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00005768-200603000-0001600005768_2006_38_513_bishop_responses_3article< 101_0_17_6 >Medicine & Science in Sports & Exercise©2006The American College of Sports MedicineVolume 38(3)March 2006pp 513-519Salivary IgA Responses to Prolonged Intensive Exercise following Caffeine Ingestion[BASIC SCIENCES: Original Investigations]BISHOP, NICOLETTE C.; WALKER, GARY J.; SCANLON, GABRIELLA A.; RICHARDS, STEPHEN; ROGERS, ELEANORSchool of Sport and Exercise Sciences, Loughborough University, Loughborough, Leicestershire, UNITED KINGDOMAddress for correspondence: Nicolette C. Bishop, School of Sport and Exercise Sciences, Loughborough University, Loughborough, Leicestershire, LE11 3TU, UK; E-mail: N.C.Bishop@lboro.ac.uk.Submitted for publication July 2005.Accepted for publication September 2005.ABSTRACTPurpose: Prolonged, intensive exercise is associated with a reduction in concentration and secretion of salivary IgA (s-IgA). Saliva composition and secretion are under autonomic nervous system control, and caffeine ingestion, a widespread practice among athletes for its ergogenic properties, is associated with increased sympathetic nervous system activation. Therefore, this study investigated the influence of caffeine ingestion on s-IgA responses to prolonged, intensive exercise.Methods: In a randomized crossover design, 11 endurance-trained males cycled for 90 min at 70% V̇O2peak on two occasions, having ingested 6 mg·kg−1 body mass of caffeine (CAF) or placebo (PLA) 1 h before exercise. Whole, unstimulated saliva samples were collected before treatment (baseline), preexercise, after 45 min of exercise (midexercise), immediately postexercise, and 1 h postexercise. Venous blood samples were collected from a subset of six of these subjects at baseline, preexercise, postexercise, and 1 h postexercise.Results: An initial pilot study found that caffeine ingestion had no effect on s-IgA concentration, secretion rate, or saliva flow rate at rest. Serum caffeine concentration was higher on CAF than PLA at preexercise, postexercise, and 1 h postexercise (P < 0.001). Plasma epinephrine concentration was higher on CAF than PLA at pre- and postexercise (P < 0.05). s-IgA concentration was higher on CAF than PLA at mid- and postexercise (P < 0.01), and s-IgA secretion rate was higher on CAF than PLA at midexercise only (P < 0.02). Caffeine ingestion did not affect saliva flow rate. Saliva α-amylase activity and secretion rate were higher on CAF than PLA (main effect for trial, P < 0.05).Conclusions: These findings suggest that caffeine ingestion before intensive exercise is associated with elevated s-IgA responses during exercise, which may be related to increases in sympathetic activation.A fall in salivary immunoglobulin A (s-IgA) concentration and secretion rate has been suggested as a risk factor for subsequent episodes of upper respiratory tract infections (URTI) in athletes (9,13). IgA is the predominant Ig in mucosal secretions and plays a major role in defense against pathogens, allergens, and antigens presented at mucosal surfaces (13). s-IgA is produced by plasma cells adjacent to ducts and acini of salivary glands. It is transported across epithelial cells into the saliva secretions via binding with the polymeric Ig receptor, endocytosis, and vesicular transport to the apical surface, where the polymeric Ig receptor is cleaved and IgA is secreted into the saliva. Therefore s-IgA is thought to provide a first line of defense against microbial pathogens in three ways: through prevention of pathogen adherence and penetration of the mucosal epithelium, by neutralizing viruses within the epithelial cells during transcytosis, and by excretion of locally formed immune complexes across mucosal epithelial cells to the luminal surface (19).A high incidence of infections is reported in individuals with selective deficiency of IgA (17) or poor saliva flow rates (10). Conversely, high levels of s-IgA are associated with low incidence of URTI (25). Acute bouts of high-intensity exercise are associated with decreases in s-IgA concentration, (29), secretion rate (28), and saliva flow rate(2,28,30). Furthermore, exercise-induced decreases in s-IgA concentration may be cumulative because over a 7-month training period, resting levels of s-IgA in a group of elite swimmers fell with each additional month of training, and resting s-IgA concentration was inversely correlated with episodes of URTI (14). More recently it has been reported that a 12-month competitive college football season was associated with a reduction in both resting s-IgA concentration and secretion rate and an increase in incidence of URTI (9). Secretion rate of s-IgA (but not concentration) was found to predict URTI incidence.Saliva secretion is regulated by the autonomic nervous system. The salivary glands are innervated by branches of both the parasympathetic and sympathetic nervous systems. Parasympathetic stimulation elicits a high volume of saliva that is low in protein content. In contrast, saliva elicited by sympathetic stimulation is low in volume and high in protein (6). In the rat model, secretion of IgA can be increased by both parasympathetic and sympathetic nerve stimulation, and epinephrine has recently been shown to increase the transport of human IgA into saliva by rat salivary cells via increased mobilization of the polymeric Ig receptor (3,4). Caffeine ingestion is associated with enhanced sympathetic nervous system activity and increases in plasma epinephrine (16,20) and is widely used among athletes for its ergogenic properties. Therefore, it may be speculated that such a practice may affect salivary responses to exercise. However, to date, any effect of caffeine on saliva flow rate, IgA concentration, and secretion rate during exercise has not been investigated.In addition to s-IgA, salivary α-amylase also plays an important role in the protection of the oral mucosa by inhibiting the adherence and growth of specific bacteria (26). Exercise increases the activity of salivary α-amylase in a manner that is dependent on exercise intensity (2,21). Salivary α-amylase activity is stimulated by increased activity of the sympathetic nervous system with the majority of this protein produced by the parotid gland (27). Because caffeine is a potent stimulator of sympathetic nervous activity, it may be that caffeine ingestion prior to intensive exercise could lead to further elevations salivary α-amylase activity.Therefore, the primary purpose of this study was to investigate the effect of prior caffeine ingestion on s-IgA and flow rate responses to prolonged, intensive exercise. In addition, the effect of caffeine on salivary α-amylase activity was assessed. An initial pilot study was also performed to determine any independent effect of caffeine ingestion on these measures at rest.METHODSSubjects.Eleven endurance-trained males (mean ± SEM: age 23 ± 1 yr; height 176 ± 2 cm; body mass 72.0± 2.6 kg; V̇O2peak 61.6 ± 1.4 mL·kg−1·min−1) volunteered to participate in the exercise trials. 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 study protocol was approved by the local university ethical committee. Subjects were required to complete a comprehensive health-screening questionnaire prior to each visit to the laboratory and did not report any symptoms of infection and had not taken any medication in the 6 wk prior to the study, nor were they currently on medication. Mean (±SEM) habitual caffeine consumption was 146 ± 41 mg·d−1 (range 10-280 mg·d−1), as categorized by their responses to a questionnaire administered at the beginning of the study.Pilot study: saliva responses to caffeine ingestion at rest.The effect of caffeine ingestion on saliva responses is unknown, and in resting subjects s-IgA values are reported to fall during the morning, before reaching a plateau around noon (30). Therefore, to determine the effect of time of day and caffeine ingestion on saliva responses at rest, we had 8 of the 11 subjects from this study reside in the laboratory on two control days, separated by 1 wk. During these visits, in a randomized crossover design, subjects ingested either 6 mg·kg−1 body mass caffeine dissolved in 3 mL·kg−1 body mass of artificially sweetened (aspartame) grapefruit-flavored water, or a placebo that contained the same volume of flavored water containing artificial sweetener but no caffeine. Saliva samples were collected at the same times of day as intended for the main exercise trials. Subjects were instructed to avoid caffeine-containing foods and drinks for 60 h before each visit, avoid alcohol and any strenuous activity during the 24 h before each visit, and eat the same foods and drinks during the 24 h before each visit in an effort to standardize their nutritional status. Subjects reported to the laboratory at 10:00 h following an overnight fast of at least 10 h, and sat quietly for 10 min before an initial baseline saliva sample was collected, as described below. Subjects then ingested either the caffeine or placebo drink, in the volume and composition as described below for the exercise trials. After drink ingestion, subjects sat quietly in the laboratory for a total of 3 h 30 min, with further saliva samples collected 1 h, 1 h 45 min, 2 h 30 min, and 3 h 30 min after drink ingestion; these corresponded to the same times of day that saliva would be collected in the main experimental trials. Mean ± SEM laboratory conditions for the resting control trials were 21.6 ± 0.9°C and 53 ± 3% relative humidity. There were no effects of time of day or caffeine ingestion on s-IgA concentration, secretion rate, or saliva flow rate (Table 1). Saliva α-amylase activity was higher with caffeine ingestion (main effect for trial; F1,7 = 13.5, P < 0.01) but was not affected by time of day. Saliva α-amylase secretion rate was higher 1 h after caffeine ingestion compared with placebo (interaction; F4,28 = 3.8, P < 0.05). Saliva cortisol concentration was unaffected by caffeine ingestion, but decreased over the duration of the trial (main effect for time; F4,24 = 14.3, P < 0.001), reflecting usual diurnal variation (Table 1). Taken together, these pilot data suggest that when collecting samples at these times of day, any effects of caffeine ingestion on s-IgA responses to exercise subsequently observed would not be artefacts of diurnal variations or of any independent effect of caffeine. However, because caffeine ingestion was associated with an increase in salivary α-amylase responses, any subsequent effect of exercise and caffeine on these measures would need to be interpreted with this in mind.TABLE 1. The effect of caffeine (CAF) or placebo (PLA) ingestion on saliva responses at rest.Preliminary exercise testing.Approximately 1 wk before the beginning of the main 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 peak oxygen uptake (V̇O2peak). 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 (Polar Beat, Polar Electro Ltd., Oy, 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 VE, V̇O2, and VCO2. Gas analyzers were calibrated according to the manufacturer's instructions using a zero gas (100% N2), a calibration mix (16% O2 and 4% CO2), and atmospheric air. The work rate equivalent to 70% V̇O2peak was interpolated from the V̇O2-work rate relationship. Subjects then returned to the laboratory 2 d later at a time of their choosing to acquaint themselves with the exercise trials. During this familiarization trial, subjects cycled on an electrically braked cycle ergometer for 90 min at 70% V̇O2peak. Expired air samples were obtained and analyzed as described above after the first 10 min and at 20-min intervals thereafter to ensure that the subjects were exercising at the required intensity. If the subject was exercising at below 70% V̇O2peak or in excess of 75% V̇O2peak, the work rate was adjusted accordingly; this occurred within 5 min of expired air sample collection. Heart rates were monitored throughout this familiarization trial.Preliminary saliva collection.The cotton swab (salivette) method of saliva collection was used because it decreases the possibility of gingival bleeding that is associated with the expectoration method of saliva collection (23). However, some concerns regarding the determination of s-IgA from cotton swab collections have been reported in sample volumes of less than 200 μL and more than 2 mL (22). To overcome these concerns, we established individual flow rate patterns before and during exercise to ensure appropriate volume collection. Therefore, during the familiarization trial saliva samples were also obtained before exercise, after 45 min of exercise, and immediately postexercise using preweighed tubes containing a cotton swab (Sarstedt Ltd, Leicester, UK). Each subject was asked to swallow in order to empty their mouths of saliva, and unstimulated whole saliva samples were collected by placing the cotton roll under the tongue for 2 min. Once collected, the swabs were reweighed to the nearest milligram and saliva volume was calculated from the difference in mass, assuming saliva density to be 1.00g·mL−1 (7). From the volume obtained over 2 min, suitable collection times for the main experimental trials were then determined for each subject to ensure a total collection volume of between 0.5 and 1.5 mL for each sample.Experimental exercise trial procedures.Each subject was given a comprehensive list of caffeine-containing foods and drinks and instructed to abstain from these products during the 60 h preceding each exercise trial. Subjects were also instructed to refrain from alcohol intake and not to participate in any sporting activity during the 24 h preceding each main experimental trial. In an effort to standardize their nutritional status, subjects were asked to eat the same foods and drinks during the 24 h prior to both experimental trials.On two occasions, separated by 1 wk, subjects reported to the laboratory at 10:00 h following an overnight fast of at least 10 h and were randomly assigned to either the caffeine (CAF) or placebo (PLA) trial. Subjects were then required to empty their bladder before body mass (in shorts only) was recorded. Subsequently, subjects sat quietly for 10 min before an initial pretreatment (baseline) saliva sample was collected into preweighed salivettes, as described above. Collection time for each subject was designed to ensure a total saliva sample of between 0.5 and 1.5 mL.Following saliva collection, in the CAF trial subjects were given 6 mg·kg−1 body mass of caffeine dissolved in 3 mL·kg−1 body mass of artificially sweetened (aspartame) grapefruit-flavored water. In the PLA trial, subjects were given the same volume of flavored water containing artificial sweetener but no caffeine. Subjects then rested quietly in the laboratory for 1 h before a preexercise saliva sample was collected. Following this, subjects began cycling on an electromagnetically braked cycle ergometer at 70% V̇O2peak (208 ± 6 W) for 90 min. To standardize fluid intake, subjects consumed a further 2 mL·kg−1 body mass of plain water at 15-min intervals throughout the exercise on both trials. Heart rates were recorded also recorded at these times. Samples of expired air were collected into Douglas bags after 20 min of exercise and every 30 min thereafter for determination of V̇O2 and VCO2 to allow determination of fat and carbohydrate oxidation and energy expenditure using stoichiometric equations (12). Further unstimulated saliva collections were obtained after 45 min of exercise (midexercise) and immediately postexercise. All saliva collections were obtained before any scheduled drink ingestion. A further 3 mL·kg−1 body mass of plain water was ingested at 5 min postexercise. A final saliva sample was collected at 1 h postexercise. No other fluid or food intake was allowed until after this time. For all saliva samples, the sample collection time for each subject varied as determined from saliva volumes obtained during the familiarization trial. Mean ± SEM laboratory conditions were 19.9 ± 0.6°C and 49 ± 5% relative humidity.Saliva analysis.Following collection, swabs were stored in their plastic containers and weighed to the nearest milligram. Saliva volume was estimated from this, as described above. Saliva flow rate was determined by dividing the volume of saliva by the collection time for each sample. The plastic containers and swabs were then stored at −20°C until analysis. After thawing, the swabs were spun at 3000 × g for 5 min at 18°C, and the saliva was subsequently analyzed for s-IgA and α-amylase using ELISA and spectrophotometric methods, respectively, as previously described (21). All samples from one subject were analyzed on the same microplate, and all samples from all subjects were analyzed on the same day. s-IgA secretion rate was calculated by multiplying s-IgA concentration (mg·L−1) by saliva flow rate (μL·min−1) and dividing by 1000 to give s-IgA secretion rate (μg·min−1). The intraassay coefficient of variation was 7.4 and 2.2% for s-IgA and α-amylase, respectively. Additionally, saliva cortisol concentration was determined using a commercially available ELISA (DX-SLV-2930, IDS, Boldon, UK); the intraassay coefficient of variation for this analysis was 1.3%.Plasma catecholamine and serum caffeine analysis.Venous blood samples were obtained from a subset of six subjects in the exercise group. Blood samples were collected into three separate monovette tubes (evacuated blood collection tubes, Sarstedt, Leicester, UK), one containing lithium heparin (1.5 IU heparin per milliliter of blood), one containing K3EDTA (1.6 mg EDTA per milliliter of blood), and one with no additive for the collection of serum; this was allowed to clot for at least 60 min. Blood taken into the K3EDTA monovette (2.7 mL) was used for hematological analysis including hemoglobin and hematocrit; these were used to estimate plasma volume changes according to Dill and Costill (8).Blood collected into the lithium heparin and serum monovettes were centrifuged at 1500 × g for 10 min at 4°C. Of the heparinized plasma obtained, 2 mL was immediately added to chilled tubes containing 200 μL of preservative (pH 6.5) containing EGTA (100 mM) and glutathione (100 mM) for later determination of norepinephrine and epinephrine concentrations. The tubes were mixed and then immediately frozen at −80°C. Plasma levels of catecholamines were determined by high-performance liquid chromatography with electrochemical detection as previously described (11). The intraassay coefficient of variation was 1.9 and 6.6% for norepinephrine and epinephrine, respectively. Serum obtained was also frozen at −80°C before subsequent determination of caffeine concentration using a commercially available spectrophotometric assay (Emit®-caffeine, Dade Behring, Milton Keynes, UK). The intraassay coefficient of variation for this analysis was <1%.Statistical analysis.Data in the text, tables, and figures are presented as mean values and the standard errors of the mean (±SEM). Data were examined using a 2(trial) × 5 (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 Greenhouse-Geisser method of correction. Any significant F-ratios subsequently shown were assessed using Student's paired t-tests, with Holm-Bonferroni adjustments for multiple comparisons applied to the unadjusted P value. Single comparisons between trials for overall exercise intensity, fat and carbohydrate oxidation rates, rate of energy expenditure, and percentage contribution of substrate to energy expenditure were made using Student's paired t-tests. Statistical significance was accepted at P < 0.05. The observed powers of the reported main and interaction effects are all >0.8.RESULTSExercise intensity did not differ between the trials. Mean % V̇O2peak during exercise was 71.8 ± 0.9 and 72.0 ± 0.6% in CAF and PLA, respectively. Likewise, heart rates were similar between trials throughout the exercise (CAF: 157 ± 1 bpm, PLA: 157 ± 2 bpm, mean of all recordings). There were no significant differences in rates of CHO oxidation (CAF: 3.4 ± 0.2 g·min−1, PLA: 3.3 ± 0.2 g·min−1; t = 0.7, P = 0.48) or rates of fat oxidation (CAF: 0.34 ± 0.05g·min−1, PLA: 0.42 ± 0.05 g·min−1; t = 1.36, P = 0.20). Mean rate of energy expenditure was similar in both trials (CAF: 68 ± 2 kJ·min−1, PLA: 67 ± 2 kJ·min−1).After exercise changes in body mass (corrected for fluid intake) were similar in both trials (−2.0 ± 0.9 and −2.1 ± 0.6 kg on CAF and PLA, respectively). There was no significant time × trial interaction for changes in plasma volume relative to the first blood sample; after exercise plasma volume had decreased by 10.4 ± 1.8 and 6.6 ± 2.0% in CAF and PLA, respectively.Serum caffeine concentration was higher in CAF than PLA at preexercise, postexercise, and 1 h postexercise (interaction; F3,12 = 59.1, P < 0.001) (Table 2). Plasma epinephrine concentration was higher in CAF than PLA at pre- and postexercise (interaction; F2,10 = 6.6, P < 0.05) (Table 2). Plasma norepinephrine concentration was unaffected by caffeine ingestion, yet increased above baseline values at pre- and postexercise (main effect for time; F2,10= 87.8, P < 0.001) (Table 2).TABLE 2. The effect of caffeine (CAF) or placebo (PLA) ingestion 1 h before cycling for 90 min at 70% V̇O2peak on serum caffeine and plasma catecholamines concentration.Saliva responses.There was a decrease in saliva flow rate with exercise duration that occurred independently of caffeine ingestion (main effect for time; F4,40 = 5.9, P<0.01) (Fig. 1). At midexercise, s-IgA concentration was approximately 50% higher on CAF than PLA and remained 40% higher at postexercise (interaction; F4,40 = 4.3, P < 0.01) (Fig. 2). At midexercise in CAF, s-IgA secretion rate was almost double that in PLA (interaction; F4,40 = 3.5, P < 0.02), but there was no difference between the trials by postexercise (Fig. 3).FIGURE 1 The effect of caffeine (CAF) or placebo (PLA) ingestion 1 h before cycling for 90 min at 70% V̇O2peak on saliva flow rate. Values are means ± SEM (N = 11). * Main effect for time, P < 0.01.FIGURE 2-The effect of caffeine (CAF) or placebo (PLA) ingestion 1 h before cycling for 90 min at 70% V̇O2peak on s-IgA concentration. Values are means ± SEM (N = 11). * Significantly higher than PLA at that time, P < 0.05; † significantly higher than baseline (within trial), P < 0.05; †† P < 0.01.FIGURE 3-The effect of caffeine (CAF) or placebo (PLA) ingestion 1 h before cycling for 90 min at 70% V̇O2peak on s-IgA secretion rate. Values are means ± SEM (N = 11). * Significantly higher than PLA at that time, P < 0.05.Saliva α-amylase activity was higher in CAF than PLA (main effect for trial; F1,10 = 6.1, P < 0.05). Values were also higher at preexercise, midexercise, and postexercise compared with baseline values (main effect for time; F4,40= 13.2, P < 0.01) (Table 3). Saliva α-amylase secretion rate was higher on CAF than PLA (main effect for trial; F1,10 = 6.4, P < 0.05) and was higher at midexercise and postexercise than at baseline (main effect for time; F4,40 = 6.0, P < 0.05) (Table 3). Saliva cortisol concentration was higher on CAF than PLA (main effect for trial; F1,10 = 5.3, P < 0.05) but was unaffected by trial duration (Table 3).TABLE 3. The effect of caffeine (CAF) or placebo (PLA) ingestion 1 h before cycling for 90 min at 70% V̇O2peak on α-amylase activity, α-amylase secretion rate, and saliva cortisol concentration.DISCUSSIONThese findings suggest that caffeine ingestion 1 h before prolonged intensive exercise is associated with elevations in s-IgA concentration and secretion rate during exercise, but does not affect saliva flow rate. Caffeine ingestion before exercise was also associated with elevations in serum caffeine and plasma epinephrine, but did not affect saliva cortisol responses. In addition, caffeine ingestion was associated with increases in salivary α-amylase, as was observed in resting subjects.It has recently been shown that adrenergic stimuli are strong inducers of s-IgA secretion in rodents, with the secretions from submandibular cells increasing in response to both α-adrenergic and β-adrenergic stimuli and those from parotid cells responding to β-adrenergic stimuli only (4). Furthermore, stimulation with epinephrine has been demonstrated to increase uptake of human IgA by rat salivary cells via increased mobilization of the polymeric Ig receptor (4). In the present study, although plasma norepinephrine concentrations increased independently of caffeine ingestion, plasma epinephrine concentrations on CAF were markedly elevated above those on PLA both before and after exercise. Therefore, it could be speculated that the greater epinephrine response observed here with exercise and caffeine ingestion resulted in increased IgA transcytosis and subsequent secretion, particularly as the saliva collected here was mixed and not from any one isolated gland. An increase in production of IgA from activated plasma cells appears to be unlikely to contribute to these responses because isolated preparations of saliva gland plasma cells have been shown to be unresponsive to adrenergic agonists (4).In contrast to these observations from exercising subjects, the results of the initial pilot study indicated that there was no effect of caffeine ingestion on s-IgA concentration and secretion rate at rest. Unfortunately, we did not determine epinephrine responses in this group, and therefore we are unable to directly relate the pattern or magnitude of any epinephrine response to the s-IgA response within this group. Nevertheless, elevations in α-amylase activity are considered a good indicator of enhanced sympathetic activity and, in particular, sympathetically evoked saliva secretion (1). Furthermore, treatment with β-adrenoreceptor antagonists significantly reduces α-amylase activity in humans (24). In the present study, although caffeine ingestion was associated with elevated α-amylase activity in both exercising and rest groups, mean values were approximately twofold higher in the exercising trials than in the resting pilot study, suggesting greater sympathetic activity with caffeine and exercise. It has been shown that additional sympathetic stimulation at high frequencies is required for the greatest increase in s-IgA secretion in rodents (3). Therefore, it may be speculated that in the present study, the stimulating effects of both exercise and caffeine ingestion together were required increase sympathetic activity to a level sufficient to influence s-IgA mobilization.Although both s-IgA concentration and secretion rate were elevated with caffeine ingestion at midexercise, at postexercise this effect was observed for s-IgA concentration only. Negative associations between resting s-IgA concentration, s-IgA secretion rate, and risk for URTI have been reported in both swimmers and college footballers over a competitive season (9,14). Of these two methods of reporting s-IgA data, secretion rate is thought to give a better indication of protection of mucosal surfaces compared with absolute IgA concentration because it takes saliva flow rate into account and reflects the amount of IgA that is available on the mucosal surface (13,30). Measures of s-IgA secretion rate also become particularly important when making comparisons during exercise when marked decreases in saliva flow rate are commonly reported (2,28,30), and elevations in s-IgA concentration could simply reflect a concentrating effect. However, in the present study, the higher s-IgA concentration observed in CAF than PLA during and after exercise could not be a reflection of altered saliva flow because caffeine ingestion did not influence saliva flow rate during exercise. Nevertheless, even though s-IgA secretion rate was also elevated with caffeine ingestion after 45 min of exercise, it would be inappropriate at this stage to interpret these responses as representing a temporary enhancement of mucosal protection because the clinical significance of these findings is unknown.Although salivary α-amylase principally acts to breakdown starch, it also plays a role in host defense by inhibiting bacterial attachment to the oral mucosa (26). In the present study α-amylase responses were elevated with caffeine ingestion in both the exercise trials and resting pilot study. Nevertheless, it must be acknowledged that any clinical relevance of these findings is unknown. Furthermore, the effects observed in this study were short-lived and were achieved using a large dose of caffeine. Although ergogenic benefits of caffeine have been reported at doses of half that used here (15), at the present time we do not know whether the positive effects of caffeine on s-IgA and α-amylase responses would still be apparent at doses of 3mg·kg−1 body mass or with the amounts found in caffeine-containing beverages.Saliva secretion is principally under parasympathetic control. However, the decrease in saliva flow rate observed during exercise is thought to be at least partly the result of increased sympathetic activity leading to vasoconstriction of blood vessels supplying saliva glands, decreasing saliva secretion (6). Additional sympathetic stimulation, for example through caffeine ingestion, may therefore be expected to cause further reductions in saliva flow rate. However, in this study, saliva flow rate decreased during exercise but was unaffected by caffeine ingestion. It may be that α-adrenergic stimulation, rather than β-adrenergic activity, plays a greater role in the control of saliva flow during exercise because plasma levels of norepinephrine increased similarly with exercise on both trials but were unaffected by caffeine ingestion. The finding that treatment with β-adrenoreceptor antagonists has little effect on saliva flow rate in humans lends further support to this idea (24).Ingesting caffeine prior to acute, strenuous exercise has been associated with an elevated cortisol response (20), and an inverse relationship between levels of salivary cortisol and s-IgA has been demonstrated in young adults (18). Thus it may be expected that caffeine ingestion would be associated with increased salivary cortisol levels and lower s-IgA responses. In this study, although salivary cortisol values were higher in CAF than PLA, this was not associated with any decrease in s-IgA concentration or secretion rate. Nevertheless, it is still possible that cortisol acted on s-IgA responses, but any effect may have been masked by the more powerful effect of increased sympathetic activity.In conclusion, these data show that ingesting caffeine 1 h before performing 90 min of strenuous exercise results in temporary elevations in s-IgA concentration and secretion rate that do not occur when caffeine is ingested without subsequent exercise. These data also show that caffeine ingestion increases in α-amylase responses. Taken together, these findings may suggest that, in addition to its well-known ergogenic properties, large doses of caffeine can enhance measures of mucosal immune function, perhaps via increased sympathetic activity. 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Research10.1519/JSC.0b013e3181c7c2992010243846-851MAR 2010Cystine and Theanine Supplementation Restores High-Intensity Resistance Exercise-Induced Attenuation of Natural Killer Cell Activity in Well-Trained MenKawada, S; Kobayashi, K; Ohtani, M; Fukusaki, Chttp://journals.lww.com/nsca-jscr/Fulltext/2010/03000/Cystine_and_Theanine_Supplementation_Restores.34.aspx120disablehttp://dx.doi.org/10.1519%2fJSC.0b013e3181c7c299InternalMedicine & Science in Sports & Exercise10.1249/MSS.0b013e31816be9c320084071228-1236JUL 2008Salivary IgA as a Risk Factor for Upper Respiratory Infections in Elite Professional AthletesNEVILLE, V; GLEESON, M; FOLLAND, JPhttp://journals.lww.com/acsm-msse/Fulltext/2008/07000/Salivary_IgA_as_a_Risk_Factor_for_Upper.5.aspx265http://pdfs.journals.lww.com/acsm-msse/2008/07000/Salivary_IgA_as_a_Risk_Factor_for_Upper.00005.pdfhttp://dx.doi.org/10.1249%2fMSS.0b013e31816be9c3