Besides the well-known beneficial effects of carbohydrate (CHO) supplementation to maintain power output during prolonged endurance exercise (1,3,6,9), possible beneficial effects have also been described for the immune system. For example, positive effects of CHO supplementation on the response of interleukin 6 (IL-6), cortisol, granulocyte and monocyte trafficking, oxidative burst and phagocytosis, or natural killer (NK) cell immunity have been described (8,10,12,14,17-19,22-25,29). Most of these studies, however, were conducted as cross-sectional studies in competitive events. Recently, Lancaster et al. (12) examined the effects of 6.4 and 12.8% CHO ingestion during 2.5 h of cycling, "without reaching exhaustion" on type 1 and type 2 T- lymphocytes in a small group of seven moderately to well endurance-trained athletes (maximal oxygen uptake ∼59 mL·min−1·kg−1) under laboratory conditions, and reported an attenuated decrease in the circulating number and percentage of CD4+ IFN-γ+ and CD8+IFN-γ+ T-lymphocytes by both CHO concentrations. Nevertheless, to our knowledge, effects of CHO supplementation on the immune system during longer periods of cycling have not been examined under standardized conditions, although cycling competition as well as training sessions of well-trained cyclists usually last longer than 2.5 h. In addition, standardized examinations with the cyclists' own bicycle in the field, which simulate a more realistic situation than laboratory conditions, have not been reported. Furthermore, moderate "short-term" endurance exercise of approximately 2 h elicits lower changes in cell concentrations and hormonal responses than strenuous or exhaustive long-term endurance exercise of >2-3 h (7).
The present study, therefore, aimed to examine the immune reactions to long-term steady-state endurance training under standardized, but realistic conditions, with varying amounts of CHO supplementation in well-trained cyclists. According to the dose-dependent shift from fat to glucose metabolism under 6 and 12% CHO supplementation during constant-load endurance rides of 4-h duration (15), we hypothesized that 6 and 12% CHO beverage ingestion leads to a dose-dependent attenuation of the exercise-induced immune reaction during 4 h of cycling as well.
A total of 14 male competitive cyclists and triathletes (age 25 ± 5 yr; height 180 ± 7 cm; weight 72 ± 9 kg; body fat 10.8 ± 2.5%; heart volume 13.8 ± 1.7 mL·kg−1; V̇O2peak 67 ± 6 mL−1·min−1·kg−1) who had an experience of 6 ± 4 yr in endurance exercise competitions, trained 6 ± 3 times per week, and completed 13,500 km (range: 8,000-20,000 km) of cycling during the last season, gave their written informed consent for the study, which was approved by the institutional review board. Athletes individual anaerobic threshold (IAT) had to exceed 3.0 W·kg−1. To exclude acute or chronic inflammatory diseases as well as cardiovascular abnormalities, each participant received a physical examination, determinations of routine blood parameters, an electrocardiogram at rest and during cycle ergometry, and a resting echocardiography.
After an initial incremental stage test on a cycle ergometer (Lode Excalibur Supersport, Groningen, Netherlands) to determine the IAT by the method of Stegmann et al. (31), cyclists had to perform three 4-h constant-load trials at an intensity of 70% IAT on their own bicycles. The prescribed workload was given in watts (W) and monitored by an ambulatory powermeter (SRM Training System, Jülich, Germany). In randomized order, cyclists received either 6 or 12% carbohydrate beverages or placebo during the trials. There was a minimum of 2 d free from strenuous exercise between all trials. All tests were performed within a maximum of 4 wk, and the subjects were not permitted to change their training routine during this period.
To determine the IAT, an incremental multistage cycle ergometry was started at a workload of 100 W and increased by 50 W every 3 min until exhaustion (31). Capillary blood samples were taken from the hyperemized earlobe at the end of each stage and 1, 3, 5, and 10 min after cessation of exercise to determine lactate concentrations (Greiner Super GL, Flacht, Germany). In addition, peak oxygen uptake (V̇O2peak) was measured by direct mixing chamber spirometry (Cortex MetaMax II, Leipzig, Germany).
Constant-load trials lasting 4 h were performed outdoors on a 400-m track at an intensity of 70% IAT. Subjects used their own bicycles, which were equipped with an SRM powermeter (Schoberer, Jülich, Germany) to monitor the workload. Capillary blood samples from the hyperemized earlobe were taken at rest, every hour during exercise (athletes had to stop for about 30 s) and 1 h after the trials to determine lactate concentrations. To determine immunological parameters, venous blood samples were taken from an antecubital vein in the supine position at rest, at the end of exercise, 1 h after exercise cessation, and on the following day between 8 and 9 a.m. The trials started at 9a.m. and took place between April and October. Weather conditions were comparable for all participants on an intraindividual basis (i.e., no between-trial differences in mean temperature and humidity and, thus, no systematic influences of the climate).
Participants were instructed to have the same breakfast at approximately 7 a.m. on all trial days. When asked if they adhered to these constructions, subjects reported perfect compliance. This was verified by qualitative analysis of nutritional protocols from the respective days.
Throughout each test, cyclists received 50 mL·kg−1 body weight fluid (10 mL·kg−1 within 60 min before the start, 5 mL·kg−1 every 30 min during the test). For CHO supplementation, 60 and 120 g, respectively, of maltodextrin (Agenamalt, Agrana, Vienna, Austria) were added to 1 L of mineral water (Nürburgquelle, Dreis, Germany). For placebo, only mineral water was used. To obscure the taste, indigestible sweetener and citric acid were added to the drinks.
Blood samples were collected from an antecubital vein into tubes containing K3 EDTA (2.7 mL) and lithium heparin (7.5 mL) while subjects were in a supine position. Samples were obtained at rest (before), immediately after cessation of exercise (end), and 1 and 19 h after exercise (1 h post and 19 h after, respectively) to analyze hematological parameters using a Sysmex K-1000 (Sysmex GmbH, Langenfeld, Germany). Leukocyte and lymphocyte subpopulations were determined by flow cytometry (FACSScan, Becton Dickinson, Heidelberg, Germany). NK cells were defined as CD3−CD16+CD56+ lymphocytes; CD94 and CD158a receptors were determined on CD3−CD16+ lymphocytes. All cell counts were adjusted for changes in plasma volume by the formula of Dill and Costill (4).
C-reactive protein, interleukin-6, cortisol, and metabolic parameters.
C-reactive protein was determined turbidimetrically (Biomed, Oberschleiβheim, Germany), IL-6 by an enzyme-linked immunoassay (R&D-Systems, Minneapolis, MN). Cortisol was determined by chemoluminescence (Magic Lyte Analyzer, CIBA-Corning Diagnostics, Fernwald, Germany). Analyzing methods (and results) of lactate, glucose, glycerol, and triglycerides of this study have been described previously by Meyer et al. (15).
All data are presented as means ± standard deviations (SD). Differences between the constant-load trials were tested by a two-factor ANOVA (concentration × time), the Scheffé test was used for post hoc testing. Pearson's coefficient of correlation was used to test correlations between selected variables. An α-error < 0.05 was considered as significant.
Ergometric data and metabolic parameters.
All 14 cyclists sufficiently maintained the constant-load trial of 4-h duration without significant differences in measured power output between the trials (mean workload at the IAT: 189 ± 21 W). The power output averaged over 4 h ranged between 187 and 189 W in the trials, the averaged oxygen uptake ranged between 61 and 63% V̇O2peak, and the averaged heart rate ranged between 137 and 139 bpm. The averaged blood glucose concentrations were significantly lower in the placebo trial (range: 4.2-5.2 mmol·L−1) than in the CHO trials (range: 4.8-7.1 mmol·L−1; P < 0.05). Further details on power output, oxygen uptake, heart rate, blood glucose, lactate concentration, concentrations of free fatty acids, and glycerol are described by Meyer et al. (15).
Leukocyte and lymphocyte subpopulations.
Exercise-induced changes of leukocytes and subpopulations are shown in Figure 1. Leukocytes and neutrophils significantly increased in all trials (Fig. 1), but concentrations were significantly higher in the placebo trial than in both CHO trials immediately and 1 h after exercise (Fig. 1). No significant differences in leukocyte and neutrophil counts could be demonstrated between the 6 and the 12% CHO trial. Monocyte concentrations significantly increased in the placebo trial only (P < 0.001), and differed significantly immediately after exercise from the concentrations in the 6 and the 12% CHO trial (Fig. 1). Concentrations of CD16+ monocytes showed no significant exercise-induced changes in none of the trials (Fig. 1).
Cell counts of total lymphocytes, CD3+CD4+ lymphocytes, and CD3+CD8+ lymphocytes demonstrated an effect of time (P < 0.001), but remained without significant differences between the trials (Fig. 1). NK cell counts were significantly elevated immediately after exercise in the placebo trial only (P < 0.001), with a significant difference to the concentration in the 12% CHO trial (P < 0.05; Fig. 1). One hour after exercise, NK cell counts had dropped significantly to similar concentrations in all trials (Fig. 1). No differences between the trials were found for absolute numbers and mean fluorescence intensities in CD3−CD16+CD158a+ lymphocytes and CD3−CD16+CD94+ lymphocytes.
C-reactive protein, interleukin-6, and cortisol.
Although mean values for CRP tended to increase in the placebo and the 6% CHO but not in the 12% CHO trial, changes were insignificant (Fig. 2A). Only a moderate significant difference between the placebo and the 12% CHO trials 1 d after exercise could be demonstrated (Fig. 2A). A significant increase in IL-6 was noted immediately after exercise in all trials (P < 0.001), which remained unchanged 1 h after cessation of exercise (Fig. 2B). In addition, the increases were significantly higher in the placebo trial than in the 6 and 12% CHO trials immediately (P < 0.01 and P < 0.001, respectively) and 1 h after exercise (P < 0.05 and P < 0.001, respectively). IL-6 concentrations did not differ between the 6 and the 12% CHO trial (Fig. 2B).
The cortisol concentration significantly increased in the placebo trial only (P < 0.001), and thereby differed significantly from the concentrations of both CHO trials immediately and 1 h after exercise (P < 0.05). Cortisol concentrations in the 6and the 12% CHO trial did not differ significantly (Fig. 2C). The cortisol concentration was negatively correlated to the blood glucose concentration (r = −0.49; P < 0.05). The cortisol concentration and the neutrocyte count were significantly correlated immediately after exercise (r = 0.72; P < 0.001; Fig. 2D). In addition, the concentrations of cortisol and IL-6 correlated significantly immediately after exercise (r = 0.47; P < 0.01).
In the present study, CHO supplementation attenuated the mobilization of leukocytes, neutrophils, and monocytes. A dose-dependent difference in the immune response between 6 and 12% CHO beverage ingestion could not be demonstrated, however. The results of this study, therefore, support the findings of Lancaster et al. (12), who examined the effects of 6.4 and 12.8% CHO ingestion during 2.5 h of cycling without reaching exhaustion, which now can be extended to longer and exhaustive exercise trials.
Leukocytes, neutrophils, monocytes, and hormones.
According to previous reports, an exercise-induced leukocytosis was demonstrated in all trials, which was about 2.5-fold in the placebo trial and about 1.5-fold in the CHO trials. In all trials, the exercise-induced leukocytosis was dominated by the increase in neutrophils (>3 times in the placebo trial, about 1.5-fold in the CHO trials). Although less in absolute cell numbers, the same behavior was found in regular monocytes, but not in CD16+ monocytes.
The different mobilization of phagocytizing cells between the placebo trial and the CHO trials can be attributed to their cortisol-induced recruitment from the marginal pool and the bone marrow, mirrored by the close relation between plasma cortisol and neutrophils after exercise (Fig. 2D). As the blood glucose concentration was significantly higher in both CHO trials than in the placebo trial (15), CHO supplementations were able to reduce the metabolic stress sufficiently, resulting in an attenuated cortisol excretion and a minor exercise-induced affection of the immune system. In addition to the attenuated mobilization of neutrophils and monocytes, the neutrophil function could be stabilized similarly by 6 and 12% CHO beverage ingestion in this study as reported recently (27). It can be postulated, therefore, that beside the metabolic stress, the immunological stress on neutrophils can be reduced by 6-12% CHO supplementation to the same extent during prolonged exercise of >2-3 h. On the other hand, CHO supplementation in shorter and more intensive exercise bouts (e.g., 1 h of high-intensity cycling (11), cycling for 2 h at 75% V̇O2max (2), or running for 3 h at 70% V̇O2max (13)) did not influence neutrophils' mobilization and function (11), total elastase content (2), or reduce the oxidative stress (13), probably because of higher concentrations of catecholamines during more intensive exercise bouts (7). In contrast to the neutrophils' oxidative burst, neutrophil and monocyte phagocytosis were not affected by 4 h of cycling at 70% IAT even without CHO supplementation in a pilot study (28) and, therefore, have not been examined in the present study.
The above-mentioned time- and intensity-dependent effect of exercise on immune cells also seems to be true for the release of IL-6. Whereas CHO ingestion before 1 h of cycling did not influence the rise in plasma IL-6 concentrations (11), CHO beverage ingestion attenuated the increase in IL-6 in the present study (about tenfold in the placebo trial and about sixfold in the CHO trials; Fig. 2B) as well as after 2.5 h of cycling at 75% V̇O2max when compared with placebo (25). The differences in IL-6 concentrations presumably result from different blood glucose concentrations because IL-6 is produced by the contracting muscle during exercise to regulate substrate delivery and to maintain the metabolic homeostasis for glycogen-depleted muscles (5,26). In addition, beside antiinflammatory properties and the induction of a CRP release from hepatocytes (26,30), IL-6 induces an increase in plasma cortisol (5,21,30), which is represented by the relation of both hormones in this study. Reports on modest increases in the plasma IL-6 concentration after 2 h of intensive resistance training, which were independent from CHO ingestion (20), further support the property of IL-6 as a metabolic hormone during prolonged endurance exercise.
Lymphocytes and natural killer cells.
In contrast to phagocytes, no different effects between placebo and CHO supplementation were found for exercise-induced alterations in total lymphocyte counts, CD4+ or CD8+ T-lymphocytes. Only a small difference was observed between the placebo and the 12% CHO trial in the increase in NK cells immediately after exercise (Fig. 1). Nevertheless, the postexercise decrease in the NK cell concentrations below preexercise values was similar in all three trials and, therefore, the open window of NK cells may not be reduced by CHO beverage ingestion.
Furthermore, CHO supplementation did not influence the expression of CD158a and CD94 receptors on NK cells, which either activate or inhibit NK cell function (16) and, consequently, it seems that CHO supplementation had no effect on NK cell activity in the present study (direct measures of NK cell activity were not performed in the present study, because NK cell activity measured with NKTEST remained unaffected by 4 h of cycling at 70% IAT in a pilot study without CHO supplementation (28)).
The present results are in accordance with previous reports of other investigators who examined the effects of CHO supplementation on lymphocytes and NK cells. Beside reductions in the concentrations of plasma cortisol, neutrophils, and monocytes, no effects of CHO supplementation were observed on the postexercise reduction in T-lymphocytes, NK cell numbers, or PHA-induced lymphocyte proliferation after a marathon (10). In another study, CHO supplementation did not influence NK cell activity after 2.5 h of intensive running, although a difference in the blood concentration of NK cells could be demonstrated (23). Comparable to the present results, a higher increase of NK cells immediately after the run in the placebo group than in the CHO group was described (23). If a greater decrease had occurred of NK cells in the placebo group 3 h after exercise, as reported by Nieman et al. (23) remains speculative because only concentrations 1 h after exercise were measured in the present study.
Together, it seems that exercise-induced alterations in lymphocytes and NK cell immunity are less influenced by CHO supplementation than neutrophils and monocytes, and that the open window of NK cells may be more a matter of numerical redistribution than of single NK cell activity (28), which presumably is predominantly regulated by other hormones than phagocytes' mobilization and function. Nevertheless, the NK cell responsiveness to IL-2 may be enhanced by CHO consumption after 1 h of cycling at 75-80% V̇O2peak, although immune system variables of lymphocyte subsets were not different between placebo and carbohydrate intake (14). Furthermore, CHO ingestion during exercise can positively influence the regulation of type 1 lymphocyte cytokine production by the attenuated exercise-induced stress hormone response (12) as well as the cortisol-independent cellular expansion and cell death rates of CD4+ and CD8+ T-lymphocytes after 2 h of cycling at 75% V̇O2max (8).
In conclusion, the acute immune response during prolonged exercise can be influenced by CHO supplementation, leading to attenuated affectations, especially in phagocytizing cells as neutrophils and monocytes. Because of the lacking dose-dependent difference in the immune response between 6 and 12% CHO beverage ingestion in the present study, the intake of at least 6% CHO beverages during prolonged exercise seems to be sufficient to reduce the metabolic stress and to attenuate the exercise-induced immune stress as well. Higher concentrations of CHO beverages do not seem to yield additional immunological advantages.
This study was supported by a grant from the National Institute for Sports Sciences (Bundesinstitut für Sportwissenschaft, VF 0407/01/11/99-2000), Bonn, Germany.
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