Reducing Oxidative Stress | Revelar


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Antioxidants Reduce Oxidative Stress in Claudicants

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Reducing Oxidative Stress | Revelar

  1. 1. 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 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 ‫؍‬ 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- strated that during the reperfusion period, production 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 Journal of Surgical Research 96, 183–187 (2001) doi:10.1006/jsre.2000.6078, available online at on 183 0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
  2. 2. 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 Ϫ20°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 Ͻ 0.05. RESULTS Vitamin E concentrations in plasma were signifi- cantly increased after a month of supplementation (P Ͻ 0.001) (see Table 2). Serum lactate was signifi- cantly increased (P ϭ 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 ϭ 0.026) during the reperfusion pe- riod (T2 to T3) and decreased significantly (P ϭ 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 ϭ 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 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 184 JOURNAL OF SURGICAL RESEARCH: VOL. 96, NO. 2, APRIL 2001
  3. 3. 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 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 ϭ 0.026) and decreased significantly after FIG. 1. Ortho-APOH/antipyrine as a percentage of the increase or decrease when compared to the prewalking test values (T1 ϭ 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. 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 ϫ 10Ϫ3 2.18 ϫ 10Ϫ3 18.4 27.5 (0.21) (0.06) (1.35) (0.51) (0.14 ϫ 10Ϫ3 ) (0.81 ϫ 10Ϫ3 ) (3.0) (4.4) P ϭ 0.038 P Ͻ 0.001 T2 2.89 2.61 6.76 7.15 1.32 ϫ 10Ϫ3 1.69 ϫ 10Ϫ3 (0.54) (0.41) (0.66) (0.66) (0.14 ϫ 10Ϫ3 ) (0.54 ϫ 10Ϫ3 ) T3 2.99 2.67 6.92 5.80 1.47 ϫ 10Ϫ3 1.89 ϫ 10Ϫ3 (0.6) (0.45) (0.87) (1.6) (0.18 ϫ 10Ϫ3 ) (0.78 ϫ 10Ϫ3 ) T4 1.42 1.31 6.15 5.92 1.38 ϫ 10Ϫ3 1.96 ϫ 10Ϫ3 (0.15) (0.10) (0.66) (1.58) (0.21 ϫ 10Ϫ3 ) (0.86 ϫ 10Ϫ3 ) 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 ϭ 16, were tested at all times, before and after supplementation. P values indicate differences between pre- and postsupplementation levels. 185WIJNEN ET AL.: ANTIOXIDANTS REDUCE OXIDATIVE STRESS IN CLAUDICANTS
  4. 4. the reperfusion period (P ϭ 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. 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