From the
It is unknown whether ascorbate alone (vitamin C), its oxidized
metabolite dehydroascorbic acid alone, or both species are transported
into human cells. This problem was addressed using specific assays for
each compound, freshly synthesized pure dehydroascorbic acid, the
specially synthesized analog 6-chloroascorbate, and a new assay for
6-chloroascorbate. Ascorbate and dehydroascorbic acid were transported
and accumulated distinctly; neither competed with the other. Ascorbate
was accumulated as ascorbate by sodium-dependent carrier-mediated
active transport. Dehydroascorbic acid transport and accumulation as
ascorbate was at least 10-fold faster than ascorbate transport and was
sodium-independent. Once transported, dehydroascorbic acid was
immediately reduced intracellularly to ascorbate. The analog
6-chloroascorbate had no effect on dehydroascorbic acid transport but
was a competitive inhibitor of ascorbate transport. The
K
Ascorbate (vitamin C) is accumulated in human tissues as much as
50-fold compared to plasma
(1) . However, the mechanism of
transport is unknown. One possibility is that ascorbate is transported
as such. Data supporting this mechanism are that ascorbate transport is
concentration dependent, saturable, energy dependent, and
sodium-dependent
(2, 3, 4, 5, 6, 7, 8) .
Ascorbate but not dehydroascorbic acid is found in plasma from healthy
volunteers
(9, 10) .
Another possibility is that
ascorbate is oxidized at or near cell membranes, enters cells as
dehydroascorbic acid, and is reduced intracellularly to
ascorbate
(3, 11, 12, 13, 14, 15) .
Dehydroascorbic acid is transported into neutrophils where it is
immediately reduced to ascorbate
(11, 12, 13) .
Dehydroascorbic acid was proposed to be transported by a glucose
transporter
(12, 13, 14, 15) , which was
identified as GLUT I expressed in Xenopus laevis oocytes
(15) . The interpretation of these results was that
dehydroascorbic acid was transported via GLUT I and that ascorbate as
such was not transported at all
(13, 14, 15) .
There are several explanations for these conflicting conclusions.
There have been no direct attempts to distinguish between ascorbate and
dehydroascorbic acid as transport substrates. Analog compounds have not
been commercially available which could potentially distinguish
transported substrates. Interpretation of experiments is also hampered
by lack of attention to these issues: substrate purity, specific assays
for both substrates, measurement of intracellular mass of both
substrates, an assay of analog mass, and the need to account for
substrate stability.
We addressed these problems in human cells by
directly investigating whether ascorbate transport alone occurs,
dehydroascorbic acid transport alone occurs, whether both occur, and
whether the carrier mechanisms are similar or different.
HPLC
For myelocyte tumor cells experiments were performed with suspended
cells in test tubes at concentrations of 1-2
For
measurement of dehydroascorbic acid reducing activity, neutrophil
homogenates were prepared. Approximately 1
Optimum
potentials for 6-deoxy-6-chloro-L-ascorbate detection were
determined using the Coulometric Electrochemical Array
System
(19) . The assay was adapted for the HPLC system described
for ascorbate quantitation using a mobile phase composed of 55%
methanol (v/v), 50 mM NaH
On-line formulae not verified for accuracy
Data points in all figures
represent the mean of
From these experiments we
could address whether ascorbate transport and accumulation occurred
independently of dehydroascorbic acid transport. Neutrophils were
incubated with 50 µM
[1-
Because 6-chloroascorbate was a
competitive inhibitor of ascorbate transport, we investigated whether
6-chloroascorbate could utilize the ascorbate transport mechanism for
accumulation against a concentration gradient with a sodium
requirement. Fibroblasts were incubated with 50 µM
6-chloroascorbate in the presence or absence of sodium for varying
times (Fig. 3, inset). 6-Chloroascorbate was accumulated
against a concentration gradient in the presence of sodium but was not
transported at all without sodium. These data suggest that
6-chloroascorbate is accumulated by the same mechanism as ascorbate.
We report here new evidence that both ascorbate and
dehydroascorbic acid are transported into human cells, but by separate
mechanisms. A number of experimental criteria distinguished the two
transport systems. Competition experiments showed that ascorbate but
not dehydroascorbic acid competed with
[1-
It is theoretically possible that a single transport protein
has two independent properties, such that ascorbate and dehydroascorbic
acid transport could be mediated by the same protein. Several lines of
evidence suggest this is not likely. GLUT I expressed in oocytes
transports glucose and dehydroascorbic acid, but not
ascorbate
(15) . Despite use of insensitive assay techniques, the
data suggest that one single transport protein (GLUT I) does not have
independent transport properties for ascorbate and dehydroascorbic
acid. Glucose transporters I-V are well
characterized
(28, 29) . They do not appear to be
sodium-dependent nor to have sites indicative of sodium dependence. As
shown here, such sodium dependence is required for ascorbate but not
dehydroascorbic acid transport. Recently, ascorbate transport activity
was expressed in oocytes using mRNA from rabbit kidney
(30) .
Activity was sodium-dependent and uptake of radiolabeled ascorbate was
inhibited by excess unlabeled ascorbate but not glucose, implying that
the putative ascorbate transporter is mediated by a different protein
than that responsible for dehydroascorbic acid transport. It is unclear
whether one or several mRNAs are responsible for ascorbate transport
activity and there are no clones for the transporter. These problems
must be solved before the transporter is expressed definitively. Until
this can be accomplished it is necessary to demonstrate functionally
that there are two distinct transport activities. The data in this
paper are the first to suggest that this is the case.
Generation of
reactive oxygen intermediates is a normal process in aerobic organisms.
However, these oxidants do not oxidize ascorbate outside resting
(unactivated) neutrophils or normal fibroblasts. The evidence is that
external ascorbate was not oxidized by resting neutrophils, and
dehydroascorbic acid was detected externally only when cells were
activated
(3, 11, 18) . Additional data are seen
in Fig. 1and . If oxidants from resting neutrophils
or fibroblasts oxidized external [1-
We and others proposed that intracellular ascorbate is utilized for
protection against permeant oxidants made by activated
neutrophils
(11, 31, 32) . Oxidants from
activated neutrophils oxidize extracellular ascorbate to
dehydroascorbic acid. Dehydroascorbic acid is more rapidly transported
than ascorbate and is immediately reduced intracellularly to ascorbate
for potential oxidant protection. High concentrations of intracellular
ascorbate would be available at the same time they would be needed to
quench newly formed oxidants generated by activated neutrophils.
Oxidant generation by neutrophils can be as high as 200 nmol
oxidant/10
There may be other explanations why
ascorbate recycling occurs. In theory, it is possible that
dehydroascorbic acid is reduced in cells so that ascorbate can be
continuously exported to replenish extracellular ascorbate consumed by
oxidants. However, ascorbate efflux does not occur from plated
fibroblasts or neutrophils (2, 18, data not shown). The results were
unaffected by mixing the incubation medium: efflux still did not occur
(data not shown). The best current explanation for ascorbate recycling
is that it rapidly provides high concentrations of ascorbate for
intracellular oxidant quenching at the time oxidants are generated by
activated neutrophils.
We found here that glucose was a
non-competitive inhibitor of dehydroascorbic acid transport, in
contrast to findings by others
(15) . There are several reasons
for the discrepancy. Incubation conditions used by others contained
ascorbate, DTT, and ascorbate oxidase to form dehydroascorbic
acid
(15) . True substrate concentrations were not measured
directly. Under these conditions external dehydroascorbic acid
concentrations would not be constant, making kinetic measurements
unreliable. Other analyses of substrate-velocity curves were performed
improperly
(15) . The two components of biphasic kinetics can be
separated by extrapolating the linear portion of the substrate-velocity
curve at higher substrate concentrations to the y axis. The
rate contribution represented by this line is then subtracted from the
original
data
(2, 3, 18, 20, 21) . Another
suitable method is to utilize non-linear regression programs, as used
in this paper. However, the method used by others
(15) of
drawing a line from the origin to a single point is invalid for
determining kinetics for a biphasic system. Other problems were that no
mass measurements were reported for kinetic analyses, and the few mass
measurements performed for other experiments utilized the insensitive
and nonspecific method of thin layer chromatography. Differences
between kinetic results here and elsewhere (15) cannot be accounted for
by diffusion limitations in the extracellular buffer. This is because
dehydroascorbic acid uptake and its inhibition by glucose were
identical when buffers were either mixed or stationary (data not
shown).
From findings by others
(15) , it was not clear
whether dehydroascorbic acid transport or its reduction was rate
limiting in the presence of glucose. For kinetic interpretations to be
correct, transport would have to be the rate-limiting step, and glucose
itself should not interfere with intracellular reduction. Using mass
measurements, we show here that transport was the rate-limiting step
only at dehydroascorbic acid concentrations
The mechanism of dehydroascorbic acid reduction in
cells is unknown. Although the isolated proteins protein disulfide
isomerase and glutaredoxin have dehydroascorbic acid reducing
activity
(25) , it is unclear whether these or any other proteins
actually mediate reduction in cells
(27) . Substantial evidence
suggests that dehydroascorbic acid reduction occurs only chemically
with glutathione as the
reductant
(24, 26, 27, 35, 36, 37) .
The data in this paper provide new evidence that dehydroascorbic acid
reduction in neutrophils is protein mediated and cannot be accounted
for via chemical reduction by glutathione. Definitive information on
the identity of the protein(s) involved will be best provided using
activity-based purification; this work is in progress.
Four different myelocyte cell lines were incubated for 30
min at 37 °C with 100 µM [1-
Homogenate preparation,
incubation conditions, and ascorbate measurements are described under
``Materials and Methods.'' Dehydroascorbic acid concentration
was 200 µM, glutathione concentration was 0.8 mM,
and NADPH concentration was 0.4 mM. The experiment was
performed using at least three different homogenates with similar
results.
for 6-chloroascorbate (2.9-4.4
µM) was similar to the K
for
ascorbate transport (9.8-12.6 µM). 6-Chloroascorbate
was itself transported and accumulated in fibroblasts by a
sodium-dependent transporter. These data provide new information that
ascorbate and dehydroascorbic acid are transported into human
neutrophils and fibroblasts by two distinct mechanisms and that the
compound available for intracellular utilization is ascorbate.
(
)
electrochemical assays were used for
ascorbate and dehydroascorbic acid, substrate purity and stability were
accounted for, an ascorbate analog was synthesized, and an assay was
developed to measure analog mass. Several distinct experimental
techniques provided novel evidence that both ascorbate and
dehydroascorbic acid were transported, but by separate mechanisms.
Materials
Ascorbate was purchased from Sigma.
[1-C]Ascorbate was purchased from DuPont NEN.
Bromine was purchased from Fluka. Anhydrous ether and
2,3-dimercapto-1-propanol were purchased from Aldrich. Dehydroascorbic
acid was prepared immediately prior to utilization using the method of
bromine oxidation of ascorbic acid as described previously
(11) .
6-Deoxy-6-chloro-L-ascorbate was synthesized as described
previously
(16) . Identity and purity were confirmed by NMR, thin
layer chromatography, mass spectral data, and by comparison of the
optical rotation with the literature value
(16) . All other
commercially obtained materials were of the highest grade available.
Cell Preparation
Neutrophils were isolated from
heparinized whole blood obtained from healthy adult male volunteers and
plated as described previously
(3) . Normal human skin fibroblast
strains CRL1497 and CRL1501 were obtained from American Tissue Cell
Collection. Fibroblast cultures were maintained as described previously
(2). Myelocyte cell lines were obtained from ATCC and were maintained
in RPMI 1640 with 10% fetal calf serum and 2 mM glutamine at
37 °C and 5% CO.
Methods
Ascorbate and dehydroascorbic acid
transport studies were performed as described
previously
(2, 11) . Experiments with neutrophils and
fibroblasts were conducted on plated cells incubated at 37 °C.
Culture plates were stationary to avoid cell detachment unless noted,
in which case incubation medium was mixed by agitation of plates. Cells
were incubated in HEPES/phosphate buffer containing 147 mM
NaCl, 5 mM KCl, 1.9 mM KHPO
,
1.1 mM Na
HPO
, 5.5 mM glucose,
0.3 mM MgSO
:7H
O, 1 mM
MgCl
:6H
O, 0.3 mM
CaCl
:2H
O, and 10 mM HEPES, pH 7.4. For
sodium-free buffer, NaCl and Na
HPO
were
replaced by choline chloride and K
HPO
.
Immediately prior to experiments cells were washed twice in
HEPES/phosphate buffer. Where indicated assays were performed in
HEPES/phosphate buffer with or without sodium. After incubation, plated
cells were washed twice with ice-cold phosphate-buffered saline, pH
7.4, which was removed by vacuum suction. The attached plated cells
were extracted using 0.5 ml of ice-cold 60% methanol, 1 mM
EDTA. After centrifugation at 15,000
g at 4 °C for
10 min, supernatants were frozen at -70 °C until analysis.
10
/ml in HEPES buffer at 37 °C. Cells were stationary
during incubations; settling did not occur. After incubation, cells
were washed by centrifugation three times with ice-cold
phosphate-buffered saline, pH 7.4. Cell pellets were extracted using
0.5 ml of ice-cold 60% methanol, 1 mM EDTA. After
centrifugation at 15,000
g at 4 °C for 10 min,
supernatants were frozen at -70 °C until analysis.
10
purified neutrophils were suspended in 10 ml of ice-cold 10
mM PIPES buffer, pH 7.4, containing 100 mM KCl, 3.5
mM MgCl
, 0.1 mM leupeptin, and 1
mM phenylmethylsulfonyl fluoride. Suspended cells were
pressurized with N
for 30 min at 350 pounds/square inch in
a nitrogen cavitation bomb. The cavitate was centrifuged at 100,000
g for 60 min, and the supernatant was used as a
homogenate. The homogenate was dialyzed for 18 h in 20 mM Tris
buffer at 4 °C, pH 7.5. Ascorbate reduced from dehydroascorbic acid
by the homogenate was measured using HPLC
(17) ; pure freshly
prepared dehydroascorbic acid was the substrate (10). The reaction was
performed in 25 µl of Tris buffer, pH 7.5, containing 0.8
mM glutathione, 0.4 mM NADPH, 200 µM
dehydroascorbic acid and 10 µl of dialyzed neutrophil homogenate at
room temperature. The reaction was performed for 3 min and was
terminated by adding 33 µl of ice-cold 90% methanol containing 1
mM EDTA. After centrifugation at 14,000
g for
10 min, 30 µl of supernatant was used immediately for measuring
dehydroascorbic acid reduction activity. Concentrated glucose solutions
prepared in 20 mM Tris buffer at pH 7.5 were added to assay
mixtures to yield the final indicated concentrations.
Assays
All measurements of intra- and
extracellular ascorbate were performed using high performance liquid
chromatography with coulometric electrochemical detection as described
previously (17). Dehydroascorbic acid was reduced to ascorbate with
2,3-dimercapto-1-propanol prior to determination by HPLC with
coulometric electrochemical detection as described
previously
(10) . [1-C]Ascorbate and
[1-
C]dehydroascorbic acid were measured using
both liquid scintillation spectrometry and HPLC with coulometric
electrochemical detection as described previously (11). Purity of
radiolabel was determined by HPLC with coulometric electrochemical
detection. Intracellular volume was determined as described previously
as a function of cell protein, which was measured using bicinchoninic
acid (Bio-Rad)
(3, 11, 18) .
PO
, 50
µM NaC
H
O
, 189
µM dodecyltrimethylammonium chloride, and 36.6
µM tetraoctylammonium bromide at a pH of 4.8 (17). Changes
in current were followed on the second electrode. The cell containing
the electrochemical detector was reversed so that electrode number two
was exposed to the solute first and the electrode was set at a
potential of 250 mV.
Data Analyses
Kinetics for all substrates were
determined when transport was linear; time points for assays were
selected as described previously
(2, 3, 18) . To
determine values for the high affinity dehydroascorbic acid transport
activity, the low affinity component was subtracted as described
previously
(2, 3, 18, 20, 21) . A
line was constructed through the linear portion of the curve from
100-800 µM. The rate for the lower affinity activity
was calculated (slope * [external dehydroascorbic acid]) and
subtracted from the observed value of v. Analysis of glucose
inhibition of dehydroascorbic acid transport was performed using the
non-linear regression program Enzfitter. Equations for competitive (i)
and non-competitive (ii) inhibition were
3 samples ± S.D.; S.D. is not
displayed when smaller than point size.
Competition between Ascorbate and Dehydroascorbic
Acid
To test whether there are separate carriers for ascorbate
and dehydroascorbic acid transport, we performed competition
experiments using 1-C-labeled substrate with increasing
concentrations of unlabeled substrate. Both intracellular isotope
amount and mass accumulation were measured. Human neutrophils were
incubated with either 50 µM
[1-
C]ascorbate or
[1-
C]dehydroascorbic acid and 0-500
µM of either unlabeled ascorbate or dehydroascorbic acid.
[1-
C]Ascorbate transport was inhibited by
extracellular unlabeled ascorbate but was unaffected by dehydroascorbic
acid (Fig. 1A). Conversely,
[1-
C]dehydroascorbic acid transport was
inhibited by increasing concentrations of extracellular unlabeled
dehydroascorbic acid but was unaffected by extracellular ascorbate
(Fig. 1B).
Figure 1:
Competition between ascorbate and
dehydroascorbic acid for transport. A, competition of
[1-C]ascorbate by ascorbate and dehydroascorbic
acid. Plated human neutrophils were incubated for 30 min at 37 °C
with 50 µM [1-
C]ascorbate with
increasing concentrations of either non-radiolabeled ascorbate
or freshly prepared non-radiolabeled dehydroascorbic acid (
).
B, competition of [1-
C]dehydroascorbic
acid by ascorbate and dehydroascorbic acid. Plated neutrophils were
incubated for 30 min at 37 °C with 50 µM
[1-
C]dehydroascorbic acid with increasing
concentrations of either non-radiolabeled ascorbate
or
non-radiolabeled dehydroascorbic acid (
). C, mass
accumulation of extracellular ascorbate and dehydroascorbic acid as
intracellular ascorbate. Plated neutrophils were incubated for 30 min
at 37 °C with 50 µM
[1-
C]dehydroascorbic acid with increasing
concentrations of either non-radiolabeled ascorbate
or
non-radiolabeled dehydroascorbic acid (
). Intracellular ascorbate
mass was determined using HPLC with coulometric electrochemical
detection. Endogenous ascorbate concentration in the absence of
extracellular ascorbate or dehydroascorbic acid was determined by HPLC
with coulometric electrochemical detection (
). Inset,
increase in intracellular ascorbate mass as a function of extracellular
ascorbate, in the presence of 50 µM external
[1-
C]dehydroascorbic acid. Accumulation from
ascorbate alone was determined at each point by subtracting
accumulation of 50 µM
[1-
C]dehydroascorbic acid from total
intracellular ascorbate mass. Data points in all figures represent the
mean of
3 samples ± S.D.; S.D. is not displayed when smaller
than point size. Assays were as described under ``Experimental
Procedures.''
Mass data for these experiments were
always consistent with labeled substrate findings. For example, we
predicted that when neutrophils were incubated with
[1-C]dehydroascorbic acid and increasing
concentrations of the same unlabeled compound, dehydroascorbic acid
transport would increase but resulting accumulation would be detected
only as ascorbate. These were the observed findings
(Fig. 1C, data not shown).
C]dehydroascorbic acid and increasing
concentrations of external unlabeled ascorbate. Accumulation from
ascorbate alone was determined at each point by subtracting
[1-
C]dehydroascorbic acid transport from total
ascorbate mass (Fig. 1C, inset). The data show
that ascorbate transport occurred independently of dehydroascorbic acid
and was concentration dependent even in the presence of dehydroascorbic
acid. Ascorbate was transported and accumulated at
10-fold slower
rate compared to dehydroascorbic acid, consistent with other
observations
(3, 11, 18) . Similar findings were
observed for competition and mass accumulation in human fibroblasts
(data not shown).
Ascorbate Analogs
To further distinguish between
ascorbate and dehydroascorbic acid transport, the analog
6-deoxy-6-chloro-L-ascorbate (6-chloroascorbate) was
synthesized. 6-Chloroascorbate has a chlorine substituted for the
hydroxyl on C-6. The remaining structure of the five-member lactone
ring and the stereochemistry at carbons 4 and 5 are
unchanged
(16) . A six-position substituted analog was chosen
because substitutions on C-5 or C-6 do not appear to change ascorbate
function
(22, 23) . Neutrophils or fibroblasts were
incubated with [1-C]ascorbate or
[1-
C]dehydroascorbic acid and increasing
concentrations of 6-chloroascorbate. 6-Chloroascorbate had no effect on
dehydroascorbic acid transport but inhibited ascorbate transport
4-10-fold (Fig. 2, A and B). Inhibition
of ascorbate transport by 6-chloroascorbate was competitive with a
K
of 4.4 µM in neutrophils
(Fig. 2A, inset) and 2.9 µM in
fibroblasts (Fig. 2B, inset). Both inhibition
constants are similar to the K
for
ascorbate of approximately 10 µM (Fig. 2, A and B,
insets)
(2, 3, 18) . Substitution of
other halogens at C-6 had similar effects on ascorbate
transport.
(
)
Figure 2:
Discrimination between ascorbate and
dehydroascorbic acid transport by 6-deoxy-6-chloroascorbate.
A, neutrophils were incubated with 50 µM
[1-C]dehydroascorbic acid for 10 min (
),
or 50 µM [1-
C]ascorbate for 10
(
) or 60 (
) min at 37 °C, with increasing
concentrations of 6-chloroascorbate. Inset, kinetics of
transport inhibition by 6-chloroascorbate. Neutrophils were incubated
with [1-
C]ascorbate (1.2-60
µM) and 6-chloroascorbate for 90 min at 37 °C. Data
are displayed in the Eadie-Hofstee format. 6-Chloroascorbate
concentrations (µM) were 0
, 12 (
), 30
(
). Assays were performed as described under ``Experimental
Procedures.'' B, human fibroblasts were incubated with 50
µM [1-
C]ascorbate for 30 (
)
or 90 (
) min, or 50 µM
[1-
C]dehydroascorbic acid for 30 min at 37
°C (left inset), with increasing concentrations of
6-chloroascorbate. Right inset, kinetics of transport
inhibition by 6-chloroascorbate. Fibroblasts were incubated for 2 h at
37 °C with [1-
C]ascorbate (1.2-60
µM) and 6-chloroascorbate. 6-Chloroascorbate
concentrations (µM) were 0
, 3 (
), 6
(▾), 12 (
), 30 (
). Assays were performed as
described under ``Materials and
Methods.''
Sodium Dependence of Transport
While
ascorbate transport has been shown to be sodium-dependent in
fibroblasts, it is unclear whether sodium is required for
dehydroascorbic acid
transport
(2, 4, 5, 6, 7, 8) .
Fibroblasts were incubated with dehydroascorbic acid in media with and
without sodium. Dehydroascorbic acid transport and subsequent reduction
to ascorbate were sodium-independent (Fig. 3).
Figure 3:
Sodium requirement for transport.
Dehydroascorbic acid transport with and without sodium. Plated
fibroblasts were incubated with 100 µM dehydroascorbic
acid with or without (
) sodium, for 0-30 min at 37
°C. Intracellular ascorbate was analyzed by HPLC with coulometric
electrochemical detection as described under ``Materials and
Methods.'' Inset, fibroblasts were incubated with 50
µM 6-chloroascorbate
or without (
) sodium for
0-300 min at 37 °C. 6-Chloroascorbate was determined by a new
coulometric electrochemical HPLC assay as described under
``Materials and Methods.''
Sodium
dependence of transport was investigated in neutrophils. Because
sodium-free buffers activated normal neutrophils but not myelocyte
tumor cells (data not shown), four cell lines were studied. Cells were
incubated with [1-C]ascorbate in the presence or
absence of sodium. Measurement of both mass and radiolabel indicated
ascorbate transport was sodium-dependent (). Cells were
also incubated with [1-
C]ascorbate, sodium, and
unlabeled dehydroascorbic acid. Dehydroascorbic acid had virtually no
effect on transport of [1-
C]ascorbate.
Nevertheless, dehydroascorbic acid produced a >15-fold increase in
intracellular ascorbate mass above that expected from
[1-
C]ascorbate alone (). These data
provide additional evidence that ascorbate and dehydroascorbic acid
transport occur by distinct mechanisms and that ascorbate transport is
sodium-dependent. In contrast to ascorbate, dehydroascorbic acid
transport and intracellular reduction in neutrophils is
sodium-independent
(11) . Taken together, the data indicate that
the requirement for sodium is another means to distinguish ascorbate
from dehydroascorbic acid transport.
Effects of Glucose on Transport and
Reduction
D-Glucose is a non-competitive inhibitor of
ascorbate transport in neutrophils
(18) , and dehydroascorbic
acid is transported via the glucose transporter GLUT I (15). Therefore,
we investigated whether glucose could be used to differentiate between
ascorbate and dehydroascorbic acid transport. To do so it was necessary
to characterize dehydroascorbic acid transport kinetics.
Dehydroascorbic acid uptake (2-800 µM) demonstrated
low and high affinity components (Fig. 4A). Kinetics of
the low affinity component were indeterminate due to two factors:
appearance of intracellular dehydroascorbic acid and dehydroascorbic
acid toxicity (Fig. 4B). At external dehydroascorbic
acid concentrations >800 µM intracellular radiolabel
exceeded intracellular ascorbate mass. Intracellular dehydroascorbic
acid which was not reduced to ascorbate accounted for the difference.
At extracellular dehydroascorbic acid concentrations >2000
µM cell death occurred. Subsequent kinetic measurements
were performed at extracellular dehydroascorbic acid concentrations
800 µM so that dehydroascorbic acid transport and not
reduction was measured. For the high affinity transport component
K
= 35.5 ± 9
µM and V
= 0.75 ± 0.4
mM/min (Fig. 4C). Contrary to previous
reports
(15) , glucose inhibition was non-competitive with
inhibition constants of K
= 2.37
± 0.79 mM and K
=
2.66 ± 0.38 mM(20, 21) . Graphic
analysis of observed and calculated uptake rates were compared and
supported these conclusions, as did algebraic analysis of the
Eadie-Hofstee plot (Fig. 4C,
inset)
(20, 21) . These data indicate that
glucose is a non-competitive inhibitor of dehydroascorbic acid
transport and that glucose cannot be used to distinguish ascorbate and
dehydroascorbic acid transport activities in neutrophils. External
dehydroascorbic acid concentrations were stable for the time course of
the experiments
(11) (data not shown).
Figure 4:
Dehydroascorbic acid transport kinetics
and inhibition by glucose. A, neutrophils were incubated for 5
min at 37 °C with freshly prepared
[1-C]dehydroascorbic acid (2-800
µM) with (
) and without
5.5 mM
glucose. Ascorbate was determined by scintillation spectroscopy as
described under ``Materials and Methods.'' B,
neutrophils were incubated for 5 min at 37 °C with freshly prepared
[1-
C]dehydroascorbic acid (2-10,000
µM) without glucose. Intracellular measurements represent
radiolabel (
) and mass
. Ascorbate was determined by
scintillation spectroscopy and HPLC with coulometric electrochemical
detection. C, inhibition equations were tested using the
non-linear regression analysis program Enzfitter (-,
non-competitive inhibition; - - - -, competitive inhibition). Data
points represent the high affinity activity in the presence of 5.5
mM glucose. Activity from the low affinity transport activity
was subtracted and the analysis was performed as described under
``Materials and Methods.'' Inset, Eadie-Hofstee
analysis of the substrate velocity plot with
and without
(
) glucose. Ascorbate was determined by scintillation
spectroscopy; all intracellular label was ascorbate. Data for the low
affinity component were subtracted as described under ``Materials
and Methods'' and replotted for the high affinity component using
the weighted means of the corrected rates.
For interpretations of
dehydroascorbic acid transport kinetics to be accurate, transport and
not reduction must be the rate-limiting step. Mass transport data
(Fig. 4B) imply that transport is the rate-limiting step
for external dehydroascorbic acid concentrations 800
µM. To address the issue further, we investigated whether
glucose inhibited dehydroascorbic acid reduction. For these experiments
it was necessary to partially characterize the dehydroascorbic acid
reduction activity of neutrophils. Dehydroascorbic acid reduction in
neutrophil homogenates was similar to that expected from the same
number of whole cells, was localized to cytosol, was dependent on cell
number, was non-dialyzable, and was found in the retentate using
centrifugal ultrafiltration (data not shown). Dehydroascorbic acid
reduction required reduced glutathione and NADPH for maximal activity
(). Activity could not be accounted for by simple chemical
reduction of dehydroascorbic acid and was
protein-mediated
(24, 25, 26, 27) .
Glucose was tested as an inhibitor of dehydroascorbic acid reduction
and had no effect (). These data together with those in
Fig. 4B indicate that for dehydroascorbic acid
concentrations
800 µM, reduction is not a
rate-limiting step for transport, and the assumptions for determining
transport kinetics are correct.
Substrate Stability
It remained possible that
inadvertent ascorbate oxidation could influence some experimental
results. External ascorbate was not oxidized under the experimental
conditions used here (Fig. 1, , data not
shown)
(3, 11, 18) . Nevertheless, additional
confirmatory experiments were performed. Thiol reagents such as DTT can
prevent ascorbate oxidation and reduce dehydroascorbic acid to
ascorbate
(9, 10) . We investigated the effects of DTT on
ascorbate and dehydroascorbic acid transport. If no oxidation of
external ascorbate occurred, DTT should not influence ascorbate
transport. By contrast, DTT will reduce dehydroascorbic acid to
ascorbate. Since the rate of ascorbate transport is >10-fold slower
than that of dehydroascorbic acid, its transport should be decreased by
DTT as reduction occurs. Neutrophils were incubated with either 200
µm dehydroascorbic acid or [1-C]ascorbate
and increasing concentrations of DTT. Both
[1-
C]ascorbate uptake and mass accumulation were
measured. As expected, DTT decreased dehydroascorbic acid transport as
measured by intracellular ascorbate accumulation
(Fig. 5A). DTT had no effect on
[1-
C]ascorbate transport and ascorbate
accumulation at extracellular DTT concentrations up to 100 µM (Fig. 5, B and C). These data provide
additional evidence that inadvertent oxidation of ascorbate did not
occur. Ascorbate transport in the presence of DTT was not observed in
neutrophils by others
(15) probably because the incubation time
was too short.
Figure 5:
Effects of DTT on ascorbate and
dehydroascorbic acid transport. Human neutrophils were incubated with
200 µM freshly prepared dehydroascorbic acid for 10 min
(A) or 200 µM
[1-C]ascorbate for 60 min (B and
C) at 37 °C with increasing concentrations of DTT. DTT
concentrations were 0 (
), 50 (
), or 100 (
)
µM. Ascorbate was determined by HPLC (A and
C) or scintillation spectroscopy (B). Assays were as
described under ``Materials and
Methods.''
C]ascorbate for transport. Conversely,
dehydroascorbic acid but not ascorbate competed with
[1-
C]dehydroascorbic acid for transport. Both
ascorbate transport and accumulation occurred in the presence of
dehydroascorbic acid and were independent of it. Likewise,
dehydroascorbic acid was transported independently of ascorbate. The
specially synthesized analog 6-chloroascorbate had no effect whatsoever
on dehydroascorbic acid transport. By contrast, this analog
competitively inhibited ascorbate transport with a
K
similar to the
K
for ascorbate transport. Ascorbate
transport was sodium-dependent, while dehydroascorbic acid transport
was sodium-independent. Finally, 6-chloroascorbate was transported and
accumulated against its concentration gradient only in the presence of
sodium.
C]ascorbate,
extracellular dehydroascorbic acid would have inhibited accumulation of
newly formed [1-
C]dehydroascorbic acid. Also, if
oxidation of external [1-
C]ascorbate occurred
outside resting cells, extracellular dehydroascorbic acid could have
changed the rate or amount of [1-
C]ascorbate
accumulation. None of these findings were observed. Possibilities to
explain why aerobic metabolites did not oxidize ascorbate outside
resting neutrophils and fibroblasts include: compartmentalization or
unavailability of oxidants to external ascorbate because of the site of
oxidant production, a slow rate of oxidant production, and quenching of
generated oxidants before they are available to external ascorbate.
cells
h
(33, 34) . It was
predicted that for ascorbate to be important for oxidant quenching,
rapid intracellular reduction of oxidized ascorbate would have to
occur
(34) . The data here and elsewhere suggest that ascorbate
recycling, or reduction within cells of newly formed extracellular
dehydroascorbic acid, occurs in neutrophils. In addition, reutilization
of extracellular dehydroascorbic acid occurs when neutrophils are
exposed to bacteria and ascorbate but not to ascorbate
alone(
).
800 µM.
Dehydroascorbic acid reduction was incomplete at extracellular
concentrations above 800 µM. We also demonstrated directly
that glucose itself did not interfere with reduction in neutrophil
homogenates. Only with this information is it possible to perform
correct kinetic calculations for high affinity dehydroascorbic acid
transport. Despite previous claims
(15) , calculations for low
affinity transport were not valid because these issues were not
accounted for.
Table:
Ascorbate and dehydroascorbic acid transport in
myelocytes
C]ascorbate with sodium, 100 µM [1-
C]ascorbate without sodium, or 100
µM [1-
C]ascorbate plus 200
µM dehydroascorbic acid with sodium in HEPES/phosphate
buffer, pH 7.4. Dehydroascorbic acid was freshly prepared to purity.
Cell concentration was 1
10
cells/assay.
[l-
C]Ascorbate uptake was determined using
scintillation spectroscopy (Scint. Spec.) and HPLC with coulometric
electrochemical detection (Mass) as described under ``Materials
and Methods.''
Table:
Effect of glucose on dehydroascorbic acid
reduction by neutrophil homogenates
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.