Effects of Carbohydrate Beverage Ingestion on the ...

Physiology & Biochemistry

Effects of Carbohydrate Beverage Ingestion on the Salivary IgA Response to Intermittent Exercise in the Heat

Authors

V. Sari-Sarraf1, D. A. Doran2, N. D. Clarke3, G. Atkinson2, T. Reilly2

Affiliations

1

Key words ▶ soccer-specific exercise ● ▶ salivary IgA ● ▶ cortisol ● ▶ CHO ●

Abstract ▼

Faculty of Physical Education and Sport Sciences, University of Tabriz, Tabriz, Islamic Republic of Iran Liverpool John Moores University, Research Institute of Sport and Exercise Sciences, Liverpool, United Kingdom 3 School of Human Sciences, Faculty of Life Sciences, London Metropolitan University, United Kingdom 2

The purpose of this study was to establish if provision of CHO altered the mucosal immune and salivary cortisol responses to intermittent exercise in the heat. In a double-blind design, 10 males undertook soccer-specific intermittent exercise on a motorized treadmill on 2 occasions, each over 90 min and separated by 1 week. During CHO and placebo trials, subjects were given either a carbohydrate solution (3 ml · kg − 1 body weight) or placebo drink, 5 min before the commencement of exercise, at 15, 30 min, at half time, 60 and 75 min into exercise. Salivary flow rate increased throughout the placebo trial and

Introduction ▼ accepted after revision January 13, 2011 Bibliography DOI http://dx.doi.org/ 10.1055/s-0031-1271698 Int J Sports Med 2011; 32: 659–665 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. Vahid Sari-Sarraf, PhD Faculty of Physical Education and Sport Sciences University of Tabriz 22 Bahman BLVD Tabriz/Iran Tabriz 5166614776 Iran Islamic Republic of Iran Tel.: + 98/411/339 3254 Fax: + 98/411/356 008 [email protected]

Under normal environmental conditions, exercise-induced changes in immune response are relatively small and short-lived, limiting the “open-window” period when the likelihood of an acute infection might be increased [45]. Heat exposure is a form of stress in which elevations in body core temperature occur with concomitant alterations in hormonal and immune responses [9, 18, 39]. Exercise in a thermally stressful environment appears to have an additive effect on the hormonal and immune system disturbances compared to heat alone [31, 46, 54]. There is evidence of an acute increase in the synthesis of immunoglobulins after performing exercise under hot conditions [8]. Horswill et al. [28] suggested that exercise decreases salivary flow rate, which is further exacerbated by increases in ambient temperature. Regular fluid intake appears to prevent the exercise-induced decrease in flow rate [29]. Athletes performing prolonged exercise in the heat, with associated increased sympathetic activity and increased fluid losses, might exhibit larger reductions in

decreased throughout the CHO treatment; the difference between conditions neared statistical significance (P = 0.055). Neither s-IgA concentration nor s-IgA to osmolality ratio was affected by 2 conditions or differed at any time-point post-exercise (P > 0.05). The s-IgA secretion rate increased, s-IgA to protein ratio decreased postexercise and salivary cortisol decreased 24 h post-exercise (P < 0.05) compared to pre-exercise. Carbohydrate supplementation whilst exercising in the heat, does not influence rating of perceived exertion, thermal sensation, salivary flow rate, sIgA concentration, s-IgA secretion rate, s-IgA to osmolality ratio or s-IgA to protein ratio and salivary cortisol but heart rate was increased.

saliva flow rate and possibly salivary IgA secretion rate than when they perform the same exercise in thermoneutral conditions. Fluid ingestion can help to keep levels of oral pathogens low and temper the drying of the airways that occurs during exercise, thereby reducing the vulnerability of the upper respiratory mucosa to pathogens [2]. Laing et al. [29] showed that a prolonged bout of cycling in the heat evoked a reduction in salivary IgA secretion rate, but did not influence salivary IgA responses to prolonged exercise with ad libitum water intake. The addition of carbohydrate to solutions and the ingestion of fluids before and at regular intervals during prolonged and high-intensity intermittent exercise may extend exercise duration [3], but it may also attenuate perturbations in immune function [4, 17, 24] and decrease the potential for developing symptoms of respiratory tract infections [4]. Carbohydrate ingestion may also attenuate the rise in cortisol concentrations during exercise by maintaining plasma glucose concentrations [3]. Cortisol is known to inhibit transepithelial transport of salivary IgA [42], to inhibit in vivo B lymphocyte antibody synthesis

Sari-Sarraf V et al. Intermittent exercise, CHO in heat and salivary IgA … Int J Sports Med 2011; 32: 659–665

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[42] and has been implicated in decreased B lymphocyte antibody synthesis after exercise [37]. Prolonged exercise in the heat is associated with a greater plasma cortisol response compared with thermoneutral conditions [13]. A larger reduction in salivary IgA concentration and secretion rate might therefore be expected when prolonged exercise is performed in hot compared with thermoneutral conditions. Laing et al. [29] did not monitor a reduction in salivary IgA concentration during 2 h recovery. Therefore, their results did not support a short-term inhibitory effect of elevated plasma cortisol (91 %) on salivary IgA after prolonged cycling in the heat. Soccer is a sport that consists of intermittent high-intensity bursts of activity leading to high rates of metabolic heat production. Soccer can be played under hot environmental conditions and opportunities for fluid replacement are limited during games. Fluid intake before and during the game would reduce the degree of dehydration. The addition of carbohydrate to the fluids may supplement the body’s limited carbohydrate stores, prevent muscle glycogen depletion and mitigate potential adverse changes in salivary IgA responses. The aim of the current study was to examine the effects of carbohydrate supplementation prior and at regular intervals during soccer-specific intermittent exercise in a hot environment on salivary IgA and cortisol responses.

Materials and Methods ▼ Subjects ▶ Table 1) provided written informed consent for the 10 males (● study, which was approved by the University’s Human Ethics Committee. The study meets the ethical standards of the IJSM [25]. The subjects were all in good health and regularly participated in physical activity (moderate level exercise 3 times a week), were non-smokers, reported no significant oral, dental or other symptoms of infection and were not taking any medication in the month prior to the experiment. Each participant’s height was determined using a stadiometer (Seca, Germany). Nude body mass (kg) was also evaluated before and after exercise using calibrated precision-weighing scales (Seca, Germany). Whole-body sweat loss was determined by measuring the change in body mass, corrected for fluid intake and urinary losses [10].

Design and exercise protocol Preliminary measurements and familiarisation The subjects were initially assessed for aerobic fitness using an incremental treadmill running protocol to volitional exhaustion to determine maximal oxygen consumption (VO2 max). The guidelines of the British Association for Sport and Exercise Sciences [1] were followed to confirm the occurrence of maximal oxygen uptake (VO2 max). Throughout the trial, expired gas volumes (mean expired fractions of oxygen and carbon dioxide) Table 1 Physiological and anthropometric characteristics of the subjects (Mean ± SD) [n = 10]. Age (years) height (m) mass (kg) BMI (kg · m − 2) VO2 max (ml · kg − 1 · min − 1) HR rest (beat · min − 1)

26 ± 4 1.8 ± 0.05 74.2 ± 5.7 23.4 ± 2 62.6 ± 5.6 61 ± 5

were recorded every 10 s using an on-line gas analyser (Metalyser 3B, Cortex Biophysik; Borsdorph, Germany). In addition, a Polar S610: HR monitor (Kemple, Finland) was used to measure heart rate continuously. The subjects attended the laboratory on 4 occasions. The first occasion, 3 days prior to determination of VO2 max, was for familiarisation to ensure that subjects were comfortable with the experimental exercise protocol at intensities ranging from standing to sprinting for 20 min and were able to cope with its demands. After the second visit to determine VO2 max, subjects reported to the environmental chamber (30 ° C, 40 ± 2 % RH) on 2 more occasions, to undertake soccer-specific intermittent exercise for 90 min on the motorized treadmill (h/p/cosmos, Germany). These 2 sessions were separated by 7 days. During the week between tests, the subjects followed their normal diets and lifestyle pattern. The subjects were asked to refrain from strenuous exercise for 48 h prior to and after each exercise trial. Alcohol and caffeine were restricted for 24 h and food for 3 h prior to exercise. On arrival at the laboratory, the subjects gave verbal confirmation of their adherence to these directions. Each test was scheduled for the same time of day (10:00 12:00 h) to negate the effects of circadian variation [40]. Each session was preceded by a general warm-up consisting of general body stretching exercises and approximately 5 min of running on the treadmill. Saliva samples were collected 4 times: these were before the exercise trials, immediately after, then 24 and 48 h post-exercise. Heart rate was monitored during exercise every 5 s; subjects rated their thermal sensation (Tsens) and perceived exertion (RPE) every 15 min using the thermal sensation (Tsens) [50] and Borg [6] scales respectively. Core body temperature was monitored continuously by means of an ingestible temperature sensor pill and external data logger (HQ inc., Florida, USA) and presented as the mean value for each 15-min block [11]. Gut fullness and thirst were also measured using 100-mm visual analogue scales (VAS) immediately before and after fluid ingestion prior to commencing exercise and also during the double static period of each 15 min block [26].

Experimental protocol Laboratory-based exercise protocols have been devised to replicate the physiological demands of intermittent exercise patterns specific to soccer. The protocol used in this study was a modified version of that designed by Drust et al. [22]. The protocol consisted of 5 different exercise categories, representing the increasing exercise intensities (standing “0”, walking “4”, jogging “10”, cruising “13” and sprinting “19 Km.h − 1”) that are observed in soccer match-play. The protocol was composed of six 15-min periods. After the first 45 min and a 15-min “half-time” intermission, the exercise was continued for a further 45 min to correspond to the temporal pattern of a soccer game.

Carbohydrate administration All subjects were given either a carbohydrate solution (CHO) or placebo (PLA). The study was double-blind, placebo controlled, and randomised. The 2 experimental drinks were a 6 % (W/V) orange flavoured carbohydrate solution and an artificially sweetened orange flavoured, carbohydrate-free solution (placebo). Each participant received a fluid volume based on his body mass (3 ml · kg − 1 body weight), 5 min before the exercise, at 15, 30 min, at half time, at 60 and 75 min into each exercise trial.


Physiology & Biochemistry Saliva collection and analysis In order to minimise the effects of circadian variation and habitual activity known to cause alterations in salivary immunoglobulin and cortisol levels [20, 21], saliva samples were collected at the same time of the day and on the same day of the week. Initially, subjects were required to rinse out their mouths with distilled water to remove potential sample contaminants that might affect IgA and/or cortisol levels [27]. Whole unstimulated saliva was collected by expectoration for 5 min into sterile pre-weighed plastic containers (Sarstedt, U.K.) with eyes open, head tilted slightly forward and making minimal orofacial movement [36]. Saliva was weighed to the nearest mg and volume was calculated assuming a saliva density of 1.00 g · mL − 1 [16]. All saliva samples were centrifuged at 5 200 × g for 20 min at room temperature, the supernatant was assayed for osmolality using a freezing point depression Osmometer (Model 3300, Advanced Instruments Inc.™, Norwood, Massachusetts, USA). Samples were then aliquoted and stored at – 80 ° C for later analysis. The saliva flow rate (μl · min − 1) was determined by dividing the volume of saliva by the collection time.Total protein concentration (g.l − 1) in saliva was analysed using albumin as standard [7]. The indirect competitive enzyme immunoassay (EIA) kit (Salimetrics, USA) was used for the quantitative measurement of IgA in saliva samples. A constant amount of goat anti-human salivary IgA conjugated to horseradish peroxidase was added to tubes containing specific dilutions of standards or saliva samples vortexed and centrifuged at 1 870 × g for 15 min. The antibody conjugate binds the salivary IgA in the standard or saliva samples. The quantity of salivary IgA present is inversely proportional to the amount of free antibody remaining. Following incubation and mixing, an equal solution from each tube was added in duplicate to a microtitre plate coated with human salivary IgA. The free or unbound antibody conjugate binds to the salivary IgA on the plate and then the unbound components are washed away after incubation and bound conjugate is measured by the reaction of the peroxidase enzyme on the substrate tetramethylbenzidine (TMB). The amount of peroxidase is inversely proportional to the quantity of salivary IgA in the samples [14]. Optical density was read on a standard automated plate reader at 450 nm (Triturus, IBL, Spain). Salivary IgA concentration was calculated based on the sigmoid minus curve fit of the related 4 parameters. The salivary cortisol ELISA kit (DRG Diagnostics, Germany) was used for the quantitative measurement of cortisol in saliva samples. Salivary flow rate was multiplied by saliva cortisol concentration for calculation of the saliva cortisol secretion rate (ng · min − 1). The coefficients of variation of the analytical methods were 1.7 % for osmolality, 1.9 % for IgA, and 2.8 % for cortisol.

Data analysis Data are presented as mean ± SD. The data were evaluated using a 2-factor (2 trial × 4 time measurement) repeated measures analysis of variance (ANOVA) design. Normality of distribution for all variables was checked with the Shapiro-Wilk test. If residuals were found to be skewed or 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 degree of freedom for the ANOVA were made. Significant differences were analysed using post hoc t-test with an appropriate Bonferroni correction factor. Statistical significance was set at an alpha of P < 0.05.

Results ▼ Physiological responses to the exercise protocol Heart rate (main effect of time: F1. 5, 13.2 = 48.6, p < 0.001), RPE (main effect of time: F2, 17.6 = 38.9, p < 0.001), core temperature (main effect of time: F2,16 = 39.6, P < 0.05), gut fullness (main effect of time: F2,17 = 33.7, P < 0.05) thirst (main effect of time: F2,14 = 35.9, P < 0.05) and Tsens (main effect of time F1.9, 17 = 19.6, p < 0.001), increased significantly throughout the exercise trials. Heart rate was significantly higher on the CHO trial than on the PLA trial (158 ± 7 vs. 155 ± 8 beats · min − 1, p < 0.05). Mean RPE was 13.7 ± 1.1 and 13.8 ± 1.3 for PLA and CHO, respectively, the difference in responses between the 2 treatment protocols was not significant (p > 0.05). Thermal sensations (5.7 ± 0.7 and 5.6 ± 0.7 for CHO and PLA, respectively) were not significantly different between the 2 conditions. There was no significant trial effect on core temperature (P > 0.05). The differences occurred between the first block and the following 5 blocks. Gut fullness was not significantly affected by the trials. Pairwise comparisons revealed that gut fullness increased significantly post-fluid ingestion prior to completing the protocol, and remained significantly (P < 0.05) elevated throughout. There was a significant difference in thirst between trials (P < 0.05). Pairwise comparisons also indicated these differences occurred between post-fluid ingestion at half-time and at all time points except following the ingestion of fluid before the start of exercise.

Body mass changes After correction for fluid intake and urine output, body mass decreased throughout the soccer-specific intermittent exercise under both conditions (F1, 9 = 82.5, p < 0.001). The net loss of body mass during the PLA condition was 2.36 ± 0.25 kg and 2.43 ± 0.6 kg during the CHO treatment. There were no differences in loss of body mass between the 2 treatments (P > 0.05).

Salivary responses 2 subjects’ data were omitted from the calculation due to deviation from the instructions for collection throughout the 48 h. Salivary. flow rate (main effect of time: F1.8, 12.6 = 4.9, P < 0.016) increased throughout the PLA treatment and decreased throughout the CHO condition (interaction: F2.1, 14.9 = 4.4, P = 0.015). Differences between conditions neared statistical significance (p = 0.055). Salivary IgA concentration (mg · l − 1) changes were not significant either between different time points, or CHO and PLA treatments. There was a main effect of time whereby salivary IgA secretion rate increased post-exercise by 51 % (F2, 14 = 3.9, P < 0.05). Salivary IgA secretion rate (μg · min − 1) showed a greater, albeit non-significant, increase post-exercise on the CHO treatment ▶ Fig. 1–4]. compared to the PLA trial (P > 0.05) [● A 70 % increase in saliva osmolality (61.4 to 104.5 mOsmol.kg − 1) after both exercise conditions approached significance (P = 0.06). However solute secretion rate (main effect of time: F2.5, 17.2 = 5.33, P = 0.012 and interaction: F2.5, 17.4 = 5.33, P = 0.042) increased throughout both treatments. Salivary IgA to osmolality ratio (μg · mOsmol − 1) demonstrated a non-significant change after ▶ Table 2]. PLA and CHO treatment (P > 0.05) [● Salivary protein concentration (g · l − 1) increased post-exercise and had returned to pre-exercise levels by 48 h post-exercise after a sharp reduction 24 h post-exercise (main effect of time: F2.2, 15.5 = 11.4, P = 0.001). Protein concentrations were significantly higher on the CHO treatment compared with the PLA con-


661

Flow rate (µl.min–1)


2 000

PLA CHO

*

1 500

*

1 000 500 0

Pre-ex

Post-ex

24 h Time Points

48 h

Salivary IgA concentration (mg.I–1)

Fig. 1 The effects of soccer-specific intermittent exercise in the heat with 2 ingestion conditions (PLA and CHO) on salivary flow rate (μl · min − 1 ). Values are means ( ± SD). Significantly higher than post-exercise, * P < 0.05.

600 500 400 300 200 100 0

PLA CHO

Pre-ex

Post-ex

24 h

48 h

Time Points

Salivary IgA secretion rate (µg.min–1)

Fig. 2 The effects of soccer-specific intermittent exercise in the heat with 2 ingestion conditions (PLA and CHO) on salivary IgA concentration (mg · l − 1 ). Values are means ( ± SD). No significant difference between conditions and time-points.

350 300 250 200 150 100 50 0

PLA CHO

* *

Pre-ex

Post-ex

24 h Time Points

48 h

Cortisol (ng.ml–1)

Fig. 3 The effects of soccer-specific intermittent exercise in the heat with 2 ingestion conditions (PLA and CHO) on salivary IgA secretion rate (μg.min − 1 ). Values are means ( ± SD). Significantly higher than pre-exercise, * P < 0.05.

4 2 0 8 6 4 2 0

PLA CHO * *

Pre-ex

Post-ex

24 h Time Points

48 h

Fig. 4 The effects of soccer-specific intermittent exercise in the heat with 2 ingestion conditions (PLA and CHO) on cortisol (ng · min − 1 ) significant difference between post-exercise and 24 h. Values are means ( ± SD). Significantly lower than post-exercise, * P < 0.05.

dition immediately post-exercise and 24 h afterwards (main effect of exercise: F2.5, 17.7 = 9.5, p = 0.018). Salivary protein secretion rate (μg · min − 1) increased after exercise (F2.5, 17.7 = 9.5, p = 0.001), being significantly higher on the CHO treatment after exercise and 24 h post-exercise than in the PLA condition (F1, 7 = 10.8, p = 0.013). Salivary IgA to protein ratio (mg · g − 1) decreased post-exercise (main effect of time: F2.3, 16.3 = 4.8, p = 0.019), with no differences apparent between the 2 condi▶ Table 2). tions (● Salivary cortisol (ng · ml − 1) decreased throughout the 24 h after PLA ingestion (7.5 ± 4.2 to 3.6 ± 0.8 ng · ml − 1). Conversely, it increased immediately post-exercise with CHO treatment (6.3 ± 3 to 6.5 ± 1.5 ng · ml − 1) and decreased 24 h later (main effect of time: F1.7, 11.6 = 4.2, p = 0.07). The difference in salivary cortisol between ▶ Fig. 1d). the 2 conditions neared statistical significance (●

Discussion ▼ The aim of the present study was to examine the effects of carbohydrate supplementation during soccer-specific intermittent exercise in the heat on mucosal immunity. Soccer-specific intermittent exercise in the heat has the potential for stimulating the sympathetic nervous system and reducing saliva flow rate, salivary IgA concentration and secretion rate to a greater extent than when the exercise is performed in thermoneutral conditions [29]. It was hypothesised that carbohydrate ingestion before and at regular intervals during the soccer-specific protocol in the heat attenuates the immunosuppressive effects of combined stressors (exercise and heat exposure). The results showed that carbohydrate supplementation before and during the soccer-specific intermittent exercise protocol in the heat neither influenced RPE, Tsens, core temperature, gut fullness, thirst or osmolality, solute secretion rate, salivary IgA concentration, salivary IgA secretion rate, salivary IgA to osmolality ratio or salivary IgA to protein ratio and salivary cortisol. It has been suggested that carbohydrate availability is not a limiting factor for exercise in the heat when the heat stress is uncompensable [23]. It also appears that the addition of carbohydrate to a solution has no impact on key thermoregulatory variables such as core temperature and heart rate [35]. At no point during any of the trials did core temperature stabilize, it increased significantly between each time point during the second half of the protocol, suggesting a large thermal strain. However, the final value at the completion of the 90 min was lower than previously reported values during similar exercise protocol [35], may be due to different sites being used to assess core temperature, rectal as opposed to intestinal used in the present study. Higher values for heart rate on the CHO trial than on the PLA may come from initial and progressive dehydration (higher saliva osmolality) [41]. There were no significant differences in RPE, Tsens, core temperature, gut fullness or thirst between trials during the soccerspecific protocol, indicating a similar level of physiological stress during each trial. These findings are consistent with the results of Davis et al. [19] who reported no differences in markers of cardiovascular and thermoregulatory stress with and without carbohydrate ingestion at concentrations of up to 10 % glucose. The results of the present study suggest that any differences in the rate of gastric emptying or intestinal absorption are not sufficient to alter physiological homeostasis during exercise of this nature when performed in the heat.



Table 2 The effect of ingesting carbohydrate (CHO) and placebo (PLA) on various salivary parameters during a soccer-specific intermittent exercise protocol in heat (Mean + SD)[n = 8]. a: P < 0.05, Significant difference compared with pre-exercise. b: P < 0.05, Significant difference compared with post-exercise. Variable osmolality (mOsmol.kg − 1)

Treatment

Pre-

Post-

24 h after

48 h after

exercise

exercise

exercise

exercise

77 ± 9

87 ± 21

PLA

74 ± 15

100 ± 28

CHO PLA

81 ± 34 38.1 ± 15.2

111 ± 48 57.3 ± 18.2a

81 ± 21 84.0 ± 44.4

80 ± 7 82.8 ± 50

CHO PLA

44.6 ± 15.8 0.87 ± 0.44

60.6 ± 13.2a 1.30 ± 0.46

63.0 ± 35.5 0.48 ± 0.28b

57.1 ± 22.4 0.70 ± 0.24b

CHO PLA

0.93 ± 0.42 402 ± 139

1.45 ± 0.42 725 ± 250a

0.91 ± 0.37b 450 ± 183

0.68 ± 0.08b 579 ± 157

CHO PLA

485 ± 142 2.11 ± 0.79

838 ± 287a 1.94 ± 0.66

616 ± 251 2.11 ± 0.94

513 ± 100 2.41 ± 1.30

IgA to protein ratio (mg.g − 1)

CHO PLA

2.14 ± 0.79 222 ± 124

2.40 ± 1.9 159 ± 53

1.46 ± 0.36 322 ± 164

2.2 ± 0.75 290 ± 117

cortisol (ng.ml1)

CHO CHO

190 ± 93 7.48 ± 4.2

187 ± 168 5.7 ± 1.54

152 ± 39 3.64 ± 0.85b

279 ± 79 4.64 ± 1.84

PLA

6.3 ± 3.1

6.48 ± 1.55

4.46 ± 0.73b

4.56 ± 1.4

Time

protein concentration (g.l − 1)

protein secretion rate (μg.min − 1)

IgA to osmolality ratio (μg.mOsmol − 1)

> 0.05

> 0.05

0.012

0.05

0.04

0.001

0.018

0.07

0.001

0.013

> 0.05

> 0.05

Body mass decreased during exercise under both conditions, in spite of fluid replacement at 3 ml · kg − 1of body mass before and at frequent intervals during the exercise protocol. The reduction in body mass indicates a progressive dehydration. A change of 1 g in body weight represents a 1 ml change in body water status and allowing for fluid intake and urine output, the sweat rate can be estimated as averaging 1.57 ± 0.2 vs. 1.60 ± 0.4 l.h − 1 for PLA and CHO treatments, respectively. Salivary IgA secretion rate increased immediately after completing the soccer-specific intermittent protocol. The increase in salivary IgA secretion rate after exercise is in agreement with others [2, 5, 44, 49] but contrasts with studies showing no change [30, 51], or a decrease in salivary IgA secretion rate after exercise [29, 32, 48, 52]. The inconsistencies between these findings may be explained by differences in the hydration status of subjects before and during the exercise trials as well as the inherent intra- and inter individual variability in IgA [28, 38]. As such current data suggest that a 1.5 % decline in body weight can be tolerated without imposing a detrimental effect on salivary IgA following high-intensity activity. This reverse pattern of changes in salivary flow rate after the CHO treatment compared to the PLA condition may have accounted for the corresponding non-significant alteration (46.7 % increase post-exercise) in salivary IgA concentration (mg · l − 1) and concurrent increase in salivary IgA secretion rate. This finding is in agreement with other studies [23, 29, 53]. The main fluid constituent of saliva is water, which enters saliva from plasma across the acinar cells and dehydration accounts for a 90 % decrease in unstimulated saliva flow rate after 24 h without fluid or food [47]. The decrease in saliva flow rate after exercise was delayed by regular fluid intake irrespective of the type of beverage (CHO or PLA). Walsh et al. [53] suggested that fluid availability compared to any neuro-endocrine effect has a greater

Time × Treatment

0.06 solute secretion rate (μOsmol · min − 1)

Treatment

> 0.05

> 0.05

0.011

> 0.05

> 0.05

P = 0.049

P = 0.07

P > 0.05

involvement in the change in saliva flow rate during exercise. However, high ambient temperature per se has been shown to exacerbate the exercise-induced decrease in saliva flow rate. Stimulation of the sympathetic nervous system, for example, by physical or psychological stress, causes vasoconstriction of blood vessels supplying the salivary glands, leading to a reduction in saliva secretion and vasodilatation of vessels due to parasympathetic stimulation results in a marked increase in regional blood flow [15]. Salivary total protein increased after the soccer-specific intermittent exercise protocol during both conditions, likely due to effects of increased β-sympathetic activity on the salivary glands [20]. Salivary IgA to protein ratio decreased significantly after both treatments, a finding which is in line with Mackinnon and Jenkins [33] and Moreira [34]. Salivary IgA concentrations relative to total protein and other proteins accounted for changes in salivary volume which may have occurred due to drying of oral surfaces as a result of exercise [32]. Salivary protein secretion rate also showed an increase after exercise using the present protocol; therefore, expressing salivary IgA concentration relative to salivary total protein concentration may be misleading for reporting salivary IgA after exercise [2, 5, 20, 51]. Expressing salivary IgA relative to osmolality would be a more appropriate correction since the increases were in proportion to the fall in saliva flow rate. However, in the present study, neither osmolality nor salivary IgA relative to osmolality changed significantly after either condition. It has been reported that cortisol concentrations are elevated above normal during a soccer match [12]. Most likely the psychological stress of a real match provides an additional stimulus for cortisol secretion besides the physiological stress of exercise. Performing exercise in hot conditions with elevated salivary cortisol concentration should result in a decrease in salivary IgA


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concentration, since cortisol has been shown to inhibit transepithelial transport of salivary IgA [42], and to have a delayed inhibitory effect on in vivo B lymphocyte antibody synthesis [43]. Nevertheless, a reduction was not observed in salivary IgA concentration after soccer-specific intermittent exercise in either condition due to the non-significant change of salivary cortisol concentration post-exercise. Therefore, a short-term or delayed effect of salivary cortisol at the concentrations noted may not have an inhibitory effect on the transepithelial transport of salivary IgA. In conclusion, these data show that soccer-specific intermittent exercise results in an increase in salivary IgA secretion rate. Additionally these results failed to show that carbohydrate supplementation before and at regular intervals during soccer-specific intermittent exercise in the heat, influenced mucosal immune response significantly in the present subjects.

Acknowledgements ▼ The Tabriz University in the Islamic Republic of Iran supported this study.

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