© 1998 American Heart Association, Inc.
Original Contributions |
Ascorbate Prevents the Interaction of Superoxide and Nitric Oxide Only at Very High Physiological Concentrations
From the Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass.
Correspondence to John F. Keaney, Jr, MD, Whitaker Cardiovascular Institute, Boston University School of Medicine, 715 Albany St, Room W507, Boston, MA 02118. E-mail jkeaney{at}bu.edu
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Abstract |
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Abstract—The bioactivity of nitric oxide (
NO) depends, in part, on its interaction
with superoxide. Usually, superoxide dismutase (SOD) preserves
NO bioactivity by limiting the availability of
superoxide. Ascorbic acid also effectively scavenges superoxide, but
the extent to which this interaction is necessary for intact
NO bioactivity is not known. Therefore, the present
study examined the effect of ascorbic acid on NO
bioactivity with isolated rabbit arterial segments. A
steady flux of superoxide (1.15 to 2.3 µmol ·
L-1 · min-1) produced either by
pyrogallol autoxidation or a hypoxanthine/xanthine oxidase system
inhibited endothelium-derived
NO-mediated arterial relaxation elicited by
acetylcholine. This effect of superoxide was completely blocked by SOD
(300 IU/mL) and the manganese SOD mimic EUK-8 (300 µmol/L) and
partially inhibited by ascorbic acid (10 mmol/L). Lower
concentrations of ascorbic acid were ineffective despite scavenging
>90% of superoxide. We increased the endogenous flux of
superoxide (3.2±0.3-fold) by inhibiting vascular copper-zinc SOD with
diethyldithiocarbamate. This increased endogenous flux of
superoxide produced an impairment of NO-mediated
arterial relaxation that was reversed by EUK-8 (300
µmol/L) but not ascorbic acid (10 mmol/L) despite equivalent
scavenging of the endogenous superoxide flux. We used
3-nitrotyrosine formation (from peroxynitrite) as an indicator of
NO interaction with superoxide and found that SOD and
EUK-8 compete more effectively with NO for superoxide
than does ascorbic acid. These data indicate that preservation of
NO bioactivity by superoxide scavengers depends not only
on superoxide scavenging activity, but also on the rate of superoxide
scavenging. Normal extracellular concentrations of ascorbic acid (30 to
150 µmol/L) are not likely to prevent the interaction of
NO with superoxide under physiological conditions.
Key Words: antioxidant • free radical • blood vessel • oxidant • peroxynitrite
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal vascular homeostasis depends on endothelial elaboration of paracrine factors that prevent both platelet adhesion to the endothelial surface and inappropriate vasospasm. One important endothelial product responsible for these functions is nitric oxide (
NO),1 a
free radical produced constitutively by the vascular
endothelium. Abnormalities in
NO action and metabolism are known
to develop in association with vascular disease and have been
implicated in the development of clinically significant vascular
events.1
In vivo, NO is subject to rapid inactivation
by the superoxide anion,2 3 4 an obligate
product of normal oxidative
metabolism.5
Endothelial cells constitutively produce both
superoxide6 and
NO,7 suggesting that the
effective release of NO from the vascular
endothelium depends on the relative concentrations of
these 2 species.
Usually, the availability of superoxide in tissues is strictly limited
by the abundant tissue concentration of superoxide dismutase (SOD) that
may approach 10 µmol/L.8 However,
superoxide and NO react rapidly with a
bimolecular rate constant that approaches the diffusion limit
(1.9x1010 mol ·
L-1 ·
s-1)9 and is similar to
the rate of superoxide dismutation by SOD (2x109
mol · -1 ·
s-1).8 These data indicate
that NO competes effectively with SOD for
superoxide. Considerable data now exist to support this position. For
example, inhibition of endothelial cell copper-zinc SOD
impairs effective release of NO from
endothelial cells.10 11 Intact
copper-zinc SOD function is also required for smooth muscle cell
relaxation in response to nitrovasodilators.10 In
addition, abnormalities in NO-mediated
arterial relaxation associated with
hypercholesterolemia,12 13
diabetes mellitus,14 and
hypertension15 have been linked to excess
vascular levels of superoxide. Thus,
NO-mediated arterial relaxation
depends on SOD activity to limit the availability of superoxide.
The tissue availability of superoxide is limited by its interaction
with other compounds in addition to SOD. Antioxidants such as
-tocopherol,16
glutathione,17 and ascorbic
acid18 are known to react with superoxide. Recent
studies have demonstrated that acute treatment with ascorbic acid
improves NO-mediated arterial
relaxation in patients with
atherosclerosis,19 but the
mechanism of this effect is not clear. The action and
metabolism of endothelium-derived
NO (EDNO) depends on vascular levels of
superoxide2 3 12 13 ; thus, it is conceivable that
ascorbic acid may exert some control over
NO-mediated arterial relaxation by
preventing the interaction of NO and
superoxide. The purpose of the present study was to determine the
extent to which physiologically relevant
concentrations of ascorbic acid influence arterial
relaxation in response to EDNO.
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Materials and Methods |
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Sodium pentobarbital was purchased from Anthony Products Co. Peroxynitrite was obtained from Alexis Corporation and diethylamine
NO (DEA-NO) was purchased from Cayman Chemical
Corp. Xanthine oxidase was purchased from Boehringer-Mannheim.
Acetylcholine hydrochloride, phenylephrine,
diethylenetriamine pentaacetic acid (DTPA), SOD (copper-zinc form from
bovine erythrocyte, 2500 to 7000 IU/mg), and all other compounds were
purchased from Sigma Chemical Co. Physiological salt solution (PSS) contained 118.3 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, 11.1 mmol/L glucose, 10 µmol/L indomethacin, and 0.026 mmol/L Na2EDTA. PBS consisted of 10 mmol/L NaH2PO4 and 0.15 mol/L NaCl (pH 7.4).
In Vitro Assay of Vascular Function
New Zealand White rabbits (2.5 to 3.5 kg) of either sex
were used for the present study (Pine Acres Rabbitry, Vt). Animals
consumed food and water ad libitum, and all animal studies were
approved by the Boston University Medical Center Institutional Animal
Care and Use Committee. The thoracic aorta was isolated from New
Zealand White rabbits killed with pentobarbital (120 mg/kg) via a
marginal ear vein. Vessel segments were prepared, suspended in organ
chambers as previously described,20 and gassed
with 95% O2/5% CO2. After
equilibration for 90 minutes, vessels were contracted with
phenylephrine (1 µmol/L) and vascular tone was
assayed in response to the addition of acetylcholine or the
endothelium-independent, cGMP-dependent vasodilator,
atrial natriuretic peptide (ANP). When pyrogallol was used
as a source of superoxide it was added 1 minute before the assessment
of vascular function. In some experiments, hypoxanthine (100
µmol/L) was added to the PSS and superoxide generation initiated with
0.02 IU/mL xanthine oxidase 1 minute before assessing vascular
function. Ascorbic acid was dissolved in PSS and the pH adjusted with
NaOH to produce a final pH of 7.4 in the organ chamber. Ascorbic acid
was added to organ chambers 10 minutes before the assessment of
arterial relaxation.
In some studies, vessels were treated for 30 minutes with 5 mmol/L diethyldithiocarbamate (DDC) to inhibit copper-zinc SOD.21 Vessels treated with DDC were subsequently washed 3 times with PSS containing 100 µmol/L DTPA to remove any residual redox-active copper liberated by DDC treatment.
Quantification of Superoxide and NO
The flux of superoxide from pyrogallol autoxidation was
quantified as the reduction of cytochrome c inhibited by SOD
with an extinction coefficient of 2.1x104 mol
· L-1 ·
cm-1 at 560 nm.22 The flux
of NO from DEA-NO decomposition was estimated
spectrophotometrically at 250 nm (
=6500 mol ·
L-1 · cm-1) noting
that each mole of DEA-NO produces 
1.5 mol
NO.23 Because ascorbate
directly reduces cytochrome c,24
scavenging of superoxide by ascorbate was estimated by the inhibition
of pyrogallol autoxidation, which is superoxide dependent at
pH<9.5.25 Pyrogallol (200 µmol/L) was
incubated in PBS, and autoxidation was estimated by monitoring the
change in absorbance at 420 nm25 with and without
ascorbic acid or SOD. All additions of ascorbic acid were adjusted to
achieve a final pH of 7.4.
Vascular SOD Activity
Segments of thoracic aorta were isolated as described above and
incubated with PSS at 37°C gently bubbled with 95%
O2/5% CO2. After 30
minutes, vessels were incubated for 10 minutes in 5-mL polyethylene
tubes containing HEPES-buffered PSS (PSS containing 20 mmol/L
HEPES) with 0.25 mmol/L lucigenin (bis-N-methylacridium
nitrate). After equilibration with lucigenin, vascular superoxide
levels were estimated from chemiluminescence recorded with a Turner
Designs Model 20e luminometer at 37°C in a dark, light-sealed room.
The integral of the chemiluminescence signal was recorded at
30-second intervals for 5 minutes, and the integral readings were
combined. Background chemiluminescence was determined from identically
processed vessel-free incubations and subtracted from the
determinations with vessels. Chemiluminescence was converted to
superoxide by a standard curve relating known quantities of superoxide
(from a xanthine/xanthine oxidase system as determined by SOD-inhibited
cytochrome c reduction) to chemiluminescence. To
inactivate copper-zinc SOD, vessels were incubated for 30
minutes with 5 mmol/L DDC, washed 3 times (10 mL) with
HEPES-buffered PSS containing DTPA (100 µmol/L), and superoxide
determined as above. The effect of ascorbic acid, SOD, and EUK-8 (a
cell-permeable manganese SOD [MnSOD] mimic; Evkaryon, Bedford,
Mass)26 on vascular superoxide scavenging
activity was determined by adding these compounds directly to the
chemiluminescence chamber and repeating the measurement. In the absence
of such additions, the chemiluminescence signal was stable during the
time of the assay.
Estimation of Peroxynitrite Formation
The interaction of NO and superoxide
results in the formation of peroxynitrite27 that,
in the presence of CO2, spontaneously reacts with tyrosine
to form 3-nitrotyrosine.28 We estimated
peroxynitrite formation as the production of 3-nitrotyrosine
with a modification of the method described by van der Vliet et
al.29 d,l-Tyrosine (1 mmol/L) in
10 mmol/L phosphate buffer with 50 µmol/L DTPA was
incubated with DEA-NO (20 µmol/L) and pyrogallol (200
µmol/L) with or without ascorbic acid (0 to 10 mmol/L), SOD (0.3
to 300 IU/mL), or EUK-8 (0.1 to 300 µmol/L) for 15 minutes. The
formation of 3-nitrotyrosine was analyzed by UV detection at
274 nm after separation on an LC-18 column (25 cmx4.6 mm,
Supelco) with a mobile phase of 50 mmol/L
KH2PO4, pH 3 and methanol
(92:8).29 Ascorbic acid (0 to 10 mmol/L) had
no effect on the yield of 3-nitrotyrosine when authentic peroxynitrite
(10 mmol/L) was added to 2 mmol/L d,l-tyrosine
(data not shown).
Data Analysis
All values are presented as mean±SEM. The vascular
responses to acetylcholine and NO are reported as the percent reduction
in tension (relaxation) compared with the contraction produced by
1 µmol/L phenylephrine. Dose responses to
acetylcholine and ANP were compared within treatment groups with
repeated-measures ANOVA and responses between treatment groups were
compared with 2-way ANOVA with a post hoc Dunn's or Dunnett's test as
appropriate. Statistical significance was accepted if the null
hypothesis was rejected with a P<0.05.
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Results |
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Effect of Superoxide and Ascorbic Acid on Arterial Relaxation
We observed dose-dependent arterial relaxation of the thoracic aorta in response to acetylcholine between the concentrations of 1 nmol/L and 10 µmol/L (Figure 1A
). In contrast, superoxide
(2.3±0.04 µmol · L-1 ·
min-1) generated from autoxidation of pyrogallol
(200 µmol/L) significantly inhibited the dose-dependent
relaxation to acetylcholine with maximal relaxation reduced from
73±3% to 48±3% (P<0.05 by 2-way repeated-measures
ANOVA). This effect of superoxide was completely reversed with 300
IU/mL SOD producing a maximal relaxation of 66±6% (P=NS
versus control). To test the effect of superoxide on smooth muscle cell
relaxation independent of NO, we examined
arterial relaxation in response to the cGMP-dependent
vasodilator, ANP. As shown in Figure 1B, superoxide generated from
pyrogallol (200 µmol/L) autoxidation had no effect on
cGMP-dependent arterial relaxation in response to ANP.
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), 200
µmol/L pyrogallol (
), or 300 IU/mL SOD (
). B, Vessels were contracted as
mentioned above and relaxation to ANP assayed in the presence of PSS
containing no additions (
To determine whether ascorbic acid can prevent the inactivation of
NO by superoxide, we incubated
arterial segments with pyrogallol and increasing doses of
ascorbic acid just before the assessment of arterial
relaxation. As expected, a steady flux of superoxide from pyrogallol
autoxidation produced significant inhibition of EDNO-mediated
arterial relaxation (P<0.05 by 2-way
repeated-measures ANOVA; Figure 2). The
impairment of EDNO-mediated arterial relaxation by
superoxide was prevented by ascorbic acid only at a concentration of
10 mmol/L (Figure 2A). We also sought to confirm these
observations with another superoxide-generating system. We used a
steady flux of superoxide (1.15±0.2 µmol ·
L-1 · min-1; n=3)
from hypoxanthine (100 µmol/L) and xanthine oxidase (0.02 IU/mL)
and observed significant inhibition of EDNO-mediated
arterial relaxation that was reversed by SOD
(P<0.05 by 2-way repeated-measures ANOVA; Figure 2B).
Similar to the situation with pyrogallol, this impairment of
EDNO-mediated arterial relaxation by superoxide was only
partially prevented by ascorbic acid at a concentration of 10
mmol/L (Figure 2B).
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), 200 µmol/L
pyrogallol (
), or 200
µmol/L pyrogallol with 0.1
(
), 1.0 (
) mmol/L ascorbic
acid. B, Vessels were prepared as in panel A, except relaxation was
assessed in the presence of PSS containing no additions
(
P<0.05 vs HX/XO alone by 2-way ANOVA.
Ascorbate and Superoxide Scavenging
To quantify the extent of superoxide scavenging by ascorbate and
SOD in our system, we determined the inhibition of pyrogallol
autoxidation, which is superoxide-dependent at
pH<9.5.25 As presented in Figure 3, the autoxidation of pyrogallol was
inhibited 91±3% by 300 IU/mL SOD (P<0.05 versus control,
n=4) compared with 85±8%, 99±4%, 98±3%, and 99±4% by 0.05, 0.1,
1, and 10 mmol/L ascorbic acid, respectively (all
P<0.05 versus control by 1-way ANOVA, n=4). Thus, ascorbate
effectively scavenges superoxide at concentrations that are
considerably lower than those needed to preserve
NO-mediated arterial
relaxation.
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Pyrogallol and Endothelial Cell Toxicity
To determine whether pyrogallol produced
endothelial damage, we assessed
acetylcholine-stimulated EDNO-mediated arterial relaxation
in vessel segments before and after a 20-minute exposure to a flux of
superoxide (2.3 µmol · L-1
· min-1) from pyrogallol (200 µmol/L)
autoxidation. As shown in Figure 4, endothelium-dependent arterial relaxation
to acetylcholine was the same before and after exposure to pyrogallol,
indicating that short-term exposure to superoxide does not result in
any permanent impairment in NO-mediated
arterial relaxation.
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), during (
Effect of Ascorbic Acid on NO-Mediated
Arterial Relaxation With an Endogenous Flux
of Superoxide
It is difficult to extrapolate observations with pyrogallol to
events that are relevant in vivo. To generate a more relevant
superoxide flux, we treated aortic segments with 5 mmol/L DDC, a
copper chelator that inactivates endogenous
copper-zinc SOD.21 As shown in Figure 5A, treatment of aortic segments with DDC
produces a significant impairment in EDNO-mediated arterial
relaxation in response to acetylcholine (P<0.001 versus no
DDC by 2-way ANOVA). This impairment in EDNO-mediated
arterial relaxation was not mitigated by ascorbic acid in
concentrations up to 10 mmol/L (all P<0.05 versus no
DDC by 2-way ANOVA; Figure 5A). Authentic SOD (300 IU/mL) only
partially restored EDNO-mediated arterial relaxation
(Figure 5B), which was likely a result of its limited cellular access.
In contrast, the MnSOD mimic EUK-8 (300 µmol/L) completely
restored EDNO-mediated arterial relaxation in response to
acetylcholine (Figure 5B). Endothelium-independent
arterial relaxation to ANP was not impaired in DDC-treated
vessels (Figure 5C).
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Vascular Superoxide Scavenging With Endogenous Superoxide
To determine the extent of superoxide scavenging with an
endogenous flux of superoxide, we estimated vascular
superoxide in vessel segments treated with DDC, ascorbate, SOD, or
EUK-8 as mentioned above. As shown in Figure 6, treatment of aortic segments with
5 mmol/L DDC resulted in a 3.2±0.3-fold increase in the
superoxide signal by lucigenin chemiluminescence (P<0.05).
This DDC-mediated increase in superoxide was reduced 55%, 81%, and
90% by 0.1, 1, and 10 mmol/L ascorbic acid, respectively
(P<0.001 for dose response by ANOVA). In fact, treatment
with either 10 mmol/L ascorbic acid, 300 µmol/L EUK-8, or
300 IU/mL SOD reduced the superoxide signal in DDC-treated aortic
segments to near control levels (P=NS versus control). Thus,
although ascorbic acid, EUK-8, and SOD all reduced vascular superoxide
in DDC-treated aortic segments, only EUK-8 and SOD improved
EDNO-mediated arterial relaxation.
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Ascorbic Acid and Superoxide Competition for
NO
The interaction of NO and superoxide
results in the formation of peroxynitrite27 that
spontaneously reacts with tyrosine to form
3-nitrotyrosine.28 Incubation of tyrosine (2
mmol/L) with an equimolar flux of superoxide (2.3 µmol ·
L-1 · min-1from
pyrogallol autoxidation) and NO (2.1
µmol · L-1 ·
min-1 from DEA-NO) for 15 minutes readily
produced 3-nitrotyrosine (2.1±0.31 µmol/L; Figure 7) indicating the formation of
peroxynitrite. Both SOD and EUK-8 were able to compete effectively with
NO for superoxide at concentrations exceeding
0.2 µmol/L and 1 µmol/L, respectively (both
P<0.05 versus PSS alone by ANOVA; Figure 7). In contrast,
ascorbic acid was only partially effective in competing with
NO for superoxide even at a concentration of
10 mmol/L (Figure 7). Thus, ascorbic acid is not as effective as
SOD or EUK-8 in competing with NO for
superoxide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data presented here indicate that ascorbic acid does not compete effectively with
NO for superoxide
at physiologically relevant concentrations.
With a flux of superoxide from pyrogallol autoxidation we found that
superoxide inhibits EDNO-mediated arterial relaxation and
that this effect is readily prevented by
physiologically relevant concentrations of SOD
(300 IU/mL or 2 µmol/L). In contrast, to achieve any effect
with ascorbic acid in our system, a concentration of 10 mmol/L was
required despite evidence of effective superoxide scavenging at much
lower concentrations. This discrepancy between superoxide scavenging
and the preservation of EDNO-mediated arterial relaxation
was even more pronounced with an endogenous flux of
superoxide induced by inhibition of vascular copper-zinc SOD with DDC.
We used peroxynitrite formation as a marker for the interaction of
NO with superoxide and were able to
demonstrate that high physiological concentrations
of ascorbic acid are required to interrupt the bimolecular combination
of NO and superoxide.
In complex biologic systems such as the arterial wall,
reactive species like NO and superoxide are
often produced within a milieu that contains several potential
scavengers and reactants. Several potential chemical interactions are
never realized because the rate at which they occur is too slow. The
data presented here serve as a case in point. We found that
both ascorbic acid (100 µmol/L) and SOD (300 IU/mL or 2
µmol/L) readily scavenged superoxide generated by pyrogallol
autoxidation (Figure 3
), yet only SOD prevented the inactivation of
EDNO by superoxide (Figures 1 and 2). Similarly, in vessels treated
with DDC to generate a (patho)-physiological
superoxide flux, ascorbic acid (10 mmol/L), SOD (300 IU/mL), and
EUK-8 (300 µmol/L) all demonstrated some superoxide scavenging
(Figure 6), but only SOD and EUK-8 inhibited EDNO inactivation by
superoxide (Figure 5). Thus, there appears to be a discrepancy between
the capacity of ascorbic acid to scavenge superoxide and its ability to
prevent the interaction of NO with
superoxide.
This discrepancy is not particularly surprising. Although ascorbic acid
is an effective scavenger of superoxide, the bimolecular rate constant
for this reaction is 2.7 to 3.3x105 mol ·
L-1 ·
s-116,18 approximately
105-fold less than the rate at which superoxide
reacts with either SOD (2x109 mol ·
L-1 ·
s-1)8 or
NO (1.9x1010 mol
· L-1 ·
s-1).4 Therefore, for
ascorbic acid to compete effectively with NO
for any given concentration of superoxide, the concentration of
ascorbic acid must exceed that of NO by a
factor of 105. Recent studies with a porphyrinic
microsensor have estimated NO concentrations
of 0.1 to 1.0 µmol/L adjacent to endothelial cells in culture and
rabbit aorta.30 31 Based on this information, one would
predict that a concentration of 105x(0.1 to
1.0) µmol/L 
10 to 100 mmol/L ascorbic acid is required
to prevent the interaction of NO and
superoxide, a value that is in excellent agreement with the data
presented here (Figure 2A and 2B). These data indicate that
although ascorbic acid is an efficient scavenger of superoxide, the
rate of this reaction is insufficient to compete effectively with
NO for superoxide at anything less than
supraphysiologic concentrations.
The effect of ascorbic acid on EDNO-mediated arterial
relaxation has been examined in human subjects. Ting et
al32 found that EDNO-mediated forearm blood flow
responses to methacholine were improved by a concomitant infusion of
ascorbic acid in patients with type II diabetes or
hypercholesterolemia.33
In chronic smokers, Heitzer et al34 found that an
acute arterial infusion of ascorbic acid normalized
EDNO-mediated forearm blow flow responses to acetylcholine. An
improvement in EDNO-mediated brachial artery dilation has also been
reported with ascorbic acid in patients with heart
failure.35 A common feature of these studies has
been the implication that ascorbic acid improves EDNO-mediated
responses through superoxide scavenging.32 33 34 35
The arterial concentration of ascorbic acid in those
studies was not determined directly but was estimated to be 10
mmol/L.32 34 These prior observations agree with
the results reported here, namely that plasma ascorbic acid
concentrations of 10 mmol/L may support competition between
ascorbic acid and NO for superoxide (Figures 2 and 6) and thus impair superoxide-mediated EDNO inactivation.
Despite the potential of ascorbic acid to prevent the interaction of
superoxide and NO, plasma concentrations of
ascorbic acid in the range of 10 mmol/L are not
physiologically relevant. Ascorbic acid is the
main water-soluble antioxidant in human plasma and extracellular fluids
with normal concentrations in the range of 50 to 150
µmol/L.36 37 We have recently examined the
effect of physiologically relevant ascorbic
acid concentrations on EDNO-mediated
vasodilation.19 In patients with documented
coronary artery disease, a single oral dose of ascorbic acid (2
grams) reversed endothelial dysfunction in the brachial
artery in 2 hours. Moreover, this dose of ascorbic acid was associated
with an increase in plasma ascorbate within the normal range (46±8 to
114±11 µmol/L).19 Therefore, it is
unlikely that changes in plasma ascorbate within the normal range are
sufficient to prevent superoxide-mediated EDNO inactivation in plasma
(Figure 2). The beneficial effect of oral ascorbic acid on
EDNO-mediated arterial relaxation in patients with
atherosclerosis must occur at the intracellular level
or involve mechanism(s) other than simple superoxide scavenging.
The antioxidant activity of ascorbic acid is not restricted to plasma
and extracellular fluids. Ascorbate is actively transported into cells
and along with glutathione is a major determinant of intracellular
redox state and antioxidant defenses.38
Intracellular concentrations of ascorbic acid have been reported in the
range of 1.3 to 2.5 mmol/L,39 40 and our
data (Figures 2 and 7) suggest these concentrations just begin to
support effective competition between ascorbic acid and
NO for superoxide. Therefore, it is not likely
that the improvement in EDNO-mediated arterial relaxation
that we previously observed with oral ascorbic
acid19 was purely a consequence of superoxide
scavenging by increased intracellular ascorbate.
The source(s) of superoxide in the blood vessel wall remains unclear.
In normal vessels, superoxide can be detected throughout the blood
vessel wall although the endothelium and adventitia are
most notable.41 In
hypercholesterolemia and
atherosclerosis, there is evidence for increased
activity of xanthine oxidase either within12 or
closely associated with42 the
endothelium. Because NO is
freely permeable in biologic tissues, any site of excess superoxide
generation will have some impact on EDNO-mediated
responses.43 In contrast, superoxide is not
membrane permeable, and our data suggest that competition with
NO for superoxide by ascorbic acid will be
highly dependent on the site of superoxide generation. For example,
impairment of EDNO-mediated arterial relaxation caused by
extracellular superoxide generation by xanthine
oxidase42 is not subject to modification by
ascorbic acid because the plasma and extracellular ascorbic acid
concentration is typically <150 µmol/L.37
Intracellular source(s) of superoxide that impair EDNO responses,
however, may be subject to the action of ascorbic acid by virtue of its
higher concentration (1 to 2.5 mmol/L)39 40
in the cytosol (Figure 2).
We observed a discrepancy in the action of ascorbic acid that depended
on the site of superoxide generation. In vessels treated with DDC to
produce an intracellular superoxide flux, ascorbic acid (10
mmol/L) did not improve EDNO-mediated arterial relaxation
(Figure 5). In contrast, 10 mmol/L ascorbic acid did improve EDNO
responses with an extracellular superoxide flux from pyrogallol or
hypoxanthine/xanthine oxidase. One potential explanation for these
observations may relate to incomplete intracellular transport of
ascorbic acid during the time course (20 minutes) of our
experiment.39 Another important point concerns
the scavenging of NO. The treatment of tissues
with DDC leads to the formation of Fe(DDC)2 and
Fe(DDC)3 complexes that bind
NO.44 In this context,
the results depicted in Figure 5 may reflect, in part, some component
of NO scavenging. It is unlikely, however,
that NO scavenging accounts for much of the
response to DDC because treatment with EUK-8 normalizes
arterial relaxation to acetylcholine.
In summary, the data presented here indicate that ascorbic acid
is not likely to prevent the interaction of NO
and superoxide at concentrations that are routinely achieved in plasma
or extracellular fluids (<150 µmol/L). These observations are
supported by kinetic data indicating that superoxide reacts with
NO at a rate that is
105-fold greater than the rate at which
superoxide reacts with ascorbic acid. Within the cell cytosol, however,
ascorbic acid concentrations (1 to 2.5 mmol/L) begin to approach
those needed to support ascorbic acid competition with
NO for superoxide. These data indicate that
effects of ascorbic acid attributed to preventing the interaction of
NO with superoxide must be interpreted with
some caution.
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Acknowledgments |
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This work was supported by grants from the American Heart Association, National Center (J.F.K.), and the National Institutes of Health (HL53398 to J.A.V. and HL55854 and HL59346 to J.F.K.). Joseph A. Vita is an Established Investigator of the American Heart Association and John F. Keaney, Jr, is the recipient of a Clinical Investigator Development Award (HL03195) from the National Institutes of Health.
Received January 27, 1998; accepted July 27, 1998.
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 N. Gaudiot, C. Ribiere, A.-M. Jaubert, and Y. Giudicelli Endogenous nitric oxide is implicated in the regulation of lipolysis through antioxidant-related effect Am J Physiol Cell Physiol, November 1, 2000; 279(5): C1603 - C1610. [Abstract] [Full Text] [PDF]
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A. C. Carr, B.-Z. Zhu, and B. Frei Potential Antiatherogenic Mechanisms of Ascorbate (Vitamin C) and {alpha}-Tocopherol (Vitamin E) Circ. Res., September 1, 2000; 87(5): 349 - 354. [Abstract] [Full Text] [PDF]
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N. Hirai, H. Kawano, O. Hirashima, T. Motoyama, Y. Moriyama, T. Sakamoto, K. Kugiyama, H. Ogawa, K. Nakao, and H. Yasue Insulin resistance and endothelial dysfunction in smokers: effects of vitamin C Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1172 - H1178. [Abstract] [Full Text] [PDF]
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D. Tomasian, J. F. Keaney Jr., and J. A. Vita Antioxidants and the bioactivity of endothelium-derived nitric oxide Cardiovasc Res, August 18, 2000; 47(3): 426 - 435. [Full Text] [PDF]
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O. Hirashima, H. Kawano, T. Motoyama, N. Hirai, M. Ohgushi, K. Kugiyama, H. Ogawa, and H. Yasue Improvement of endothelial function and insulin sensitivity with vitamin C in patients with coronary spastic angina: Possible role of reactive oxygen species J. Am. Coll. Cardiol., June 1, 2000; 35(7): 1860 - 1866. [Abstract] [Full Text] [PDF]
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A. Virdis, L. Ghiadoni, S. Pinto, M. Lombardo, F. Petraglia, A. Gennazzani, S. Buralli, S. Taddei, and A. Salvetti Mechanisms Responsible for Endothelial Dysfunction Associated With Acute Estrogen Deprivation in Normotensive Women Circulation, May 16, 2000; 101(19): 2258 - 2263. [Abstract] [Full Text] [PDF]
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O. T. Raitakari, M. R. Adams, R. J. McCredie, K. A. Griffiths, R. Stocker, and D. S. Celermajer Oral vitamin C and endothelial function in smokers: short-term improvement, but no sustained beneficial effect J. Am. Coll. Cardiol., May 1, 2000; 35(6): 1616 - 1621. [Abstract] [Full Text] [PDF]
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D. L. Sherman, J. F. Keaney Jr, E. S. Biegelsen, S. J. Duffy, J. D. Coffman, and J. A. Vita Pharmacological Concentrations of Ascorbic Acid Are Required for the Beneficial Effect on Endothelial Vasomotor Function in Hypertension Hypertension, April 1, 2000; 35(4): 936 - 941. [Abstract] [Full Text] [PDF]
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Y. S. Chang, J. A. Yaccino, S. Lakshminarayanan, J. A. Frangos, and J. M. Tarbell Shear-Induced Increase in Hydraulic Conductivity in Endothelial Cells Is Mediated by a Nitric Oxide-Dependent Mechanism Arterioscler Thromb Vasc Biol, January 1, 2000; 20(1): 35 - 42. [Abstract] [Full Text] [PDF]
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N. Gokce, J. F. Keaney Jr, B. Frei, M. Holbrook, M. Olesiak, B. J. Zachariah, C. Leeuwenburgh, J. W. Heinecke, and J. A. Vita Long-Term Ascorbic Acid Administration Reverses Endothelial Vasomotor Dysfunction in Patients With Coronary Artery Disease Circulation, June 29, 1999; 99(25): 3234 - 3240. [Abstract] [Full Text] [PDF]
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A. Huang, J. A. Vita, R. C. Venema, and J. F. Keaney Jr. Ascorbic Acid Enhances Endothelial Nitric-oxide Synthase Activity by Increasing Intracellular Tetrahydrobiopterin J. Biol. Chem., June 2, 2000; 275(23): 17399 - 17406. [Abstract] [Full Text] [PDF]
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S. Gupta, E. Chough, J. Daley, P. Oates, K. Tornheim, N. B. Ruderman, and J. F. Keaney Jr. Hyperglycemia increases endothelial superoxide that impairs smooth muscle cell Na+-K+-ATPase activity Am J Physiol Cell Physiol, March 1, 2002; 282(3): C560 - C566. [Abstract] [Full Text] [PDF]
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S. Mak, Z. Egri, G. Tanna, R. Colman, and G. E. Newton Vitamin C prevents hyperoxia-mediated vasoconstriction and impairment of endothelium-dependent vasodilation Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2414 - H2421. [Abstract] [Full Text] [PDF]
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