- Email: [email protected]
Effects of environmental temperature on saliva flow rate and secretion of protein, amylase and mucin 5B
Archives of Oral Biology 109 (2020) 104593
Contents lists available at ScienceDirect
Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio
Effects of environmental temperature on saliva flow rate and secretion of protein, amylase and mucin 5B
T
A.J.M. Ligtenberg*, M. Meuffels, E.C.I. Veerman Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam, the Netherlands
A R T I C LE I N FO
A B S T R A C T
Keywords: Heat Viscosity Amylase saliva secretion
Objective: The aim of this study is to evaluate the short term effects of environmental temperature on saliva flow rate and composition. Methods: In a cross-over study design 20 subjects (18–25 years old, 14 women, 6 men) were exposed in randomized order at different days to three temperatures (4 °C, 21 °C and 37 °C). Five minutes after a subject was exposed to the test temperature, collection of resting saliva was started for 5 min at the same temperature. Saliva flow rate, pH, viscosity, protein concentration, mucin 5B concentration and amylase activity were measured. Results: Exposure to 4 °C resulted in an increase of the saliva flow rate (p < 0.01), protein output and amylase output (p < 0.001) compared to exposure to 21 °C or 37 °C. Although the figures for mucin 5B output at 4 °C were higher than at higher temperatures, this was not significant. There were no significant differences in the salivary mucin 5B concentration and viscosity between saliva samples collected at the indicated temperatures. Conclusions: Lowering of the temperature induces an increase in saliva flow rate, as well as protein and amylase output.
1. Introduction Saliva is important for oral health. It plays a major role in lubrication, taste experience, moistening and protection of the oral mucosa against infection and the protection of teeth against abrasion. Low saliva flow rate may lead to problems such as dental caries and disturbed taste experience and is usually accompanied by an unpleasant sensation of dry mouth, called xerostomia (Dawes et al., 2015). Dehydration has been indicated as one of the causes of a diminished salivary flow and the subsequent development of xerostomia (Brunstrom, Tribbeck, & MacRae, 2000; Ship & Fischer, 1997). The feeling of a dry mouth increases the tendency to drink and functions as a thirst signal (Brunstrom, 2002). As a consequence, people with xerostomia have the urge to drink, even when the body is maximally hydrated (Brunstrom, 2002). On the other hand, in a cold environment the thirst feeling is attenuated, even when the body is in need of rehydration (Kenefick, St Pierre, Riel, Cheuvront, & Castellani, 2008; Shannon, 1966). Dehydration may therefore be a problem during long term exposure to low temperatures (O’Brien, Young, & Sawka, 1998). The reason for the attenuated thirst feeling at low temperatures is not known, but in view of the role played by the mouth in the perception of thirst, it is possible that temperature-dependent changes in saliva secretion may be one of
the factors underlying this phenomenon. That the environmental temperature may have an impact on the secretion rate of saliva has been shown in previous studies (Dawes, 1975; Louridis, Demetriou, & Bazopoul, 1970; Shannon, 1966). Secretion of parotid saliva was higher in December/January, with an average outdoor temperature of 10 °C, than in June/July, when the outdoor temperature was 30 °C (Shannon, 1966). The lower secretion rate was attributed to dehydration in the summer months. Seasonal differences in flow rate for parotid and submandibular/sublingual secretions were found when saliva was collected in rooms that reflected the outdoor temperatures of 16 vs. 27.2 °C (Elishoov, Wolff, Kravel, Shiperman, & Gorsky, 2008). This difference disappeared when subjects collected saliva in temperature-adjusted rooms of 21.7 and 24.8 °C suggesting that there is a direct effect of the temperature on the saliva flow rate. In the present study we have examined the influence of temperature on the flow rate and composition of saliva. Participants were exposed to temperatures of 4 °C, 21 °C and 37 °C, and saliva was collected. Saliva flow rate, pH, protein, amylase, mucin 5B (MUC5B) and viscosity were determined. Flow rate and the output of protein and amylase increased with decreasing temperature.
⁎ Corresponding author at: Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Gustav Mahlerlaan 3004, 1081 LA Amsterdam, the Netherlands. E-mail address: [email protected] (A.J.M. Ligtenberg).
https://doi.org/10.1016/j.archoralbio.2019.104593 Received 16 July 2019; Received in revised form 22 October 2019; Accepted 23 October 2019 0003-9969/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Archives of Oral Biology 109 (2020) 104593
A.J.M. Ligtenberg, et al.
Table 1 Effect of different room temperatures on the different saliva parameters. Saliva was collected at 4 °C, 21 °C and 37 °C. The mean, median values and interquartile range (IQR) of the different parameters are shown respectively. Significance was tested using the Friedman test for related samples and the Wilcoxon test for related samples as post-hoc procedure. Room temperature
4 °C
Saliva flow rate (ml/min)** pH Protein concentration (μg/ml)***(a,c) Protein-output (μg/min)***(a,b,c) Mucin concentration (units/ml) Mucin-output (units/min) Amylase activity (units/ml)***(a,b,c) Amylase-output (units/min)***(a,b,c) Viscosity (mP/s) (b,c)
21 °C
37 °C
Mean
Median (IQR)
Mean
Median (IQR)
Mean
Median (IQR)
0.64 6.97 1534 947 1.1 0.66 280 186 2.83
0.57 (0.27) 7.00 (0.03) 1479 (230) 869 (317) 0.70 (1.02) 0.53 (0.24) 195.6(130.6) 127.5(73.7) 2.32 (0.73)
0.59 6.98 1122 611 0.8 0.46 124 72 2.63
0.61 (0.11) 7.05(0.08) 1214 (170) 622 (151) 0.48 (0.48) 0.27 (0.20) 100.1 (58.8) 53.6 (41.7) 2.18 (0.87)
0.46 7.02 1191 492 0.8 0.30 97 36 2.49
0.49 (0.09) 7.00(0.23) 1065(531) 431 (178) 0.52 (0.46) 0.26 (0.19) 63.9 (32.3) 28.0 (26.9) 2.15 (0.43)
Significance is displayed as * p < 0.05; **p < 0.01; ***p < 0.001. a: 4 °C vs 21 °C; b:21 °C vs 37 °C; c: 4 °C vs 37 °C.
2. Materials and methods
concentration was determined with the bicinchoninic acid (BCA) protein assay of Pierce (Rockford, Il., USA) as described previously (Prodan et al., 2015) with bovine serum albumin as a standard. The MUC5B concentration was determined by an enzyme linked immunosorbent assay (ELISA) as described previously (Prodan et al., 2015). A reference saliva sample of one and the same person was included on each plate as a standard. MUC5B concentrations were expressed against this standard, which had an arbitrary MUC5B concentration of 1 unit/ml (U/ ml). Amylase activity was measured using the chromogenic substrate 2chloro-4-nitrophenyl-α-D-maltotrioside (Henskens et al., 1996). 10 μl 100-fold diluted saliva was mixed with 90 μl substrate in microplates. Color development was measured at 405 nm every minute during 10 min with a Multiscan FC microplate reader (Thermo Scientific). 1 U/ ml human amylase (Sigma-Aldrich) was used as a standard.
2.1. Participants 20 participants, 14 women and 6 men, were recruited, all students between 18 and 25 years old from the Academic Centre for Dentistry Amsterdam (ACTA), the Netherlands. The exclusion criteria were: use of any medication, except of oral contraceptives, complete or partial dentures, a history of problems with a dry mouth, smoking and systemic diseases. Saliva was collected according to the Code of Ethics of the World Medical Association for experiments involving humans. Participants were informed about the experimental procedure verbally and signed an informed consent. 2.2. Experimental design
2.5. Statistics In a crossover study participants were in randomized order exposed to three experimental conditions on separate days; a cold room (4 °C), a standard room (21 °C) and a warm room (37 °C). Participants sat in a standard office chair for 10 min in the different rooms. After 5 min acclimation they collected saliva for 5 min (Kariyawasam & Dawes, 2005). To minimize the influence of circadian variations all experiments were conducted between 9:00 and 12:00 AM.
Statistical analyses were performed with IBM SPSS Statistics 21. Normality of the data was tested with Shapiro-Wilk tests. The nonparametric Friedman test for related samples was used for comparison of the different temperature regimes. The significance level was set at p < 0.05. When the test revealed a significant difference, the Wilcoxon signed-rank test for related samples was applied as post-hoc procedure.
2.3. Saliva collection and storage
3. Results
The participants were asked not to drink or eat 1 h before the experiment. After 5 min at the experimental temperature, resting saliva was collected as described by Prodan et al. (Prodan et al., 2015) by expectorating every 30 s for 5 min in pre-weighed polypropylene tubes that were kept on ice. The tubes with saliva were weighed and flow rate was calculated, assuming a specific weight for saliva of 1 g/ml. Saliva pH and viscosity were determined directly after collection. Before storage, the saliva samples were diluted 1:1 with 150 mM NaCl and homogenized on a Vortex mixer for 30 s. This lowered the viscosity of saliva facilitating pellet formation after centrifugation and it inhibited the formation of protein precipitates that formed after freezing and thawing of saliva (Francis, Hector, & Proctor, 2000; Prodan et al., 2015). After centrifugation (10 min at 10,000 x g) the supernatant was transferred to 1.5 ml tubes (Greiner Bio-one, Germany) and stored at −20 °C until use.
After exposure of the participants to 4, 21, and 37 °C saliva was collected and a number of salivary parameters, including flow rate, pH, viscosity and the concentrations and output of total protein, amylase and MUC5B was determined (Table 1). All the variables showed large differences between individuals and significant deviations from normality. Therefore, variables at the different temperatures were compared by non-parametric Friedman tests. An overall increase in several parameters with decreasing temperature was observed. Saliva flow rate, protein concentration and -output, as well as amylase concentration and -output in individuals at 4 °C were statistically significantly higher than at 37 °C and, with the exception of flow rate, also higher than those at 21 °C. MUC5B concentration and output, as well as viscosity, showed a similar increasing trend with decreasing temperature but this did not reach statistical significance. 4. Discussion
2.4. Analytical assays This study suggests that exposure to different temperatures has an immediate effect on saliva flow rate and composition. With decreasing temperature saliva flow rate and output of total protein and amylase increase. MUC5B output shows a similar trend, although not significant, but this might be due to the limited number of participants. Future
The saliva pH was measured directly after collection with a pH meter (Radiometer, Copenhagen, Denmark). Viscosity was measured with a Vilastic 3 viscoelasticity analyzer (Vilastic Scientific Inc., Austin, Texas, USA)(van der Reijden, Veerman, & Amerongen, 1993). Protein 2
Archives of Oral Biology 109 (2020) 104593
A.J.M. Ligtenberg, et al.
References
studies with a higher number of participants might confirm this trend. During exposure to cold two effects may play a role; the physiological response of the body to maintain homeostasis in a cold environment and an effect of cold on receptors in the mouth that directly may have an effect on saliva secretion (Eccles, 2000; Eccles, Du-Plessis, Dommels, & Wilkinson, 2013). Transient membrane potential cation receptor Melastatin8 (TRPM8) is the cold receptor in the mouth. This receptor is stimulated by cold sensation and menthol (Patel, Ishiuji, & Yosipovitch, 2007) Stimulation of cold receptors leads to an increase in saliva flow as has been described previously (Haahr et al., 2004). In a study where subjects put a tube of fixed temperature on the tongue, a tube of 10 °C gave a stimulation of parotid saliva flow, whereas 22 and 44 °C had no effect (Lee, Guest, & Essick, 2006). Also, intake of cold water increased the saliva flow rate more than did the intake of warm water (Brunstrom et al., 2000). Saliva flow rate increased during exercise in a cold (1 °C) environment, but decreased in a thermoneutral (24 °C) environment (Mylona, Fahlman, Morgan, Boardley, & Tsivitse, 2002). In line with our results Chatterton et al. (Chatterton, Vogelsong, Lu, Ellman, & Hudgens, 1996) reported increases in amylase output after exposure to cold air. Increases in protein and amylase output might be explained by stimulation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. Increased norepinephrine levels were reported after 5 min residence in a cold room of 5 °C (Pettit, Marchand, & Graham, 1999). This could lead to increases in amylase output as changes in plasma norepinephrine levels correlated well with changes in saliva amylase (Rohleder, Nater, Wolf, Ehlert, & Kirschbaum, 2004). Compared to 21 °C a hot environment of 37 °C resulted in a decrease in saliva flow and output of protein and amylase. Exposure to 66 °C in a sauna resulted in increases in amylase output (Chatterton et al., 1996), but this temperature may be experienced more stressful than 37 °C. General complaints in hot environments are thirst and dry mouth, which have been associated with low saliva flow (Nayha et al., 2014; Sreebny, 2000; van Loenhout et al., 2016). Thirst in hot environments has been attributed to dehydration (Louridis et al., 1970; Shannon, 1966), but in our study the participants were exposed to 37 °C for only 10 min. This exposure time is too short for dehydration, but still the saliva flow rate decreased suggesting a quick response of saliva secretion on higher temperatures. The physiological background of this observation remains to be investigated. To support this observation, future studies should monitor the hydration status of the participants. In addition, core body temperature of the participants should be measured. In conclusion, there is an immediate effect of temperature on saliva flow rate and protein output.
Brunstrom, J. M. (2002). Effects of mouth dryness on drinking behavior and beverage acceptability. Physiology & Behavior, 76, 423–429. Brunstrom, J. M., Tribbeck, P. M., & MacRae, A. W. (2000). The role of mouth state in the termination of drinking behavior in humans. Physiology & Behavior, 68, 579–583. https://doi.org/10.1016/S0031-9384(99)00210-3. Chatterton, R. T., Jr, Vogelsong, K. M., Lu, Y. C., Ellman, A. B., & Hudgens, G. A. (1996). Salivary alpha-amylase as a measure of endogenous adrenergic activity. Clinical Physiology, 16, 433–448. Dawes, C. (1975). Circadian-rhythms in flow-rate and composition of unstimulated and stimulated human submandibular saliva. Journal of Physiology-London, 244, 535–548. Dawes, C., Pedersen, A. M. L., Villa, A., Ekstrom, J., Proctor, G. B., Vissink, A., et al. (2015). The functions of human saliva: A review sponsored by the World Workshop on Oral Medicine VI. Archives of Oral Biology, 60, 863–874. https://doi.org/10.1016/ j.archoralbio.2015.03.004. Eccles, R. (2000). Role of cold receptors and menthol in thirst, the drive to breathe and arousal. Appetite, 34(1), 29–35. Eccles, R., Du-Plessis, L., Dommels, Y., & Wilkinson, J. E. (2013). Cold pleasure. Why we like ice drinks, ice-lollies and ice cream. Appetite, 71, 357–360. Elishoov, H., Wolff, A., Kravel, L. S., Shiperman, A., & Gorsky, M. (2008). Association between season and temperature and unstimulated parotid and submandibular/ sublingual secretion rates. Archives of Oral Biology, 53, 75–78. Francis, C. A., Hector, M. P., & Proctor, G. B. (2000). Precipitation of specic proteins by freeze-thawing of human saliva. Archives of Oral Biology, 45, 601–606. Haahr, A. M., Bardow, A., Thomsen, C. E., Jensen, S. B., Nauntofte, B., Bakke, M., et al. (2004). Release of peppermint flavour compounds from chewing gum: Effect of oral functions. Physiology & Behavior, 82, 531–540. Henskens, Y. M., van der Weijden, F. A., van den Keijbus, P. A., Veerman, E. C., Timmerman, M. F., van der Velden, U., et al. (1996). Effect of periodontal treatment on the protein composition of whole and parotid saliva. Journal of Periodontology, 67, 205–212. Kariyawasam, A. P., & Dawes, C. (2005). A circannual rhythm in unstimulated salivary flow rate when the ambient temperature varies by only about 2 degrees C. Archives of Oral Biology, 50, 919–922. Kenefick, R. W., St Pierre, A., Riel, N. A., Cheuvront, S. N., & Castellani, J. W. (2008). Effect of increased plasma osmolality on cold-induced thirst attenuation. European Journal of Applied Physiology, 104, 1013–1019. https://doi.org/10.1007/s00421-0080857-9. Lee, A., Guest, S., & Essick, G. (2006). Thermally evoked parotid salivation. Physiology & Behavior, 87, 757–764. Louridis, O., Demetriou, N., & Bazopoul, E. (1970). Environmental temperature effect on secretion rate of resting and stimulated human mixed saliva. Journal of Dental Research, 49. https://doi.org/10.1177/00220345700490052301 1136-+. Mylona, E., Fahlman, M. M., Morgan, A. L., Boardley, D., & Tsivitse, S. K. (2002). s-IgA response in females following a single bout of moderate intensity exercise in cold and thermoneutral environments. International Journal of Sports Medicine, 23, 453–456. https://doi.org/10.1055/s-2002-33744. Nayha, S., Rintamaki, H., Donaldson, G., Hassi, J., Jousilahti, P., Laatikainen, T., et al. (2014). Heat-related thermal sensation, comfort and symptoms in a northern population: The National FINRISK 2007 study. European Journal of Public Health, 24, 620–626. https://doi.org/10.1093/eurpub/ckt159. O’Brien, C., Young, A. J., & Sawka, M. N. (1998). Hypohydration and thermoregulation in cold air. Journal of Applied Physiology, 84, 185–189. Patel, T., Ishiuji, Y., & Yosipovitch, G. (2007). Menthol: A refreshing look at this ancient compound. Journal of the American Academy of Dermatology, 57, 873–878. Pettit, S. E., Marchand, I., & Graham, T. (1999). Gender differences in cardiovascular and catecholamine responses to cold-air exposure at rest. Canadian Journal of Applied Physiology-Revue Canadienne De Physiologie Appliquee, 24, 131–147. https://doi.org/ 10.1139/h99-011. Prodan, A., Brand, H. S., Ligtenberg, A. J., Imangaliyev, S., Tsivtsivadze, E., van der Weijden, F., et al. (2015). Interindividual variation, correlations, and sex-related differences in the salivary biochemistry of young healthy adults. European Journal of Oral Sciences, 123, 149–157. https://doi.org/10.1111/eos.12182. Rohleder, N., Nater, U. M., Wolf, J. M., Ehlert, U., & Kirschbaum, C. (2004). Psychosocial stress-induced activation of salivary alpha-amylase - An indicator of sympathetic activity? Annals of the New York Academy of Sciences, 1032, 258–263. Shannon, I. L. (1966). Climatological effects on human parotid gland function. Archives of Oral Biology, 11. https://doi.org/10.1016/0003-9969(66)90109-9 451-&. Ship, J. A., & Fischer, D. J. (1997). The relationship between dehydration and parotid salivary gland function in young and older healthy adults. The Journals of Gerontology Series A, Biological Sciences and Medical Sciences, 52, M310–M319. Sreebny, L. M. (2000). Saliva in health and disease: An appraisal and update. International Dental Journal, 50, 140–161. https://doi.org/10.1111/j.1875-595X.2000.tb00554.x. van der Reijden, W. A., Veerman, E. C., & Amerongen, A. V. (1993). Shear rate dependent viscoelastic behavior of human glandular salivas. Biorheology, 30, 141–152. van Loenhout, J. A. F., le Grand, A., Duijm, F., Greven, F., Vink, N. M., Hoek, G., et al. (2016). The effect of high indoor temperatures on self-perceived health of elderly persons. Environmental Research, 146, 27–34.
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest The authors declare that they have no conflict of interest Acknowledgments We thank Kamran Nazmi for technical support and dr. Henk Brand for statistical advice.
3
Contents lists available at ScienceDirect
Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio
Effects of environmental temperature on saliva flow rate and secretion of protein, amylase and mucin 5B
T
A.J.M. Ligtenberg*, M. Meuffels, E.C.I. Veerman Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam, the Netherlands
A R T I C LE I N FO
A B S T R A C T
Keywords: Heat Viscosity Amylase saliva secretion
Objective: The aim of this study is to evaluate the short term effects of environmental temperature on saliva flow rate and composition. Methods: In a cross-over study design 20 subjects (18–25 years old, 14 women, 6 men) were exposed in randomized order at different days to three temperatures (4 °C, 21 °C and 37 °C). Five minutes after a subject was exposed to the test temperature, collection of resting saliva was started for 5 min at the same temperature. Saliva flow rate, pH, viscosity, protein concentration, mucin 5B concentration and amylase activity were measured. Results: Exposure to 4 °C resulted in an increase of the saliva flow rate (p < 0.01), protein output and amylase output (p < 0.001) compared to exposure to 21 °C or 37 °C. Although the figures for mucin 5B output at 4 °C were higher than at higher temperatures, this was not significant. There were no significant differences in the salivary mucin 5B concentration and viscosity between saliva samples collected at the indicated temperatures. Conclusions: Lowering of the temperature induces an increase in saliva flow rate, as well as protein and amylase output.
1. Introduction Saliva is important for oral health. It plays a major role in lubrication, taste experience, moistening and protection of the oral mucosa against infection and the protection of teeth against abrasion. Low saliva flow rate may lead to problems such as dental caries and disturbed taste experience and is usually accompanied by an unpleasant sensation of dry mouth, called xerostomia (Dawes et al., 2015). Dehydration has been indicated as one of the causes of a diminished salivary flow and the subsequent development of xerostomia (Brunstrom, Tribbeck, & MacRae, 2000; Ship & Fischer, 1997). The feeling of a dry mouth increases the tendency to drink and functions as a thirst signal (Brunstrom, 2002). As a consequence, people with xerostomia have the urge to drink, even when the body is maximally hydrated (Brunstrom, 2002). On the other hand, in a cold environment the thirst feeling is attenuated, even when the body is in need of rehydration (Kenefick, St Pierre, Riel, Cheuvront, & Castellani, 2008; Shannon, 1966). Dehydration may therefore be a problem during long term exposure to low temperatures (O’Brien, Young, & Sawka, 1998). The reason for the attenuated thirst feeling at low temperatures is not known, but in view of the role played by the mouth in the perception of thirst, it is possible that temperature-dependent changes in saliva secretion may be one of
the factors underlying this phenomenon. That the environmental temperature may have an impact on the secretion rate of saliva has been shown in previous studies (Dawes, 1975; Louridis, Demetriou, & Bazopoul, 1970; Shannon, 1966). Secretion of parotid saliva was higher in December/January, with an average outdoor temperature of 10 °C, than in June/July, when the outdoor temperature was 30 °C (Shannon, 1966). The lower secretion rate was attributed to dehydration in the summer months. Seasonal differences in flow rate for parotid and submandibular/sublingual secretions were found when saliva was collected in rooms that reflected the outdoor temperatures of 16 vs. 27.2 °C (Elishoov, Wolff, Kravel, Shiperman, & Gorsky, 2008). This difference disappeared when subjects collected saliva in temperature-adjusted rooms of 21.7 and 24.8 °C suggesting that there is a direct effect of the temperature on the saliva flow rate. In the present study we have examined the influence of temperature on the flow rate and composition of saliva. Participants were exposed to temperatures of 4 °C, 21 °C and 37 °C, and saliva was collected. Saliva flow rate, pH, protein, amylase, mucin 5B (MUC5B) and viscosity were determined. Flow rate and the output of protein and amylase increased with decreasing temperature.
⁎ Corresponding author at: Department of Periodontology and Oral Biochemistry, Academic Centre for Dentistry Amsterdam (ACTA), Gustav Mahlerlaan 3004, 1081 LA Amsterdam, the Netherlands. E-mail address: [email protected] (A.J.M. Ligtenberg).
https://doi.org/10.1016/j.archoralbio.2019.104593 Received 16 July 2019; Received in revised form 22 October 2019; Accepted 23 October 2019 0003-9969/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Archives of Oral Biology 109 (2020) 104593
A.J.M. Ligtenberg, et al.
Table 1 Effect of different room temperatures on the different saliva parameters. Saliva was collected at 4 °C, 21 °C and 37 °C. The mean, median values and interquartile range (IQR) of the different parameters are shown respectively. Significance was tested using the Friedman test for related samples and the Wilcoxon test for related samples as post-hoc procedure. Room temperature
4 °C
Saliva flow rate (ml/min)** pH Protein concentration (μg/ml)***(a,c) Protein-output (μg/min)***(a,b,c) Mucin concentration (units/ml) Mucin-output (units/min) Amylase activity (units/ml)***(a,b,c) Amylase-output (units/min)***(a,b,c) Viscosity (mP/s) (b,c)
21 °C
37 °C
Mean
Median (IQR)
Mean
Median (IQR)
Mean
Median (IQR)
0.64 6.97 1534 947 1.1 0.66 280 186 2.83
0.57 (0.27) 7.00 (0.03) 1479 (230) 869 (317) 0.70 (1.02) 0.53 (0.24) 195.6(130.6) 127.5(73.7) 2.32 (0.73)
0.59 6.98 1122 611 0.8 0.46 124 72 2.63
0.61 (0.11) 7.05(0.08) 1214 (170) 622 (151) 0.48 (0.48) 0.27 (0.20) 100.1 (58.8) 53.6 (41.7) 2.18 (0.87)
0.46 7.02 1191 492 0.8 0.30 97 36 2.49
0.49 (0.09) 7.00(0.23) 1065(531) 431 (178) 0.52 (0.46) 0.26 (0.19) 63.9 (32.3) 28.0 (26.9) 2.15 (0.43)
Significance is displayed as * p < 0.05; **p < 0.01; ***p < 0.001. a: 4 °C vs 21 °C; b:21 °C vs 37 °C; c: 4 °C vs 37 °C.
2. Materials and methods
concentration was determined with the bicinchoninic acid (BCA) protein assay of Pierce (Rockford, Il., USA) as described previously (Prodan et al., 2015) with bovine serum albumin as a standard. The MUC5B concentration was determined by an enzyme linked immunosorbent assay (ELISA) as described previously (Prodan et al., 2015). A reference saliva sample of one and the same person was included on each plate as a standard. MUC5B concentrations were expressed against this standard, which had an arbitrary MUC5B concentration of 1 unit/ml (U/ ml). Amylase activity was measured using the chromogenic substrate 2chloro-4-nitrophenyl-α-D-maltotrioside (Henskens et al., 1996). 10 μl 100-fold diluted saliva was mixed with 90 μl substrate in microplates. Color development was measured at 405 nm every minute during 10 min with a Multiscan FC microplate reader (Thermo Scientific). 1 U/ ml human amylase (Sigma-Aldrich) was used as a standard.
2.1. Participants 20 participants, 14 women and 6 men, were recruited, all students between 18 and 25 years old from the Academic Centre for Dentistry Amsterdam (ACTA), the Netherlands. The exclusion criteria were: use of any medication, except of oral contraceptives, complete or partial dentures, a history of problems with a dry mouth, smoking and systemic diseases. Saliva was collected according to the Code of Ethics of the World Medical Association for experiments involving humans. Participants were informed about the experimental procedure verbally and signed an informed consent. 2.2. Experimental design
2.5. Statistics In a crossover study participants were in randomized order exposed to three experimental conditions on separate days; a cold room (4 °C), a standard room (21 °C) and a warm room (37 °C). Participants sat in a standard office chair for 10 min in the different rooms. After 5 min acclimation they collected saliva for 5 min (Kariyawasam & Dawes, 2005). To minimize the influence of circadian variations all experiments were conducted between 9:00 and 12:00 AM.
Statistical analyses were performed with IBM SPSS Statistics 21. Normality of the data was tested with Shapiro-Wilk tests. The nonparametric Friedman test for related samples was used for comparison of the different temperature regimes. The significance level was set at p < 0.05. When the test revealed a significant difference, the Wilcoxon signed-rank test for related samples was applied as post-hoc procedure.
2.3. Saliva collection and storage
3. Results
The participants were asked not to drink or eat 1 h before the experiment. After 5 min at the experimental temperature, resting saliva was collected as described by Prodan et al. (Prodan et al., 2015) by expectorating every 30 s for 5 min in pre-weighed polypropylene tubes that were kept on ice. The tubes with saliva were weighed and flow rate was calculated, assuming a specific weight for saliva of 1 g/ml. Saliva pH and viscosity were determined directly after collection. Before storage, the saliva samples were diluted 1:1 with 150 mM NaCl and homogenized on a Vortex mixer for 30 s. This lowered the viscosity of saliva facilitating pellet formation after centrifugation and it inhibited the formation of protein precipitates that formed after freezing and thawing of saliva (Francis, Hector, & Proctor, 2000; Prodan et al., 2015). After centrifugation (10 min at 10,000 x g) the supernatant was transferred to 1.5 ml tubes (Greiner Bio-one, Germany) and stored at −20 °C until use.
After exposure of the participants to 4, 21, and 37 °C saliva was collected and a number of salivary parameters, including flow rate, pH, viscosity and the concentrations and output of total protein, amylase and MUC5B was determined (Table 1). All the variables showed large differences between individuals and significant deviations from normality. Therefore, variables at the different temperatures were compared by non-parametric Friedman tests. An overall increase in several parameters with decreasing temperature was observed. Saliva flow rate, protein concentration and -output, as well as amylase concentration and -output in individuals at 4 °C were statistically significantly higher than at 37 °C and, with the exception of flow rate, also higher than those at 21 °C. MUC5B concentration and output, as well as viscosity, showed a similar increasing trend with decreasing temperature but this did not reach statistical significance. 4. Discussion
2.4. Analytical assays This study suggests that exposure to different temperatures has an immediate effect on saliva flow rate and composition. With decreasing temperature saliva flow rate and output of total protein and amylase increase. MUC5B output shows a similar trend, although not significant, but this might be due to the limited number of participants. Future
The saliva pH was measured directly after collection with a pH meter (Radiometer, Copenhagen, Denmark). Viscosity was measured with a Vilastic 3 viscoelasticity analyzer (Vilastic Scientific Inc., Austin, Texas, USA)(van der Reijden, Veerman, & Amerongen, 1993). Protein 2
Archives of Oral Biology 109 (2020) 104593
A.J.M. Ligtenberg, et al.
References
studies with a higher number of participants might confirm this trend. During exposure to cold two effects may play a role; the physiological response of the body to maintain homeostasis in a cold environment and an effect of cold on receptors in the mouth that directly may have an effect on saliva secretion (Eccles, 2000; Eccles, Du-Plessis, Dommels, & Wilkinson, 2013). Transient membrane potential cation receptor Melastatin8 (TRPM8) is the cold receptor in the mouth. This receptor is stimulated by cold sensation and menthol (Patel, Ishiuji, & Yosipovitch, 2007) Stimulation of cold receptors leads to an increase in saliva flow as has been described previously (Haahr et al., 2004). In a study where subjects put a tube of fixed temperature on the tongue, a tube of 10 °C gave a stimulation of parotid saliva flow, whereas 22 and 44 °C had no effect (Lee, Guest, & Essick, 2006). Also, intake of cold water increased the saliva flow rate more than did the intake of warm water (Brunstrom et al., 2000). Saliva flow rate increased during exercise in a cold (1 °C) environment, but decreased in a thermoneutral (24 °C) environment (Mylona, Fahlman, Morgan, Boardley, & Tsivitse, 2002). In line with our results Chatterton et al. (Chatterton, Vogelsong, Lu, Ellman, & Hudgens, 1996) reported increases in amylase output after exposure to cold air. Increases in protein and amylase output might be explained by stimulation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. Increased norepinephrine levels were reported after 5 min residence in a cold room of 5 °C (Pettit, Marchand, & Graham, 1999). This could lead to increases in amylase output as changes in plasma norepinephrine levels correlated well with changes in saliva amylase (Rohleder, Nater, Wolf, Ehlert, & Kirschbaum, 2004). Compared to 21 °C a hot environment of 37 °C resulted in a decrease in saliva flow and output of protein and amylase. Exposure to 66 °C in a sauna resulted in increases in amylase output (Chatterton et al., 1996), but this temperature may be experienced more stressful than 37 °C. General complaints in hot environments are thirst and dry mouth, which have been associated with low saliva flow (Nayha et al., 2014; Sreebny, 2000; van Loenhout et al., 2016). Thirst in hot environments has been attributed to dehydration (Louridis et al., 1970; Shannon, 1966), but in our study the participants were exposed to 37 °C for only 10 min. This exposure time is too short for dehydration, but still the saliva flow rate decreased suggesting a quick response of saliva secretion on higher temperatures. The physiological background of this observation remains to be investigated. To support this observation, future studies should monitor the hydration status of the participants. In addition, core body temperature of the participants should be measured. In conclusion, there is an immediate effect of temperature on saliva flow rate and protein output.
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Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest The authors declare that they have no conflict of interest Acknowledgments We thank Kamran Nazmi for technical support and dr. Henk Brand for statistical advice.
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