Journal of Surgical Research 96, 183–187 (2001)

doi:10.1006/jsre.2000.6078, available online at http://www.idealibrary.com on

Antioxidants Reduce Oxidative Stress in Claudicants

M. H. W. A. Wijnen,* S. A. J. Coolen,† H. L. Vader,* ,‡ J. C. Reijenga,† F. A. Huf,† and R. M. H. Roumen*
*Department of Surgery and ‡Clinical Laboratory, Sint Joseph Hospital, P. O. Box 7777, 5500 MB Veldhoven, The Netherlands; and
†Laboratory of Instrumental Analysis, Eindhoven University of Technology, P. O. Box 513, 5600 MB, Eindhoven, The Netherlands

Submitted for publication July 10, 2000

strated that during the reperfusion period, production

Background. Low-grade ischemia–reperfusion in
claudicants leads to damage of local tissues and re-
mote organs. Since this damage is partly caused by
oxygen-derived free radicals (ODFR), scavenging
these ODFR could reduce the local and remote injury.
Methods. Using a new method by which a free radi-
cal reaction product (ortho-APOH) of the exogenous
marker antipyrine is measured to quantify the oxida-
tive stress, 16 stable claudicants performed a standard
walking test before and after administration of vita-
min E (200 mg) and vitamin C (500 mg) daily for 4
weeks.
Findings. Ortho-APOH was significantly increased
during the reperfusion period (P ⴝ 0.026) before ad-
ministration of the vitamins. After 4 weeks of vitamin
supplementation no rise was found in the reperfusion
period. Malondialdehyde showed no changes in either
group.
Interpretation. These findings indicate that admin-
istering extra antioxidants to claudicants reduces ox-
idative stress in these patients. This may also have an
effect on the remote ischemia–reperfusion damage
and reduce cardiovascular morbidity in this
group. © 2001 Academic Press
Key Words: ischemia–reperfusion; vitamin E; vitamin
C; antipyrine; intermittent claudication; malondial-
dehyde.
INTRODUCTION
Intermittent claudication can be considered an im-
portant health problem since 5% of men over 50 years
of age suffer from it [1]. Usually located in the lower
extremities, claudication is caused by narrowing or
obstruction of arteries in the aorto-iliacal region or in
peripheral arteries, resulting in hypoxia during exer-
cise. This results in repetitive low-grade ischemia with
calf or buttock pain that subsides when the exercise is
stopped and reperfusion starts. It has been demon-
of oxygen-derived free radicals (ODFR) and neutrophil
activation can cause additional damage [2– 4]. This not
only results in local changes in the ischemic and reper-
fused tissues, but can also cause systemic effects [5].
Some authors have suggested that this systemic in-
flammatory response was responsible for additional
atherosclerosis, one of the reasons for an increase in
ischemic heart disease observed in claudicants [1, 6, 7].
There is reason to believe that an increase in scav-
enging activity is beneficial in claudicants and that
scavengers can reduce the systemic and local inflam-
matory response seen after ischemia–reperfusion (I–R)
injury in humans [8 –15]. Modulation of the ODFR
activity in claudicants would therefore be an attractive
option in the treatment of these patients.
In vivo, measuring ODFR activity has always been a
problem due to the extremely short half-life of oxygen
radicals. Therefore, most studies use metabolites of the
ODFR-induced lipid peroxidation, such as malondial-
dehyde, as markers for ODFR activity. Other measure-
ments are aimed at neutrophils that are activated dur-
ing reperfusion or at the total antioxidant capacity that
is lowered after oxidative stress [16].
Others have reported remote organ damage during
I–R that is caused by direct oxygen radical reactions or
activated neutrophils. The target organ most fre-
quently studied is the kidney, where an increase in the
albumin creatinine ratio in urine is thought to be an
indicator of endothelial damage and reperfusion injury
[3, 17–21]. All of these methods have their limitations
and are very susceptible to interference from other
reactions in vivo [22].
Recently, we developed a new method for measuring
oxidative stress in humans, using antipyrine (2,3-
dimethyl-phenyl-3-pyrazolyn-5-one) as a marker sub-
stance and measuring its free radical reaction product
ortho-hydroxyantipyrine (o-APOH) as an indicator for

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184 JOURNAL OF SURGICAL RESEARCH: VOL. 96, NO. 2, APRIL 2001
TABLE 1
Time Frame for the Sampling of Blood and Urine in
Intermittent Claudicants Performing a Standard
Walking Test
Time Event
T0 Blank blood sample; administration of antipyrine
T1 Blood sample; urine sample; start of walking test; 60
min after T0
T2 Blood sample; 1 min after walking test; 66 min after T0
T3 Blood sample; 5 min after walking test; 70 min after T0
T4 Blood sample; urine sample; 60 min after walking test;
125 min after T0
the amount of oxidative stress encountered [submitted
for publication]. The enzymatic metabolism of anti-
pyrine is well known since antipyrine has widely been
used as a marker for the enzymatic p450 activity in the
liver [23]. From this research it is known that o-APOH
cannot be formed by a natural biological pathway,
which makes it an ideal marker substance for oxidative
stress. Previous research showed that o-APOH is one of
the free radical products that is formed when anti-
pyrine is exposed to hydroxyl radicals [24].
This study was performed to assess the influence of
free radical scavenger administration on oxidative
stress during reperfusion in stable intermittent clau-
dicants during a standard treadmill test before and
after daily administration of 200 mg vitamin E and 500
mg vitamin C for 4 weeks.
MATERIALS AND METHODS
After approval by the local ethics committee 16 claudicants were
included in this pilot study, 14 males and 2 females. Mean age was
66 years (range 51–74 years). Seven patients were claudicants in one
leg and nine patients were claudicants in both legs. Twelve people
smoked more than five cigarettes per day and all used 80 mg aspirin
daily. All had been stable for 1 year regarding brachial ankle (b-a)
index and walking distance. Included were patients with a b-a index
of less than 0.8 and a drop in b-a index of more than 0.3, in one or
both legs, after a standard walking test (5 minutes, 3 km/h, 8°
incline). Excluded were patients with preexistent renal dysfunction
and those who were not able to perform a standard walking test.
On arrival in the vascular laboratory, blood was drawn from an
indwelling catheter inserted in the medial cubital vein to ascertain
the blank values (T0). Then, the patients were given 15 mg/kg
antipyrine orally. Following this, they were seated for 1 h to exclude
preexistent ischemia during the treadmill test and ensure full ab-
sorption of the antipyrine.
After a urine sample was produced, blood was drawn at four
different points in time (see Table 1). The b-a index was measured
before, 1 min after, and 5 min after the walking test to assess
whether the patients met the inclusion criteria.
The blood and urine samples were put on melting ice and analyzed
at the hospital laboratory. The blood samples for the determination
of antipyrine and malondialdehyde were put on melting ice and after
being centrifuged were snap-frozen and stored at ⫺20°C. After the
first test all patients received 200 mg of vitamin E and 500 mg of
vitamin C daily for 4 weeks. After 4 weeks the second walking test
was performed and sampling of blood and urine was repeated as
described above. The determination of the malondialdehyde or, more
specifically, thiobarbituric acid reactive species (TBARS) in plasma
was carried out on an UV–VIS spectrophotometer at 532 nm (Spec-
tronic 1001, Meyvis, Bergen op Zoom, The Netherlands). Nine hun-
dred microliters of 0.1 M HCl solution (containing 0.6 g thiobarbitu-
ric acid) was mixed with 100 ␮l plasma, vortexed, and heated to 95°C
for 1 h. After the sample was cooled to room temperature, the
absorbance was measured at 532 nm.
Antipyrine and o-APOH were measured using an optimized
HPLC-Tandem-MS method (LC-10AT, Shimadzu Ltd. Kyoto, Japan;
API-300, Perkin–Elmer Sciex Instruments, Thornhill, Canada) [25].
The sample pretreatment consisted of an optimized C-18 solid-phase
extraction (a solid-phase extraction procedure) [26]. To correct for
changes in the antipyrine concentration during the experiment, the
ratio of o-APOH and antipyrine was used for the statistical analyses.
The serum lactate concentration was measured on a Vitros 950
analyzer (Ortho Clinical Diagnostics) using standard Ektachem
Slide technology.
Vitamin E in serum was determined as ␣-tocopherol. The reverse-
phase HPLC method involved protein precipitation with ethanol
followed by hexane extraction of the supernatant. A fluorometric
detector was used.
To compare the groups we used a nonparametric test for paired
samples (Wilcoxon signed-rank test). Statistical significance was set
at P ⬍ 0.05.
RESULTS
Vitamin E concentrations in plasma were signifi-
cantly increased after a month of supplementation
(P ⬍ 0.001) (see Table 2). Serum lactate was signifi-
cantly increased (P ⫽ 0.002) after the walking test in
all claudicants with no difference between tests before
and after vitamins were administered (see Table 2).
Malondialdehyde (TBARS) concentration showed no
significant change after the walking test before and
after vitamins were used (see Table 2).
During the exercise and reperfusion period the anti-
pyrine concentration in plasma did not change before
and after antioxidant administration, suggesting that
the maximum serum concentration was reached before
the walking test. The change in concentration was
tested as the difference in concentrations between
measuring points. Before administration of the vita-
mins the ratio of o-APOH and antipyrine increased
significantly (P ⫽ 0.026) during the reperfusion pe-
riod (T2 to T3) and decreased significantly (P ⫽ 0.039)
after the reperfusion period (T3 to T4). After a month of
vitamin E and C administration no significant increase
in o-APOH concentration was seen (see Fig. 1). There
was a difference initial APOH levels after a month of
antioxidant supplementation, with the postsupplemen-
tation levels being higher (P ⫽ 0.038). Only four pa-
tients showed a detectable increase in albumin creati-
nine ratio after the first walking test. None of these
showed an increase after 1 month of vitamin E and C
administration.
DISCUSSION
Claudicants suffer from chronic ischemia reperfu-
sion injury. This not only damages the muscles and
 WIJNEN ET AL.: ANTIOXIDANTS REDUCE OXIDATIVE STRESS IN CLAUDICANTS 185

TABLE 2
Values of Serum Lactate (mmol/liter), Malondialdehyde (TBARS) (␮mol/liter), ortho-Hydroxy Antipyrine/
Antipyrine Ratio (APOH), and Vitamin E (␮mol/liter) Concentration at Four Different Sampling Times in 16
Intermittent Claudicants Performing a Standard Walking Test

Lactate T-bars APOH Vitamin E

Pre Post Pre Post Pre Post Pre Post

T1 1.59 1.31 7.07 7.34 1.26 ⫻ 10 ⫺3 2.18 ⫻ 10 ⫺3 18.4 27.5

(0.21) (0.06) (1.35) (0.51) (0.14 ⫻ 10 ⫺3) (0.81 ⫻ 10 ⫺3) (3.0) (4.4)
P ⫽ 0.038 P ⬍ 0.001
T2 2.89 2.61 6.76 7.15 1.32 ⫻ 10 ⫺3 1.69 ⫻ 10 ⫺3
(0.54) (0.41) (0.66) (0.66) (0.14 ⫻ 10 ⫺3) (0.54 ⫻ 10 ⫺3)
T3 2.99 2.67 6.92 5.80 1.47 ⫻ 10 ⫺3 1.89 ⫻ 10 ⫺3
(0.6) (0.45) (0.87) (1.6) (0.18 ⫻ 10 ⫺3) (0.78 ⫻ 10 ⫺3)
T4 1.42 1.31 6.15 5.92 1.38 ⫻ 10 ⫺3 1.96 ⫻ 10 ⫺3
(0.15) (0.10) (0.66) (1.58) (0.21 ⫻ 10 ⫺3) (0.86 ⫻ 10 ⫺3)

Note. T1, before walking test; T2, 1 min after walking test; T3, 5 min after walking test; T4, 60 min after walking test. Pre and post vitamin
supplementation values are shown. Values are given as means and (SEM). All patients, n ⫽ 16, were tested at all times, before and after
supplementation. P values indicate differences between pre- and postsupplementation levels.

other tissues that are being submitted to these periods
of ischemia–reperfusion but also causes a systemic re-
sponse and remote organ impairment.
Studies have been performed in which this remote
damage in claudicants has been measured and it ap-
pears to be a constant finding that some form of dam-
age takes place [3, 5, 7, 16, 18, 19, 21, 27, 28]. To
indicate that oxidative stress is responsible for the
damage found in remote organs in claudicants after
exercise and that we can reduce the damage by admin-
istering extra antioxidants to the patients [29], we
must be able to measure the level of free radical dam-
age. We have measured oxidative stress using malon-
dialdehyde as a product of lipid peroxidation. Malon-
dialdehyde or, more accurately, the TBARS as a
FIG. 1. Ortho-APOH/antipyrine as a percentage of the increase
or decrease when compared to the prewalking test values (T1 ⫽
100%). T1, before walking test, T2, 1 min after walking test, T3, 5
min after walking test, T4, 60 min after walking test. Uninterrupted
line represents the period before antioxidant supplementation; dot-
ted line represents the period after antioxidant supplementation.
marker for oxidative stress, however, has several dis-
advantages that can lead to misinterpretation of the
results. Malondialdehyde is very unstable and will be
metabolized rapidly in vivo. Another disadvantage of
the TBARS measurement is the cross-reactivity with
other products. The concentration of TBARS, which is
often used in the literature as a marker for oxidative
stress, is shown in Table 2. We found no statistically
significant decrease over the measuring period. There
is a nonsignificant rise in the concentration of the
TBARS during the period, just after the exercise pe-
riod. The use of TBARS concentration as a marker for
the level of free radical damage can lead to an under-
estimation of the oxidative stress in vivo.
We have used a new method using antipyrine as a
marker substance and measuring its free radical reac-
tion products. Antipyrine has been extensively tested
and its metabolism is well known [23]. It is absorbed
readily, the peak concentration is reached within 1 h,
and it is divided evenly in all body compartments. The
fact that no change in antipyrine concentration was
found during the exercise and reperfusion period is
important, since the amount of the free radical prod-
ucts that is formed is dependent on the concentration
of antipyrine present at the site of radical formation,
which is assumed to be equal to the plasma concentra-
tion. Thus, a rise in the amount of free radicals that is
produced in patients will lead to a higher formation of
the nonenzymatic free radical product of antipyrine,
o-APOH.
Regarding the level of o-APOH formed in the walk-
ing test, before administration of vitamins (see Table 2
and Fig. 1), we find significant changes in the o-APOH
concentrations. The ratio of o-APOH and antipyrine
increased significantly during the reperfusion period
(T2–T3) (P ⫽ 0.026) and decreased significantly after
186 JOURNAL OF SURGICAL RESEARCH: VOL. 96, NO. 2, APRIL 2001
the reperfusion period (P ⫽ 0.039). So it seems, as
expected, that free radical damage occurs during the
reperfusion period. Since the antipyrine concentration
is constant in time, the balance of formation and
breakdown/excretion of the free radical product
o-APOH is in favor of the latter after the reperfusion
period (T3–T4). This means that the highest degree of
free radical damage occurs for only a short period of
time immediately after exercise (T2–T3).
These results indicate an increase in oxidative stress
with a significant rise in the antipyrine free radical
product o-APOH after a standard walking test. After
the patients were given vitamin E and C for 1 month,
the vitamin E concentration was significantly in-
creased. There is no significant change in the level of
o-APOH following a standard walking test after 4
weeks of additional vitamin E and C supplementation.
Comparing this with the situation without antioxidant
supplementation, a decrease in free radical damage is
found.
With this study we have shown a decrease in oxida-
tive stress after a month of antioxidant supplementa-
tion in intermittent claudicants. The fact that all clau-
dicants had been stable for 1 year and that during
these 4 weeks no change in lifestyle or exercise pattern
was recorded makes it very unlikely that this change
was induced by exercise training alone. Although not
all patients showed a detectable increase in albumin
creatinine ratio in urine, the few patients that did
show an increase after the first walking test did not do
so after the month of vitamin supplementation. More
research must be carried out to prove the influence of
the antioxidant medication and the reduction of oxida-
tive stress on organ function in this group of patients.
Claudicants are four times more likely to develop
additional cardiovascular diseases [30]. A very plausi-
ble explanation for this is that claudication is just one
of the symptoms of generalized atherosclerosis. Others
argue that the oxidative stress in claudicants aggra-
vates the atherosclerosis [31]. What if we can reduce
the remote organ damage and endothelial activation in
claudicants by giving them antioxidants? In a large
study in the general population a beneficial effect has
been found regarding cardiovascular mortality for vi-
tamin E when taken over a long period of time [32].
If vitamin E has been shown to reduce heart disease
in the general population and one of the mechanisms
might be the reduction of oxidative stress, there is
certainly a case to be made for administering extra
antioxidants to claudicants, since the amount of oxida-
tive stress appears to be larger in this group of pa-
tients. This would then add a new dimension to the
treatment of claudicants, not only treating the limb but
reducing the risk of cardiovascular disease and possi-
bly death.
In conclusion we have shown that with a new and
possibly more accurate marker for oxidative stress,
using ortho-hydroxyantipyrine as a free radical prod-
uct of the exogenous marker antipyrine, oxidative
stress in claudicants can be measured and that admin-
istration of vitamin E and C for 4 weeks diminishes
this stress. Further work should be done to prove the
influence of antioxidant supplementation on remote
organ functions and the influence on cardiovascular
comorbidity.
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