Many hypotheses have been put forward to explain the relationship between physical activity and the incidence of infections among people who are physically active (14,16,21). Previous reports suggest that strenuous exercise triggers inflammatory responses. The observed inflammatory responses occurring after exercise are in many respects similar, although often lesser in magnitude than those seen in septic patients and patients with the systemic inflammatory response syndrome (SIRS) with activated complement and coagulation cascades, as well as increased phagocyte release of inflammatory mediators (2).
Free radicals are highly reactive atoms or molecules with one or more unpaired electrons. The reactivity of molecular oxygen can be increased by reduction or excitations giving rise to highly reactive oxygen species (ROS), for example, the superoxide anion (·O2−), hydrogen peroxide (H2O2), the hydroxyl radical (·OH), and singlet oxygen (·O2). The principal cellular source of ROS is the neutrophils in which ROS is produced by the NADPH oxidase-catalyzed oxidative burst reaction (3,12,15). Neutrophil-generated ROS are produced primarily in order to destroy invading micro-organisms (3,15). This effect is normally advantageous, but inadvertent extracellular release of ROS may induce undue inflammatory reactions in surrounding tissues (15).
Many disease states are linked to ROS damage as a result of an imbalance between radical-generating and radical-scavenging systems—a condition termed oxidative stress (19). It has also been suggested that physical exercise stimulates the neutrophils to increased synthesis of ROS (12,15), but this exercise-induced ROS production varies profoundly with sex, diet, type, and duration of activity, aerobic and anaerobic energy production, and the number of muscle groups involved (12,18). In general, it is assumed that the body has adequate antioxidant reserves to cope with the production of ROS under physical conditions, but these relationships are not well documented (9,10,11,12).
The protective mechanisms against oxidative stress can be divided into two categories: the endogenously produced antioxidants, for example, superoxide dismutase, glutathione peroxidase, catalase, glutaredoxin and thioredoxin, and the nonenzymatic antioxidants including nutritionally derived vitamins and provitamins (vitamin E and C and β-carotene), polyphenols and flavonoids, peptides or proteins containing thiol groups, and various other low molecular weight compounds (20). These substances can either prevent ROS formation or scavenge radical species and convert them into less active molecules. The enzymatic antioxidants are synthesized in the body, and there are several lines of evidence that their production can be upregulated in response to chronic oxidant exposure (10,11). There have also been reports on upregulation of antioxidant enzyme activity after chronic physical exercise (9,10). This may explain the findings of less oxidative stress in trained individuals (12). These phenomena are, however, not well documented, and there are several unanswered questions.
Flow cytometry (FCM) has the unique ability within seconds to supply quantitative measurements of individual cells in large numbers. The cellular content of constituents that have been labeled using specific fluorescence probes can be determined. In this study, we used the ROS-sensitive fluorescence probe dihydroethidium for instant determination of intracellular ROS levels in leukocytes harboring their natural habitat (whole blood). This constitutes an improvement compared with other methods used for detecting ROS, where time-consuming and potentially cell-activating isolation procedures followed by a period of in vitro cell culture are necessary before ROS production can be studied. With knowing that ROS responses are “bursty” in nature, maximal efforts should be executed to minimize the flaws of excessive in vitro handling.
The objective of this study was to examine the effect of marathon and half-marathon running on leukocyte ROS production measured with flow cytometry. We wanted to study both how the basal ROS levels as well as the in vitro stimulated ROS production capacity and the total antioxidant status were affected by a marathon and a half-marathon race, and if any gender differences could be noted.
MATERIALS AND METHODS
Study Design/Blood Sampling
Flow cytometry was used to study basal intracellular leukocyte ROS levels as well as ROS levels after in vitro stimulation with phorbol myristate acetate (PMA). Results obtained in whole-blood samples collected after marathon/half-marathon races were compared with results in prerace samples from the same individuals. Unpublished data from our laboratory indicate that there are no diurnal fluctuations in leukocyte ROS responses during a 24-h period, so we did not include a control group in this study. Blood samples were collected within 1 h before the start of the races, before the participants had started to warm up, and immediately (within 10 min) after the races. Venous blood was sampled into anticoagulated (EDTA) vacuum tubes (Becton Dickinson, Plymouth, UK). The blood samples were kept on ice and as soon as possible transported to the laboratory for further preparation within a 1 h.
Fourteen men participating in Oslo marathon race (year 2000) and eight women and eight men participating in Oslo half-marathon race (yr 2001) were recruited to the study. They were selected based on last year’s result list with the criterion that their running time was 3 h 30 min at the marathon race, and 1 h 30 min for men and 1 h 45 min for women at the half-marathon race. All subjects were informed about the study and gave their written consent for participation. The Regional Committee for Medical Ethics had approved the test protocols. Background characteristics of the athletes are presented in Table 1.
Platelet and white blood cell counts, hemoglobin, and hematocrit were assessed in EDTA blood using the Technicon H2·TM System (New York, NY) at the Department of Clinical Chemistry, Ullevaal University Hospital.
Preparation of Blood Leukocytes for Flow Cytometry
Basal ROS levels.
Aliquots of 50-μL whole blood were incubated for 15 min at 37°C in a 5% CO2/humidified air atmosphere in 5-mL polystyrene round-bottom tubes (2052 Falcon, Oxnard, CA) with dihydroethidium (DHE) (Sigma, St. Louis, MO) (final concentration 5 μmol·L−1), recognizing mainly the oxygen species superoxide anion (O2−) (24) or phosphate-buffered saline (PBS) (Sigma) (10 mmol·L−1 phosphate buffer, 2.7 mmol·L−1 KCl, 137 mmol·L−1NaCl, pH 7.4) as autofluorescence control. After ended incubation, 1.5-mL red cell lysing solution containing 156 mmol·L−1 NH4Cl (Merck, Darmstadt, Germany), 10 NH4Cl mmol·L−1 NaHCO3 (Merck), and 0.12 mmol·L−1 NaEDTA (Sigma) was added to the tubes. The tubes were incubated in the dark at room temperature for 15 min and centrifuged at 300 g for 5 min at 4°C. Subsequently, the supernatant was discarded and the leukocyte pellet was gently resuspended and washed once with 2-mL cold PBS. The cells were finally resuspended in 0.5-mL 1% w/v paraformaldehyde (PFA) (Merck) in PBS, pH 7.4. The samples were stored in the dark at 0–4°C until flow cytometry could be performed.
In vitro stimulated ROS production.
Aliquots of 1.5-mL whole blood were incubated in polystyrene tubes with ventilation caps with 15-μL phorbol myristate acetate (PMA) (final concentration 100 ng·mL−1) (Sigma) or PBS (control) at 37°C for 60 min in a CO2 incubator. Aliquots of 50-μL blood were then DHE labeled as described for basal ROS levels. In the half-marathon protocol, aliquots of 50-μL whole blood were simultaneously incubated with PMA and DHE for 60 min before red cell lysing and washing as described.
Flow Cytometry Analysis
The labeled samples were analyzed within 24 h in a FACSortTM flow cytometer (Becton Dickinson, San Jose, CA). The flow cytometer was equipped with an argon laser and CellQuestTM software (Becton Dickinson). Ten thousand events were collected from each sample. The leukocyte subpopulations, monocytes and granulocytes, were identified by their light scatter characteristics, enclosed in electronic gates, and separately analyzed for fluorescence intensity from the fluorescent probe. The results were calculated as the difference mean fluorescence intensity (dMFI), which is the mean fluorescence intensity (MFI) (arbitrary units) obtained in the specific fluorescent probe sample subtracted the MFI value obtained when the sample was incubated with PBS. It should be noted that the dMFI values after in vitro PMA stimulation were about 100 times higher than the dMFI values at basal level. The intra-assay coefficient of variation (CV) was < 5% in unstimulated and < 10% in PMA-stimulated samples.
Total Antioxidant Status
The Randox Total Antioxidant Status kit (Crumlin, UK) was used according to the manufacturer’s instructions to measure the total antioxidant status of the athletes before and after the races. Blood samples were collected in heparin tubes, centrifuged at 2300 g, 4°C for 12 min and plasma stored at −70°C until analysis. The inter- and intra-assay coefficients of variations (CV) were 2.4 and 1.2%, respectively.
The dMFI values before the races are defined to be 100%, and the values after the races are reported relative to this. Before choosing the statistical method, the SPSS (version 11.0) skewness plot was calculated for all data sets, in order to check whether or not there was a reason to believe that the observed data came from a normally distributed population. On this basis, it was decided to use parametric methods. Difference within groups (marathon and half-marathon) (from pre- to posttest) was analyzed using two-tailed paired-sample t-test. An independent sample t-test was used to compare men and women within the half-marathon group and to compare the results in the marathon and half-marathon group. The results are given as mean value and standard error of the mean (SEM). P values less than 0.05 were considered statistically significant.
The average running time in the marathon race was 3 h 40 min (range: 3 h 2 min to 4h 20 min) and the half-marathon race was finished in 1 h 53 min (range: 1 h 47 min to 2 h 15 min) and 1 h 41 min (range: 1 h 30 min to 1 h 59 min) for women and men, respectively.
The total number of leukocytes was within the normal range (3.0–11.0 × 109·L−1) before both races (Table 2). After the marathon race, it had increased 3.2-fold compared with the prerace situation, and a 2.4-fold increase was seen as a result of the half-marathon race. The main increases in white blood cells were due to the increase in neutrophils (Table 2). Platelets increased 1.2-fold after both races, whereas hematocrit and hemoglobin did not change.
Basal leukocyte ROS levels.
In men, the basal levels of intracellular ROS in granulocytes and monocytes (as measured by the DHE probe) did not change as a result of neither the marathon nor the half-marathon race (3–25% reduction, NS) (Fig. 1). In women, on the other hand, the basal level of ROS in granulocytes was reduced to 33% (P < 0.01) of the prerace level after the half-marathon race (Fig. 1).
In vitro stimulated leukocyte ROS production.
After the marathon race, the capacity of both granulocytes and monocytes to respond with ROS production when whole blood was stimulated with PMA was markedly reduced when compared with the prerace situation, that is 6% (P < 0.001) and 23% (P < 0.01) residual capacity in granulocytes and monocytes, respectively (Fig. 2). In women, the monocyte ROS production was reduced by 30% (P < 0.05) as a result of the half-marathon race, whereas a 22% (P < 0.05) reduction was observed in men (Fig. 2). The granulocyte ROS response to PMA was not significantly changed (19% (women) and 15% (men) reduction, NS) as a consequence of the half-marathon run. No gender differences were observed in leukocyte ROS levels, either basally or after in vitro PMA stimulation as a consequence of the half-marathon race.
Total antioxidant status.
The total antioxidant status during the marathon and half-marathon races increased in all three athlete groups. A 19% (P < 0.05) increase was observed after the marathon race and 11% (P < 0.05) increase was observed in both male and female half-marathon runners (Fig. 3).
In the present study, we have investigated the changes in selected hematologic parameters, the leukocyte expression of ROS, and the total blood plasma antioxidant capacity in female and male athletes after a marathon and a half-marathon race.
We observed a 3.2-fold increase in circulating leukocytes during the marathon race and a 2.4-fold increase after the half-marathon race. The increase in total circulating leukocytes was mainly due to the increase in granulocytes, in particular neutrophils, immediately after the half-marathon and marathon races, respectively (7,8,13). Platelet counts increased 1.2-fold after both races, whereas hematocrit, hemoglobin, and red blood cell counts showed no significant changes after the races.
There is a broad evidence that physical exercise affects the generation of ROS in leukocytes (3,15) and, further, for the influence of free radicals on the occurrence of exercise-induced muscle damage (12,23) explaining phenomena like decreased physical performance, muscular fatigue, and overtraining. Detrimental influences of free radicals are due to their oxidizing effects on lipids, proteins, nucleic acids, and the extracellular matrix. However, the available data to support the role of ROS in relation to physical exercise are highly inconsistent and partly controversial. These controversies are probably for the major part due to the different methodologies being used for the assessment of ROS, generally including time-demanding and laborious cell isolation procedures and subsequent cell culturing that most certainly affects the ROS status of these cells in an uncontrolled and unpredictable manner. The type of physical activity studied also varied considerably and probably influenced the results presented. In the present study, we therefore, for the first time in relation to physical exercise, used flow cytometry and a ROS-sensitive probe (DHE) to monitor the intracellular levels of ROS in leukocytes. The advantage of this technique is that leukocytes can be studied in their natural habitat (whole blood) and further that it provides a “snap-shot” of the intracellular ROS levels in leukocytes immediately after bleeding thus avoiding undue ex vivo blood cell handling. This minimizes the confounding effect of ex vivo and in vitro leukocyte activation. The present technique thus more exactly reflects the in vivo situation because the cells are fixed and the ROS levels are “frozen” at the moment of exsanguination. The additional approach performed by us is the in vitro stimulation by PMA (still in a whole blood milieu) to examine the capacity of neutrophils/monocytes to respond to a defined and strong stimulus. This tells us how large is the cells residual capacity for ROS synthesis, which in turn illustrates to which degree the cells have been exhausted with regard to ROS production as a result of the exercise. The combination of determining as exact as possible the true intracellular ROS levels with the acquired knowledge of the cells capacity to produce ROS upon maximal stimulation with PMA gives a total understanding of the ROS-generating mechanisms, which out ranges the methods applied in earlier studies. It is also noteworthy that our technique exclusively measures the intracellular levels of ROS. This is essential in relation to the microbicidal properties of leukocytes, much more so than measuring the overflow of ROS to the extracellular milieu, which most other applied methods relies on.
By and large, we demonstrated equal or reduced basal leukocyte ROS levels when comparing the postrace with the prerace (marathon and half-marathon) situation. At least five different explanations can be forwarded to explain this finding. First, there is reason to believe that exercise-induced activated leukocytes to a large extent (and rapidly) become sequestered in the microcirculation and will thus not be available for analysis in circulating blood. As evidence of this, both the marathon and the half-marathon races lead to changes in the leukocyte expression of both CD11b and CD62L (unpublished observations). This changed pattern of adhesion molecule expression, which is prototypical for leukocyte activation favors leukocyte/endothelium interaction and withdrawal of activated leukocytes from the circulation. Second, intracellularly produced ROS may be rapidly equilibrated with and released to the extracellular (plasmatic) milieu. The occurrence of thiobarbituric acid-reactive substances (TBARS) in runner’s serum postexercise reported in previous studies (1,6,22) most probably is the result of lipid peroxidation due to ROS released from activated leukocytes (and endothelium). There are also reports of increased oxidative stress in skeletal muscles after exercise (12,15,17). Third, recruitment of new (and nonactivated) leukocytes to the circulation is evidenced by the vast increase in circulating neutrophils. Fourth, adaptive changes may have occurred in the organism’s antioxidative system, for example, induction of endogenous enzymatic antioxidants like superoxide dismutase and mobilization of nonenzymatic antioxidants like some of the nutritionally derived vitamins. In fact, we demonstrated a significant increase in total antioxidant status as a consequence of strenuous exercise, more so in the marathon than in the half-marathon runners, indicating that the body’s antioxidant system has been activated as a response to the exercise-induced burst of ROS production. The rise in total antioxidant status did obviously not prevent the exercise-induced lipid peroxidation that has been demonstrated by earlier authors (1,5). This might indicate inadequacies of the antioxidant defense to fully counteract the increased oxidative burden generated as a consequence of half-marathon running and even more so during a full marathon run. The advantageous role of antioxidants in preventing exercise-induced systemic or muscular oxidative stress has been suggested (4,11), but this relation certainly needs further investigations. In our investigation, we did not notice any significant differences in leukocyte ROS levels or ROS production capacity between those individuals regularly taking dietary antioxidant supplements and those who did not. Nor was there any significant difference in total antioxidant status between those two groups. Our study was, however, too small and not designed to adequately address this particular relationship. A fifth, but less likely, explanation would be that the intensity of activity during the races was not high enough to trigger ROS-generating mechanisms.
After the marathon race, the capacity of leukocytes (both monocytes and granulocytes) to respond with ROS synthesis to a standardized in vitro PMA stimulus was heavily suppressed. The half-marathon race also lead to a moderate decline in the residual capacity for ROS production in monocytes, whereas the granulocytes retained their full PMA responsiveness. These findings indicate that the cellular machinery for ROS generation was partly exhausted, much more so as a result of a full marathon than the shorter-lasting half-marathon race. No obvious gender differences were noted except for the slightly reduced basal ROS levels seen in women’s granulocytes after the half-marathon run. The nearly complete exhaustion of the ROS-generating systems demonstrated after the marathon run might explain the observation that the defense against bacteria and other micro-organisms is temporarily impaired as a result of heavy physical stress. In view of the role of ROS in microbial killing, the present data strengthen the hypothesis forwarded by Pedersen and Ullum (16) that athletes are more sensitive to infectious diseases in the immediate period after intensive physical activity. The fact we have stated by our investigation is that the microbicidal capacity of leukocytes is substantially reduced in the immediate postexercise period. If the individual is challenged by a pathogenic agent during that interval, a potential pathological outcome is to be expected. How long this reduced capacity resides before the cells regain full ROS-generating capacity is a matter of future research. The present findings also add to the validity of the proposed J-curve, that illustrates how the resistance against upper respiratory infection is affected by the level of physical training (14). Further, the ROS exhaustion of leukocytes seen after endurance activity strongly indicates a preceding burst of ROS release, which is partly responsible for the several negative effects on muscle function reported as a result of strenuous exercise (12,15).
In conclusion, we have shown that endurance activity with moderate intensity and long duration leads to a burst of ROS synthesis and subsequent exhaustion of leukocyte ROS-generating mechanisms. This may partly explain the observation that athletes are more sensitive to infectious diseases in the immediate period after intensive physical activity. However, we still do not know how long this critical period lasts and thus when a new bout of exercise is justifiable. Moreover, the effect of different types and intensities of physical activity, the physical training level of the subjects, and the importance of age and gender remains to be investigated.
The authors would like to thank Trude Aspelin, Hilde Eid, Anne Marie Ransve, and Lisbeth Saetre for technical and practical assistance.
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