Share this article on:

00005768-200707000-0001100005768_2007_39_1107_michailidis_measurement_7article< 94_0_15_4 >Medicine & Science in Sports & Exercise©2007The American College of Sports MedicineVolume 39(7)July 2007pp 1107-1113Sampling Time is Crucial for Measurement of Aerobic Exercise-Induced Oxidative Stress[BASIC SCIENCES: Original Investigations]MICHAILIDIS, YIANNIS1; JAMURTAS, ATHANASIOS Z.2,3; NIKOLAIDIS, MICHALIS G.2,3,4; FATOUROS, IOANNIS G.1; KOUTEDAKIS, YIANNIS2,5; PAPASSOTIRIOU, IOANNIS6; KOURETAS, DIMITRIS41Department of Physical Education and Sport Science, Democritus University of Thrace, Komotini, GREECE; 2Department of Physical Education and Sport Science, University of Thessaly, Trikala, GREECE; 3Institute of Human Performance and Rehabilitation, Centre for Research and Technology-Thessaly, Trikala, GREECE; 4Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, GREECE; 5School of Sport, Performing Arts and Leisure, Wolverhampton University, Walshall, UNITED KINGDOM; and 6Department of Clinical Biochemistry, Aghia Sophia Children's Hospital, Athens, GREECEAddress for correspondence: Dimitris Kouretas, Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, 41221, Greece; E-mail: for publication December 2006.Accepted for publication February 2007.ABSTRACTPurpose: To thoroughly investigate the time-course changes of several commonly used markers of oxidative stress by performing serial measurements during a 24-h period after an acute bout of strenuous cardiovascular exercise.Methods: Eleven untrained men performed two trials. In the experimental trial, the subjects exercised for 45 min at 70-75% V˙O2max and then at 90% V˙O2max to exhaustion on a treadmill; in the control trial, the subjects remained at rest. Blood samples were drawn before and after exercise (immediately after exercise and at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 24 h). Reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG, thiobarbituric acid-reactive substances (TBARS), protein carbonyls, catalase activity, and total antioxidant capacity (TAC) were determined.Results: The time to lowest concentration after exercise was 1.7 ± 0.7 h (mean ± SD) for GSH/GSSG, and the time to highest concentration after exercise was 1.2 ± 0.6 h for TBARS, 4.4 ± 0.5 h for protein carbonyls, 0.5 ± 0.4h for catalase, and 2.2 ± 0.9 h for TAC. The greatest change after exercise was −74 ± 9% for GSH/GSSG, 129 ± 29% for TBARS, 135 ± 53% for protein carbonyls, 51 ± 16% for catalase, and 24 ± 10% for TAC.Conclusion: There is no best time point applying to all markers for collecting blood samples after aerobic exercise. The optimum postexercise time points for blood collection in untrained individuals are immediately after exercise for catalase, 1 h for TBARS, 2 h for TAC, GSH, and GSSG, and 4 h after exercise for protein carbonyls.The effect of aerobic exercise on the levels of oxidative stress biomarkers in many tissues has been studied extensively (10). The most frequently studied tissue is blood, because of its ease in sampling and analysis of its constituents. It is now well established that strenuous exercise results in oxidative stress in blood (10). In some diseases, oxidative stress contributes significantly to tissue injury; therefore, increased oxidative stress may pose a health problem in the general population (12). Additionally, there is evidence that oxidative stress is linked to fatigue, muscle damage, and reduced immune function (12,17).Despite the hundreds of human studies that have investigated the effects of aerobic exercise on oxidative stress, the vast majority of them have collected blood samples just immediately after exercise (5,16) or at some other early postexercise point (1,29). Few studies have collected more than two blood samples after non-muscle-damaging exercise (3,6,24,30), and the most detailed of these have taken four blood samples (6,21,22). The relevant studies have reported inconsistent results concerning the effect of exercise on the levels of specific oxidative stress biomarkers, a fact that, at least partially, may be attributable to differences in subjects (trained vs untrained), type of protocol (differences in duration and intensity), blood-sampling time points, and assays employed among the studies.The aim of the present study was to thoroughly investigate the time-course changes of several commonly used markers of oxidative stress by performing serial measurements during a 24-h period after an acute bout of cardiovascular exercise. For this purpose, we measured a battery of oxidative stress biomarkers, because currently there is no single biomarker that can reliably describe oxidative damage. We believe that this is an important preliminary step in ascertaining the possible biological function of oxidative stress during and after aerobic exercise. In addition, useful information can be derived regarding the design of human experiments in the field of oxidative stress and exercise.MATERIALS AND METHODSSubjects.Eleven untrained men (age, 23 ± 6 yr; height, 175 ± 3 cm; weight, 75 ± 5 kg; body fat, 14 ± 3%; maximal oxygen consumption (V˙O2max), 47 ± 6 mL·kg−1·min−1) participated. Most of the subjects were students in our sports science department, participating no more than three times per week in low- to moderate-intensity and short- to moderate-duration physical education classes. Subjects visited the laboratory three times in the morning after an overnight fast and abstained from alcohol and caffeine for 24 h. V˙O2max measurement ensured that subjects ran at similar exercise intensities. A written informed consent to participate in the study was provided by all participants after they had been informed of all risks, discomforts, and benefits involved in the study. The procedures were in accordance with the Helsinki declaration of 1975, and approval was received from the institutional review board.Design.Each subject participated in two trials in a random, counterbalanced design. Subjects visited the laboratory for a second time 7-14 d after V˙O2max determination (0800-0900 h) and either exercised (experimental trial) or rested (control trial). Exercise included 45 min of running on a treadmill at an intensity corresponding to 70-75% of their V˙O2max. After 45 min, speed was increased to 90% V˙O2max, and exercise was terminated at exhaustion (18). Volunteers had access to water ad libitum during exercise. During the testing, the heart rate was 164.3 ± 6.2, the rating of exertion on Borg's scale (6-20) was 13.1 ± 2.3, and the respiratory exchange ratio was 0.95 ± 0.05. Each subject consumed a banana during the first hour of the postexercise period and had lunch between 1300 and 1400 h and dinner between 2000 and 2100 h, in both trials (resting and exercise). Each meal contained, per kilogram of body mass, 0.53 g of fat, 1.71 g of carbohydrate, and 0.50 g of protein (energy content 13.9 kcal·kg−1; 30% fat, 55% carbohydrate, and 15% protein). Subjects remained seated or lying in the laboratory during the 10-h postexercise period (between 2000 and 2100 h). Subjects visited the laboratory for a third time 7-14 d after the second visit, to perform the second trial (resting or exercise). Subjects followed exactly the same diet before each trial (resting or exercise).Blood handling and analyses.A catheter was inserted into a forearm vein and was kept patent by flushing with normal saline. Blood samples were drawn at rest and after exercise (immediately after exercise and at 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 24 h). Directly after taking the blood sample, 5% trichloroacetic acid (TCA) was added to whole blood collected in EDTA tubes for reduced glutathione (GSH) analysis. For oxidized glutathione (GSSG) analysis, 5% TCA and 2-vinyl pyridine were added to whole blood collected in EDTA tubes. The whole-blood samples were centrifuged at 4000g for 10 min at 4°C. Two hundred microliters of the supernatant were dispensed in tubes and mixed with 60 μL of 5% TCA. The samples were centrifuged again at 28,000g for 5 min at 4°C, and the clear supernatants were collected and stored at −30°C until GSH and GSSG analysis. Another portion of blood was collected in plain tubes, left on ice for 20 min to clot, and centrifuged at 1500g for 10 min at 4°C for serum separation. Serum was transferred in Eppendorf tubes and was used for the determination of thiobarbituric acid-reactive substances (TBARS), protein carbonyls, catalase, and total antioxidant capacity (TAC). Blood samples were stored in multiple aliquots at −30°C and were thawed only once before analysis.For GSH, 20 μL of whole blood treated with TCA was mixed with 660 μL of 67 mM sodium potassium phosphate (pH 8.0) and 330 μL of 1 mM 5,5′-dithiobis-2-nitrobenzoate (DTNB). The samples were incubated in the dark at room temperature for 45 min, and the absorbance was read at 412 nm. GSSG was assayed by treating 260 μL of whole blood with TCA, to be neutralized up to pH 7.0-7.5 with NaOH. Four microliters of 2-vinyl pyridine was added, and the samples were incubated for 2 h at room temperature. Five microliters of whole blood treated with TCA was mixed with 600 μL of 143 mM sodium phosphate (6.3 mM EDTA, pH 7.5), 100 μL of 3 mM NADPH, 100 μL of 10 mM DTNB, and 194 μL of distilled water. The samples were incubated for 10 min at room temperature. After the addition of 1 μL of glutathione reductase, the change in absorbance at 412 nm was read for 3 min.For TBARS, 100 μL of serum was mixed with 500 μL of TCA 35% and 500 μL of Tris-HCl (200 mM, pH 7.4) and was incubated for 10 min at room temperature. One milliliter of 2 M Na2SO4 and 55 mM thiobarbituric acid solution was added, and the samples were incubated at 95°C for 45 min. The samples were cooled on ice for 5 min and were vortexed after adding 1 mL of TCA 70%. The samples were centrifuged at 15,000g for 3 min, and the absorbance of the supernatant was read at 530 nm. A baseline shift in absorbance was taken into account by running a blank along with all samples during the measurement. A standard curve was constructed by using malondialdehyde as a standard at concentrations of 0, 1.25, 2.5, 5, and 10 μM.For protein carbonyls, 50 μL of 20% TCA was added to 50 μL of serum, and this mixture was incubated in an ice bath for 15 min and centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 500 μL of 10 mM 2,4-dinitrophenylhydrazine (in 2.5 N HCL) for the sample, or 500 μL of 2.5 N HCL for the blank, was added in the pellet. The samples were incubated in the dark at room temperature for 1 h, with intermittent vortexing every 15 min, and were centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 1 mL of 10% TCA was added, vortexed, and centrifuged at 15,000g for 5 min at 4°C. The supernatant was discarded, and 1 mL of ethanol-ethyl acetate (1:1 v/v) was added, vortexed, and centrifuged at 15,000g for 5 min at 4°C. The washing step was repeated two more times. The supernatant was discarded, and 1 mL of 5 M urea (pH 2.3) was added, vortexed, and incubated at 37°C for 15 min. The samples were centrifuged at 15,000g for 3 min at 4°C, and the absorbance was read at 375 nm.For catalase activity, 2975 μL of 67 mM sodium potassium phosphate (pH 7.4) were added to 20 μL of serum, and the samples were incubated at 37°C for 10 min. Five microliters of 30% hydrogen peroxide were added to the samples, and the change in absorbance was immediately read at 240 nm for 1.5 min.For TAC, 480 μL of 10 mM sodium potassium phosphate (pH 7.4) and 500 μL of 0.1 mM 2,2-diphenyl-1-picrylhydrazyl free radical were added to 20 μL of serum, and the samples were incubated in the dark for 30 min at room temperature. The samples were centrifuged for 3 min at 20,000g, and the absorbance was read at 520 nm.Total protein in serum was assayed using a Bradford reagent. Each assay was determined spectrophotometrically in duplicates (except triplicates for GSSG). The intra- and interassay CV for each measurement were 3.1 and 4.5% for GSH, 6.0 and 7.3% for GSSG, 3.9 and 5.9% for TBARS, 4.3 and 7.0% for protein carbonyls, 6.2 and 10.0% for catalase, and 2.9 and 5.4% for TAC, respectively. Postexercise plasma volume changes were calculated on the basis of hematocrit and hemoglobin.Dietary analysis.To control for the effect of previous diet on the outcome measures of the study, and to establish that participants after both trials had similar levels of macronutrient and antioxidant intake, they were asked to record their diet for 3 d before the control trial and to repeat this diet before the exercise trial. Each subject had been provided with a written set of guidelines for monitoring dietary consumption and a record sheet for recording food intake. Diet records were analyzed using the nutritional analysis system Science Fit Diet 200A (Sciencefit, Greece).Statistical analysis.Data were log-transformed before being analyzed by a two-way repeated-measures (trial × time) ANOVA. If a significant interaction was obtained, pair wise comparisons were performed through simple contrasts and simple main-effects analysis. The estimates of the uncertainty in the timing and magnitude of the exercise effects were determined through calculation of 95% confidence intervals (CI). To determine the meaningfulness of the differences in daily energy, macronutrient, and antioxidant intake between the trials, effect size was calculated as the difference between trial 1 and trial 2 mean concentrations divided by the mean standard deviation of trial 1 and trial 2. According to a modified Cohen scale ( ), effect sizes of 0.2, 0.6, 1.2, 2.0, and 4.0 were considered small, moderate, large, very large, and nearly perfect, respectively. For comparison, the values of the original Cohen scale are 0.2 for small, 0.5 for moderate, and 0.8 for large effects. The level of significance was set at α = 0.05. SPSS version 13.0 was used for all analyses (SPSS Inc., Chicago, IL). Data are presented as means ± SD.RESULTSIn general, nutrition was similar between the two trials, except for carbohydrate and fat intake, where the difference can be considered moderate (i.e., an effect size between 0.6 and 1.2) (Table 1). The total duration of the exercise protocol was 51.4 ± 2.9 min. Plasma volume did not change during the 24-h postexercise period (P > 0.05); nevertheless, the values were corrected for any nonsignificant plasma volume changes.TABLE 1. Analysis of daily energy intake of individuals during the control and the exercise trials (mean ± SD).Glutathione status.The main effects of trial and time, as well as their interaction, were significant with regard to GSH (P = 0.0065, P = 0.001, and P = 0.002, respectively; Fig. 1A), GSSG (P = 0.047, P = 0.002, and P = 0.011, respectively; Fig. 1B), and GSH/GSSG (all at P < 0.001; Fig. 1C). GSH concentration significantly decreased immediately after exercise (−30%), remained depressed for 5 h (−19%), and demonstrated its lowest value 2 h after exercise (−63%). Conversely, GSSG significantly increased 30 min (22%) after exercise, remained elevated for 4 h (16%), and peaked 2 h after exercise (38%). Consequently, their ratio (GSH/GSSG) significantly declined immediately after exercise (−34%), remained depressed until 5 h after exercise, (−23%), reached a nadir at 2 h after exercise (−73%), and returned to baseline values thereafter. The time to highest or lowest concentration after exercise was 2.0 ± 0.6 h (CI: 1.6-2.4) for GSH, 1.8 ± 0.7 h (CI: 1.3-2.3) for GSSG, and 1.7 ± 0.7 h (CI: 1.3-2.2) for GSH/GSSG. The greatest percent change after exercise was 61 ± 12 (CI: 53-69) for GSH, 39 ± 8 (CI: 34-44) for GSSG, and −74 ± 9 (CI: 66-78) for GSH/GSSG.FIGURE 1-Glutathione (A), oxidized glutathione (B), and glutathione/oxidized glutathione (C) concentrations during control (open rectangles) and exercise trials (closed rectangles). Values are means ± SD. Error bars at the side of each figure represent 95% confidence limits of the difference between rest and exercise for the maximum or minimum concentration.TBARS and protein carbonyls.The interaction of trial and time, as well as trial and time main effects, were significant with regard to TBARS concentration (P < 0.001, P = 0.022, and P < 0.001, respectively; Fig. 2A). TBARS significantly increased immediately after exercise (46%), peaked 1 h after exercise (123%), remained elevated for 3 h (31%), and returned toward baseline levels thereafter. The time to highest TBARS concentration after exercise was 1.2± 0.6 h (CI: 0.8-1.6). The greatest percent increase in TBARS after exercise was 129 ± 12 (CI: 110-149). Regarding protein carbonyls, the main effects of trial and time, as well as their interaction, were significant (P = 0.003, P < 0.001, and P < 0.001, respectively; Fig. 2B). Protein oxidation significantly increased 30 min (32%) after exercise, peaked 4 h after exercise (96%), remained elevated for 8 h (55%), and declined thereafter. The time to highest protein carbonyl concentration after exercise was 4.4 ± 0.5 h (CI: 4.2-4.6). The greatest percent increase in protein carbonyl after exercise was 135 ± 53 (CI: 70-191).FIGURE 2-Thiobarbituric acid-reactive substances (A) and protein carbonyl (B) concentration during control (open rectangles) and exercise trials (closed rectangles). Values are means ± SD. Error bars at the side of each figure represent 95% confidence limits of the difference between rest and exercise for the maximum concentration.Catalase and TAC.Regarding catalase activity, the interaction of trial and time, as well as both main effects, were significant (P = 0.002, P = 0.041, and P < 0.001, respectively; Fig. 3A). Catalase activity significantly peaked immediately after exercise (50%), remained elevated for 1 h after exercise (20%), and returned to preexercise values thereafter. The time to highest catalase activity after exercise was 0.5 ± 0.4 h (CI: 0.2-0.7). The greatest percent increase in catalase activity after exercise was 51 ± 16 (CI: 39-60). The main effects of trial and time, as well as their interaction, were significant with regard to TAC (P = 0.024, P < 0.001, and P < 0.001, respectively; Fig. 3B). TAC significantly increased immediately after exercise (6%), peaked 2 h after exercise (21%), remained elevated for 3 h (11%), and declined thereafter. The time to highest TAC after exercise was 2.2 ± 0.9 h (CI: 1.6-2.8). The greatest percent increase in TAC after exercise was 24± 10 (CI: 17-30).FIGURE 3-Catalase activity (A) and total antioxidant capacity (B) concentrations during control (open rectangles) and exercise trials (closed rectangles). Values are means ± SD. Error bars at the side of each figure represent 95% confidence limits of the difference between rest and exercise for the maximum concentration.DISCUSSIONTo our knowledge, this is the first attempt to characterize the influence of exercise on the time-course changes of several commonly used blood oxidative stress markers. Although hundreds of studies have determined the effects of acute exercise on blood oxidative stress, most of them have collected only a few samples after non-muscle-damaging exercise (at best, up to four samples (6,21,22)), explaining, at least in part, the large discrepancy in the relevant literature. The present results clearly indicate that sampling time after exercise may lead to different conclusions regarding exercise-induced oxidative stress responses. In fact, our findings depict nonuniform changes in these markers because both transient (i.e., catalase) and prolonged (i.e., protein carbonyls) changes were observed.Exercise decreased GSH, increased GSSG, and decreased their ratio. These results denote that after exercise, hepatic GSH supply may not be sufficient to match the enhanced use resulting in reduction of blood GSH concentration. There might also be an increase in blood GSH clearance (e.g., increased consumption by muscle) for several hours after exercise. The fact that exercise decreased GSH by 63% and increased GSSG by 38% at 2 h after exercise implies that only a small part of GSH decrease can be attributed to its oxidation into GSSG. The majority of studies that investigated the effects of acute aerobic exercise on glutathione redox status suggest that GSH and GSH/GSSG decrease and that GSSG increases during exercise, at least partly because of GSH use against free radicals and its consumption to regenerate ascorbic acid and alpha tocopherol (10). In the present study, the maximum effect of exercise on glutathione redox status appeared at 2 h after exercise. The most detailed study that has measured glutathione redox status is that of Bloomer et al. (6), which collected four blood samples the first 24 h after aerobic exercise in trained humans. In that study, GSH/GSSG experienced a decrease only immediately after, but not at 1, 6, or 24 h after, aerobic cycling lasting 30 min (6). It is difficult to interpret the more long-lasting effects of exercise on glutathione redox status found in the present study. However, the fact that Bloomer et al. (6) employed trained individuals and a nonexhaustive exercise protocol, in contrast to the untrained individuals and exhaustive exercise employed in the present study, may partly explain this discrepancy. Indeed, several studies have found that trained individuals responded differently to oxidative stress induced by an acute exercise bout compared with untrained subjects (16,27), and that exhaustive exercise induced higher oxidative stress than nonexhaustive exercise (10).Results from the present study indicate that lipid peroxidation, as measured by TBARS, was elevated immediately until 3 h after exercise. The main probable mechanism through which exercise increased lipid peroxidation after its cessation is the increased susceptibility to peroxidation of unsaturated fatty acids (13), because exercise markedly increases the concentration and unsaturation degree of nonesterified fatty acids in blood (19). Many studies have provided indications for substantial increases in plasma lipid-peroxidation levels after aerobic exercise (28,29), although others have reported no changes (6).Albumin makes up approximately 55% of total serum protein content, whereas 10 other abundant proteins account for more than 90% of all serum proteins (2). Therefore, the increased protein carbonyls after exercise should be derived mainly from oxidation of albumin and the other major serum proteins. Removal of oxidized proteins from blood is, presumably, a time-consuming process, considering that protein carbonyl concentration remained elevated for a prolonged period (8 h) after exercise. Certainly, increased production of reactive oxygen and nitrogen species after cessation of exercise also may have contributed to the elevated levels of protein carbonyls after exercise. We are aware of four studies that have investigated the effects of aerobic exercise on plasma protein carbonyls (1,3,5,6). These studies generally have reported increases similar to ours immediately after exercise, whereas the increases in protein carbonyls mostly disappeared after 0.5 to 6 h of recovery (1,3,5,6). The shorter-lived response of protein carbonyls reported in the literature compared with that in the present study is difficult to interpret. It may be partly attributed to the generally higher intensity and more muscle-damaging exercise mode (treadmill vs cycling) used in our study, and also to differences in the physical fitness of the participants employed (untrained in the present study vs trained in the literature).Catalase has no apparent function in serum, because it is an intracellular enzyme. Therefore, its increased activity in serum after exercise probably indicates an increased damage of muscle fibers or erythrocytes that results in its increased leakage into the circulation. Several studies have examined the effect of a single bout of aerobic exercise on blood catalase activity and have generally reported increases similar to ours (15,25), although others have reported no differences (16).The increase of TAC in serum after exercise suggests that acute exercise activates the body's antioxidant defenses. Mobilization of tissue antioxidant stores into plasma, such as uric acid (9), is probably one mechanism responsible for the marked increase (and not decrease, as might be expected intuitively) of TAC after exercise. This is a widely accepted phenomenon that would help maintain or even increase serum antioxidant status in times of need (20). Increased catalase activity after exercise also could have contributed to the increased TAC. Nevertheless, this increase in the antioxidant capacity of serum did not prove efficient at inhibiting the increase in glutathione, lipid, and protein oxidation of the blood. Most studies agree that exercise increases TAC, even though the majority of them have measured it only up to 1 h after exercise (1,26), making almost impossible to compare our time-course data with those of the literature.Certainly, the findings of the present study apply only to untrained male individuals performing exhaustive aerobic exercise of moderate duration. Other types of exercise, such as anaerobic exercise or longer-duration exercise, may affect blood oxidative stress in a different way. For example, there is evidence that aerobic and anaerobic exercise affect several oxidative stress biomarkers differently (6) and that elevation of protein carbonyls after aerobic exercise is greater after longer-duration protocols (3). There is also a body of evidence suggesting that the degree of oxidative stress may be attenuated by chronic aerobic and anaerobic training, through an increase in endogenous antioxidant production, a decrease in free-radical generation, or a combination of both processes (4,27). Finally, whether or not exercise induces muscle damage probably plays major role in determining the effect of exercise on the time course of oxidative stress. For example, there is convincing evidence that disturbances in some indices of blood oxidative stress may persist for and/or appear several days after muscle-damaging exercise (7,11), which is in contrast to the return to the resting values a few hours after an acute bout of non-muscle-damaging exercise reported by us in the present study and in other studies (7,23).The main aim of the present study was to monitor the time course of several commonly used biomarkers to provide guidelines for the study design of similar experiments. On the basis of these findings, there is no best time point applying to all markers for collecting blood samples after aerobic exercise. Consequently, the optimum postexercise time points for blood collection in which the greatest magnitude of change will likely be noted are immediately for catalase, 1 h for TBARS, 2 h for TAC, GSH and GSSG, and 4 h for protein carbonyls.Four postexercise blood samples up to 4 h should be enough to satisfactory describe the changes in oxidative stress after exhaustive aerobic exercise of moderate duration (~50 min) performed by untrained male subjects. The frequently collected blood sample at 24 h after aerobic exercise or at some other (more delayed) time point after exercise (8,14) does not seem to offer important information. The practice of the vast majority of the relevant studies to collect one blood sample immediately after exercise can potentially lead to inaccurate deductions. There are many markers available to assess oxidative stress, and, in general, every assay has its advantages and disadvantages. For example, there are some reservations about the validity of the TBARS assay in detecting lipid peroxidation in vivo, because the TBARS assay is not specific to malondialdehyde, and lipid peroxidation is not the exclusive source of malondialdehyde (27). Although almost all oxidative stress biomarkers have been criticized for their reliability (including those used in the present study), it is apparent that all change in a way indicating increased oxidative stress after exercise, indicating that it is possible to describe exercise-induced oxidative stress to some extent. Certainly, the fact that the oxidative stress markers employed increased or decreased as expected does not necessarily validate their use.REFERENCES1. Alessio, H. M., A. E. Hagerman, B. K. Fulkerson, J. Ambrose, R. E. Rice, and R. L. Wiley. Generation of reactive oxygen species after exhaustive aerobic and isometric exercise. Med. Sci. Sports Exerc. 32:1576-1581, 2000. [CrossRef] [Full Text] [Medline Link] [Context Link]2. Anderson, N. L., and N. G. Anderson. The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 11:845-867, 2002. [Context Link]3. Bloomer, R. J., P. G. Davis, L. A. Consitt, and L. Winderman. Plasma protein carbonyl response to increasing exercise duration in aerobically trained men and women. Int. J. Sports Med. 28:21-25, 2007. [CrossRef] [Medline Link] [Context Link]4. Bloomer, R. J., and A. H. Goldfarb. Anaerobic exercise and oxidative stress: a review. Can. J. Appl. Physiol. 29:245-263, 2004. [Context Link]5. Bloomer, R. J., A. H. Goldfarb, and M. J. McKenzie. Oxidative stress response to aerobic exercise: comparison of antioxidant supplements. Med. Sci. Sports Exerc. 38:1098-1105, 2006. [CrossRef] [Full Text] [Medline Link] [Context Link]6. Bloomer, R. J., A. H. Goldfarb, L. Wideman, M. J. McKenzie, and L. A. Consitt. Effects of acute aerobic and anaerobic exercise on blood markers of oxidative stress. J. Strength Cond. Res. 19:276-285, 2005. [CrossRef] [Full Text] [Medline Link] [Context Link]7. Close, G. L., T. Ashton, T. Cable, D. Doran, and D. P. Maclaren. Eccentric exercise, isokinetic muscle torque and delayed onset muscle soreness: the role of reactive oxygen species. Eur. J. Appl. Physiol. 91:615-621, 2004. [CrossRef] [Medline Link] [Context Link]8. Duthie, G. G., J. D. Robertson, R. J. Maughan, and P. C. Morrice. Blood antioxidant status and erythrocyte lipid peroxidation following distance running. Arch. Biochem. Biophys. 282:78-83, 1990. [CrossRef] [Medline Link] [Context Link]9. Fatouros, I. G., A. Destouni, K. Margonis, et al. Cell-free plasma DNA as a novel marker of aseptic inflammation severity related to exercise overtraining. Clin. Chem. 52:1820-1824, 2006. [CrossRef] [Medline Link] [Context Link]10. Finaud, J., G. Lac, and E. Filaire. Oxidative stress: relationship with exercise and training. Sports Med. 36:327-358, 2006. [Context Link]11. Goldfarb, A. H., R. J. Bloomer, and M. J. McKenzie. Combined antioxidant treatment effects on blood oxidative stress after eccentric exercise. Med. Sci. Sports Exerc. 37:234-239, 2005. [CrossRef] [Full Text] [Medline Link] [Context Link]12. Halliwell, B. Free radicals and other reactive species in disease. In: Encyclopedia of Life Science, London: Nature Publishing Group, pp. 1-7, 2001. [Context Link]13. Hulbert, A. J. On the importance of fatty acid composition of membranes for aging. J. Theor. Biol. 234:277-288, 2005. [CrossRef] [Medline Link] [Context Link]14. Ilhan, N., A. Kamanli, R. Ozmerdivenlic, and N. Ilhana. Variable effects of exercise intensity on reduced glutathione, thiobarbituric-acid reactive substance levels, and glucose concentration. Arch. Med. Res. 35:294-300, 2004. [Context Link]15. Inal, M., F. Akyuz, A. Turgut, and W. M. Getsfrid. Effect of aerobic and anaerobic metabolism on free radical generation swimmers. Med. Sci. Sports Exerc. 33:564-567, 2001. [Context Link]16. Miyazaki, H., S. Oh-Ishi, T. Ookawara, et al. Strenuous endurance training in humans reduces oxidative stress following exhausting exercise. Eur. J. Appl. Physiol. 84:1-6, 2001. [Context Link]17. Mylonas, C., and D. Kouretas. Lipid peroxidation and tissue damage. In Vivo 13:295-309, 1999. [Medline Link] [Context Link]18. Nikolaidis, M. G., A. Z. Jamurtas, V. Paschalis, et al. Exercise-induced oxidative stress in G6PD-deficient individuals. Med. Sci. Sports Exerc. 38:1443-1450, 2006. [CrossRef] [Full Text] [Medline Link] [Context Link]19. Nikolaidis, M. G., and V. Mougios. Effects of exercise on the fatty-acid composition of blood and tissue lipids. Sports Med. 34:1051-1076, 2004. [CrossRef] [Full Text] [Medline Link] [Context Link]20. Prior, R. L., and G. Cao. In vivo total antioxidant capacity: comparison of different analytical methods. Free Radic. Biol. Med. 27:1173-1181, 1999. [CrossRef] [Medline Link] [Context Link]21. Radak, Z., H. Ogonovszky, J. Dubecz, et al. Super-marathon race increases serum and urinary nitrotyrosine and carbonyl levels. Eur. J. Clin. Invest. 33:726-730, 2003. [CrossRef] [Full Text] [Medline Link] [Context Link]22. Radak, Z., J. Pucsuk, S. Boros, L. Josfai, and A. W. Taylor. Changes in urine 8- hydroxydeoxyguanosine levels of supermarathon runners during a four-day race period. Life Sci. 66:1763-1767, 2000. [CrossRef] [Medline Link] [Context Link]23. Sacheck, J. M., E. A. Decker, and P. M. Clarkson. The effect of diet on vitamin E intake and oxidative stress in response to acute exercise in female athletes. Eur. J. Appl. Physiol. 83:40-46, 2000. [CrossRef] [Medline Link] [Context Link]24. Schneider, M., A. M. Niess, F. Rozario, et al. Vitamin E supplementation does not increase the vitamin C radical concentration at rest and after exhaustive exercise in healthy male subjects. Eur. J. Nutr. 42:195-200, 2003. [CrossRef] [Medline Link] [Context Link]25. Tauler, P., A. Aguilo, I. Gimeno, et al. Differential response of lymphocytes and neutrophils to high intensity physical activity and to vitamin C diet supplementation. Free Radic. Res. 37:931-938, 2003. [CrossRef] [Medline Link] [Context Link]26. Vider, J., J. Lehtmaa, and T. Kullisaar. Acute immune response in respect to exercise-induced oxidative stress. Pathophysiology 7:263-270, 2001. [Context Link]27. Vollaard, N. B., J. P. Shearman, and C. E. Cooper. Exercise-induced oxidative stress: myths, realities and physiological relevance. Sports Med. 35:1045-1062, 2005. [CrossRef] [Full Text] [Medline Link] [Context Link]28. Waring, W. S., A. Convery, V. Mishra, A. Shenkin, D. J. Webb, and S. R. Maxwell. Uric acid reduces exercise-induced oxidative stress in healthy adults. Clin. Sci. (Lond.) 105:425-430, 2003. [CrossRef] [Medline Link] [Context Link]29. Watson, T. A., R. Callister, R. D. Taylor, D. W. Sibbritt, L. K. Macdonald-Wicks, and M. L. Garg. Antioxidant restriction and oxidative stress in short-duration exhaustive exercise. Med. Sci. Sports Exerc. 37:63-71, 2005. [Context Link]30. Weiss, C., A. Bierhaus, R. Kinscherf, et al. Tissue factor-dependent pathway is not involved in exercise-induced formation of thrombin and fibrin. J. Appl. Physiol. 92:211-218, 2002. [Medline Link] [Context Link] EXERCISE; TIME COURSE; FREE RADICALS; REACTIVE OXYGEN SPECIES; BLOOD|00005768-200707000-00011#xpointer(id(R1-11))|11065213||ovftdb|00005768-200009000-00008SL00005768200032157611065213P58[CrossRef]|00005768-200707000-00011#xpointer(id(R1-11))|11065404||ovftdb|00005768-200009000-00008SL00005768200032157611065404P58[Full Text]|00005768-200707000-00011#xpointer(id(R1-11))|11065405||ovftdb|00005768-200009000-00008SL00005768200032157611065405P58[Medline Link]|00005768-200707000-00011#xpointer(id(R3-11))|11065213||ovftdb|SL000043552007282111065213P60[CrossRef]|00005768-200707000-00011#xpointer(id(R3-11))|11065405||ovftdb|SL000043552007282111065405P60[Medline Link]|00005768-200707000-00011#xpointer(id(R5-11))|11065213||ovftdb|00005768-200606000-00013SL00005768200638109811065213P62[CrossRef]|00005768-200707000-00011#xpointer(id(R5-11))|11065404||ovftdb|00005768-200606000-00013SL00005768200638109811065404P62[Full Text]|00005768-200707000-00011#xpointer(id(R5-11))|11065405||ovftdb|00005768-200606000-00013SL00005768200638109811065405P62[Medline Link]|00005768-200707000-00011#xpointer(id(R6-11))|11065213||ovftdb|00124278-200505000-00007SL0012427820051927611065213P63[CrossRef]|00005768-200707000-00011#xpointer(id(R6-11))|11065404||ovftdb|00124278-200505000-00007SL0012427820051927611065404P63[Full Text]|00005768-200707000-00011#xpointer(id(R6-11))|11065405||ovftdb|00124278-200505000-00007SL0012427820051927611065405P63[Medline Link]|00005768-200707000-00011#xpointer(id(R7-11))|11065213||ovftdb|SL0000364720049161511065213P64[CrossRef]|00005768-200707000-00011#xpointer(id(R7-11))|11065405||ovftdb|SL0000364720049161511065405P64[Medline Link]|00005768-200707000-00011#xpointer(id(R8-11))|11065213||ovftdb|SL0000072819902827811065213P65[CrossRef]|00005768-200707000-00011#xpointer(id(R8-11))|11065405||ovftdb|SL0000072819902827811065405P65[Medline Link]|00005768-200707000-00011#xpointer(id(R9-11))|11065213||ovftdb|SL00003030200652182011065213P66[CrossRef]|00005768-200707000-00011#xpointer(id(R9-11))|11065405||ovftdb|SL00003030200652182011065405P66[Medline Link]|00005768-200707000-00011#xpointer(id(R11-11))|11065213||ovftdb|00005768-200502000-00009SL0000576820053723411065213P68[CrossRef]|00005768-200707000-00011#xpointer(id(R11-11))|11065404||ovftdb|00005768-200502000-00009SL0000576820053723411065404P68[Full Text]|00005768-200707000-00011#xpointer(id(R11-11))|11065405||ovftdb|00005768-200502000-00009SL0000576820053723411065405P68[Medline Link]|00005768-200707000-00011#xpointer(id(R13-11))|11065213||ovftdb|SL00005354200523427711065213P70[CrossRef]|00005768-200707000-00011#xpointer(id(R13-11))|11065405||ovftdb|SL00005354200523427711065405P70[Medline Link]|00005768-200707000-00011#xpointer(id(R17-11))|11065405||ovftdb|SL0000174919991329511065405P74[Medline Link]|00005768-200707000-00011#xpointer(id(R18-11))|11065213||ovftdb|00005768-200608000-00012SL00005768200638144311065213P75[CrossRef]|00005768-200707000-00011#xpointer(id(R18-11))|11065404||ovftdb|00005768-200608000-00012SL00005768200638144311065404P75[Full Text]|00005768-200707000-00011#xpointer(id(R18-11))|11065405||ovftdb|00005768-200608000-00012SL00005768200638144311065405P75[Medline Link]|00005768-200707000-00011#xpointer(id(R19-11))|11065213||ovftdb|00007256-200434150-00004SL00007256200434105111065213P76[CrossRef]|00005768-200707000-00011#xpointer(id(R19-11))|11065404||ovftdb|00007256-200434150-00004SL00007256200434105111065404P76[Full Text]|00005768-200707000-00011#xpointer(id(R19-11))|11065405||ovftdb|00007256-200434150-00004SL00007256200434105111065405P76[Medline Link]|00005768-200707000-00011#xpointer(id(R20-11))|11065213||ovftdb|SL00003949199927117311065213P77[CrossRef]|00005768-200707000-00011#xpointer(id(R20-11))|11065405||ovftdb|SL00003949199927117311065405P77[Medline Link]|00005768-200707000-00011#xpointer(id(R21-11))|11065213||ovftdb|00003653-200308000-00015SL0000365320033372611065213P78[CrossRef]|00005768-200707000-00011#xpointer(id(R21-11))|11065404||ovftdb|00003653-200308000-00015SL0000365320033372611065404P78[Full Text]|00005768-200707000-00011#xpointer(id(R21-11))|11065405||ovftdb|00003653-200308000-00015SL0000365320033372611065405P78[Medline Link]|00005768-200707000-00011#xpointer(id(R22-11))|11065213||ovftdb|SL00005557200066176311065213P79[CrossRef]|00005768-200707000-00011#xpointer(id(R22-11))|11065405||ovftdb|SL00005557200066176311065405P79[Medline Link]|00005768-200707000-00011#xpointer(id(R23-11))|11065213||ovftdb|SL000036472000834011065213P80[CrossRef]|00005768-200707000-00011#xpointer(id(R23-11))|11065405||ovftdb|SL000036472000834011065405P80[Medline Link]|00005768-200707000-00011#xpointer(id(R24-11))|11065213||ovftdb|SL0012620120034219511065213P81[CrossRef]|00005768-200707000-00011#xpointer(id(R24-11))|11065405||ovftdb|SL0012620120034219511065405P81[Medline Link]|00005768-200707000-00011#xpointer(id(R25-11))|11065213||ovftdb|SL0001951920033793111065213P82[CrossRef]|00005768-200707000-00011#xpointer(id(R25-11))|11065405||ovftdb|SL0001951920033793111065405P82[Medline Link]|00005768-200707000-00011#xpointer(id(R27-11))|11065213||ovftdb|00007256-200535120-00004SL00007256200535104511065213P84[CrossRef]|00005768-200707000-00011#xpointer(id(R27-11))|11065404||ovftdb|00007256-200535120-00004SL00007256200535104511065404P84[Full Text]|00005768-200707000-00011#xpointer(id(R27-11))|11065405||ovftdb|00007256-200535120-00004SL00007256200535104511065405P84[Medline Link]|00005768-200707000-00011#xpointer(id(R28-11))|11065213||ovftdb|SL00003111200310542511065213P85[CrossRef]|00005768-200707000-00011#xpointer(id(R28-11))|11065405||ovftdb|SL00003111200310542511065405P85[Medline Link]|00005768-200707000-00011#xpointer(id(R30-11))|11065405||ovftdb|SL0000456020029221111065405P87[Medline Link]11744662Sampling Time is Crucial for Measurement of Aerobic Exercise-Induced Oxidative StressMICHAILIDIS, YIANNIS; JAMURTAS, ATHANASIOS Z.; NIKOLAIDIS, MICHALIS G.; FATOUROS, IOANNIS G.; KOUTEDAKIS, YIANNIS; PAPASSOTIRIOU, IOANNIS; KOURETAS, DIMITRISBASIC SCIENCES: Original Investigations739InternalMedicine & Science in Sports & Exercise10.1249/MSS.0b013e31817d1cce200840101772-1780OCT 2008Gender-Related Differences in Muscle Injury, Oxidative Stress, and ApoptosisKERKSICK, C; TAYLOR, L; HARVEY, A; WILLOUGHBY, D,.9.aspx298,.00009.pdf & Science in Sports & Exercise10.1249/MSS.0b013e3181824dab200840122119-2128DEC 2008No Indications of Persistent Oxidative Stress in Response to an Ironman TriathlonNEUBAUER, O; KÖNIG, D; KERN, N; NICS, L; WAGNER, K & Science in Sports & Exercise10.1249/MSS.0b013e3181ac7a452010421142-151JAN 2010Ergogenic and Antioxidant Effects of Spirulina Supplementation in HumansKALAFATI, M; JAMURTAS, AZ; NIKOLAIDIS, MG; PASCHALIS, V; THEODOROU, AA; SAKELLARIOU, GK; KOUTEDAKIS, Y; KOURETAS, D & Science in Sports & Exercise10.1249/MSS.0b013e31818338b72009411155-163JAN 2009Protein Modification Responds to Exercise Intensity and Antioxidant SupplementationLAMPRECHT, M; OETTL, K; SCHWABERGER, G; HOFMANN, P; GREILBERGER, JF