Nine, healthy male trained cyclists (mean ± SEM: age 25 ± 2 yr; height 191 ± 4 cm; body mass 76.8 ± 2.8 kg; V̇O2max 64.7 ± 1.7 mL·kg−1·min−1) volunteered to participate in the study. All subjects were informed about the rationale for the study and the nature of the exercise tests to be performed before providing written informed consent. The Ethics Committee of Loughborough University approved the study. The subjects were all members of cycle clubs either at Loughborough University or locally. Mean weekly training mileage was 253 ± 14 miles (range 220–300 miles·wk−1). Subjects were required to complete a comprehensive health-screening questionnaire before each exercise test and did not report any symptoms of infection and had not taken any medication in the 6 wk before the study, nor were they currently on medication. A further three subjects were excluded from the study for showing symptoms of upper respiratory tract infection either at the preliminary testing stage or before the main trials.
Approximately 1 wk before the start of the study, each subject performed a continuous incremental exercise test on an electromagnetically braked cycle ergometer (Lode Excalibur Sport, Groningen, The Netherlands) to volitional exhaustion to determine maximal oxygen uptake (V̇O2max). Subjects began cycling at 95 W, with increments of 35 W every 3 min until volitional fatigue. Samples of expired gas were collected in Douglas bags during the third minute of each work rate increment, and heart rates were measured continuously using short-range radio telemetry (Sportstester®, Polar, Kempele, Finland). A paramagnetic oxygen analyzer (Servomex 1420B, Crowborough, UK) and an infrared carbon dioxide analyzer (Servomex 1415B) were used along with a dry gas meter (Harvard Apparatus, Edenbridge, UK) for determination of V̇E, V̇O2, and V̇CO2. The work rate equivalent to 75% V̇O2max was interpolated from the V̇O2-work rate relationship.
Experimental trial procedures.
During the 24-h period before each exercise trial, subjects were required to refrain from training and recorded their food intake in an effort to standardize their nutritional status. They were required to eat the same foods during the 24-h period before the second trial. Energy intake and dietary composition were subsequently analyzed using a dietary analysis computer program (Nutritionist Five version 1.7 (1998), First DataBank, San Bruno, CA).
Each subject completed two main trials that were separated by at least 7 d. In one trial, subjects consumed a CHO-containing beverage (6.4%, i.e., 64 g·L−1, glucose and maltodextrin drink, “Lucozade Sport,” GlaxoSmithKline, Coleford, UK; CHO trial) before, during, and after exercise. For the other trial, the drink consumed was an artificially sweetened placebo solution (PLA trial). Each drink was identical in flavor (lemon) and appearance, and the subjects were unaware of the content of the drinks in each trial. The order of the trials was randomized. For each main trial, subjects arrived at the laboratory at 08:00 h after an overnight fast of at least 10 h. Subjects were then required to empty the bladder before body mass (in shorts only) was recorded. After sitting quietly for 10 min, an initial resting blood sample was obtained from an antecubital vein by venepuncture. Immediately after this, subjects consumed 5 mL·kg−1 body mass of the prescribed drink (CHO or placebo) and then began cycling on an electromagnetically braked cycle ergometer (Lode Excalibur Sport) at 75% V̇O2max for 2 h. Heart rates were recorded at 15-min intervals during the exercise using short-range radio telemetry (Sportstester®) as were subjective ratings of perceived exertion (RPE) (5). After these data had been recorded, subjects ingested a further 2 mL·kg−1 body mass of the prescribed solution. Samples of expired air were collected into Douglas bags after 20 min of exercise and every 30 min thereafter for determination of V̇O2 and nonprotein respiratory exchange ratio (RER) to allow estimation of rates of CHO oxidation (10). Immediately postexercise, a blood sample was obtained before body mass (in shorts only) was recorded. Subjects then consumed an additional 5 mL·kg−1 body mass of the prescribed drink. A further blood sample was obtained at 1 h postexercise. For all samples, 10 mL of blood was collected, and all samples were obtained with the subject in a seated position. No other fluid or food intake was allowed until the blood sample had been collected at 1 h postexercise. Laboratory conditions were 22.4 ± 0.7°C and 58 ± 3% relative humidity.
Blood samples were collected into two separate monovette tubes (evacuated blood collection tubes, Sarstedt, Leicester, UK), one containing K3EDTA (1.6 mg EDTA·mL−1 blood) and the other containing lithium heparin (1.5 IU heparin·mL−1 blood). Blood taken into the K3EDTA monovette (2.7 mL) was used for hematological analysis including hemoglobin, hematocrit, and total and differential leukocyte counts using a Sysmex SE9000 cell counter (Sysmex UK Ltd, Milton Keynes, UK) at the Chemical Pathology laboratories of Leicester Royal Infirmary NHS Trust. Plasma volume changes were estimated according to Dill and Costill (8). Of the 7.5 mL of blood collected into the lithium heparin monovette, 500 μL was added to 500 μL of 20 mM deoxycholate (Sigma, Poole, UK) and lysed by vigorous shaking. This sample was then stored at −70°C before determination of elastase concentration.
A further 1.0 mL of each heparinized blood sample was immediately added to a separate snap-seal microcentrifuge tube (1.5-mL capacity) containing 50 μL of 10 mg·mL−1 bacterial LPS solution (Stimulant, Sigma). Blood and LPS were mixed by gentle inversion and then incubated for 1 h at 37°C, being gently mixed again after 30 min. After incubation the mixture was centrifuged for 2 min at 5000 g. The supernatant was immediately stored at −70°C before analysis of elastase concentration.
Elastase concentration in plasma before (baseline elastase) and after treatment with LPS (stimulated elastase release) and deoxycholate (total elastase content) was determined using a sandwich-type enzyme-linked immunosorbent assay specific for polymorphonuclear cell elastase using antihuman elastase antibody coated tubes (Merck, Lutterworth, UK), according to the manufacturer’s instructions.
The remaining heparinized whole blood was spun at 1500 g for 10 min in a refrigerated centrifuge (4°C). This occurred within 15 min of sampling. The plasma obtained was immediately stored at −70°C. After thawing, 100-μL aliquots of heparinized plasma were deproteinized by mixing with 1.0 mL of ice-cold perchloric acid (0.3 M). The suspension was vortex mixed and then centrifuged for 1 min at 5000 g. The supernatant was analyzed for lactate using a standard spectrophotometric method, as described by Fink and Costill (9). Briefly, this method uses a reagent mix of lactate dehydrogenase (10,000 U·mL−1, Sigma), NAD (10 ng·mL−1, Sigma) and hydrazine buffer (1.1 M, pH 9.0). The subsequent change in NADH concentration (equimolar to lactate concentration) is determined by the absorption of the sample at 340 nm, assuming that the absorption of a 0.1 M solution of NADH is 0.622 at this wavelength. Aliquots of plasma were also analyzed to determine the concentration of glucose and cortisol using hexokinase (No. 16-50 Kit, Sigma) and 125I radioimmunoassay (ICN Pharmaceuticals, Costa Mesa, CA) methods, respectively. For each assay, all samples were analyzed on the same day, with quality controls included with each set of analysis.
The intra-assay coefficient of variation was 2.4%, 2.8%, 1.8%, and 6.3% for glucose, lactate, cortisol, and elastase, respectively.
Data in the text, tables, and figures are presented as mean values and the standard errors of the mean (± SEM). The data were examined using a two-factor (trial × time of measurement) ANOVA with repeated measures design. If a data set was not normally distributed, statistical analysis was performed on the logarithmic transformation of the data. Assumptions of homogeneity and sphericity in the data were checked, and, where appropriate, adjustments in the degrees of freedom for the ANOVA were made using the Huynh-Feldt method of correction. Any significant F-ratios subsequently shown were assessed using post hoc Tukey tests and paired t-tests, where appropriate. Statistical significance was accepted at P < 0.05. To determine this sample size of 9, analyses of power and effect size were performed using data from our previous work looking at the effect of carbohydrate supplementation on neutrophil degranulation responses to prolonged intense exercise (1). The calculations indicated that a sample size of 9 would be sufficient to detect a significant difference in the major dependent variables. Accordingly, in the present study, the observed powers for the trial × time interactions for glucose, neutrophil count, plasma elastase, and LPS-stimulated elastase release per neutrophil were 0.89, 0.86, 0.77, and 0.80, respectively. Those for trial × time interactions for total elastase content in hemolyzed whole blood and % of elastase released in response to LPS were 0.78 and 0.73, respectively. In addition, the observed powers for the reported main effects of time were 0.92 and 0.99 for lactate and total LPS-stimulated elastase release, respectively. Finally, the observed power for the reported main effect of trial for cortisol was 0.62.
Dietary energy intake during the 24-h period before both trials averaged 11.6 ± 0.6 MJ (2780 ± 150 kcal), with the proportion of CHO, fat, and protein being 64.1 ± 3.7%, 19.6 ± 3.0%, and 17.3 ± 0.8%, respectively. Exercise intensity did not differ between the trials: mean percent V̇O2max during exercise was 74.2 ± 1.3% (CHO trial) and 75.2 ± 1.4% (PLA trial). Mean RER was 0.91 ± 0.01 on the CHO trial and 0.92 ± 0.01 on the PLA trial, and estimated rates of CHO oxidation were 3.2 ± 0.3 g·min−1 and 3.4 ± 0.4 g·min−1 on the CHO and PLA trials, respectively. Heart rate was similar on both trials throughout the exercise (CHO: 159 ± 1 beats·min−1, PLA: 157 ± 1 beats·min−1; mean of all recordings). RPE increased as a function of exercise duration on both trials, after 15 min of cycling values were 12.2 ± 0.6 and 12.1 ± 0.6 on the CHO and PLA trials, respectively. This increased to 14.9 ± 0.7 (CHO) and 15.6 ± 0.6 (PLA) after 2 h of cycling (P < 0.05). After the exercise, changes in body mass (corrected for fluid intake) were similar on both trials (−2.6 ± 0.4 kg and −2.7 ± 0.4 kg on the CHO and PLA trials, respectively) as was the fall in plasma volume (−8.2 ± 1.1% and −8.0 ± 1.1% on the CHO and PLA trials, respectively).
Plasma glucose concentration was significantly higher on the CHO trial compared with the PLA trial immediately postexercise (P < 0.05;Fig. 1). There were no significant trial × time interaction effects observed for plasma cortisol; nevertheless, there was a main effect of trial, with values on the CHO trial significantly lower than on the PLA trial (P < 0.01;Table 1). Plasma lactate concentration increased significantly in response to the exercise (CHO: 1.3 ± 0.1 mmol·L−1 to 3.7 ± 0.6 mmol·L−1; PLA: 1.7 ± 0.2 mmol·L−1 to 5.0 ± 0.9 mmol·L−1; main effect of time, P < 0.01).
After the exercise, a marked neutrophilia was observed on both trials that was sustained at 1 h postexercise (P < 0.01;Fig. 2). The magnitude of this neutrophilia was significantly greater on the PLA trial than on the CHO trial at postexercise and 1 h postexercise (P < 0.01;Fig. 2).
Plasma elastase concentration increased significantly above preexercise values on the PLA trial immediately postexercise and at 1 h postexercise (P < 0.05;Table 1). Values remained close to preexercise levels on the CHO trial. Total LPS-stimulated elastase release increased significantly above preexercise values immediately and at 1 h postexercise (main effect of time; P < 0.01;Table 1). Adjusting these values to take into account changes in numbers of circulating neutrophils revealed a 47% fall in LPS-stimulated elastase release per cell immediately postexercise compared with preexercise values on the PLA trial with values remaining 45% lower than at preexercise at 1 h postexercise (P < 0.01;Fig. 3). LPS-stimulated elastase release per neutrophil did not change significantly from preexercise values on the CHO trial (Fig. 3). Total elastase content in hemolyzed whole blood increased significantly after the exercise on both trials (P < 0.01;Table 1). When correcting these data for changes in numbers of circulating neutrophils (i.e., total elastase content of neutrophils), no significant interaction or group effects were observed (Table 1). From the total elastase content of neutrophils and the LPS-stimulated neutrophil elastase release data, it was calculated that the percentage of elastase released in response to LPS did not change from preexercise values on either trial (Table 1).
The main finding of the present study is that total elastase content of neutrophils (elastase availability) is unaffected by prolonged intense exercise. Nevertheless, exercise was associated with a fall in LPS-stimulated neutrophil elastase release compared with resting values. In addition, CHO ingestion had no influence on total elastase content of neutrophils but was associated with a blunting of the postexercise fall in LPS-stimulated neutrophil elastase release, in agreement with our previous work (1). Furthermore, there was an exercise induced rise in plasma elastase levels that was attenuated by CHO ingestion; this is consistent with the findings of Smith (23).
Falls in LPS-stimulated neutrophil degranulation capacity after intense exercise were hypothesized to be associated with an increase in the proportion of immature neutrophils in the blood. Because these would contain less cytotoxic granules (21), less elastase would be available to be released in response to stimulation with LPS. Ingesting CHO during exercise is associated with a reduced stress hormone response (7) and therefore would be expected to reduce the stimulus for the mobilization of these less mature cells from the bone marrow, thereby minimizing the fall in neutrophil function. The findings of the present study refute this hypothesis. There was a marked increase in whole blood total elastase content after the exercise, but this most likely reflects increases in the number of circulating neutrophils (i.e., more cells, more elastase). The bone marrow contains a 3-d store of mature neutrophils for release (18); hence, it is questionable whether a single exercise bout of this nature would impact significantly on this store. However, the subjects in this study were all actively training and competing endurance cyclists, and it is arguable that repeated prolonged, intense bouts of exercise could exhaust the bone marrow stores of mature neutrophils, leading to the release of less competent cells into the circulation (19), potentially impairing innate host defense and widening the “window of opportunity” for infectious agents to enter the body. However, at rest for both trials combined, the percentage of elastase released in response to LPS was only ∼11% of the total available, suggesting that granular enzyme availability is far from limited in endurance-trained cyclists. Exercise was associated with marked falls in LPS-stimulated elastase release per neutrophil on the PLA trial compared with the CHO trial, yet the percentage of elastase released in response to LPS on the PLA trial fell to only ∼7–10% of the total enzyme available. Although these findings agree with previous reports that neutrophil degranulation capacity is decreased in comparison with resting samples by prolonged intense exercise (20) and that CHO ingestion can attenuate this response (1), they suggest that elastase availability per se is not the limiting factor. Perhaps future attention should be directed at the influence of exercise and CHO ingestion on intracellular calcium signaling, because increases in Ca2+ are required for the steps that lead to degranulation (and the oxidative burst) (6,13) and a recent study reports of an inhibition of intracellular Ca2+ signaling in neutrophils after exhaustive exercise (14).
Although the findings of the present study do not support the idea that a higher proportion of immature neutrophils (as indicated by a fall in neutrophil elastase content) are released after prolonged exercise in endurance-trained cyclists, this is not to say that endurance training may not lead to an increased proportion of less mature neutrophils in the blood at rest. In support of this, the blood of elite cyclists has been shown to have a higher proportion of immature neutrophils compared with that of sedentary controls (11). Moreover, this may influence neutrophil functional capacity at rest; our previous work may suggest that resting LPS-stimulated elastase release per neutrophil is lower in endurance-trained athletes (mean value of 166 fg·cell−1) (20) compared with recreationally active individuals (mean value of 404 fg·cell−1) (2). The mean preexercise LPS-stimulated elastase release of the endurance-trained cyclists in the present study was 218 fg·cell−1 (mean of all resting measurements). However, it should be noted that variations in resting in vitro neutrophil function do not necessarily reflect a suppressed immune system in vivo or an increased risk of systemic infection.
Although neutrophil granular enzyme availability could not account for the exercise-induced fall in neutrophil LPS-stimulated elastase release in the present study, it may be that the influence of exercise (and CHO) on neutrophil function is associated with the activation status of neutrophils. Neutrophils exist in several states of activation that exist on a continuum between dormant and fully activated, with primed cells able to become fully activated rapidly (18). Exercise induces a shift toward a higher percentage of primed neutrophils (24). In the present study, a higher percentage of primed cells in response to exercise would mean enhanced elastase release after stimulation with LPS. Cortisol is known to suppress neutrophil activation (4), and a greater cortisol response to exercise may be associated with a smaller proportion of primed cells and therefore a lower elastase release. In the present study, there was a significant main effect of trial on plasma cortisol concentration, with overall values higher on the PLA trial than on the CHO trial. This may have contributed to the marked postexercise decline in LPS-stimulated elastase release on the PLA trial compared with resting values and for the blunting of this response on the CHO trial observed here.
In conclusion, these findings suggest that prolonged intense exercise does not influence the total elastase content of neutrophils, regardless of CHO ingestion. Therefore, granular enzyme availability does not account for the decrease in LPS-stimulated neutrophil elastase release observed after prolonged intense exercise in endurance-trained cyclists or for the attenuation of this response when CHO is ingested during exercise. Nevertheless, given the key role that neutrophils play in innate host defense, the influence of CHO ingestion on neutrophil function may contribute to a smaller “window of opportunity” for infection after prolonged strenuous exercise. However, it is important to acknowledge that cellular measurements do not necessarily reflect the situation in vivo or potential risk of systemic infection, and so extrapolations should be made with due caution. Further research is required to clarify the clinical significance of exercise-induced falls in neutrophil degranulation.
We would like to express our sincere thanks to Professor Mike Gleeson for his help and advice in the preparation of this manuscript.
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