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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
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 marker for oxidative stress, however, has several dis-
of ischemia–reperfusion but also causes a systemic re- advantages that can lead to misinterpretation of the
sponse and remote organ impairment. results. Malondialdehyde is very unstable and will be
Studies have been performed in which this remote metabolized rapidly in vivo. Another disadvantage of
damage in claudicants has been measured and it ap- the TBARS measurement is the cross-reactivity with
pears to be a constant finding that some form of dam- other products. The concentration of TBARS, which is
age takes place [3, 5, 7, 16, 18, 19, 21, 27, 28]. To often used in the literature as a marker for oxidative
indicate that oxidative stress is responsible for the stress, is shown in Table 2. We found no statistically
damage found in remote organs in claudicants after significant decrease over the measuring period. There
exercise and that we can reduce the damage by admin- is a nonsignificant rise in the concentration of the
istering extra antioxidants to the patients [29], we TBARS during the period, just after the exercise pe-
must be able to measure the level of free radical dam- riod. The use of TBARS concentration as a marker for
age. We have measured oxidative stress using malon- the level of free radical damage can lead to an under-
dialdehyde as a product of lipid peroxidation. Malon- estimation of the oxidative stress in vivo.
dialdehyde or, more accurately, the TBARS as a 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-
FIG. 1. Ortho-APOH/antipyrine as a percentage of the increase ing test, before administration of vitamins (see Table 2
or decrease when compared to the prewalking test values (T1 ⫽ and Fig. 1), we find significant changes in the o-APOH
100%). T1, before walking test, T2, 1 min after walking test, T3, 5
min after walking test, T4, 60 min after walking test. Uninterrupted
concentrations. The ratio of o-APOH and antipyrine
line represents the period before antioxidant supplementation; dot- increased significantly during the reperfusion period
ted line represents the period after antioxidant supplementation. (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 possibly more accurate marker for oxidative stress,
expected, that free radical damage occurs during the using ortho-hydroxyantipyrine as a free radical prod-
reperfusion period. Since the antipyrine concentration uct of the exogenous marker antipyrine, oxidative
is constant in time, the balance of formation and stress in claudicants can be measured and that admin-
breakdown/excretion of the free radical product istration of vitamin E and C for 4 weeks diminishes
o-APOH is in favor of the latter after the reperfusion this stress. Further work should be done to prove the
period (T3–T4). This means that the highest degree of influence of antioxidant supplementation on remote
free radical damage occurs for only a short period of organ functions and the influence on cardiovascular
time immediately after exercise (T2–T3). comorbidity.
These results indicate an increase in oxidative stress
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