The effect of physical exercise on salivary secretion of MUC5B ...

Archives of Oral Biology 60 (2015) 1639–1644

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The effect of physical exercise on salivary secretion of MUC5B, amylase and lysozyme Antoon J.M. Ligtenberg* , Henk S. Brand, Petra A.M. van den Keijbus, Enno C.I. Veerman Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, Amsterdam, The Netherlands

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 January 2015 Received in revised form 4 June 2015 Accepted 13 July 2015

Objectives: Saliva secretion is regulated by the autonomic nervous system. Parasympathic stimuli increase the secretion of water and mucin MUC5B, whereas sympathetic stimuli such as physical exercise increase the secretion of amylase and other proteins. In the present study we investigated the effect of physical exercise, as a sympathetic stimulus, on salivary flow rate and output of MUC5B, amylase, lysozyme and total protein. Design: Unstimulated whole saliva was collected before exercise (1), after 10 min exercise with moderate intensity by running with a heart rate around 130 beats per minute (2), followed by 10 min exercise with high intensity by running to exhaustion (3) and after 30 min recovery (4). Salivary flow rate, protein and MUC5B concentration, and amylase and lysozyme activity were determined. Saliva protein composition was analysed using SDS-PAGE and immunoblotting. Results: Salivary flow rate, protein and lysozyme secretion increased after exercise with moderate intensity and increased further after exercise with high intensity (p < 0.01). Amylase and MUC5B increased after exercise with moderate intensity (p < 0.0001), but did not differ significantly between moderate and high exercise intensity. SDS-PAGE analysis and immunoblotting showed that, especially after exercise with high intensity, the concentrations of several other salivary proteins, including MUC7, albumin, and extra-parotid glycoprotein, also increased. Conclusions: Exercise may not only lead to the anticipated increase in amylase and protein secretion, but also to an increase in salivary flow rate and MUC5B secretion. ã 2015 Elsevier Ltd. All rights reserved.

Keywords: Mucins Amylase Sympathetic stimulation Anaerobic threshold

1. Introduction Saliva secretion is controlled by the autonomic nervous system, with sympathetic activation resulting in the secretion of proteinrich saliva whereas parasympathetic activation results in the secretion of water and mucins (Garrett, 1987; Proctor & Carpenter, 2007). The sublingual and minor salivary glands are mainly innervated parasympathetically, and these glands produce most of the lysozyme and MUC5B, the high molecular weight mucin responsible for the viscosity of saliva (Proctor and Carpenter, 2007; Bosch, Veerman, de Geus, & Proctor, 2011; Veerman, van den Keybus, Vissink, & Nieuw Amerongen, 1996). The parotid and submandibular glands are innervated both sympathetically and parasympathetically. The submandibular glands also produce mucins, but the parotid glands secrete serous saliva without

* Corresponding author at: Department of Oral Biochemistry, Academic Centre for Dentistry Amsterdam, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands. Fax: +31 20 5980333. E-mail address: [email protected] (A.J.M. Ligtenberg). http://dx.doi.org/10.1016/j.archoralbio.2015.07.012 0003-9969/ ã 2015 Elsevier Ltd. All rights reserved.

mucins that is characterized by high concentrations of amylase (Veerman et al., 1996). Saliva composition depends on the relative contribution of the different salivary glands. Therefore, secretion and composition of whole saliva show large inter- and intraindividual variations. Physical exercise is a strong activator of the sympathetic nervous system (Paterson, 1996), which may affect saliva composition. Especially above the anaerobic threshold, the exercise intensity above which the blood lactate concentration increases exponentially, the concentrations of a number of salivary constituents, including electrolytes, lactate, catecholamine, amylase, lysozyme, lactoferrin, LL-37, defensin HNP1-3 and chromogranin A increase (Calvo et al., 1997; Chicharro, Legido, Alvarez, Serratosa, Bandres, & Gamella, 1994; Davison, Allgrove, & Gleeson, 2009; Lehmann, Schmid, & Keul, 1985; West, Pyne, Kyd, Renshaw, Fricker, & Cripps, 2010). During exercise the viscosity of saliva increases (Dawes, 1981). Salivary viscosity is determined by the concentration of the high molecular weight mucin MUC5B (Thornton, Rousseau, & McGuckin, 2008). Increases in viscosity may be due to systemic

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(Navazesh, 1993). Before start of the saliva collection, subjects were instructed to swallow the residual saliva in their mouth. Unstimulated whole saliva was collected by expectoration every 30 s during 2 min. Immediately after collection salivary pH was measured with indicator strips ranging from pH 6.5–10.0 and pH 4.0–7.0 (Merck, Darmstadt, Germany). Saliva volume was measured gravimetrically, assuming that 1 ml saliva equals 1 g. Hereafter, an equal volume of 150 mM NaCl solution was added, and the saliva was homogenized for 1 min on a Vortex mixer (Snijders analysers, Tilburg, Netherlands). Saliva was centrifuged for 5 min at 10,000 g and the supernatant was stored at 20 C.

dehydration after prolonged exercise, which leads to a reduced salivary flow with a concomitant increase in protein and MUC5B concentration (Ljungberg, Ericson, Ekblom, & Birkhed, 1997). Heavy mouth breathing during exercise may also lead to enhanced evaporation and subsequently to elevated concentrations of proteins and MUC5B (Walsh, Montague, Callow, & Rowlands, 2004). Another possibility, which has not been studied before, is that exercise may increase the secretion of salivary mucins. In the present study the effect of exercise on salivary secretion of MUC5B, total protein, amylase and lysozyme was studied. For this purpose volunteers ran for 10 min at moderate and high exercise intensity after which saliva samples were collected and analysed.

2.4. Protein concentration 2. Materials and methods Protein concentration was measured by the bicinchoninic acid method with the Pierce BCA assay (Thermo Scientific, Rockford, IL) (Smith et al., 1985). Bovine serum albumin (0–1.5 mg/ml) was used as a standard. Saliva was diluted two-, four- and eightfold in 150 mM NaCl solution. 20 ml of each dilution was added in duplicate to Greiner non affinity microtiterplates (Greiner Bio-One, Frickenhausen, Germany). Subsequently, 180 ml bicinchoninic acid reagent was added and incubated for 30 min at 37 C. Colour development was measured with an ELISA reader (Thermo Scientific) at 570 nm.

2.1. Participants Twenty-nine volunteers were recruited among the students and staff members of the Academic Centre for Dentistry Amsterdam (age 21.6 1.5 years). All volunteers, 12 males and 17 females, were non-smokers with no oral or systemic disease and not taking medication, except for oral contraceptives. The experiment was performed according to the guidelines of the Medical Ethical Committee of the VU University Medical Center (protocol OB-96-01). All volunteers provided written informed consent.

2.5. Amylase and lysozyme activity

2.2. Experimental design

Amylase activity was determined with the EnzChek Amylase kit (Molecular Probes, Leiden, The Netherlands). This kit contains a starch derivative that is labeled with a dye to such a degree that fluorescence is quenched. Hydrolysis by amylase abolishes this quenching yielding fluorescent fragments. The accompanying increase in fluorescence is proportional to amylase activity and was monitored with a fluorescence microplate reader (Fluostar, Galaxy, BMG laboratories, Offenburg, Germany) at 485 nm excitation wavelength and 520 nm emission. The following adaptations were made to the protocol of the manufacturer: human salivary amylase (Fluka, Buchs, Germany) was used as control enzyme in concentrations of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 U/ml to produce a calibration curve. 0.1% BSA was added to the provided reaction buffer. The saliva samples were used in a dilution of 1:100,000. The reaction was measured every minute for 18 min. Enzyme activity per minute was determined using the calibration curve. Amylase activity was expressed in units per ml (U/ml). Lysozyme activity was measured using an EnzChek Lysozyme Activity kit (ThermoScientific, West Palm Beach, U.S.A.) according to the manufacturer’s specifications.

Exercise was performed by running outdoors at a temperature between 7 and 15 C. Saliva was collected at four different time points. Participants walked slowly to the running course, where the first saliva sample was collected (point 1). Hereafter, they performed 10 min of exercise at moderate intensity by jogging with a heart rate around 130 beats per minute, after which the second saliva sample was collected (moderate exercise intensity, point 2). Then participants performed exercise at high intensity by running for 10 min as fast as they could. Hereafter, the third saliva sample was collected (high exercise intensity, point 3). Participants slowly walked back to the laboratory where the fourth saliva sample was collected after 30 min (point 4). At each time point, the heart rate was determined manually at the radial artery. 2.3. Saliva collection and handling The experiment was performed between 9.00 h and 11.00 h a.m. to exclude circadian variations in salivary flow rate and composition (Dawes & Ong, 1973). All saliva samples of a participant were collected at the same day thus excluding daily variations (Dawes & Ong, 1973). Since saliva composition may change during collection, with a higher protein concentration in the first minute, for all collections time was set constant at two minutes (Dawes, 1981). The participants were instructed not to eat at least 1 h before the experiment (Hoek, Brand, Veerman, & Nieuw Amerongen, 2002). Unstimulated saliva was collected as described by Navazesh

2.6. MUC5B concentration MUC5B concentration was determined by ELISA using the monoclonal antibody 5G2, as described previously (Veerman et al., 1997). Resting saliva of one person was used as a standard. The MUC5B concentration of the saliva samples was expressed as a percentage of the standard sample.

Table 1 Effect of exercise intensity on heart rate and salivary parameters. Data are presented as mean SD (n = 29).

Heart rate (beats/min)*** Salivary pH* Saliva secretion rate (ml/min)**

Before exercise

Moderate exercise intensity

High exercise intensity

30 min recovery

80 16 6.9 0.3 0.62 0.24

131 17 a 7.0 0.3 a 0.78 0.38

178 21 ab 7.2 0.4 a 0.94 0.75

80 16 bc 7.2 0.4a 0.60 0.26bc

*

**

a

***

Friedman test for exercise related differences: p < 0.01, p < 0.001; p < 0.0001. Wilcoxon signed-ranks test p < 0.05: a vs before exercise, b vs moderate exercise intensity and

c

vs high exercise intensity.

a


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Table 2 Effect of exercise intensity on salivary protein concentrations and enzyme activities. Data are presented as mean SD (n = 29).

Protein concentration (mg/ml)** Amylase activity (units/ml)** Lysozyme activity (units/ml)*** MUC5B concentration (units/ml)***

Before exercise



1518 582

1653 707

2244 1145

86.9 50.1 3512 3160 4.16 4.75

93.8 52.0 4547 3854a 8.80 14.61a

112.9 67.9 6901 5605ab 9.86 10.55ab

Friedman test for exercise related differences: *p < 0.01; **p < 0.001; ***p < 0.0001. Wilcoxon signed-ranks test p < 0.05: a vs before exercise, b vs moderate intensity exercise and

c

ab

30 mins recovery 1604 682

c

64.8 35.5abc 3498 4053bc 4.65 6.32bc

vs high intensity exercise.

Table 3 Effect of exercise intensity on relative changes in protein concentrations and in enzyme activities in saliva. Protein values before exercise were set at 100%. Data are presented as mean percentage SD (n = 29).

Relative Relative Relative Relative

protein concentration (%) amylase activity (%) lysozyme activity (%) MUC5B concentration (%)

Before exercise



30 min recovery

100 100 100 100

114 31 118 45 153 98 145 70

156 62 174 139 309 292 377 314

109 27 86 38 102 46 129 113

Table 4 Effect of exercise intensity on secretion rate of salivary proteins. Data are presented as mean SD (n = 29).

Protein secretion rate (mg/min)*** Amylase secretion rate (Units/min)*** Lysozyme secretion rate (U/min.)*** MUC5B secretion rate (U/min.)***

Before exercise



30 recovery min

924.1 437.1 53.1 37.0 2373 2389 2.19 1.73

1243.5 656.8a 69.6 47.1a 3578 3116a 5.02 5.56a

1899.0 1557.8ab 97.2 91.2a 5353 3297ab 6.96 5.65a

898.5 399.2bc 38.2 26.2abc 2041 2606bc 2.18 2.59bc

Friedman test for exercise related differences: * p < 0.01; **p < 0.001; *** p < 0.0001. Wilcoxon signed-ranks test P < 0.05: a vs before exercise, b vs moderate intensity exercise and

c


Table 5 Effect of exercise intensity on the specific concentration or activity of salivary proteins. Data are presented as mean SD (n = 29).

Amylase activity (U/mg protein)* Lysozyme activity (U/mg protein)*** MUC5B concentration (U/mg protein)*

Before exercise



30 min recovery

58 28 2.38 1.92 2.77 3.49

59 23 2.52 1.54 4.07 5.01a

55 29 3.03 1.54ab 4.48 4.99a

43 22abc 2.13 1.88c 2.73 3.47bc

Friedman test for exercise related differences: * p < 0.01; **p < 0.001; *** p < 0.0001. Wilcoxon signed-ranks test p < 0.05: a vs before exercise, b vs moderate intensity exercise,

2.7. Protein secretion rate and specific protein concentrations The secretion rate of a specific protein was calculated by multiplying the saliva flow rate by the concentration of the analyte and expressed in mg/min or Units/min. The specific activity or concentration was defined as the activity or concentration of an analyte relative to the total protein concentration and was expressed as U/mg protein. 2.8. Gelelectrophoresis Electrophoresis of saliva was performed on NuPAGE 4–12% BIS/ TRIS gels (Life technologies, Carlsbad, CA, USA). Samples were prepared by boiling for 5 min in NuPAGE LDS sample buffer (Life technologies). After electrophoresis, gels were stained with Coomassie Phastgel Blue R-350 dye (GE Healthcare, Diegem, Belgium) and destained in 10% (v/v) acetic acid. Carbohydrates were stained with periodic acid and Schiff reagent according to the following procedure. After electrophoresis, gels were incubated in 20% trichloroacetic acid for 30 min at room temperature. After extensive washing in water gels were incubated in 2% periodic acid in 10% acetic acid for 30 min at room temperature. Hereafter gels

c


were washed in 10% acetic acid and incubated for 10 min in Schiff reagent (Merck), followed by destaining in 10% acetic acid. 2.9. Western blotting After SDS-PAGE, proteins were transferred to nitrocellulose with an iBlot system (Life technologies). Hereafter, membranes were blocked by incubation in PBS-tween20 (PBST) with 1% gelatine. All incubations were performed in PBST with 1% gelatine and between incubations membranes were washed three times in PBST. MUC7 was identified with a polyclonal rabbit antibody developed at our laboratory (Bolscher et al., 1999) and alkaline phosphatase-conjugated goat-anti-rabbit antibodies (DAKO, Glostrup, Denmark). Extra-parotid glycoprotein was identified with mouse monoclonal antibody 7E5 and horse radish peroxidase conjugated rabbit anti-mouse antibodies (DAKO). Albumin was identified with a polyclonal rabbit antibody (DAKO) and horse radish peroxidase-conjugated goat anti-rabbit antibodies. Lysozyme was identified with a polyclonal rabbit antibody (DAKO) and an alkaline phosphatase conjugated goat-anti-rabbit antibody (DAKO). For visualisation blots with alkaline phosphatase were stained with 5-bromo-4-choro-3 indolyl phosphate (Roche

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Fig. 1. Effect of exercise on salivary proteins. Saliva samples of 4 different individuals were collected at rest (1), after moderate exercise intensity (2), high exercise intensity (3) and 30.

Diagnostics, Mannheim, Germany) and blots with horse radish peroxidase were stained with the visualisation kit SuperSignal Westfemto1 (Thermo Scientific, Rockford IL, USA).

Salivary pH increased with exercise and remained elevated after 30 min recovery. 3.2. Protein concentrations

2.10. Statistical analysis Statistical analysis was performed using the statistical software package PASW Statistics 18.0.2 for Windows (IBM SPSS, Chicago, USA). For overall analysis the Friedman test was used, followed by Wilcoxon signed-ranks tests as posthoc procedure for pairwise comparisons. All levels of significance were set at p < 0.05. 3. Results 3.1. Physiologic parameters The effect of exercise on heart rate, salivary secretion and pH is presented in Table 1. Heart rate increased significantly with increasing exercise intensity and returned to baseline values after 30 min recovery. Saliva secretion also increased significantly with exercise and returned to baseline values after 30 min recovery.

Salivary protein concentration and amylase activity both increased with exercise, but the observed patterns of increase were not similar (Table 2). The protein concentration showed a slight non-significant increase with moderate exercise intensity compared to baseline values, followed by a strong significant increase for high exercise intensity. The amylase activity showed a gradual non-significant increase both at moderate and high exercise intensity. After 30 min recovery, however, amylase activity was significantly reduced compared to baseline values and values obtained during exercise. Salivary MUC5B concentration and lysozyme activity both increased significantly with moderate exercise intensity and showed a further significant increase with high exercise intensity. Protein concentrations and enzyme activities showed large individual variations at baseline resulting in high standard deviations. Therefore, to investigate the effect of exercise on protein concentrations and enzyme activities, the data were also


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4. Discussion

Fig. 2. Effect of exercise on MUC7, albumin, extra-parotid glycoprotein and lysozyme. Saliva samples of individual d in Fig. 1 were applied on 4–12% NuPAGE Bis-Tris gels, transferred to nitrocellulose and developed with specific antibodies.

expressed as relative changes compared to baseline values (Table 3). This reduced the standard deviations for the protein concentrations, but the standard deviations of amylase activity, lysozyme activity and MUC5B concentrations remained high. 3.3. Protein secretion rate and specific concentrations The secretion rate of all the proteins increased significantly, both with moderate and high exercise intensity (Table 4). To investigate whether any of the proteins was specifically enhanced, the specific values per mg of total protein was calculated (Table 5). Specific amylase activity did not change significantly with exercise, but decreased significantly after 30 min recovery. The specific MUC5B concentration and lysozyme activity increased with both moderate and high exercise intensity. 3.4. SDS-PAGE and western blotting To explore further the effect of exercise on salivary protein composition, saliva samples were analyzed with SDS-PAGE. Fig. 1 shows representative SDS-PAGE profiles of saliva samples of four different individuals collected at the four different time points. In addition to the increases in amylase and mucin concentrations, several other salivary proteins increased in concentration during exercise. Salivary samples taken before exercise, after moderate exercise intensity and after recovery showed relatively little variation in protein profiles, but major changes were observed after high exercise intensity. At this time point, the concentrations of MUC7, albumin, extra-parotid glycoprotein and lysozyme were increased (Fig. 2).

The results of this study show that physical exercise leads to an increase in salivary secretion and increased secretion of protein, amylase, lysozyme and MUC5B. The increase in salivary secretion in the present study is in contrast to previous studies that reported a decreased or unaltered salivary secretion after exercise (Bishop, Blannin, Armstrong, Rickman, & Gleeson, 2000; Chicharro, Lucia, Perez, Vaquero, & Urena, 1998; Ljungberg et al., 1997). A decrease in salivary secretion may be caused by dehydration after prolonged exercise as has been observed after marathon running and 2 h of cycling (Ljungberg et al., 1997; Bishop et al., 2000). In our experimental set up, the volunteers practiced for only 20 mins at an average temperature of 10 C. Under these circumstances dehydration is expected to be limited. The relatively low temperature during the physical exercise may have been a cold stimulus to the salivary glands, which might explain the increase in the salivary secretion (Kavanagh, O’Mullane, & Smeeton, 1998). As in most other studies dealing with the effects of exercise on saliva secretion, also in this study saliva was collected under nonstimulating conditions (Davison et al., 2009; Mckune, Bach, Semple, & Dyer, 2014; Cullen, Thomas, Webb, & Hughes, 2015). In this way the observed effects are only caused by the physical exercise, and are not superimposed on those of the mechanical stimulus or flavor (Neyraud, Sayd, Morzel, & Dransfield, 2006). The average secretion rate we found for unstimulated saliva collected before exercise (0.62 ml/min) is within the range of what other exercise experiments have reported for unstimulated saliva collected before exercise. (0.38–0.75 ml/min) (Davison et al., 2009; Kunz et al., 2015; Allgrove, Oliveira, & Gleeson, 2014; Laing, Gwynne, Blackwell, Williams, Walters, & Walsh, 2005). We observed a moderate increase in protein concentration after aerobic exercise and a strong increase after intense exercise (Table 2). These data confirm the results of Bortolini et al., (Bortolini, De Agostini, Reis, Lamounier, Blumberg, & Espindola, 2009) who showed that the salivary protein concentration sharply increases above the anaerobic threshold. This may be explained by direct sympathetic stimulation of the salivary glands by plasma catecholamines that strongly increase above the anaerobic threshold (Green & Hughson, 1985). de Oliveira, Bessa, Lamounier, de Santana, de Mello, and Espindola (2010) suggested that the increased protein concentration above the anaerobic threshold was solely due to an increased secretion of amylase. Our data and other reports show that the increase is not limited to amylase, but that other protein concentrations also increase during exercise (Calvo et al., 1997; Davison et al., 2009; Allgrove, Gomes, Hough, & Gleeson, 2008). An important difference with our study is that Oliveira and co-workers used chewing stimulated saliva which increases the contribution of amylase-rich parotid saliva (Bosch et al., 2011; Veerman et al., 1996), while we refrained from mechanical stimulation of the saliva secretion. Strenuous exercise is associated with immune suppression and a higher incidence of upper respiratory tract infections in athletes (Nieman, 1994; Gleeson & Pyne, 2000). In the present study, however, secretion of antimicrobial proteins like amylase and lysozyme was increased after high exercise intensity. Thirty minutes after exercise a significant decrease in amylase activity was observed and also MUC7 concentration seemed to be lower than before exercise (Fig. 2) (Schenkels, Walgreen-Weterings, Oomen, Bolscher, Veerman, & Nieuw Amerongen, 1997; Lis, Liu, Barker, Rogers, & Bobek, 2010). The increased susceptibility for upper respiratory tract infections after intense exercise might be due to a reduced immune defense in the period after exercise. MUC5B increases with exercise intensity (Table 2), just like the total protein concentration. This is counterintuitive as MUC5B is considered to be secreted primarily by the sublingual glands that

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are parasympathetically innervated, whereas intense exercise is a strong sympathetic inducer (Proctor & Carpenter, 2007; Veerman et al., 1996; Nater & Rohleder, 2009). The increase in salivary viscosity after exercise is usually explained by a decreased saliva secretion or evaporation of water during heavy mouth breathing (Walsh et al., 2004; Chicharro et al., 1998). An increased secretion of MUC5B during exercise provides an alternative explanation. The increased viscosity may hamper oral clearance thereby increasing the susceptibility to upper airway infection (Nieman, 1994; Bardon, Ceder, & Kollberg, 1983; Walsh et al., 2011a,b). On the other hand, MUC5B plays an important role in airway defense (Roy et al., 2014). In conclusion, high intensity exercise not only leads to the anticipated increase in protein secretion, but also to an increase in salivary flow and MUC5B secretion. The increased MUC5B secretion might explain the increased salivary viscosity after exercise and thereby play a role in the increased susceptibility of athletes to upper respiratory tract infections. Acknowledgments We thank Amanda Laret, Yonne Geldof, Sarah Said Afellad and Francis Verburg for their assistance in the collection and biochemical analysis of the saliva samples. This study was financed by the Dutch Research School for Dentistry. References Allgrove, J. E., Gomes, E., Hough, J., & Gleeson, M. (2008). Effects of exercise intensity on salivary antimicrobial proteins and markers of stress in active men. Journal of Sports Sciences, 26(6), 653–661. Allgrove, J. E., Oliveira, M., & Gleeson, M. (2014). Stimulating whole saliva affects the response of antimicrobial proteins to exercise. Scandinavian Journal of Medicine & Science in Sports, 24(4), 649–655. Bardon, A., Ceder, O., & Kollberg, H. (1983). Cystic fibrosis-like changes in saliva of healthy persons subjected to anaerobic exercise. Clinica Chimica Acta, 133(3), 311–316. Bishop, N. C., Blannin, A. K., Armstrong, E., Rickman, M., & Gleeson, M. (2000). Carbohydrate and fluid intake affect the saliva flow rate and IgA response to cycling. Medicine & Science in Sports & Exercise, 32(12), 2046–2051. Bolscher, J. G. M., Groenink, J., van der Kwaak, J. S., van den Keijbus, P. A. M., van't Hof, W., Veerman, E. C. I., & Nieuw Amerongen, A. V. (1999). Detection and quantification of MUC7 in submandibular, sublingual, palatine, and labial saliva by anti-peptide antiserum. Journal of Dental Research, 78(7), 1362–1369. Bortolini, M. J., De Agostini, G. G., Reis, I. T., Lamounier, R. P., Blumberg, J. B., & Espindola, F. S. (2009). Total protein of whole saliva as a biomarker of anaerobic threshold. Research Quarterly for Exercise and Sport, 80(3), 604–610. Bosch, J. A., Veerman, E. C. I., de Geus, E. J., & Proctor, G. B. (2011). Alpha-amylase as a reliable and convenient measure of sympathetic activity: don’t start salivating just yet!. Psychoneuroendocrinology, 36(4), 449–453. Calvo, F., Chicharro, J. L., Bandres, F., Lucia, A., Perez, M., Alvarez, J., Mojares, L. L., Vaquero, A. F., & Legido, J. C. (1997). Anaerobic threshold determination with analysis of salivary amylase. Canadian Journal of Applied Physiology, 22(6), 553– 561. Chicharro, J. L., Legido, J. C., Alvarez, J., Serratosa, L., Bandres, F., & Gamella, C. (1994). Saliva electrolytes as a useful tool for anaerobic threshold determination. European Journal of Applied Physiology and Occupational Physiology, 68(3), 214– 218. Chicharro, J. L., Lucia, A., Perez, M., Vaquero, A. F., & Urena, R. (1998). Saliva composition and exercise. Sports Medicine, 26(1), 17–27. Cullen, T., Thomas, A. W., Webb, R., & Hughes, M. G. (2015). The relationship between interleukin-6 in saliva, venous and capillary plasma, at rest and in response to exercise. Cytokine, 71(2), 397–400. Davison, G., Allgrove, J., & Gleeson, M. (2009). Salivary antimicrobial peptides (LL-37 and alpha-defensins HNP1-3), antimicrobial and IgA responses to prolonged exercise. European Journal of Applied Physiology, 106(2), 277–284. Dawes, C., & Ong, B. Y. (1973). Circadian rhythms in the flow rate and proportional contribution of parotid to whole saliva volume in man. Archives of Oral Biology, 18(9), 1145–1153. Dawes, C. (1981). The effects of exercise on protein and electrolyte secretion in parotid saliva. The Journal of Physiology, 320, 139–148. Garrett, J. R. (1987). The proper role of nerves in salivary secretion: a review. Journal of Dental Research, 66(2), 387–397. Gleeson, M., & Pyne, D. B. (2000). Exercise effects on mucosal immunity. Immunology & Cell Biology, 78(5), 536–544.

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