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Journal of the American College of Cardiology
© 2000 by the American College of Cardiology
Published by Elsevier Science Inc.
Vol. 36, No. 5, 2000
ISSN 0735-1097/00/$20.00
PII S0735-1097(00)00916-5
Neutrophil Superoxide Anion–Generating
Capacity, Endothelial Function and
Oxidative Stress in Chronic Heart Failure:
Effects of Short- and Long-Term Vitamin C Therapy
Gethin R. Ellis, MRCP,* Richard A. Anderson, MRCP,* Derek Lang, PHD,* Daniel J. Blackman, MRCP,*
R. H. Keith Morris, MSC,† Jayne Morris-Thurgood, PHD,* Ian F. W. McDowell, MD,*
Simon K. Jackson, PHD,* Malcolm J. Lewis, MB, DSC,* Michael P. Frenneaux, MD, FRCP, FACC*
Cardiff, Wales, United Kingdom
First, we sought to study the effects of short- and long-term vitamin C therapy on oxidative
stress and endothelial dysfunction in chronic heart failure (CHF), and second, we sought to
investigate the role of neutrophils as a cause of oxidative stress in CHF.
BACKGROUND Oxidative stress may contribute to endothelial dysfunction in CHF. Vitamin C ameliorates
endothelial dysfunction in CHF, presumably by reducing oxidative stress, but this is
unproven.
METHODS
We studied 55 patients with CHF (ischemic and nonischemic etiologies) and 15 control
subjects. Flow-mediated dilation (FMD) in the brachial artery was measured by ultrasound
wall-tracking, neutrophil superoxide anion (O2⫺) generation by lucigenin-enhanced chemiluminescence and oxidative stress by measurement of free radicals (FRs) in venous blood
using electron paramagnetic resonance (EPR) spectroscopy and plasma thiobarbituric acid
reactive substances (TBARS). Measurements were performed at baseline in all subjects. The
effects of short-term (intravenous) and long-term (oral) vitamin C therapy versus placebo
were tested in patients with nonischemic CHF.
RESULTS
At baseline, FRs were higher in patients with CHF than in control subjects (p ⬍ 0.01),
TBARS were greater (p ⬍ 0.005), neutrophil O2⫺-generating capacity was enhanced (p ⬍
0.005) and FMD was lower (p ⬍ 0.0001). Compared with placebo, short-term vitamin C
therapy reduced FR levels (p ⬍ 0.05), tended to reduce TBARS and increased FMD (p ⬍
0.05), but did not affect neutrophil O2⫺-generating capacity. Long-term vitamin C therapy
reduced FR levels (p ⬍ 0.05), reduced TBARS (p ⬍ 0.05) and improved FMD (p ⬍ 0.05),
but also reduced neutrophil O2⫺-generating capacity (p ⬍ 0.05). Endothelial dysfunction was
not related to oxidative stress, and improvements in FMD with vitamin C therapy did not
relate to reductions in oxidative stress.
CONCLUSIONS Oxidative stress is increased in ischemic and nonischemic CHF, and neutrophils may be an
important cause. Vitamin C reduces oxidative stress, increases FMD and, when given long
term, decreases neutrophil O2⫺ generation, but the lack of a correlation between changes in
endothelial function and oxidative stress with vitamin C implies possible additional
non-antioxidant benefits of vitamin C. (J Am Coll Cardiol 2000;36:1474 – 82) © 2000 by the
American College of Cardiology
OBJECTIVES
The prevalence of chronic heart failure (CHF) is increasing
alarmingly against the trend in coronary artery disease
(CAD) prevention, and this phenomenon is not entirely
explained by demographic changes (1,2). Furthermore,
despite the impressive gains resulting from advances in
medical therapy over the past few years, CHF remains a
disease with a high mortality rate and severe impairment of
quality of life (1,2). Therefore, we need to better understand
the pathophysiology, with a view to further improvements
in therapy.
From the *Cardiovascular Sciences Research Group, Wales Heart Research
Institute, University of Wales College of Medicine, Cardiff, Wales, United Kingdom;
and †School of Biomedical Sciences, University of Wales Institute of Cardiff, Cardiff,
Wales, United Kingdom. This work was supported by the British Heart Foundation
(BHF). Drs. Ellis and Anderson are BHF Junior Research Fellows; Dr. Frenneaux
holds the BHF Sir Thomas Lewis Chair of Cardiology; and Dr. Morris-Thurgood is
supported by the BHF.
Manuscript received October 28, 1999; revised manuscript received April 24, 2000;
accepted June 9, 2000.
It has been postulated that oxidative stress plays a key role
in the genesis and progression of CHF (3– 8) and that, as a
consequence, vitamin C may have an important potential
therapeutic role (8). Although there is considerable evidence
that oxidative stress is increased in CHF, previous studies
have generally been based on patients with underlying
CAD, which is a cause of increased oxidative stress, or have
used indirect, nonspecific markers (3–7). The underlying
origin and species of oxygen free radicals (FRs) in CHF
have also not been defined, and the mechanism(s) of action
of vitamin C on endothelial dysfunction in CHF remain(s)
unclear.
The aims of this study were: 1) to assess the role of
oxidative stress as a mechanism of endothelial dysfunction
in CHF, using a novel, direct technique involving electron
paramagnetic resonance (EPR) spectroscopy and a standard
marker of lipid peroxidation; 2) to assess the effects of
vitamin C therapy on these variables and on endothelial
JACC Vol. 36, No. 5, 2000
November 1, 2000:1474–82
Ellis et al.
Oxidative Stress and Therapy With Vitamin C in Heart Failure
Abbreviations and Acronyms
CHF
⫽ chronic heart failure
CAD
⫽ coronary artery disease
eNOS
⫽ endothelial nitric oxide synthase
EPR
⫽ electron paramagnetic resonance
FMD
⫽ flow-mediated dilation
FRs
⫽ free radicals
O2⫺
⫽ superoxide anion
NO
⫽ nitric oxide
NYHA ⫽ New York Heart Association
TBARS ⫽ thiobarbituric acid reactive substances
function; and 3) to investigate neutrophil superoxide anion–
generating capacity as a potentially important source of free
radicals (FRs) in CHF and to test their response to vitamin
C therapy.
METHODS
Subjects. We studied 55 patients with New York Heart
Association (NYHA) functional class II-IV symptoms of
heart failure who were recruited from a cardiology clinic, as
well as 15 healthy control subjects (Table 1). Patients were
clinically stable, with no signs of pulmonary or peripheral
congestion, and were stable (unchanged for three months)
on medical therapy. Those patients with evidence of diabetes or significant concurrent medical illness were excluded.
Patients had objective left ventricular systolic impairment
(ejection fraction ⬍35%), and all had recently undergone
exercise testing and coronary arteriography. The diagnosis
of ischemic CHF was based on evidence of reversible
ischemia on stress testing or a history of myocardial infarction and significant coronary disease on the angiogram, or
both. The subjects gave written, informed consent, and
approval was obtained from the local Research Ethics
Committee.
Study protocol. Baseline EPR spectroscopy and analysis of
plasma thiobarbituric acid reactive substances (TBARS) and
assessment of neutrophil NADPH oxidase activity and
endothelial function were performed in all patients and
control subjects at 9 AM after an overnight fast. Blood
samples were obtained by venipuncture. Neutrophils were
separated from whole blood for measurement of neutrophil
superoxide anion– generating capacity. Plasma was also
obtained after centrifugation at 2,500 rpm at 4°C for
10 min. Measurement of lipid-derived FRs by EPR spectroscopy was performed on fresh plasma samples. Plasma
was also stored frozen for subsequent measurement of
TBARS.
The effects of short-term (intravenous) and long-term
(oral) vitamin C therapy were assessed separately in two
groups of patients with CHF of nonischemic etiology,
because our aim was to study the effects on CHF, per se,
rather than on CAD. Twenty of these patients were given
2 g of vitamin C (Evans Medical Ltd., Surrey, United
Kingdom) intravenously over 5 min. Over a week later, 10
of the patients (matched for age and functional status) were
given intravenous saline. Baseline measures were repeated
30 min later. We had previously established that plasma
vitamin C levels increased by over 500% at 30 min when
given intravenously.
In a separate arm of the study, we also tested the effects
of long-term (oral) vitamin C therapy in patients with CHF
of nonischemic etiology. Ten patients were given oral
vitamin C, 2 g twice daily, and 10 different patients matched
for NYHA status received placebo. Repeat measurements of
endothelial function, oxidative stress and neutrophil superoxide anion (O2⫺)-generating capacity were performed after
one month of therapy in each group and again after a
six-week washout period in the active treatment group.
Measurement of oxidative stress. LIPID-DERIVED FREE
RADICALS. It is recognized that the formation of lipid
carbon radicals follows oxidative damage in vivo (9), and in
this study, we used the technique of ex vivo spin trapping
and EPR spectroscopy to measure these radicals in venous
blood samples. This technique has been recently described
and validated by ourselves (10). The EPR spectra were obtained on a Varian E104 spectrometer operating at 9 GHz
with 100 kHz field modulation. Typical spectrometer settings
were: power 10 mW, modulation amplitude 1 G, time constant 0.25 s, scan range 100 G and scan time 4 min. We
employed the spin trap reagent ␣-phenyl-tertbutylnitrone
(Sigma Co., Dorset, United Kingdom). Venous samples
were taken directly into spin-trap– containing vacutainer
bottles, and the resultant spin adduct was extracted from
plasma using toluene. The spin adduct was then dissolved in
100 ␮l of chloroform and put into sealed Pasteur pipettes
for EPR analysis. The EPR spectral peak heights, recorded
in arbitrary units, were taken as a measure of spin adduct
concentration.
Table 1. Clinical Characteristics of Patients With Chronic Heart Failure and Control Subjects
Patients With CHF
n (females)
Mean ⫾ SEM age (yrs)
Passive heavy smoking (n)
Mean ⫾ SEM total cholesterol (mmol/liter)
Mean ⫾ SEM triglyceride (mmol/liter)
Mean NYHA class
1475
DCM
CAD
Control Subjects
40 (7)
53 ⫾ 2
5
15 (4)
65 ⫾ 2
1
15 (4)
55 ⫾ 2
1
5.8 ⫾ 1.8
1.2 ⫾ 0.6
—
2.8
6.0 ⫾ 2.4
1.7 ⫾ 1.0
3.0
CAD ⫽ coronary artery disease; DCM ⫽ dilated cardiomyopathy; NYHA ⫽ New York Heart Association.
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Oxidative Stress and Therapy With Vitamin C in Heart Failure
THIOBARBITURIC ACID REACTIVE SUBSTANCES. The
plasma level of TBARS (an indicator of lipid peroxides) was
determined as previously described using a colorimetric
assay available as a standard commercially available kit (Oxis
International Inc., Oxford, United Kingdom) (11). Briefly,
the assay is based on the reaction of a chromogenic reagent
with malonaldehydes and 4-hydroxyalkenals. One molecule
of either malonaldehyde or 4-hydroxyalkenals reacts with
two molecules of this reagent to yield a stable chromophore
with maximal absorbance at 568 nm. The results are
expressed as ␮mol/liter, with the values for malonaldehyde
and 4-hydroxyalkenals combined.
SUPEROXIDE ANION GENERATION BY POLYMORPHONU-
We employed a refined lucigenin-based
chemiluminescent assay of NADPH oxidase activity to
measure basal and post-activation levels of O2⫺ generation
by neutrophils (12). Neutrophils were separated from venous blood using a Ficoll-Hypaque density gradient, and
activation of the neutrophil respiratory burst was achieved
using 1 ␮ mol/liter of N-formyl-methionyl-leucylphenylalanine (Sigma Co.). The peak level of O2⫺ generated after activation was recorded in mV/5 ⫻ 105 neutrophils.
Measurement of flow-related endothelial function.
Changes in brachial artery diameter in response to reactive
hyperemia were measured noninvasively using a high resolution ultrasonic wall-tracking system (Vadirec Wall-Track
System, resolution ⫾ 3 ␮m). This technique was previously
described and validated by our group (13). The brachial
artery was imaged using a 7.5-MHz transducer, and reactive
hyperemia was produced by releasing a sphygmomanometer
cuff inflated to suprasystolic pressure (systolic pressure ⫹
50 mm Hg) for 5 min. Internal brachial artery diameter was
measured at baseline and 60 s after cuff release. The
flow-mediated dilation (FMD) data are presented as the
percent change in brachial artery diameter from baseline.
Plasma vitamin C (ascorbic acid). Ascorbate was measured by reverse phase high performance liquid chromatography with the use of an ion pairing agent and electrochemical detection (14). This assay measures ascorbate in the
reduced form. Levels were checked in a subset of five
patients to assess their response to therapy at baseline and
after intravenous (30 min after dose) and oral (1 h after
dose) vitamin C therapy, as well as after a six-week washout
period.
Statistical analysis. For our statistical analysis, we used
SPSS 9.0. All data are presented as the mean value ⫾ SEM,
unless otherwise stated. At baseline, measures of oxidative
stress, neutrophil O2⫺-generating capacity and FMD were
compared in patients with CHF (ischemic vs. nonischemic)
and control subjects, using one-way analysis of variance
(ANOVA) followed by the post hoc Bonferroni multiple
comparisons test. The relations between NYHA status and
these variables at baseline were also assessed using
ANOVA. We used multi-way ANOVA and applied
CLEAR CELLS.
JACC Vol. 36, No. 5, 2000
November 1, 2000:1474–82
Tukey’s multiple comparisons test to compare the effects of
short-term vitamin C therapy versus placebo on oxidative
stress, neutrophil O2⫺-generating capacity and FMD. The
effects of long-term vitamin C therapy versus placebo were
assessed using analysis of covariance. Relations between
changes in FMD and measures of oxidative stress and
neutrophil O2⫺-generating capacity were assessed individually using linear regression analysis. Correlation coefficients
were calculated using Pearson’s formula. Statistical significance was accepted at the 95% confidence level (p ⬍ 0.05).
RESULTS
The clinical characteristics of the study group are shown in
Table 1. All patients were on stable medical therapy,
including maximally tolerated angiotensin-converting enzyme inhibitor therapy and a diuretic agent.
Baseline
Oxidative stress. Analysis of the EPR spectra and comparison with published values indicated trapping of lipid
alkoxyl and carbon-centered FRs derived from lipid peroxidation (15). These lipid-derived FRs were significantly
higher in patients with CHF than in control subjects
(0.41 ⫾ 0.03 vs. 0.27 ⫾ 0.04 U, p ⬍ 0.01). Plasma TBARS
were also higher in patients with CHF than in control
subjects (7.0 ⫾ 0.6 vs. 3.8 ⫾ 0.6 ␮mol/liter, p ⬍ 0.005).
Post hoc analysis showed that lipid-derived FR levels were
higher in patients with ischemic versus nonischemic CHF
(0.53 ⫾ 0.09 vs. 0.36 ⫾ 0.06 U, p ⬍ 0.05), but not in levels
of plasma TBARS (8.2 ⫾ 1.2 vs. 6.5 ⫾ 0.5 ␮mol/liter). In
the patients with nonischemic CHF, severity (NYHA
status) did not correlate with lipid-derived FR levels (p ⫽
NS), nor with TBARS (p ⫽ NS).
Neutrophil superoxide-generating capacity. Neutrophil
O2⫺-generating capacity was significantly increased in patients with CHF versus control subjects (44.6 ⫾ 4.3 vs.
17.7 ⫾ 2.1 mV/5 ⫻ 105 neutrophils, p ⬍ 0.005), but was
similar in patients with both nonischemic and ischemic
etiologies (44.4 ⫾ 3.8 vs. 45.1 ⫾ 8.1 mV/5 ⫻ 105
neutrophils, respectively, p ⫽ NS). The NYHA status was
related in the patients with nonischemic CHF to neutrophil
O2⫺-generating capacity (p ⬍ 0.0001) (Fig. 1).
Endothelial function. Flow-mediated dilation was significantly impaired in patients with CHF versus control
subjects (2.5 ⫾ 0.4% vs. 6.5 ⫾ 0.9%, p ⬍ 0.0001), but did
not differ between patients with ischemic and those with
nonischemic etiologies of CHF (1.3 ⫾ 0.4% vs. 2.9 ⫾ 0.4%,
p ⫽ NS). In the patients with nonischemic CHF, FMD did
not significantly relate to levels of oxidative stress (p ⫽ NS),
neutrophil O2⫺-generating capacity (p ⫽ NS) or NYHA
status (p ⫽ NS).
Effects of Vitamin C
In the subsets of patients studied, plasma levels of vitamin C
rose significantly after 1 month of oral administration
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Oxidative Stress and Therapy With Vitamin C in Heart Failure
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Figure 1. Relation between neutrophil O2⫺-generating capacity and NYHA functional class in patients with ischemic and nonischemic CHF. *p ⬍ 0.0001.
(measured at 1 month) and after intravenous administration
(measured at 30 min) (from 28.6 ⫾ 1.4 to 74.8 ⫾
1.6 ␮mol/liter and from 20.6 ⫾ 0.9 to 134.5 ⫾ 31.6
␮mol/liter, respectively). In the group given long-term
vitamin C therapy, levels then fell after a six-week washout
period, back to 41.4 ⫾ 0.9 ␮mol/liter.
Short-term vitamin C therapy, as compared with placebo,
reduced lipid-derived FRs (from 0.35 ⫾ 0.03 to 0.23 ⫾
0.03 U, p ⬍ 0.05) (Fig. 2a), but only tended to reduce
TBARS (from 6.4 ⫾ 0.7 to 5.2 ⫾ 0.7 ␮mol/liter) (Fig. 2b).
Neutrophil O2⫺-generating capacity was not affected by
short-term vitamin C therapy (from 38.3 ⫾ 7.6 to 39.0 ⫾
8.0 mV/5 ⫻ 105 neutrophils, p ⫽ NS) (Fig. 2b). Flowmediated dilation increased after intravenous vitamin C
administration (from 3.1 ⫾ 0.6% to 6.7 ⫾ 1.0%, p ⬍ 0.05).
Improvements in FMD with short-term vitamin C therapy
did not correlate with reductions in oxidative stress (FRs:
r ⫽ 0.05; TBARS: r ⫽ 0.04) (Fig. 3), nor with reductions
in neutrophil O2⫺-generating capacity (r ⫽ 0.04).
Long-term oral treatment with vitamin C, as compared
with placebo, reduced lipid-derived FRs (from 0.38 ⫾ 0.03
to 0.25 ⫾ 0.02 U, p ⬍ 0.05) (Fig. 4a) and reduced plasma
TBARS (from 7.1 ⫾ 0.8 to 5.2 ⫾ 0.6 ␮mol/liter, p ⬍ 0.05)
(Fig. 4b). Neutrophil O2⫺-generating capacity was also
reduced by long-term vitamin C therapy (from 43.4 ⫾ 6.5
to 23.7 ⫾ 2.9 mV/5 ⫻ 105 neutrophils, p ⬍ 0.05) (Fig. 4c).
Flow-mediated dilation increased after oral vitamin C
administration (from 3.0 ⫾ 0.6% to 8.2 ⫾ 1.1%, p ⬍ 0.05).
Improvements in FMD after long-term vitamin C therapy
did not correlate with reductions in oxidative stress (FRs:
r ⫽ ⫺0.04; TBARS: r ⫽ ⫺0.2), nor with reductions in
neutrophil O2⫺-generating capacity (r ⫽ 0.2). After a
six-week washout period in the active treatment group,
lipid-derived FRs tended to increase toward baseline levels
(from 0.23 ⫾ 0.02 to 0.36 ⫾ 0.04 U, p ⫽ 0.09), but plasma
TBARS increased (from 5.2 ⫾ 0.7 to 7.1 ⫾ 1.0 ␮mol/liter,
p ⬍ 0.05), and neutrophil O2⫺-generating capacity returned
toward baseline levels after withdrawal of vitamin C therapy
(from 23.7 ⫾ 2.9 to 40.4 ⫾ 7.9 mV/5 ⫻ 105 neutrophils,
p ⬍ 0.05). There was no change in NYHA status during
long-term vitamin C therapy as compared with placebo
(2.7 ⫾ 0.2 to 3 ⫾ 0.2 vs. 2.6 ⫾ 0.2 to 2.8 ⫾ 0.2,
respectively, p ⫽ NS). The study was not powered to assess
this, however.
DISCUSSION
This study makes the following important new observations.
Firstly, lipid-derived FR are increased in patients with CHF
even in the absence of underlying CAD, though the
magnitude of oxidative stress was greater in those patients
with an ischemic etiology. Secondly, these lipid-derived FR
are reduced significantly by vitamin C, both acutely when
given intravenously and chronically with oral therapy.
Thirdly, neutrophils from patients with CHF have an
enhanced capacity to generate O2⫺ via the NADPH oxidase
enzyme system and this relates to NYHA status. Fourthly,
vitamin C has no effect on the ability of neutrophils to
generate superoxide anions acutely, but during long-term
therapy there was a marked suppression. We confirm the
earlier observation of impaired conduit artery endotheliumdependent FMD in patients with CHF (8,13) and the
recent observations that this may be improved by treatment
with vitamin C, both acutely and chronically (8).
Finally, the absence of a relationship between the impairment in FMD and the magnitude of elevation of FR at
baseline, or between change in endothelial function and the
change in FR with short-term (Fig. 3) and long-term
vitamin C argues against oxidative stress as the sole
mechanism of endothelial dysfunction in CHF and
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Oxidative Stress and Therapy With Vitamin C in Heart Failure
JACC Vol. 36, No. 5, 2000
November 1, 2000:1474–82
Figure 2. (a) Mean plasma lipid-derived FR levels in patients with nonischemic CHF before and after short-term vitamin C (VC) therapy versus those
subjects receiving placebo. *p ⬍ 0.05. (b) Mean plasma TBARS in patients with nonischemic CHF before and after short-term VC therapy versus those
subjects receiving placebo. (c) Mean O2⫺ generation by activated neutrophils in patients with nonischemic CHF before and after short-term VC therapy
versus those subjects receiving placebo. Data are shown as the mean value ⫾ SEM. Solid bar ⫽ baseline; open bar ⫽ after treatment.
suggests the possibility that the beneficial effects of vitamin
C may not relate solely to its FR-scavenging actions.
Oxidative stress in heart failure. Oxidative stress is known
to be increased in atherosclerotic vascular disease (16). In
this study, we demonstrate that oxidative stress, as assessed
using both the novel and relatively direct marker of plasma
lipid-derived FRs and plasma TBARS, is increased in both
ischemic and nonischemic CHF, although it tends to be
more elevated in the former. Increased oxidative stress in
CHF has previously been implied from very indirect measures (3–7), which generally lack both specificity and sensitivity. The alkoxyl and carbon-centered FRs, as measured
using EPR spectroscopy in this study, provide a much more
direct and physiologically relevant measure of oxidative
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Ellis et al.
Oxidative Stress and Therapy With Vitamin C in Heart Failure
1479
Figure 3. Relation between changes in FMD and lipid-derived FRs in patients with nonischemic CHF given intravenous vitamin C.
stress than those previously reported in heart failure, as
alkoxyl radicals are capable of reacting directly with nitric
oxide (NO) (17). Short-term vitamin C therapy reduced
EPR-measured FRs significantly, although there was only a
trend toward a reduction in plasma TBARS. This would
also suggest that our ex vivo EPR technique provided a
more direct measure of oxidative stress.
Oxygen FRs may contribute importantly to the pathophysiology of CHF by initiating myocyte apoptosis through the
nuclear factor ␬B-like transcription factors (18) and by exerting
direct negatively inotropic effects through depressed calcium
uptake and reduced calcium-stimulated magnesiumdependent adenosine triphosphatase activity of the cardiac
sarcoplasmic reticulum (19,20). Oxidative stress may also
contribute to endothelial dysfunction in CHF through direct
toxic effects on endothelial cells, by reducing NO synthase
activity and by reacting with NO to form peroxynitrite (3,21–
23). Endothelial dysfunction may limit cardiac and skeletal
muscle blood flow (particularly on exercise), impair pulmonary
blood flow regulation (resulting in ventilation-perfusion mismatching) and may lead to impaired diastolic cardiac performance (24). Despite these likely effects of oxidative stress on
endothelial function, we found no relation between the impairment of FMD and the magnitude of oxidative stress. This
may support the involvement of additional factors in the
genesis of endothelial dysfunction, including the effects of
asymmetric dimethylarginine, which competes with arginine
for endothelial nitric oxide synthase (eNOS) binding and is
elevated in CHF (25).
The NADPH/NADH oxidase system in endothelial
cells, smooth muscle cells and phagocytes has been suggested as an important potential source of FRs in heart
failure (26). Angiotensin II, acting through type I receptors,
may modulate the activity of this enzyme (27). Our data
demonstrate that, despite the fact that all patients were
being treated with angiotensin-converting enzyme inhibitors, their neutrophils exhibited an enhanced capacity to
generate O2⫺ through the NADPH oxidase system. How-
ever, recent data suggest that tissue angiotensin II levels may
be increased in CHF, despite angiotensin-converting enzyme inhibitor therapy, owing to alternative angiotensin II
generating pathways (28) or to increased angiotensin II
generation through the incompletely blocked classic pathway when angiotensin I levels become markedly elevated
(29). Tumor necrosis factor-␣, which is elevated in CHF
(30), also appears to modulate the activity of the NADPH/
NADH oxidase system (31).
Effects of vitamin C. In this study, vitamin C reduced
lipid-derived FR levels during both the short-term and
long-term treatment studies and increased brachial artery
FMD to levels similar to those of healthy control subjects.
After a washout period of six weeks, FR levels rose again but
failed to return to pretreatment levels. Interestingly, plasma
vitamin C levels remained higher than baseline levels after
the washout period (either as a result of continued vitamin
supplementation, although this was denied, or because of
improvements in dietary vitamin C intake), implying an
enhanced antioxidant state.
Although vitamin C is recognized as a potent watersoluble free radical scavenger (32), the rate constant of its
interaction with O2⫺ is approximately five orders of magnitude lower than those for the interaction of O2⫺ with the
endogenous antioxidants superoxide dismutase and NO,
and vitamin E exhibits similar differential kinetics (33).
Therefore, it seems unlikely that the antioxidant effects of
these vitamins rely solely on their extracellular scavenging
effects. In a recent study in the pig coronary angioplasty
model, it was suggested that these vitamins may decrease
O2⫺ generation by inhibiting NADPH oxidase activity
(34). Our data confirm this, in that long-term therapy with
vitamin C reduced the ability of neutrophils to generate
O2⫺. However, intravenous vitamin C administration did
not have this effect. Nevertheless, it reduced lipid-derived
FRs and normalized brachial artery endothelial function.
Vitamin C is actively transported into cells through an
insulin-dependent transport system and can achieve intra-
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JACC Vol. 36, No. 5, 2000
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Figure 4. (a) Mean plasma lipid-derived FR levels in patients with nonischemic CHF before and after long-term vitamin C (VC) therapy versus those
subjects receiving placebo. *p ⬍ 0.05. (b) Mean plasma TBARS in patients with nonischemic CHF before and after long-term VC therapy versus those
subjects receiving placebo. (c) Mean O2⫺ generation by activated neutrophils in patients with nonischemic CHF before and after long-term VC therapy
versus those subjects receiving placebo. Data are shown as the mean value ⫾ SEM. *p ⬍ 0.05. Solid bar ⫽ baseline; open bar ⫽ after treatment.
cellular concentrations in the 1 to 10 mmol/liter range, at
which it is an effective scavenger of O2⫺ (35). Thus, its
scavenging effects may be largely intracellular. Aside from
its scavenging actions, vitamin C spares intracellular glutathione from oxidative degeneration (36). Depletion of
intracellular-reduced thiols impairs NO synthase activity in
cultured endothelial cells, hence vitamin C may increase
endothelial NO production (37). Reduced thiols stabilize
NO (and increase its bioavailability) by reacting to form
5-nitrosothiol species (38). Vitamin C also functions synergistically with vitamin E, promoting its regeneration and
leading to enhanced intracellular antioxidant capability (39).
Two recent in vitro studies suggest that vitamin C may
have additional important actions. One study reported that
vitamin C increased eNOS gene expression in cultured
endothelial cells (40), and in another study, vitamin C
increased NO generation from eNOS by increasing tetrahydrobiopterin availability or increasing the affinity of tet-
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Oxidative Stress and Therapy With Vitamin C in Heart Failure
rahydrobiopterin binding (41). The relevance of these observations to the in vivo situation is unknown; however, our
observation that the improvement in FMD did not correlate
with the fall in FRs supports the additional effects of
vitamin C. A recent study of vitamin C in patients with
CAD demonstrated a marked improvement in endothelial
function with no detectable change in oxidative stress (42).
The discrepant response of the neutrophil NADPH
oxidase system to long-term oral versus short-term intravenous vitamin C therapy is also unexplained. There are a
number of potential stages at which vitamin C may act,
including the priming process and NO feedback of
NADPH oxidase activity. These processes may be time
dependent, and understanding them may be important in
the development of vitamin C as a therapeutic tool in the
prevention and treatment of CHF.
Study limitations. Endothelium-independent dilation was
not assessed in this study. All patients were maintained on
stable, maximal medical therapy for the duration of the
study, including, in the majority of cases, long-acting nitrate
preparations. Therefore, testing endothelium-independent
dilation using a NO donor would have been unreliable.
Other investigators have shown that the endotheliumindependent response to a supramaximal dose of nitroglycerin is preserved in patients with CHF (8,13), and that
vitamin C, given both long term and short term, has no
effect on this variable (8).
Although a direct measure of O2⫺ in vivo is, at present,
technically impossible in human subjects, the ex vivo spintrapping technique provides good evidence for the production of lipid radicals in vivo. Furthermore, although it is
likely that a reduction in oxidative stress coupled with
improvements in endothelial function will lead to clinical
benefits, this study was not powered to assess changes in
important clinical variables, such as exercise capacity and left
ventricular function. A study of the longer term effects of
vitamin C therapy on such clinical variables, with a larger
patient group, ideally those with nonischemic CHF, would
seem to be clearly warranted.
Conclusions. Oxidative stress is increased in CHF, even
when the etiology is nonischemic. Neutrophils may be an
important source of FRs. Vitamin C reduces oxidative
stress, both short and long term. In the latter case, reduced
neutrophil-generating capacity may contribute to this effect.
However, the beneficial effects of vitamin C therapy on
endothelial function do not seem to be explicable solely on
the basis of reduced oxidative stress.
Acknowledgments
The authors thank Mrs. Heather Wheatley, Mrs. Moira
Ashton and Mr. Peter Gapper for providing technical support.
Reprint requests and correspondence: Prof. M.P. Frenneaux,
Wales Heart Research Institute, University of Wales College of
Medicine, Heath Park, Cardiff, Wales, United Kingdom CF14
4XN. E-mail: [email protected].
1481
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