Time-related Changes in pH, Buffering Capacity and Phosphate and Urea Concentration of Stimulated Saliva Lea Vuletica/Kristina Perosb/Stjepan Spaljc/Dunja Rogicd/Ivan Alajbege Purpose: To quantify changes in pH, buffering capacity and hydrogen carbonate, phosphate, protein and urea concentrations of stimulated saliva which occur during a 30-min measurement delay after saliva collection. The correlation between time-related chemical changes and changes of salivary pH and buffering capacity was assessed in order to explain the observed changes in salivary pH and buffering capacity. Materials and Methods: Stimulated saliva samples were collected from 30 volunteers after inducing salivation by chewing a piece of parafilm. Measurements of salivary variables were made immediately after saliva collection and again 30 min later, during which time the specimens were exposed to the atmosphere in collection cups at room temperature. Results: Postponement of measurements resulted in a significant increase in pH and a significant decrease of buffering capacity, phosphate and urea concentration. The results suggest that the time-related pH increase could primarily be attributed to loss of dissolved carbon dioxide from saliva, and confirm the importance of hydrogen carbonate in the neutralisation of hydrogen ions, but they do not support the principle of catalysed phase-buffering for the hydrogen carbonate buffer system in saliva. A decrease in phosphate and urea concentration affects salivary buffering capacity. Conclusion: This study emphasises the importance of the standardisation of measurement time when measuring salivary pH, buffering capacity, phosphate and urea concentrations following the collection of saliva in order to obtain comparable results. It also provides a partial explanation of the mechanisms underlying the observed changes of pH and buffering capacity over time. Key words: hydrogen carbonate, phosphate, salivary buffering capacity, salivary pH, urea Oral Health Prev Dent 2014;1:45-53
Submitted for publication: 27.04.12; accepted for publication: 21.12.12
he buffering capacity of saliva, especially stimulated, is a salivary variable measured both in scientific research and in clinical practice in order to assess the protective power of saliva against tooth demineralisation. Rapid normalisation of the oral pH opposes chemical erosion that is likely to a
Research and Teaching Fellow, Department of Physiology, School of Dental Medicine, University of Zagreb, Zagreb, Croatia.
Senior Research and Teaching Fellow, Department of Pharmacology, School of Dental Medicine, University of Zagreb, Zagreb, Croatia.
Assistant Professor, Department of Paediatric Dentistry and Orthodontics, School of Medicine, University of Rijeka, Rijeka, Croatia.
Assistant Professor, Clinical Institute for Laboratory Diagnostics, University Hospital Centre Zagreb, Zagreb, Croatia.
Associate Professor, Department of Oral Medicine, School of Dental Medicine, University of Zagreb, and Department of Dental Medicine, University Hospital Centre Zagreb, Zagreb, Croatia.
Correspondence: Lea Vuletic, Department of Physiology, School of Dental Medicine, University of Zagreb, Salata 3, 10 000 Zagreb, Croatia. Tel: +385-1-459-0243. Email: [email protected]
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happen under the influence of erosive foods and beverages (Puy, 2006). Further, salivary buffering mechanisms are important modulators of dental plaque pH. A number of studies reported an inverse correlation between the buffering capacity of stimulated saliva and caries susceptibility (Ravald and Birkhed, 1991; Guivante-Nabet et al, 1998; Kitasako et al, 2006; Varma et al, 2008). The hydrogen carbonate buffer pair (CO2/ HCO3 -) is considered to be the principal buffer of stimulated saliva. Additionally, protein and phosphate (in the form of monohydrogen phosphate and dihydrogen phosphate) buffering systems contribute to the buffering capacity of whole saliva (Lilienthal, 1955; Bardow et al, 2000b). These systems have different pH ranges of maximal buffering capacity depending on their pK values (-log of the dissociation constant). The hydrogen carbonate and phosphate systems in human saliva have a pK value of approximately 6.1 (Bardow et al, 2000a) and 7.0 (Bar-
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dow et al, 2008), respectively. Proteins contribute to the salivary buffering capacity only at very low pH values (Bardow et al, 2000b; Lamanda et al, 2007). The increased ability of stimulated saliva to neutralise acids is the result of a significant rise in the concentration of hydrogen carbonate ions (HCO3 -) upon stimulation of the salivary flow during food intake and mastication (Bardow et al, 2000b; Bardow et al, 2008). The protein concentration in saliva does not change significantly upon paraffin stimulation of salivation, while the total phosphate concentration shows a flow-dependent decrease (Bardow et al, 2000b). Besides an increase of HCO3 - in stimulated saliva, another advantage often pointed out in favour of the hydrogen carbonate buffer system is its ability to act as an open buffer system or a phase-buffer, i.e. its buffering effect involves a conversion of carbon dioxide (CO2) from a dissolved into a gaseous state (Kivelä et al, 1999; Bardow et al, 2000b; Lenander-Lumikari and Loimaranta, 2000; Bardow et al, 2008). When acid is added, this catalysed (Bardow et al, 2000b) phase conversion considerably increases the efficacy of the neutralisation reaction, as there is no accumulation of end products and thus a complete removal of the acid occurs (Kivelä et al, 1999). Delayed determination of pH and buffering capacity after saliva sample collection can influence the measurement results due to time-related changes in the chemical composition of saliva. It has been suggested that exposure of saliva to the atmosphere during eating or breathing, or collecting and keeping saliva in an open system (open collection cup or tube), shifts the equilibrium (reaction 1) for the hydrogen carbonate buffer system to the left due to CO2 loss because of the higher partial pressure of this compound in saliva than in the atmosphere (Bardow et al, 2000b; Schipper et al, 2007). CO2 + H2O H2CO3 HCO3 - + H+ (reaction 1) This results in a loss of HCO3 - and protons and a change of salivary pH toward the alkaline (Bardow et al, 2000b; Schipper et al, 2007). A change of pH and buffering capacity of saliva samples over time may also be caused by (bacterial) degradation of the salivary organic compounds such as proteins, amino acids and urea (Lamanda et al, 2007; Schipper et al, 2007). The purpose of this study was to quantify changes of the pH, buffering capacity and HCO3 -, inorganic phosphate, total protein and urea concentrations of stimulated saliva which were postulated to occur due to a short (half-hour) delay of their meas-
urement during which the saliva samples were exposed to the atmosphere in collection cups at room temperature. The results represent a contribution to the standardisation of methods for collection and storage of stimulated saliva samples when used to measure the previously mentioned salivary variables in scientific research and clinical practice. The determination of HCO3 -, phosphate, protein and urea concentration, salivary compounds known to influence the salivary pH and/or which act as physiological regulators of the salivary pH, was also used to assess the correlation between timerelated changes in their concentrations and timerelated changes of the salivary pH value and buffering capacity.
MATERIALS AND METHODS Ethical approval This study was approved by the Ethics Committee of the School of Dental Medicine, University of Zagreb, and all participants signed an informed consent form. All experimental procedures were conducted in accordance with the Declaration of Helsinki’s recommendations guiding physicians in biomedical research involving human subjects.
Subjects Second- and fourth-semester students of the School of Dental Medicine, University of Zagreb, were invited to voluntarily participate in the study. Students who used antibacterial mouthwashes, those that had been taking antibiotics eight weeks prior to the beginning of the study, smokers, students with fixed orthodontic appliances, active carious lesions, damaged tooth fillings, poor oral hygiene and gingival or periodontal inflammatory lesions were excluded from participation in the study. Thirty healthy volunteers, 15 females and 15 males aged 19 to 26 years (mean age 21.3 years) were included in the study.
Collection of stimulated whole saliva Saliva specimens were collected between 08:00 and 10:00 a.m. in fasting conditions and without morning toothbrushing. The participants were instructed to drink at least one glass (1 dl) of water
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before going to sleep to ensure adequate hydration. Salivation was stimulated by chewing a piece (ca 0.56 g) of unflavoured wax, parafilm (Parafilm, Heinz Herenz; Hamburg, Germany). Saliva secreted in the first 30 s was swallowed. The collection of saliva began thereafter and lasted for 5 min. Six participants were unable to collect at least 4.5 ml of stimulated saliva during the first 5 min, so the collection time was extended until the required volume was gathered in a maximum of 10 min (N = 2). Mean collection time was 5.8 min. The participants were asked to chew at a regular rate of approximately 55 cycles/min (guided by a metronome) with their mouth closed, to alternate chewing sides and to spit saliva into a graduated collection cup (GC Europe, Leuven, Belgium) at regular intervals.
Saliva specimen processing Saliva specimens were used to measure salivary pH, buffering capacity, HCO3 -, inorganic phosphate and total protein and urea concentrations. These measurements were made immediately after saliva collection and after a 30-min delay. During this delay, the saliva samples were exposed to the atmosphere in collection cups at room temperature.
Salivary pH and buffering capacity measurement A volume of 1.5 ml of stimulated saliva was transferred from the collection cups to plastic test tubes for the determination of the pH of stimulated saliva and its buffering capacity. Salivary pH was measured with a hand-held pH meter (Piccolo Plus ATC pH-meter, Hanna Instruments; Kehl am Rhein, Germany). Buffering capacity was determined thereafter by titration of samples with 0.4 ml of 0.01M HCl. A change in pH was recorded after each addition of 0.4 ml of acid until the pH reached the first value lower than 3.0. Buffering capacity was calculated using the formula: BC = – Δn(acid) ⁄ (V1 × ΔpH) where BC denotes salivary buffering capacity, − Δn(acid) is the titrating acid volume, V1 is the volume of saliva and ΔpH is the change of salivary pH after the addition of acid. These measurements were performed at the Department of Physiology, School of Dental Medicine, University of Zagreb, by the same investigator.
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Sialochemistry The volume of saliva required for measurements of salivary HCO3 -, phosphate, protein and urea concentrations (0.6 ml) was transferred from the collection cups to Eppendorf tubes (Safe-Lock Tubes 1.5 ml, Eppendorf; Hamburg, Germany), frozen (-20°C) and stored until biochemical analysis was possible. Saliva samples were thawed at room temperature and centrifuged for 10 min at 3000 rpm (relative centrifugal force = 2000 g) before sialochemical analysis. Salivary HCO3 - concentration was determined spectrophotometrically by the enzymatic method with phosphoenolpyruvate (Forrester et al, 1976). Total inorganic phosphate was determined by the molybdate method (Fossati, 1985). The total protein concentration was determined by staining with pirogalol red (Polkinghorne, 2006). The concentration of urea was determined by the UV enzymatic urease and glutamate dehydrogenase method (Talke and Schubert, 1965). All sialochemical procedures were performed on an Olympus AU 2700 immunochemistry analyzer (Beckman Coulter; Brea, CA, USA) with appropriate calibrators and controls purchased from the same manufacturer. Laboratory measurements were performed at the Department of Laboratory Diagnostics, University Hospital Centre, Zagreb.
Statistical analysis The Shapiro-Wilk test was used to test the assumption of normality, and Levene‘s test was used to test for homogeneity of variance. Paired samples t-test was used to test differences in pH, buffering capacity and sialochemistry between time intervals. One-way and two-way repeated measures ANOVA with the Bonferroni post-hoc test was used to assess the influence of time interval and acid volume on buffering capacity. Eta squared (d2) was used to estimate the size of the effect, that is, the share of total variability of the dependent variable explained by the factor tested. Pearson‘s correlation was used as a measure of association. Data were analysed using commercial software SPSS 10.0 (SPSS; Chicago, IL, USA), with significance preset at _ < 0.05 for a two-sided test.
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Table 1 Stimulated salivary pH, buffering capacity and concentration of hydrogen carbonate, inorganic phosphate, total protein and urea measured immediately after saliva collection and after a 30-min delay Time point t0 measurement made immediately after saliva collection
t1 measurement made after a 30-min delay
Stimulated saliva variable
Mean ± SD
Mean ± SD
7.38 ± 0.26
7.67 ± 0.26