 mM, but no effect on
transport was observed with fructose, sucrose,
-methyl-D-glucoside, or L-glucose (Fig. 2D).
-Methyl-D-glucoside is a
substrate of the sodium-dependent glucose cotransporter (41) that is not transported by the facilitative glucose
transporters, and sucrose and L-glucose do not interact with
the glucose transporters(40) . Cytochalasin B, but not
cytochalasin E, inhibited the transport of methylglucose with an
IC
of 200 nM (data not shown).
Figure 3: Kinetics of the uptake of deoxyglucose by HL-60 cells and the effect of competition with different sugars. A, time course of the uptake of 2 and 5 mM deoxyglucose. B, time course of the uptake of 5 mM deoxyglucose. C, dose-response of the transport of deoxyglucose using 30-s uptake assays. D, double-reciprocal plot of the substrate dependence for deoxyglucose transport. Uptake assay, 30 s. E, semi-log plot of the concentration dependence for inhibition of deoxyglucose transport in HL-60 cells by different sugars. Measurements were performed at 0.2 mM deoxyglucose using 30-s uptake assays. DOG, deoxyglucose; OMG, methylglucose.
Figure 4:
Kinetics of the uptake of dehydroascorbic
acid by HL-60 cells and the effect of competition with different
sugars. A, time course of the uptake of 50 µM dehydroascorbic acid. B, short time course of the uptake
of 50 µM dehydroascorbic acid. C, dose-response
of the transport of dehydroascorbic acid. , 10-min uptake assay.
, 30-s uptake assay. D, double-reciprocal plot of the
substrate dependence for dehydroascorbic acid transport.
, 10-min
uptake assay.
, 30-s uptake assay. E, dose-response of
the transport of dehydroascorbic acid using 30-s uptake assays. F, dose-response of the transport of dehydroascorbic acid
using 10-min uptake assays. G, semi-log plot of the
concentration dependence for inhibition of dehydroascorbic acid
transport in HL-60 cells by different sugars. Measurements were
performed at 50 µM dehydroascorbic acid using 30-s uptake
assays. DHA, dehydroascorbic acid; DOG, deoxyglucose; OMG, methylglucose;
MethylG,
-methyl-D-glucopyranoside.
Dose-response studies using 30-s uptake assays revealed that
the transport of dehydroascorbic acid by the HL-60 cells saturated at
about 4 mM (Fig. 4C). These studies also
revealed the presence of a single component with an apparent K of 0.85 ± 0.12 mM (n = 8), and a V
of 4 nmol/min/10
cells for the transport of dehydroascorbic acid by the HL-60
cells (Fig. 4D). Parallel studies measuring uptake of
dehydroascorbic acid using 10-min assays revealed the presence of one
component saturating at about 15 mM of dehydroascorbic acid. A
more detailed examination of the dose-response curve, however, revealed
the presence of two components, one saturating at about 4 mM
dehydroascorbic acid, and a second one saturating at about 15 mM dehydroascorbic acid (Fig. 4C). The
Lineweaver-Burk analysis revealed the presence of two components, with
apparent uncorrected K
values of 0.9 and 3.5
mM and V
of 1.6 and 2 nmol/min/10
cells, respectively (Fig. 4D). HL-60 cells
incubated for 10 min with dehydroascorbic acid showed the presence of
an additional high affinity component involved in uptake, but the
kinetic analysis failed to reveal the presence of the high affinity
component when measuring transport of dehydroascorbic acid using 30-s
assays (Fig. 4, E and F).
Competition
experiments using 30-s transport assays indicated that deoxyglucose,
methylglucose, and maltose inhibited the transport of dehydroascorbic
acid by the HL-60 cells with IC of 2, 8, and 15
mM, respectively, but no effect of L-glucose,
fructose, sucrose, or
-methyl-D-glucoside was observed (Fig. 4G). Cytochalasin B, but not cytochalasin E,
inhibited transport with an IC
of approximately 200 nM (data not shown). Dehydroascorbic acid, but not ascorbic acid,
inhibited the transport of methylglucose by the HL-60 cells in a
dose-dependent manner, with an IC
of about 1 mM (Fig. 5A). The inhibition of the transport of
methylglucose by dehydroascorbic acid was competitive, with a K
of 0.8 mM (Fig. 5, B and C). We also determined the effect of deoxyglucose on
the cellular uptake of concentrations of dehydroascorbic acid, from 20
µM to 5 mM, using incubation periods from 30 s to
10 min. Deoxyglucose completely blocked the uptake of dehydroascorbic
acid under every condition tested, confirming that GLUT1 is the only
pathway for the transport of dehydroascorbic acid by HL-60 cells (data
not shown).
Figure 5:
Competitive inhibition of methylglucose
uptake by dehydroascorbic acid. A, semi-log plot of the
concentration dependence for inhibition of methylglucose transport in
HL-60 cells by dehydroascorbic acid () and reduced ascorbic acid
(
). Measurements were performed at 1 mM methylglucose
using 30-s uptake assays. B, double-reciprocal plot of the
effect of different concentrations of dehydroascorbic acid on the
substrate dependence for methylglucose transport using 30-s assays.
Uptake was measured in the absence (
) or in the presence of 0.5
(
), 1 (
), or 3 mM (
) dehydroascorbic acid. C, secondary plot of the effect of dehydroascorbic acid on the
substrate dependence for methylglucose transport. DHA,
dehydroascorbic acid; OMG,
methylglucose.
Figure 6:
Sodium-independence of the uptake of
dehydroascorbic acid by HL-60 cells. A, time course of the
uptake of dehydroascorbic acid in the presence of NaCl () or
choline chloride (
). B, time course of the uptake of
dehydroascorbic acid in the presence of NaCl (
) or choline
chloride (
). C, time course of the uptake of
dehydroascorbic acid in the presence of NaCl (
) or sucrose
(
). D, time course of the uptake of dehydroascorbic acid
in the presence of NaCl (
) or LiCl (
). E, time
course of the uptake of ascorbic acid in the presence of Na
(
) or choline
(
). F, time
course of the uptake of ascorbic acid in the presence of Na
(
) or choline
(
). G, time
course of the uptake of ascorbic acid in the presence of Na
(
) or choline
(
). H, time
course of the uptake of ascorbic acid (AA,
) or
dehydroascorbic acid (DHA,
).
We further analyzed this
issue by measuring uptake in HL-60 cells incubated with ascorbic acid
in the absence of Na. HL-60 cells incubated with
ascorbic acid accumulate lower levels of ascorbate (as compared to
cells incubated with dehydroascorbic acid) due to the oxidation of
ascorbic acid to the transported form, dehydroascorbic
acid(26) . Uptake in the presence of Na
proceeded in a linear fashion for the length of the incubation
period (120 min), but accounted for less than 5-7% of the uptake
of dehydroascorbic acid under similar experimental conditions (Fig. 6, E and A). A decrease of approximately
40% in uptake was observed in the absence of Na
at 120
min, but no effect was evident during the first 45 min of incubation (Fig. 6E). In the presence of choline
,
there was a small but reproducible stimulation of uptake during the
first 30 min of incubation, as compared to control cells (Fig. 6F). Short uptake experiments indicated that, in
the presence of Na
, the cells failed to accumulate any
radioactivity during the first 30 s of incubation, but uptake increased
rapidly afterwards, compatible with the time-dependent generation of
the transported substrate dehydroascorbic acid (Fig. 6G). Similar uptake kinetics were observed in
cells incubated in the presence of choline
, but uptake
was always greater than in the control cells (Fig. 6G).
Again, uptake under these conditions (cells incubated with ascorbic
acid) was only a fraction of the uptake observed in cells incubated
with dehydroascorbic acid (Fig. 6H). Similar results
were obtained when sucrose or LiCl were used to replace NaCl in the
incubation buffer (data not shown). Overall, these data are consistent
with the concept that GLUT1, a transporter whose functional activity is
Na
-independent, is the only pathway involved in the
cellular uptake of dehydroascorbic acid by the HL-60 cells.
Our data indicate that the accumulation of ascorbic acid in HL-60 cells is a process that can be dissociated into at least two components with characteristic kinetics. The initial step consists of the facilitated transport of dehydroascobate, followed by its reduction and intracellular accumulation as ascorbic acid. The transport component of uptake was very rapid and could be detected only by using very short uptake assays. At extended incubation periods, the rate-limiting step of uptake was the cellular trapping/reduction and accumulation of ascorbic acid, and it was no longer possible to measure transport separately and as distinct from accumulation. Transport was mediated by GLUT1, the member of the family of facilitative glucose transporters expressed in the HL-60 cells.
Although six different
facilitative glucose transporters have been identified in mammalian
cells(39) , we identified GLUT1 as the main facilitative
glucose transporter expressed by HL-60 cells. The HL-60 cells were able
to transport deoxyglucose, a substrate specific for the facilitated
glucose transporters that is not transported by the sodium-glucose
cotransporters(40, 41) . In addition,
-methyl-D-glucoside, the specific substrate of the
sodium-glucose cotransporter(41) , did not inhibit the
transport of deoxyglucose or methylglucose by the HL-60 cells. The
specificity of the transporter was further confirmed by the lack of
effect of L-glucose and sucrose and the inhibitory effect of
maltose on the transport of deoxyglucose and methylglucose. The HL-60
cells were unable to transport fructose, and transport of methylglucose
and deoxyglucose was not affected by fructose. These findings are
consistent with the immunological evidence indicating that the HL-60
cells do not express GLUT2 and GLUT5. The inhibitory effect of
nanomolar concentrations of cytochalasin B on the transport of
deoxyglucose and methylglucose confirmed the lack of expression of
GLUT2 and GLUT5 by HL-60 cells. GLUT5 transports fructose but not
deoxyglucose, and its functional activity is not affected by
cytochalasin B(43) . GLUT2 transports fructose and
deoxyglucose, and its functional activity is affected by micromolar but
not by nanomolar concentrations of cytochalasin B(44) .
Furthermore, the K
for the transport of
deoxyglucose or methylglucose by GLUT2 is in the range of 15 to 30
mM, as opposed to 3-8 mM for the transporter
expressed by the HL-60 cells. Direct evidence for the presence of GLUT1
in HL-60 cells was provided by the results of immunoblotting and
immunolocalization experiments with specific antibodies that indicated
a clear reactivity with anti-GLUT1 and an absence of reactivity with
anti-GLUT2, -GLUT3, -GLUT4, and -GLUT5 antibodies.
Our transport
data are consistent with the models presented in Fig. 7. Short
uptake assays enabled us to carry out a detailed kinetic analysis of
the transport of dehydroascorbic acid as distinct from the accumulation
of ascorbic acid. Only one functional component, with an apparent K of 0.75 mM, was involved in the
transport of dehydroascorbic acid by HL-60 cells. The competition
studies indicated that dehydroascorbic acid was transported by a
facilitative mechanism which, together with the identification of GLUT1
as the main glucose transporter present in the HL-60 cells, strongly
supports the concept that GLUT1 mediates the transport of
dehydroascorbic acid in these cells. The competition studies,
especially the absence of effect of
-methylglucoside on the
transport of dehydroascorbic acid, indicated the lack of participation
of a transporter with the functional characteristics of the
sodium-glucose cotransporter in the transport of dehydroascorbic acid
by HL-60 cells.
Figure 7: Models for differentiating transport from accumulation during uptake experiments. A, the initial rate of transport of a substrate, such as methylglucose, that is not metabolized in the interior of the cell, corresponds to the early linear phase of the uptake curve. As the intracellular concentration of the recently transported substrate increases, efflux becomes significant until its rate equals the rate of influx. Under steady state conditions, the intracellular and extracellular concentrations of the transported substrate are identical, and the net rate of transport in any direction is zero. The kinetic analysis is done using data obtained at short uptake times in which the rate-limiting step of uptake is transport. OMG, methylglucose. B, the situation is more complex for a substrate such as dehydroascorbic acid that is transported intracellularly and then trapped by reduction to the non-transported species ascorbic acid. The early linear phase of the uptake curve represents the initial rate of transport, but a second linear phase that represents the trapping component and may last for a long period of time is also evident. Trapping is the rate-limiting step when using long uptake assays because the rate of transport exceeds the rate of trapping, and part of the transported dehydroascorbic acid will be transported back out of the cell. At steady state, the intracellular concentration of the trapped, non-transported species, may exceed by severalfold the extracellular concentration of the transported substrate. In a typical uptake experiment using radioactive dehydroascorbic acid, this result may give the false impression that the transported substrate, dehydroascorbic acid, is accumulating against a concentration gradient. Thus, long uptake experiments measure the accumulation of the non-transported species and its respective associated kinetic constants, rather than the transport of the substrate handled by the transporter. Similar considerations apply to the uptake of deoxyglucose. AA, ascorbic acid; DHA, dehydroascorbic acid.
The existence of several components involved in the uptake of dehydroascorbic acid by HL-60 cells was evident only in long uptake experiments that are a complex function of the transport of dehydroascorbic acid and the intracellular trapping of ascorbic acid. We failed to detect the presence of a second, high-affinity component involved in the transport of dehydroascorbic acid by HL-60 cells. We observed, however, a high-affinity component when measuring the accumulation of ascorbic acid in cells incubated with dehydroascorbic acid for periods of time equal to or longer than 10 min. Under these conditions, the intracellular trapping of ascorbic acid rather than transport is the rate-limiting step for uptake. An additional low-affinity component was also observed in these studies, adding another level of complexity to the overall process of accumulation of ascorbic acid. Competition experiments showed that the transport of dehydroascorbic acid was inhibited by methylglucose and deoxyglucose, and the specificity of this inhibition was demonstrated by the results indicating that other sugars unable to interact with the glucose transporters had no effect on the transport of dehydroascorbic acid. Competition experiments, using 10-min incubation assays to measure accumulation, indicated that hexoses completely blocked the accumulation of ascorbic acid in HL-60 cells by a noncompetitive mechanism. We interpreted these results as indicating that glucose acts directly on the primary event responsible for the entry of dehydroascorbic acid, that is GLUT1-mediated transport, not on the functional component(s) involved in the intracellular accumulation of ascorbic acid. Therefore, the high- and low-affinity components detected in the 10-min uptakes are likely associated with the reduction of dehydroascorbic acid permitting the intracellular accumulation of ascorbic acid. Support for the interpretation that the high-affinity component is not related to the glucose transporter is provided by the observation that low concentrations of dehydroascorbic acid (<50 µM) did not affect the transport of methylglucose or deoxyglucose. A high-affinity component has been identified in experiments measuring the accumulation of ascorbic acid in different cellular systems and the hypothesis has been advanced that it corresponds to a high-affinity transporter of reduced ascorbic acid (27) . These studies were performed, however, under experimental conditions that led to the oxidation of ascorbic acid with generation of the transported form, dehydroascorbic acid. Of major concern is the fact that in such studies the kinetic constants were derived from long uptake experiments (90 min), raising the question of the identity of the rate-limiting step under those conditions(27) . Our data indicating that deoxyglucose was able to completely inhibit the cellular uptake of dehydroascorbic acid in a range of experimental conditions, including different concentrations of dehydroascorbic acid and different times of incubation, are consistent with the concept that GLUT1 is the only means by which the HL-60 cells transport dehydroascorbic acid.
Our results also indicate that HL-60
cells do not express a Na-sensitive ascorbate
transporter. Although we observed a major decrease in uptake of
dehydroascorbic acid by HL-60 cells in the absence of
Na
, the kinetic data clearly showed that the step
inhibited in the absence of Na
was the accumulation of
ascorbic acid, and not the transport of dehydroascorbic acid via GLUT1.
Na
-sensitive ascorbate transporters have been proposed
to exist in several tissues and cells such as adrenomedullary
chromaffin cells, retinal epithelial cells, osteoblasts, and the
brush-border membrane of reabsorbing renal
epithelia(5, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 42) .
In these studies, incubating the cells in the absence of external
Na
produced a marked decrease in the uptake of
ascorbic acid. Evidence for Na
-sensitive uptake of
ascorbate was recently provided by expression studies in X. laevis oocytes injected with poly(A)
RNA
extracted from rabbit kidney cortex(24) .
Taken together, our data indicate that the transport of dehydroascorbic acid via the glucose transporters is kinetically and biologically separable from the reduction of dehydroascorbic acid to ascorbic acid and its subsequent intracellular accumulation. These findings have major significance for our understanding of the biological control mechanisms involved in the homeostasis of the cellular content of ascorbic acid in human cells. We can envision the existence of separate mechanisms that regulate the ability of a cell to transport dehydroascorbic acid and its capacity to reduce the transported substrate intracellularly and accumulate ascorbic acid. The issue of the mechanisms involved in the intracellular reduction of dehydroascorbic acid and the concomitant accumulation of ascorbic acid has been controversial. No dehydroascorbic acid reductase activity has been consistently detected in cells or human tissues that accumulate intracellularly millimolar concentrations of ascorbic acid when incubated in the presence of dehydroascorbic acid(45, 46) . The existence of a dehydroascorbate reductase has been clearly demonstrated in plants that use reduced glutathione to catalyze the reduction of dehydroascorbate to ascorbate, and a similar activity has been partially characterized in bovine iris-ciliary body(47) . In humans, it has been proposed that the tripeptide glutathione, which is present at millimolar concentrations in cells, may play a central role in maintaining extracellular and intracellular ascorbic acid in its reduced state(48) . Experimental observations in animal models have revealed a close interrelationship between the cellular content of ascorbate and glutathione. Guinea pigs and newborn rats cannot synthesize ascorbic acid and it must be provided in the diet(4) . Newborn rats, made glutathione deficient by treatment with L-buthionine-(S,R)-sulfoximine, showed a decreased content of ascorbic acid in tissues, and supplementation with ascorbic acid in the diet induced increased tissue levels of ascorbic acid and glutathione(49) . In guinea pigs fed an ascorbic acid-free diet, supplementation with glutathione monoester, a compound that increases the cellular levels of glutathione, delayed the onset and the development of the pathologic complications of scurvy (50) . These observations established a close functional correlation between the respective in vivo levels of glutathione and ascorbic acid, but did not provide information about the mechanisms involved. On the other hand, in vitro observations have established that glutathione is able to reduce dehydroascorbic acid to ascorbic acid in the absence of any enzymatic activity through a direct chemical reaction(37, 51) . No direct evidence is available, however, indicating that glutathione is the physiological reducer of dehydroascorbic acid in cells or that it is directly involved in the ability of cells to take up dehydroascorbic acid and accumulate ascorbic acid(52) . Recently, it was described that two well known and widely expressed enzymes, protein disulfide isomerase and glutaredoxin, possess dehydroascorbate reductase activity(53) , raising the possibility that they could be involved in the reduction-dependent intracellular accumulation of ascorbic acid in human cells. In fact, the characterization and purification of two different dehydroascorbate reductase activities present in rat liver led to the identification of glutaredoxin as one of the proteins (54) . The ability to differentiate the transport of dehydroascorbic acid from its accumulation as ascorbic acid offers an opportunity to address these and related issues in a controlled cellular system amenable to experimentation.