The ph change after HCl titration into resting and stimulated saliva for a buffering capacity test

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1 SCIENTIFIC ARTICLE Australian Dental Journal 2006;51:(2): The ph change after HCl titration into resting and stimulated saliva for a buffering capacity test M Moritsuka,* Y Kitasako,* MF Burrow, M Ikeda,* J Tagami* Abstract Background: Saliva collection can provide clinical information about individual patients. However, a correlation between ranking buffering capacity using resting and stimulated saliva is still unknown. The aim of this study was to evaluate the ph change after HCl titration into resting and stimulated saliva for a salivary buffering capacity test. Methods: Resting and stimulated saliva (by chewing paraffin wax) were collected from 80 patients. After the ph of both saliva samples was measured using a hand-held ph meter, the saliva samples were titrated with 0.1N HCl to evaluate the buffering capacity. Correlations of ranking buffering capacity (high, medium, low) between stimulated saliva and resting saliva with 30µL HCl titration and between stimulated saliva and resting saliva with 40µL HCl titration were statistically analysed by Spearman Rank Correlation Test (p<0.05). Results: At 50µL HCl titration, stimulated saliva buffering capacities were ranked into high (above ph 5.5), medium (ph from 5.5 to 4.5) and low (below ph 4.5). At 30 40µL HCl titration, the resting saliva buffering capacities were ranked into the same categories. Spearman Rank Correlation indicated significant positive coefficients for the stimulated saliva and resting saliva buffering capacity at 30µL titration and the stimulated saliva and resting saliva at 40µL titration. Conclusion: Stimulated saliva is more resistant to variation in ph change during HCl titration than resting saliva. Stimulated saliva sampling is a good method to determine buffering capacity during a comprehensive oral health assessment. Key words: Buffering capacity, resting saliva, stimulated saliva, ph. Abbreviation: CPP-ACP = casein phosphopeptideamorphous calcium phosphate. (Accepted for publication 15 August 2005.) *Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan. School of Dental Science, The University of Melbourne, Victoria. Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, Japan. INTRODUCTION Saliva collection can provide oral health information about individual patients. 1,2 Commercially available saliva collection kits are used for determining an individual s saliva buffering capacity level as part of caries risk factor assessment. During the demineralization and remineralization that occurs in the caries process, saliva is effective in helping to maintain a neutral ph in the oral cavity. 3 The major regulator of ph is salivary bicarbonate from parotid saliva. 4 Because the concentration of salivary bicarbonate depends on flow rate, the capacity for saliva to act as a buffer will vary directly with saliva flow rate. 4,5 Saliva flow rates can be measured in either a resting or stimulated state. Resting saliva flow is characterized as the flow occurring in the absence of any obvious oral or physiological stimulation. 6 Although stimulated secretions represent the major contribution to daily gland fluid output, resting saliva bathes the oral cavity for more than 90 per cent of the day. 2 When whole resting and stimulated salivary flow measurements were compared, White 1 found that they were significantly correlated. On the other hand, the ph value of resting saliva varies during the day. 7 Therefore, most studies investigating saliva buffering capacity have focused on whole stimulated saliva. 4,8 Ericsson 9 reported that there were distinct differences in the stimulated whole saliva buffering capacity of caries-free and caries-active patients when using expensive equipment and complex laboratory techniques for a saliva buffering capacity test. In the case of commercially available caries risk kits, a colour code chart is used as the means to determine saliva buffering ability. However, there could be cases where the level of the buffering capacity is difficult to classify using a colour coding method. Recently, a new saliva buffering capacity test has been introduced into dental practice which uses a small hand-held ph meter. 10 A quantitative ph analysis can be performed by a simplified process of placing saliva samples and titrating HCl onto the flat glass electrode of the ph meter. Although this simplified, quantitative saliva buffering capacity test can be used for a chair- 170 Australian Dental Journal 2006;51:2

2 Fig 1. a: Hand-held ph meter (B-212, Horiba Ltd. Japan). b: A flat-surface sensor (flat glass electrodes) is constructed of several layers of plastic glued to each other. c: The flat ph sensor is protected by a sliding sensor guard. d: the lid of sensor guard can be slid back. e: It is easy for evaluating the ph changing under the acid titration into saliva samples to just slide back the lid. f: This hand-held ph meter is very similar in construction to common glass electrodes. side evaluation of individual saliva buffering capacities, the buffering capacity of resting and stimulated saliva in similar individuals has still to be clarified. The aim of this study was to evaluate the ph change during HCl titration into resting and stimulated saliva samples to determine buffering capacity. The null hypothesis of this study was that there are no significant differences in the buffering capacity of resting and stimulated saliva in similar individuals. MATERIALS AND METHODS Study population The study population consisted of 80 patients (aged years, mean age 38.3 years) who attended for treatment at the Cariology and Operative Dentistry Department of Tokyo Medical and Dental University, Japan. Written informed consent, based on the Code of Ethics of the World Medical Association (Declaration of Helsinki), was obtained prior to participation in the study. The main criteria for selection were, besides being healthy, their ability to cooperate and to follow the instructions given regarding food intake and oral hygiene. Saliva sampling method Saliva samples from patients were collected once between 9am and 11am or 2pm and 4pm. Saliva collections were taken at least two hours after meals and at least one hour after brushing to minimize the effects of diurnal variability in salivary composition. 11,12 After collecting resting saliva by direct expectoration, each patient was given a one-gram piece of unflavoured paraffin wax (CRT test, Ivoclar Vivadent, Liechtenstein) to chew for five minutes to obtain the stimulated saliva. ph meter Saliva ph was measured directly using a small hand-held ph meter (B-212, Horiba Ltd., Tokyo, Japan) (Fig 1a). This compact ph meter (dimensions 165 x 29 x 19mm; weight 53g) is able to determine the ph value of a single drop of saliva (0.1mL) using a flatsurface sensor (flat glass electrodes). These glass electrodes are constructed of several layers of plastic glued to each other, and are very similar in construction to common glass electrodes (Figs 1b and f). The electrodes are not dipped into the sample, but the sample is applied to the surface directly. The flat ph sensor is protected by a sliding cover which also acts to form a closed chamber (Figs 1b, c and d). It is easy to evaluate the ph change with acid titration into saliva samples by sliding back the cover (Fig 1e). For calibration, ph 4 and ph 7 standard buffers are used where the ph meter automatically makes the calibration by pushing of the CAL button. The Australian Dental Journal 2006;51:2. 171

3 individual was constructed to classify individual saliva buffering capacities. a b Fig 2. a: ph change of stimulated whole saliva after titration with 0.1N HCl. At the point of the addition of 50µL, the individual saliva buffer capacities were ranked into the one of the following three categories; high buffer capacity (above ph 5.5), medium buffer capacity (ph from 5.5 to 4.5) and low buffer capacity (below ph 4.5). b: At the point of 30-40µL of titrated HCl, the individual resting saliva buffer capacities were ranked into the same three categories as the stimulated saliva. accuracy of the ph meter was checked at regular intervals to ensure that the readings were consistent. Saliva buffering capacity test After collection of the resting and stimulated whole saliva, 0.5mL of each saliva sample was placed onto the ph-sensitive electrode to immediately measure the early ph value within 30 seconds. 10 In this study, 10µL of 0.1mol/L hydrochloric acid (Wako Corp., Tokyo, Japan) was titrated into the saliva sample. After closing the cover, the ph meter was gently shaken to mix the tested saliva and titrated HCl. The digital reading was allowed to stabilize for a few seconds and then the ph reading taken. Up to a total of 160µL of HCl was titrated into a sample. From this, the ph curve for each Statistical analysis The correlations of ranking results of buffering capacity (high, medium, low) between stimulated saliva with 50µL titration of HCl and resting saliva with 30µL titration, and between stimulated saliva with 50µL titration and resting saliva with 40µL titration were statistically analysed by the Spearman Rank Correlation Test 13 at the confidence level of 95 per cent. All statistical calculations were performed using the SPSS statistical software programme (Chicago, Illinois, USA). RESULTS Figure 2a shows the ph change of the stimulated saliva with 0.1N HCl acid for buffering capacity. At the point of 50µL of titrated HCl, individual stimulated saliva buffering capacities were ranked into one of the following three categories: high (above ph 5.5), medium (ph from 5.5 to 4.5) and low (below ph 4.5). In this study, 40 out of 80 patients showed high buffering capacities, 14 showed medium buffering capacities, and the remaining 26 showed low buffering capacities (Table 1). At the end of the titration (i.e., at 160µL titrated HCl), the ph data ranged from ph 2.3 to ph 1.6. On the other hand, at the point of 30 40µL of titrated HCl, individual resting saliva buffering capacities were ranked into the same three categories (Fig 2b). The percentage of concordance rates (resting saliva/stimulated saliva) for the different volumes of HCl titration into resting saliva (30µL and 40µL respectively) were 71 and 46 per cent for total rate, 83 and 25 per cent for high buffering capacity, 79 and 43 per cent for medium buffering capacity, 50 and 80 per cent for low buffering capacity. Analysis of Spearman Rank Correlation indicated significant strong positive coefficients for the relation between stimulated saliva with 50µL titration and resting saliva with 30µL titration ( =0.735) and significant moderate coefficients for the relation between stimulated saliva with 50µL titration and resting saliva with 40µL titration ( =0.608). DISCUSSION Using a commercially available saliva buffering test, the Dentbuff colorimetric system (Orion Diagnostica Table 1. The distributions of patients in the stimulated and resting saliva groups according to buffering capacity rank Type of saliva HCl titration(µl) Number of each buffering capacity rank Stimulated saliva High Medium Low (n=80) % Concordance rate of total and each buffering rank (number : resting saliva/stimulated saliva) Total High Medium Low Resting saliva (57/80) 83 (33/40) 79 (11/14) 50 (13/26) (n=80) (37/80) 25 (10/40) 43 (6/14)1 80 (21/26) 172 Australian Dental Journal 2006;51:2.

4 Co. Ltd, Epsom, Finland) and Ericsson s electrometric method, 9 Wikner and Nedlich 14 compared the results from patients classified with different caries risks and with different buffering capacities. They reported that the frequency distribution of a three-graded scale indicating low (ph ), intermediate (ph ) and high (ph 5.6 to over ph 7.0) buffering capacities was similar for both the electrometric and colourimetric methods. In this study, 10µL aliquots of 0.1N HCl was titrated into the saliva samples up to a total of 160µL to determine individual ph curves. For stimulated saliva, at the point of 50µL of titrated HCl, the individual saliva buffering capacities were ranked into the three categories; high (above ph 5.5), medium (ph from 5.5 to 4.5) and low (below ph 4.5) buffering capacity. These ph results closely follow the outcomes of previous reports. 14,15 On the other hand, the resting saliva buffering capacities were also ranked into the same three categories at 30 40µL of titrated HCl. The percentages of concordance rates (resting saliva/ stimulated saliva) for the different volumes of HCl titrated into resting saliva (30µL and 40µL) were different for each buffering category. Although there was a stronger positive correlation of the ranking results between simulated saliva and resting saliva with 30µL titration and the total concordance rate at 30µL of titrated HCl was higher than that of 40µL of titrated HCl, it was lower than that of 40µL of titrated HCl for evaluating low buffering capacity. For chair-side caries risk diagnosis, it is more important to detect those patients that exhibit a low buffering capacity. Further clinical and laboratory studies are required to evaluate the ph change of resting saliva between 30 40µL of HCl titration. Caries diagnostic methods provide the clinician and patient with tangible information to assist with preventive programmes. For treatment of caries based on minimally invasive dentistry concepts, it is important for clinicians to understand caries progression including rates and ways of stopping and preventing caries before restoring teeth. 16 For each buffering capacity level, caries prevention treatments must be designed individually. According to minimally invasive dentistry concepts, healing of caries lesions for patients with a high buffering capacity may be the first treatment option providing cavitation has not occurred. In the case of the low buffering capacity group, restorations should be repaired or caries treated by surgical means to prevent gross destruction of teeth as well as institute a comprehensive preventive programme. 17 Many of those patients who have xerostomia suffer from a high incidence of caries 3 and the caries risk of these patients needs careful assessment. However, conventional buffering capacity tests are not able to be used due to the very small quantity of saliva secreted by this group of patients. On the other hand, the hand-held ph meter used in this study can determine the ph of 0.1mL of saliva, making it possible to determine the buffering capacity. In conjunction with oral hygiene and other caries prevention measures, such as plaque control by professional tooth cleaning, dietary modification and stimulation of salivary flow using gum, the ph meter and saliva buffering capacity makes an important contribution to the multifaceted strategy for patients who fall into the low buffering capacity group. Moreover, supplementing fluoride toothpaste use with chair-side fluoride applications might also be needed to enhance caries prevention. Zimmer et al. 18 suggested that if increased caries protection was required for high caries risk patients, a highly concentrated fluoride gel could be used weekly. Multiple uses of various fluoride products such as mouthwashes and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) containing GC Tooth Mousse (GC Corp., Tokyo, Japan) leads are likely to enhance protection against caries as well as heal lesions. Resting and stimulated salivary flow rates have been reported to decrease with age, 19,20 and these functional changes are consistent with histological findings. 21,22 However, recent studies have also demonstrated that both stimulated and unstimulated parotid salivary flow rates are not reduced in healthy elderly subjects. 23,24 In the present study, 11 out of 80 patients were over 60 years of age. Only three out of 11 patients taking prescribed medication showed a low buffering capacity for both resting and stimulated saliva. Although five out of 11 patients showed a medium or low buffering capacity for resting saliva with 40µL HCl titration, they showed a high buffering capacity for stimulated saliva with 50µL HCl titration. These results suggest that aging does not necessarily result in degeneration of salivary gland function, i.e., saliva buffering capacity and medications may have a significant influence on the buffering capacity of resting saliva. It is suggested that resting saliva output is not sufficient for saliva analysis because of its daily variation. 25,26 Moreover, the measurement of resting saliva is difficult to standardize. 5 Although saliva ph parameters are very difficult to eliminate completely in common dental practice, stimulated saliva is known to vary less throughout the day. 26,27 Therefore, stimulated saliva rather than resting saliva should be used for saliva analysis since a more consistent correlation between salivary factors and the oral health of individuals is likely. CONCLUSION A strong positive correlation of the ranking buffering capacity results between stimulated saliva with 50µL HCl titration and resting saliva with 30µL HCl titration was observed. A moderately positive correlation of the ranking buffering capacity results between stimulated saliva with 50µL HCl titration and resting saliva with 40µL HCl titration was observed. Because stimulated saliva is more resistant to ph change with HCl titration, rather than resting saliva, stimulated saliva could be used in saliva buffering Australian Dental Journal 2006;51:2. 173

5 capacity testing to reveal more about the oral health of individuals. ACKNOWLEDGEMENTS This project was supported by Grant # from the Japan Society for the Promotion of Science and the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone at Tokyo Medical and Dental University. The authors would like to thank Dr. Hiroshi Nagai and Dr. Satoshi Nomura for their contributions. REFERENCES 1. White KD. Salivation: a review and experimental investigation of major techniques. Psychophysiology 1977;14: Mandel ID. The functions of saliva. J Dent Res 1987;66: Navazesh M, Mulligan RA, Kipnis V, Denny PA, Denny PC. Comparison of whole saliva flow rates and mucin concentrations in healthy Caucasian young and aged adults. J Dent Res 1992;71: Mandel ID, Wotman S. The salivary secretions in health and disease. Oral Sci Rev 1976;8: Larsen MJ, Jensen AF, Madsen DM, Pearce EI. Individual variations of ph, buffer capacity, and concentrations of calcium and phosphate in unstimulated whole saliva. Arch Oral Biol 1999;44: Navazesh M. Christensen CM. A comparison of whole mouth resting and stimulated salivary measurement procedures. J Dent Res 1982;61: Grossman LI, Brickman BM. Some observations on the ph of saliva. J Dent Res 1937;16: Shellis RP, Dibdin GH. Analysis of the buffering systems in dental plaque. J Dent Res 1988;67: Ericsson Y. Clinical investigations of the salivary buffering action. Acta Odontol Scand 1959;17: Kitasako Y, Moritsuka M, Foxton RM, Ikeda M, Tagami J, Nomura S. Simplified and quantitative saliva buffer capacity test using a hand-held ph meter. Am J Dent 2005;18: Dogon IL, Amdur BH, Bell K. Observations on the diurnal variation of some inorganic constituents of human parotid saliva in smokers and non-smokers. Arch Oral Biol 1971;16: Kho HS, Lee SW, Chung SC, Kim YK. Oral manifestations and salivary flow rate, ph, and buffer capacity in patients with endstage renal disease undergoing hemodialysis. Oral Surg Oral Med Oral Pathol 1999;88: Atwood CS, James IR, Keil U, Roberts NK, Hartmann PE. Circadian changes in salivary constituents and conductivity in women and men. Chronobiologia 1991;18: Wikner S, Nedlich U. A clinical evaluation of the ability of the Dentobuff method to estimate buffer capacity of saliva. Swed Dent J 1985;9: Ericson D, Bratthall D. Simplified method to estimate salivary buffer capacity. Scand J Dent Res 1989;97: Tyas MJ, Anusavice KJ, Frencken JE, Mount GJ. Minimal intervention dentistry - a review. FDI Commission Project Int Dent J 2000;50: Mjör IA, Dahl JE, Moorhead JE. Age of restorations at replacement in permanent teeth in general dental practice. Acta Odontol Scand 2000;58: Zimmer S, Jahn KR, Barthel CR. Recommendations for the use of fluoride in caries prevention. Oral Health Prev Dent 2003;1: Yaegaki K, Ogura R, Kameyama T, Sujaku C. Biochemical diagnosis of reduced salivary gland function. Int J Oral Surg 1985;14: Ben-Aryeh H, Shalev A, Szargel R, Laor A, Laufer D, Gutman D. The salivary flow rate and composition of whole and parotid resting and stimulated saliva in young and old healthy subjects. Biochem Med Metab Biol 1986;36: Waterhouse JP, Chisholm DM, Winter RB, Patel M, Yale RS. Replacement of functional parenchymal cells by fat and connective tissue in human submandibular salivary glands: an age-related study. J Oral Path 1973;2: Scott J. Quantitative age changes in the histological structure of human submandibular salivary glands. Arch Oral Biol 1977;22: Parvinen T, Parvinen I, Larmas M. Stimulated salivary flow rate, ph and lactobacillus and yeast concentrations in medicated persons. Scand J Dent Res 1984;92: Gandara BK, Izutsu KT, Truelove EL, Ensign WY, Sommers EE. Age-related salivary flow rate changes in controls and patients with oral lichen planus. J Dent Res 1985;64: Ferguson DB, Fort A. Circadian variation in human resting submandibular saliva flow rate and composition. Arch Oral Biol 1974;19: Ben-Aryeh H, Fisher M, Szargel R, Laufer D. Composition of whole unstimulated saliva of healthy children: changes with age. Arch Oral Biol 1990;35: Dawes C. The effects of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human submandibular saliva. Arch Oral Biol 1974;19: Address for correspondence/reprints: Dr. Michiyo Moritsuka Cariology and Operative Dentistry Department of Restorative Sciences Graduate School Tokyo Medical and Dental University 5-45 Yushima 1-chome, Bunkyo-ku Tokyo, Japan moritsuka.ope@tmd.ac.jp 174 Australian Dental Journal 2006;51:2.