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  • 1. 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 of oxygen-derived free radicals (ODFR) and neutrophil claudicants leads to damage of local tissues and re- activation can cause additional damage [2– 4]. This not mote organs. Since this damage is partly caused by only results in local changes in the ischemic and reper- oxygen-derived free radicals (ODFR), scavenging fused tissues, but can also cause systemic effects [5]. these ODFR could reduce the local and remote injury. Methods. Using a new method by which a free radi- Some authors have suggested that this systemic in- cal reaction product (ortho-APOH) of the exogenous flammatory response was responsible for additional marker antipyrine is measured to quantify the oxida- atherosclerosis, one of the reasons for an increase in tive stress, 16 stable claudicants performed a standard ischemic heart disease observed in claudicants [1, 6, 7]. walking test before and after administration of vita- There is reason to believe that an increase in scav- min E (200 mg) and vitamin C (500 mg) daily for 4 enging activity is beneficial in claudicants and that weeks. scavengers can reduce the systemic and local inflam- Findings. Ortho-APOH was significantly increased matory response seen after ischemia–reperfusion (I–R) during the reperfusion period (P 0.026) before ad- injury in humans [8 –15]. Modulation of the ODFR ministration of the vitamins. After 4 weeks of vitamin activity in claudicants would therefore be an attractive supplementation no rise was found in the reperfusion option in the treatment of these patients. period. Malondialdehyde showed no changes in either In vivo, measuring ODFR activity has always been a group. problem due to the extremely short half-life of oxygen Interpretation. These findings indicate that admin- radicals. Therefore, most studies use metabolites of the istering extra antioxidants to claudicants reduces ox- idative stress in these patients. This may also have an ODFR-induced lipid peroxidation, such as malondial- effect on the remote ischemia–reperfusion damage dehyde, as markers for ODFR activity. Other measure- and reduce cardiovascular morbidity in this ments are aimed at neutrophils that are activated dur- group. © 2001 Academic Press ing reperfusion or at the total antioxidant capacity that Key Words: ischemia–reperfusion; vitamin E; vitamin is lowered after oxidative stress [16]. C; antipyrine; intermittent claudication; malondial- Others have reported remote organ damage during dehyde. I–R that is caused by direct oxygen radical reactions or activated neutrophils. The target organ most fre- INTRODUCTION quently studied is the kidney, where an increase in the albumin creatinine ratio in urine is thought to be an Intermittent claudication can be considered an im- indicator of endothelial damage and reperfusion injury portant health problem since 5% of men over 50 years [3, 17–21]. All of these methods have their limitations of age suffer from it [1]. Usually located in the lower and are very susceptible to interference from other extremities, claudication is caused by narrowing or reactions in vivo [22]. obstruction of arteries in the aorto-iliacal region or in Recently, we developed a new method for measuring peripheral arteries, resulting in hypoxia during exer- oxidative stress in humans, using antipyrine (2,3- cise. This results in repetitive low-grade ischemia with dimethyl-phenyl-3-pyrazolyn-5-one) as a marker sub- calf or buttock pain that subsides when the exercise is stance and measuring its free radical reaction product stopped and reperfusion starts. It has been demon- ortho-hydroxyantipyrine (o-APOH) as an indicator for 183 0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
  • 2. 184 JOURNAL OF SURGICAL RESEARCH: VOL. 96, NO. 2, APRIL 2001 TABLE 1 described above. The determination of the malondialdehyde or, more specifically, thiobarbituric acid reactive species (TBARS) in plasma Time Frame for the Sampling of Blood and Urine in was carried out on an UV–VIS spectrophotometer at 532 nm (Spec- Intermittent Claudicants Performing a Standard tronic 1001, Meyvis, Bergen op Zoom, The Netherlands). Nine hun- Walking Test 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 Time Event for 1 h. After the sample was cooled to room temperature, the absorbance was measured at 532 nm. T0 Blank blood sample; administration of antipyrine Antipyrine and o-APOH were measured using an optimized T1 Blood sample; urine sample; start of walking test; 60 HPLC-Tandem-MS method (LC-10AT, Shimadzu Ltd. Kyoto, Japan; min after T0 API-300, Perkin–Elmer Sciex Instruments, Thornhill, Canada) [25]. T2 Blood sample; 1 min after walking test; 66 min after T0 The sample pretreatment consisted of an optimized C-18 solid-phase T3 Blood sample; 5 min after walking test; 70 min after T0 extraction (a solid-phase extraction procedure) [26]. To correct for changes in the antipyrine concentration during the experiment, the T4 Blood sample; urine sample; 60 min after walking test; ratio of o-APOH and antipyrine was used for the statistical analyses. 125 min after T0 The serum lactate concentration was measured on a Vitros 950 analyzer (Ortho Clinical Diagnostics) using standard Ektachem Slide technology. the amount of oxidative stress encountered [submitted Vitamin E in serum was determined as -tocopherol. The reverse- for publication]. The enzymatic metabolism of anti- phase HPLC method involved protein precipitation with ethanol followed by hexane extraction of the supernatant. A fluorometric pyrine is well known since antipyrine has widely been detector was used. used as a marker for the enzymatic p450 activity in the To compare the groups we used a nonparametric test for paired liver [23]. From this research it is known that o-APOH samples (Wilcoxon signed-rank test). Statistical significance was set cannot be formed by a natural biological pathway, at P 0.05. which makes it an ideal marker substance for oxidative stress. Previous research showed that o-APOH is one of RESULTS the free radical products that is formed when anti- Vitamin E concentrations in plasma were signifi- pyrine is exposed to hydroxyl radicals [24]. cantly increased after a month of supplementation This study was performed to assess the influence of (P 0.001) (see Table 2). Serum lactate was signifi- free radical scavenger administration on oxidative cantly increased (P 0.002) after the walking test in stress during reperfusion in stable intermittent clau- all claudicants with no difference between tests before dicants during a standard treadmill test before and and after vitamins were administered (see Table 2). after daily administration of 200 mg vitamin E and 500 Malondialdehyde (TBARS) concentration showed no mg vitamin C for 4 weeks. significant change after the walking test before and after vitamins were used (see Table 2). MATERIALS AND METHODS During the exercise and reperfusion period the anti- After approval by the local ethics committee 16 claudicants were pyrine concentration in plasma did not change before included in this pilot study, 14 males and 2 females. Mean age was and after antioxidant administration, suggesting that 66 years (range 51–74 years). Seven patients were claudicants in one the maximum serum concentration was reached before leg and nine patients were claudicants in both legs. Twelve people the walking test. The change in concentration was smoked more than five cigarettes per day and all used 80 mg aspirin tested as the difference in concentrations between 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 measuring points. Before administration of the vita- of less than 0.8 and a drop in b-a index of more than 0.3, in one or mins the ratio of o-APOH and antipyrine increased both legs, after a standard walking test (5 minutes, 3 km/h, 8° significantly (P 0.026) during the reperfusion pe- incline). Excluded were patients with preexistent renal dysfunction riod (T2 to T3) and decreased significantly (P 0.039) and those who were not able to perform a standard walking test. On arrival in the vascular laboratory, blood was drawn from an after the reperfusion period (T3 to T4). After a month of indwelling catheter inserted in the medial cubital vein to ascertain vitamin E and C administration no significant increase the blank values (T0). Then, the patients were given 15 mg/kg in o-APOH concentration was seen (see Fig. 1). There antipyrine orally. Following this, they were seated for 1 h to exclude was a difference initial APOH levels after a month of preexistent ischemia during the treadmill test and ensure full ab- antioxidant supplementation, with the postsupplemen- sorption of the antipyrine. After a urine sample was produced, blood was drawn at four tation levels being higher (P 0.038). Only four pa- different points in time (see Table 1). The b-a index was measured tients showed a detectable increase in albumin creati- before, 1 min after, and 5 min after the walking test to assess nine ratio after the first walking test. None of these whether the patients met the inclusion criteria. showed an increase after 1 month of vitamin E and C The blood and urine samples were put on melting ice and analyzed administration. 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 DISCUSSION 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 Claudicants suffer from chronic ischemia reperfu- was performed and sampling of blood and urine was repeated as sion injury. This not only damages the muscles and
  • 3. 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 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
  • 4. 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. 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