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Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?
+
MODEL
Journal of Dental Sciences (2013) xx, 1e5
Available online at www.sciencedirect.com
journal homepage: www.e-jds.com
ORIGINAL ARTICLE
Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores? ¨kru ¨ Beydemir b, Cenk Fatih C ¸ic ¸ek a, S‚u Taner Arabacı a*, Yasin C ¸anakc ¸ı a, Varol C ¸anakc ¸ı a a b
Department of Periodontology, Faculty of Dentistry, Atatu¨rk University, Erzurum, Turkey Department of Chemistry, Science and Arts Faculty, Atatu¨rk University, Erzurum, Turkey
Received 19 April 2012; Final revision received 29 November 2012
KEYWORDS carbonic anhydrase; dental calculus; oral hygiene; saliva
Abstract Background/purpose: Carbonic anhydrase (CA) VI is a unique salivary isoenzyme that regulates the pH in the oral environment. Its increased levels result in a more-alkaline saliva, hence it was hypothesized to contribute to dental calculus formation together with salivary inorganic salts. In this study, we evaluated the relationship between salivary CA VI levels and the amount of supragingival dental calculus formation on human teeth. Materials and methods: Seventy-five volunteers were grouped according to their oral hygiene status, and the amount of supragingival dental calculus was measured by the VolpeeManhold Index (VMI). Group I included control individuals who had no dental calculus formation clinically (VMI Z 0). Group II included light calculus formers (VMI < 7), and group III included heavy calculus formers (VMI 7). Clinical periodontal indices and salivary CA VI levels were measured in all individuals, and were compared among the groups and correlated within groups. Results: Differences among the groups according to clinical scores and CA VI levels were found to be statistically significant (P < 0.05). The correlation between the oral hygiene index and salivary CA VI levels was found statistically significant in group III (P < 0.05), but was not statistically significant in group II (P > 0.05). Significant positive correlations were found between salivary CA levels and amount of dental calculus in groups II and III (P < 0.05 and < 0.01, respectively). Conclusion: This study found positive correlations between increased salivary CA VI activity and the amount of supragingival dental calculus; however, further investigations are needed to clarify the exact association between these parameters. Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.
¨ niversitesi, Erzurum 25240, Turkey. * Corresponding author. Periodontoloji Anabilim Dalı, Dis‚ Hekimligi Faku ¨ltesi, Atatu ¨rk U E-mail address: [email protected] (T. Arabacı). 1991-7902/$36 Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jds.2012.12.014
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
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Introduction Carbonic anhydrase (CA) is a zinc-containing metalloenzyme that participates in a variety of physiological processes. CA enzymes catalyze various reactions including carbon dioxide and bicarbonate transport, pH regulation, and water/electrolyte balance in various tissues.1e6 Among many of the active mammalian isozymes, CA VI is the specific form secreted by salivary glands.7e9 It was demonstrated that this isoform accelerates the removal of carbon dioxide and results in alkalinization of the saliva.10,11 Although CA was originally considered to regulate the salivary pH or buffer capacity, it was demonstrated that these variables are not directly associated with the CA VI concentration in saliva.12e14 Saliva helps maintain the oral health, and one of its important functions is to regulate pH changes in the oral cavity. Salivary pH is closely related to the buffering capacity, which is essential for neutralization of acidity in dental plaque,6,13 and affects the caries process and calculus formation.15 Although the formation of dental calculus is primarily influenced by individual oral hygiene habits, many factors including the diet, age, ethnic origin, systemic diseases, inorganic contents, and the pH of the saliva are also related to calculus indices. It was revealed that calcification of dental plaque biofilm with mineral ions was provided by bathing saliva or crevicular fluids.16 On the other hand, the viscosity and contents of the saliva were reported to be related to calcification of organic deposits on tooth surfaces.13 Furthermore, several authors suggested that increased levels of some salivary enzymes, such as protease and alkaline phosphatase, can also contribute to the formation of sub- and supragingival dental calculus together with the inorganic contents of oral fluids.17,18 Although the influence of salivary CA levels on pH and caries formation was previously evaluated, there is a lack of information on the contribution to dental calculus formation in the current literature. In the present study, we investigated the correlation between salivary CA VI activity and the amount of supragingival dental calculus on human teeth.
Materials and methods Study population and clinical examination This study was approved by the Ethical Committee of the Faculty of Dentistry, Ataturk University, and informed consent was obtained from each participant. In total, 75 voluntary patients (36 males and 39 females, aged 18e51 years) who were referred to the Periodontology Department were included in the study. None of the individuals was on medication, and none had any teeth with active caries. All clinical evaluations in all individuals at all time points were carried out by the same examiner. Periodontal conditions and the oral hygiene status of the individuals were assessed by means of recording the scores of the plaque index (PI),19 gingival index (GI),20 and oral hygiene index (OHI).21 Levels of supragingival dental calculus were scored
T. Arabacı et al with the VolpeeManhold Index22 (VMI) using a periodontal probe graduated in millimeters. Participants were divided into three groups according to their oral hygiene status and the amount of supragingival dental calculus. Group I included individuals who had no dental calculus formation clinically (VMI Z 0), and were presumed to be individuals with good oral hygiene. Based on previous reports,23,24 individuals with VMI scores of <7 were considered to be light calculus formers (presumed to have moderate oral hygiene, group II), and those with VMI scores of 7 were considered to be heavy calculus formers (presumed to have poor oral hygiene, group III). Individuals were recalled the following day for saliva collection. To measure salivary CA activity, 4 mL of an unstimulated whole-saliva sample was collected from individuals 1 hour after the last food or drink consumption at 8:00e10:00 AM, according to a spitting method previously described by Navazesh.25 Prior to sample collection, individuals were asked to thoroughly rinse their mouth with distilled water to remove any food debris. Individuals were seated comfortably with their eyes open and head tilted slightly forward, and were instructed to rest for 10 min and minimize orofacial movements. Saliva was allowed to accumulate in the floor of the mouth, and the individual spat it out into a graduated plastic tube containing 1 mL 0.1 M Tris SO4 and 0.2 M Na2SO4 pH 8.7 buffer solution every 60 seconds. Forcible spitting was avoided. Salivary samples were then centrifuged at 16,000 rpm for 20 minutes and kept at 4 C until the laboratory assays were run.
Measurement of CA VI levels CA activity was assayed following the hydration of CO2 according to a calorimetric method described by Gulcin et al.26 CO2ehydratase activity of the enzyme was determined at room temperature in veronal buffer (pH 8.6) with Bromo Timol blue as an indicator and a saturated carbon dioxide solution as a substrate in a final volume of 4.2 mL. The time (in seconds) taken for the solution to change from blue to yellow was measured. The enzyme unit (EU) was calculated using the equation (t0 e tc/tc) where t0 and tc are the times for the pH change of the nonenzymatic and enzymatic reactions, respectively.
Statistical analysis Statistical analysis was performed using PASW Statistics 18 software for Windows (IBM Corp., PASW Statistics for windows, version 18.0 (SPSS Inc., Chicago, USA)). Statistical significance between clinical scores and demographic features of the groups were analyzed by Fisher’s least significant difference test. Spearman rank tests were used to correlate the age, clinical scores, and salivary CA VI levels in groups II and III.
Results Comparisons of demographic features and clinical scores among the groups are given in Table 1. There were no statistically significant differences (P > 0.05) in the age or gender distribution among the groups. Differences among
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
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Carbonic anhydrase VI and dental calculus Table 1
3
Comparison of the demographic features and the clinical scores of the participants.
Age Mean Range Gender (n) Male Female PI GI OHI VMI CA VI (EU/mL)
Group I (Good OH) Control (VMI Z 0)
Group II (Moderate OH) Light calculus formers (VMI < 7)
Group III (Poor OH) Heavy calculus formers (VMI 7)
P*
35.4 4.34 18e49
37.1 5.21 20e48
38.3 5.37 22e51
NS d
11 14 0.67 0.59 0.28 0 0.18
10 15 1.08 1.13 0.98 5.53 0.30
15 10 1.82 1.71 1.84 9.78 0.53
d d P< P< P< P< P<
0.38 0.33 0.16 0.11
0.71 0.64 0.42 1.12 0.11
0.94 0.96 0.93 1.94 0.24
0.05 0.05 0.05 0.05 0.05
Analysis of variance (ANOVA) and a post-hoc Fisher’s LSD test were used. *Differences were considered to be significant at P < 0.05. CA VI Z carbonic anhydrase VI; CI Z calculus index; EU Z enzyme unit; GI Z gingival index; NS Z not significant; OH Z oral hygiene; OHI Z oral hygiene index; PI Z plaque index; VMI Z ValpeeManhold index.
the groups according to the mean scores of the PI, GI, OHI, and VMI were statistically significant (P < 0.05). Results showed that the mean CA VI level in group I (individuals with good oral hygiene) was significantly lower than those of the other groups (P < 0.05). However, comparisons between groups II and III showed that salivary CA VI levels were significantly higher in group III (0.53 0.24 EU/mL saliva) compared to group II (0.30 0.11 EU/mL saliva) (P < 0.05). Table 2 shows the correlative analysis between the clinical parameters, age, and salivary CA levels in groups II and III (all individuals of which had clinical dental calculus formation). It was found that the OHI was significantly correlated with VMI scores in groups II (r Z 0.521, P < 0.05) and III (r Z 0.568, P < 0.05). The correlation between OHI and salivary CA VI levels was statistically
Table 2 Correlative analysis between the clinical parameters, age, and salivary CA VI levels in groups II and III. VMI
PI
Group II Age 0.123 PI GI OHI VMI Group III Age 0.163 PI GI OHI VMI
GI
OHI
VMI
CA VI
0.180 0.513*
0.267 0.487* 0.402
0.224 0.496* 0.506* 0.521*
0.123 0.256 0.173 0.357 0.461*
0.122 0.535*
0.160 0.556* 0.503*
0.321 0.605* 0.460* 0.568*
0.151 0.451* 0.453* 0.631* 0.763**
Spearman rank correlation was used. *P < 0.05. **P < 0.01. CA VI Z carbonic anhydrase VI; EU Z enzyme unit; GI Z gingival index; OHI Z oral hygiene index; PI Z plaque index; VMI Z ValpeeManhold index.
significant in group III (r Z 0.631, P < 0.05), while it was not statistically significant in group II (P > 0.05). Significant positive correlations were found between salivary CA levels and the amount of dental calculus in group III (patients with poor oral hygiene) (r Z 0.763, P < 0.01). The relationship between these parameters in group II (patients with moderate oral hygiene) was also statistically significant (r Z 0.461, P < 0.05).
Discussion Isoenzymes of CA catalyze the reversible hydration of carbon dioxide and participate in regulating the acidebase balance in various tissues and biological fluids.27e29 CA VI is an important protein among active mammalian isozymes, and is described as a salivary constituent produced by serous acinar cells.30,31 This isoform is present in the secretion of the parotid and submandibular glands, and has direct activity in the bicarbonate buffering system of saliva.32e34 The salivary buffering capacity controls the pH of dental plaque after exposure to fermentable carbohydrates and hence helps prevent dental caries.14,35 With this buffering action, it was demonstrated that CA VI contributes to neutralizing plaque acids and reducing enamel demineralization.36 Leinonen et al37 and Hannig et al38 reported that active CA VI in the enamel pellicle was assumed to prevent caries by accelerating the removal of hydrogen ions and acidic metabolic products of bacterial plaque in the local microenvironment of tooth surfaces. However, various studies determined lower CA VI activity in salivary samples of carious individuals compared to noncarious individuals.14,35 Contents and the physicochemical structure of saliva affect caries formation and plaque and calculus formation, which are the primary factors responsible for periodontal diseases. The formation and amount of dental calculus are reasonably related to the mineralization of dental plaque by inorganic salts generated from oral fluids. Moreover, it is known that supragingival dental calculus tends to be
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
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4 restricted to tooth surfaces adjacent to salivary ducts, and the inorganic salts in saliva, responsible for the primary stage of calculus formation, rapidly precipitate in an alkaline pH.39 Several studies investigated whether salivary enzymes affected the calcification process of dental plaque together with the inorganic components of the saliva. Watanabe et al17 analyzed protease activities of saliva, and they reported significant positive correlations between suband supragingival calculus indices and salivary protease activity. They also suggested that salivary protease enhanced calculus formation. Morita and Watanabe40 later demonstrated that the protease activity of dental plaque was significantly higher in patients with an increased supragingival calculus index. In one study, the correlation between salivary alkaline phosphatase levels and the formation of supragingival calculus was analyzed, but no significant correlations were found between these parameters.18 However, various enzymes such as lactate dehydrogenase, alkaline phosphatase, and acid phosphatase were found in most calculus sections.41e43 Even though several studies postulated a possible relation between increased salivary CA activities and supragingival dental calculus formation, no significant investigation was found in the current literature. In this study, CA VI levels were measured in unstimulated whole-saliva samples obtained from individuals with good, moderate, and poor oral hygiene habits classified according to their VMI and OHI scores. It was previously reported that saliva is closely associated with the distribution pattern of supragingival calculus. It was also stated that plaque absorbs calcium and phosphate from the saliva to form supragingival calculus and from crevicular fluid to form subgingival calculus.44 Therefore, we correlated salivary CA activity with the amount of supragingival dental calculus. In this study, a colorimetric method was used for the salivary CA assay. However, it was reasonably difficult to detect salivary CA activity by a colorimetric method, especially at low concentrations of the enzyme, and the measurements had to be performed more precisely due to the disadvantages of the method. The results of this study indicated that salivary CA VI concentrations were significantly higher in individuals with poor oral hygiene compared to individuals with a good or moderate oral hygiene status. The results showed a positive relationship between higher PI and GI scores and salivary CA VI levels in patients with poor oral hygiene, and supported the findings of Kivela et al.14 In a neglected oral environment, the salivary pH is reduced to acidic levels, and the salivary buffering system is immediately activated in order to neutralize the pH. Salivary CA is an important component of this buffering system, and its salivary levels would be expected to be elevated in individuals with poor oral hygiene. Our findings also confirmed that there were significant positive correlations between CA VI activity and OHI scores in patients with poor oral hygiene. Previously, it was suggested that higher CA levels increased the salivary pH and the tendency to precipitate calcium salts.10,16 Thus, it is reasonable to consider that the level of CA VI in saliva of patients with poor oral hygiene would increase to prevent caries processes. As mentioned above, several studies reported that some salivary enzymes facilitate the accumulation of inorganic salts in saliva with a basic pH.17,40 The
T. Arabacı et al hypothesis of this study suggested a potential relationship between higher levels of salivary CA and the amount of supragingival dental calculus. Consequently, significant positive correlations were found between CA VI levels and VMI scores in patients with supragingival dental calculus. Furthermore, the correlation was more significant in heavy calculus formers. Since there are few investigations of the relationship between these parameters, data of this study could not be compared with the previous literature. Although the correlation between increased salivary CA activity and the amount of supragingival dental calculus was statistically significant, especially in a neglected oral hygiene status, many factors including individual variations and the methodology of this study might have influenced this finding. However, if these parameters were correlated with the other components and the pH of saliva samples, we believe that the findings would be more accurate. Therefore, further investigations are needed to define the exact role of salivary CA in calculus formation, including an analysis of ion transport between the saliva and deposits on tooth surfaces.
Funding statement This study was self-funded by our institution.
References 1. Parkkila S, Parkkila A- K. Carbonic anhydrase in the alimentary tract. Roles of the different isozymes and salivary factors in the maintenance of optimal conditions in the gastrointestinal canal. Scand J Gastroenterol 1996;31:305e17. 2. Bardow A, Moe D, Nyvad B, Nauntofte B. The buffer capacity and buffer systems of human whole saliva measured without loss of CO2. Arch Oral Biol 2000;45:1e12. 3. Supuran CT, Scozzafava A. Applications of carbonic anhydrase inhibitors and activators in therapy. Exp Opin Ther Patents 2002;12:217e42. 4. Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors. Med Res Rev 2003;23:146e89. 5. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors and their therapeutic potential. Exp Opin Ther Patents 2000;10: 575e600. 6. Kimoto M, Kishino M, Yura Y, Ogawa Y. A role of salivary carbonic anhydrase VI in dental plaque. Arch Oral Biol 2006;51: 117e22. 7. Ogawa Y, Matsumoto K, Maeda T, Tamai R, Suzuki T, Sasano H, et al. Characterization of lacrimal gland carbonic anhydrase VI. J Histochem Cytochem 2002;50:821e7. 8. Kimoto M, Iwai S, Maeda T, Yura Y, Fernley RT, Ogawa Y. Carbonic anhydrase VI in the mouse nasal gland. J Histochem Cytochem 2004;52:1057e62. 9. Fleming RE, Parkkila S, Parkkila A-K, Rajaniemi H, Waheed A, Sly WS. Carbonic anhydrase IV in rat and human gastrointestinal tract: regional cellular and subcellular localization. J Clin Invest 1995;96:2907e13. 10. Rapp GW. The biochemistry of oral calculus. II. The presence of carbonic anhydrase in human saliva. J Am Dental Assoc 1946; 33:191. 11. McConnell D, Frajola WJ, Deamer DW. Relation between the inorganic chemistry and biochemistry of bone mineralization. Science 1960;133:281e2. 12. Parkkila S, Parkkila AK, Vierjoki T, Sta ˚hlberg T, Rajaniemi H. Competitive time-resolved immunofluorometric assay for
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
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MODEL
Carbonic anhydrase VI and dental calculus
13.
14.
15. 16.
17.
18.
19.
20. 21. 22.
23.
24.
25. 26.
27.
28.
quantifying carbonic anhydrase VI in saliva. Clin Chem 1993;39: 2154e7. Kivela J, Parkkila S, Metteri J, Parkkila A-K, Toivanen A, Rajaniemi H. Salivary carbonic anhydrase VI concentrations and its relation to basic characteristics of saliva in young men. Acta Physiol Scand 1997;161:221e5. Kivela J, Parkkila S, Parkkila AK, Rajaniemi H. A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence. Caries Res 1999;33:178e84. Szabo I. Carbonic anhydrase activity in the saliva of children and its relation to caries activity. Caries Res 1974;8:187e91. Lang NP, Mombelli A, Attstro ¨m R. Dental plaque and calculus. In: Lindhe J, ed. Clinical Periodontology and Implant Dentistry, 9th ed. Philadelphia: W.B. Saunders Company, 2002:106e49. Watanabe T, Toda K, Morishita M, Iwamoto Y. Correlations between salivary protease and supragingival or subgingival calculus index. J Dent Res 1982;61:1048e51. Yamalık N, Ozer N, Caglayan F, Caglayan G. Salyadaki alkalen fosfataz du ¨zeyi ve supragingival dis‚tas‚ı olus‚umu arasındaki ilis‚kinin incelenmesi. Hacettepe Dis Hek Fak Derg 1990;14:65e7. Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:121e35. Loe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand 1963;21:533e51. Greene JC, Vermillion JR. The simplified oral hygiene index. J Am Dental Assoc 1964;68:7e13. Volpe AR, Manhold JH, Hazen SP. In vivo calculus assessment: Part I. A method and its examiner reproducibility. J Periodontol 1965;36:32e44. Volpe AR, Kupczak LJ, King WJ. In vivo calculus assessment: Part III: scoring techniques, rate of calculus formation, partial mouth exams vs. full mouth exams, and intra-examiner reproducibility. Periodontics 1967;5:184e93. Beiswanger BB, Segreto VA, Mallatt ME, Pfeiffer HJ. The prevalence and incidence of dental calculus in adults. J Clin Dent 1989;1:55e8. Navazesh M. Methods for collecting saliva. Ann NY Acad Sci 1993;694:72e7. Gulcin I, Beydemir S, Buyukokuroglu ME. In vitro and in vivo effects of dantrolene on carbonic anhydrase enzyme activities. Biol Pharm Bull 2004;27:613e6. Tashian RE. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioassays 1989;10: 186e92. Tashian RE. Genetics of the mammalian carbonic anhydrases. Adv Genet 1992;30:321e56.
5 29. Parkkila S, Parkkila A-K, Rajaniemi H. Circadian periodicity in salivary carbonic anhydrase VI concentration. Acta Physiol Scand 1995;154:205e11. 30. Thatcher BJ, Doherty AE, Orvisky E, Martin BM, Henkin RI. Gustin from human parotid saliva is carbonic anhydrase VI. Biochem Biophys Res Commun 1998;250:635e41. 31. Kivela J, Parkkila S, Parkkila A-K, Leinonen J, Rajaniemi H. Salivary carbonic anhydrase isoenzyme VI. Topical review. J Physiol 1999;520:315e20. 32. Fernley RT, Wright RD, Coghlan JP. Radioimmunoassay of carbonic anhydrase VI in saliva and sheep tissues. Biochem J 1991; 274:313e6. 33. Fernley RT, Farthing J, Cooper EJ. Radioimmunoassay for salivary carbonic anhydrase in human parotid saliva. Arch Oral Biol 1995;40:567e9. 34. Parkkila S, Kaunisto K, Rajaniemi L, Kumpulainen T, Jokinen K, Rajaniemi H. Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II and I in human parotid and submandibular glands. J Histochem Cytochem 1990;38: 941e7. 35. Parkkila S, Parkkila A-K, Lehtola J, Reinila ¨ A, So ¨dervik HJ, Rannisto M, et al. Salivary carbonic anhydrase protects gastroesophageal mucosa from acid injury. Dig Dis Sci 1997;42: 1013e9. 36. Yildiz M, Nalbantoglu B. The comparison of saliva carbonic anhydrase activities in carrious and noncarrious subjects. J Dent Fac Atatu¨rk Univ 2000;10:1e3. 37. Leinonen J, Kivela J, Parkkila S, Parkkila AK, Rajaniemi H. Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle. Caries Res 1999;33:185e90. 38. Hannig C, Hannig M, Attin T. Enzymes in the acquired enamel pellicle. Eur J Oral Sci 2005;113:2e13. 39. Sharma NC, Galustians HC, Qaqish J, Cugini M, Warren PR. The effect of two power toothbrushes on calculus and stain formation. Am J Dent 2002;15:71e6. 40. Morita M, Watanabe T. Relation between the presence of supragingival calculus and protease activity in dental plaque. J Dent Res 1986;65:703e5. 41. Friskopp J, Hammarstrom L. An enzyme histochemical study of dental plaque and calculus. Acta Odontol Scand 1982;40: 459e66. 42. Isacsson G, Hammarstrom L. An enzyme histochemical study of human salivary duct calculi. J Oral Pathol 1983;12:217e22. 43. Draus FJ, Tarbet WJ, Miklos FL. Salivary enzymes and calculus formation. J Periodontal Res 1968;3:232e5. 44. Jin Y, Yip H- K. Supragingival calculus: formation and control. Crit Rev Oral Biol Med 2002;13:426e41.
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
MODEL
Journal of Dental Sciences (2013) xx, 1e5
Available online at www.sciencedirect.com
journal homepage: www.e-jds.com
ORIGINAL ARTICLE
Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores? ¨kru ¨ Beydemir b, Cenk Fatih C ¸ic ¸ek a, S‚u Taner Arabacı a*, Yasin C ¸anakc ¸ı a, Varol C ¸anakc ¸ı a a b
Department of Periodontology, Faculty of Dentistry, Atatu¨rk University, Erzurum, Turkey Department of Chemistry, Science and Arts Faculty, Atatu¨rk University, Erzurum, Turkey
Received 19 April 2012; Final revision received 29 November 2012
KEYWORDS carbonic anhydrase; dental calculus; oral hygiene; saliva
Abstract Background/purpose: Carbonic anhydrase (CA) VI is a unique salivary isoenzyme that regulates the pH in the oral environment. Its increased levels result in a more-alkaline saliva, hence it was hypothesized to contribute to dental calculus formation together with salivary inorganic salts. In this study, we evaluated the relationship between salivary CA VI levels and the amount of supragingival dental calculus formation on human teeth. Materials and methods: Seventy-five volunteers were grouped according to their oral hygiene status, and the amount of supragingival dental calculus was measured by the VolpeeManhold Index (VMI). Group I included control individuals who had no dental calculus formation clinically (VMI Z 0). Group II included light calculus formers (VMI < 7), and group III included heavy calculus formers (VMI 7). Clinical periodontal indices and salivary CA VI levels were measured in all individuals, and were compared among the groups and correlated within groups. Results: Differences among the groups according to clinical scores and CA VI levels were found to be statistically significant (P < 0.05). The correlation between the oral hygiene index and salivary CA VI levels was found statistically significant in group III (P < 0.05), but was not statistically significant in group II (P > 0.05). Significant positive correlations were found between salivary CA levels and amount of dental calculus in groups II and III (P < 0.05 and < 0.01, respectively). Conclusion: This study found positive correlations between increased salivary CA VI activity and the amount of supragingival dental calculus; however, further investigations are needed to clarify the exact association between these parameters. Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved.
¨ niversitesi, Erzurum 25240, Turkey. * Corresponding author. Periodontoloji Anabilim Dalı, Dis‚ Hekimligi Faku ¨ltesi, Atatu ¨rk U E-mail address: [email protected] (T. Arabacı). 1991-7902/$36 Copyright ª 2013, Association for Dental Sciences of the Republic of China. Published by Elsevier Taiwan LLC. All rights reserved. http://dx.doi.org/10.1016/j.jds.2012.12.014
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
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2
Introduction Carbonic anhydrase (CA) is a zinc-containing metalloenzyme that participates in a variety of physiological processes. CA enzymes catalyze various reactions including carbon dioxide and bicarbonate transport, pH regulation, and water/electrolyte balance in various tissues.1e6 Among many of the active mammalian isozymes, CA VI is the specific form secreted by salivary glands.7e9 It was demonstrated that this isoform accelerates the removal of carbon dioxide and results in alkalinization of the saliva.10,11 Although CA was originally considered to regulate the salivary pH or buffer capacity, it was demonstrated that these variables are not directly associated with the CA VI concentration in saliva.12e14 Saliva helps maintain the oral health, and one of its important functions is to regulate pH changes in the oral cavity. Salivary pH is closely related to the buffering capacity, which is essential for neutralization of acidity in dental plaque,6,13 and affects the caries process and calculus formation.15 Although the formation of dental calculus is primarily influenced by individual oral hygiene habits, many factors including the diet, age, ethnic origin, systemic diseases, inorganic contents, and the pH of the saliva are also related to calculus indices. It was revealed that calcification of dental plaque biofilm with mineral ions was provided by bathing saliva or crevicular fluids.16 On the other hand, the viscosity and contents of the saliva were reported to be related to calcification of organic deposits on tooth surfaces.13 Furthermore, several authors suggested that increased levels of some salivary enzymes, such as protease and alkaline phosphatase, can also contribute to the formation of sub- and supragingival dental calculus together with the inorganic contents of oral fluids.17,18 Although the influence of salivary CA levels on pH and caries formation was previously evaluated, there is a lack of information on the contribution to dental calculus formation in the current literature. In the present study, we investigated the correlation between salivary CA VI activity and the amount of supragingival dental calculus on human teeth.
Materials and methods Study population and clinical examination This study was approved by the Ethical Committee of the Faculty of Dentistry, Ataturk University, and informed consent was obtained from each participant. In total, 75 voluntary patients (36 males and 39 females, aged 18e51 years) who were referred to the Periodontology Department were included in the study. None of the individuals was on medication, and none had any teeth with active caries. All clinical evaluations in all individuals at all time points were carried out by the same examiner. Periodontal conditions and the oral hygiene status of the individuals were assessed by means of recording the scores of the plaque index (PI),19 gingival index (GI),20 and oral hygiene index (OHI).21 Levels of supragingival dental calculus were scored
T. Arabacı et al with the VolpeeManhold Index22 (VMI) using a periodontal probe graduated in millimeters. Participants were divided into three groups according to their oral hygiene status and the amount of supragingival dental calculus. Group I included individuals who had no dental calculus formation clinically (VMI Z 0), and were presumed to be individuals with good oral hygiene. Based on previous reports,23,24 individuals with VMI scores of <7 were considered to be light calculus formers (presumed to have moderate oral hygiene, group II), and those with VMI scores of 7 were considered to be heavy calculus formers (presumed to have poor oral hygiene, group III). Individuals were recalled the following day for saliva collection. To measure salivary CA activity, 4 mL of an unstimulated whole-saliva sample was collected from individuals 1 hour after the last food or drink consumption at 8:00e10:00 AM, according to a spitting method previously described by Navazesh.25 Prior to sample collection, individuals were asked to thoroughly rinse their mouth with distilled water to remove any food debris. Individuals were seated comfortably with their eyes open and head tilted slightly forward, and were instructed to rest for 10 min and minimize orofacial movements. Saliva was allowed to accumulate in the floor of the mouth, and the individual spat it out into a graduated plastic tube containing 1 mL 0.1 M Tris SO4 and 0.2 M Na2SO4 pH 8.7 buffer solution every 60 seconds. Forcible spitting was avoided. Salivary samples were then centrifuged at 16,000 rpm for 20 minutes and kept at 4 C until the laboratory assays were run.
Measurement of CA VI levels CA activity was assayed following the hydration of CO2 according to a calorimetric method described by Gulcin et al.26 CO2ehydratase activity of the enzyme was determined at room temperature in veronal buffer (pH 8.6) with Bromo Timol blue as an indicator and a saturated carbon dioxide solution as a substrate in a final volume of 4.2 mL. The time (in seconds) taken for the solution to change from blue to yellow was measured. The enzyme unit (EU) was calculated using the equation (t0 e tc/tc) where t0 and tc are the times for the pH change of the nonenzymatic and enzymatic reactions, respectively.
Statistical analysis Statistical analysis was performed using PASW Statistics 18 software for Windows (IBM Corp., PASW Statistics for windows, version 18.0 (SPSS Inc., Chicago, USA)). Statistical significance between clinical scores and demographic features of the groups were analyzed by Fisher’s least significant difference test. Spearman rank tests were used to correlate the age, clinical scores, and salivary CA VI levels in groups II and III.
Results Comparisons of demographic features and clinical scores among the groups are given in Table 1. There were no statistically significant differences (P > 0.05) in the age or gender distribution among the groups. Differences among
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
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Carbonic anhydrase VI and dental calculus Table 1
3
Comparison of the demographic features and the clinical scores of the participants.
Age Mean Range Gender (n) Male Female PI GI OHI VMI CA VI (EU/mL)
Group I (Good OH) Control (VMI Z 0)
Group II (Moderate OH) Light calculus formers (VMI < 7)
Group III (Poor OH) Heavy calculus formers (VMI 7)
P*
35.4 4.34 18e49
37.1 5.21 20e48
38.3 5.37 22e51
NS d
11 14 0.67 0.59 0.28 0 0.18
10 15 1.08 1.13 0.98 5.53 0.30
15 10 1.82 1.71 1.84 9.78 0.53
d d P< P< P< P< P<
0.38 0.33 0.16 0.11
0.71 0.64 0.42 1.12 0.11
0.94 0.96 0.93 1.94 0.24
0.05 0.05 0.05 0.05 0.05
Analysis of variance (ANOVA) and a post-hoc Fisher’s LSD test were used. *Differences were considered to be significant at P < 0.05. CA VI Z carbonic anhydrase VI; CI Z calculus index; EU Z enzyme unit; GI Z gingival index; NS Z not significant; OH Z oral hygiene; OHI Z oral hygiene index; PI Z plaque index; VMI Z ValpeeManhold index.
the groups according to the mean scores of the PI, GI, OHI, and VMI were statistically significant (P < 0.05). Results showed that the mean CA VI level in group I (individuals with good oral hygiene) was significantly lower than those of the other groups (P < 0.05). However, comparisons between groups II and III showed that salivary CA VI levels were significantly higher in group III (0.53 0.24 EU/mL saliva) compared to group II (0.30 0.11 EU/mL saliva) (P < 0.05). Table 2 shows the correlative analysis between the clinical parameters, age, and salivary CA levels in groups II and III (all individuals of which had clinical dental calculus formation). It was found that the OHI was significantly correlated with VMI scores in groups II (r Z 0.521, P < 0.05) and III (r Z 0.568, P < 0.05). The correlation between OHI and salivary CA VI levels was statistically
Table 2 Correlative analysis between the clinical parameters, age, and salivary CA VI levels in groups II and III. VMI
PI
Group II Age 0.123 PI GI OHI VMI Group III Age 0.163 PI GI OHI VMI
GI
OHI
VMI
CA VI
0.180 0.513*
0.267 0.487* 0.402
0.224 0.496* 0.506* 0.521*
0.123 0.256 0.173 0.357 0.461*
0.122 0.535*
0.160 0.556* 0.503*
0.321 0.605* 0.460* 0.568*
0.151 0.451* 0.453* 0.631* 0.763**
Spearman rank correlation was used. *P < 0.05. **P < 0.01. CA VI Z carbonic anhydrase VI; EU Z enzyme unit; GI Z gingival index; OHI Z oral hygiene index; PI Z plaque index; VMI Z ValpeeManhold index.
significant in group III (r Z 0.631, P < 0.05), while it was not statistically significant in group II (P > 0.05). Significant positive correlations were found between salivary CA levels and the amount of dental calculus in group III (patients with poor oral hygiene) (r Z 0.763, P < 0.01). The relationship between these parameters in group II (patients with moderate oral hygiene) was also statistically significant (r Z 0.461, P < 0.05).
Discussion Isoenzymes of CA catalyze the reversible hydration of carbon dioxide and participate in regulating the acidebase balance in various tissues and biological fluids.27e29 CA VI is an important protein among active mammalian isozymes, and is described as a salivary constituent produced by serous acinar cells.30,31 This isoform is present in the secretion of the parotid and submandibular glands, and has direct activity in the bicarbonate buffering system of saliva.32e34 The salivary buffering capacity controls the pH of dental plaque after exposure to fermentable carbohydrates and hence helps prevent dental caries.14,35 With this buffering action, it was demonstrated that CA VI contributes to neutralizing plaque acids and reducing enamel demineralization.36 Leinonen et al37 and Hannig et al38 reported that active CA VI in the enamel pellicle was assumed to prevent caries by accelerating the removal of hydrogen ions and acidic metabolic products of bacterial plaque in the local microenvironment of tooth surfaces. However, various studies determined lower CA VI activity in salivary samples of carious individuals compared to noncarious individuals.14,35 Contents and the physicochemical structure of saliva affect caries formation and plaque and calculus formation, which are the primary factors responsible for periodontal diseases. The formation and amount of dental calculus are reasonably related to the mineralization of dental plaque by inorganic salts generated from oral fluids. Moreover, it is known that supragingival dental calculus tends to be
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
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4 restricted to tooth surfaces adjacent to salivary ducts, and the inorganic salts in saliva, responsible for the primary stage of calculus formation, rapidly precipitate in an alkaline pH.39 Several studies investigated whether salivary enzymes affected the calcification process of dental plaque together with the inorganic components of the saliva. Watanabe et al17 analyzed protease activities of saliva, and they reported significant positive correlations between suband supragingival calculus indices and salivary protease activity. They also suggested that salivary protease enhanced calculus formation. Morita and Watanabe40 later demonstrated that the protease activity of dental plaque was significantly higher in patients with an increased supragingival calculus index. In one study, the correlation between salivary alkaline phosphatase levels and the formation of supragingival calculus was analyzed, but no significant correlations were found between these parameters.18 However, various enzymes such as lactate dehydrogenase, alkaline phosphatase, and acid phosphatase were found in most calculus sections.41e43 Even though several studies postulated a possible relation between increased salivary CA activities and supragingival dental calculus formation, no significant investigation was found in the current literature. In this study, CA VI levels were measured in unstimulated whole-saliva samples obtained from individuals with good, moderate, and poor oral hygiene habits classified according to their VMI and OHI scores. It was previously reported that saliva is closely associated with the distribution pattern of supragingival calculus. It was also stated that plaque absorbs calcium and phosphate from the saliva to form supragingival calculus and from crevicular fluid to form subgingival calculus.44 Therefore, we correlated salivary CA activity with the amount of supragingival dental calculus. In this study, a colorimetric method was used for the salivary CA assay. However, it was reasonably difficult to detect salivary CA activity by a colorimetric method, especially at low concentrations of the enzyme, and the measurements had to be performed more precisely due to the disadvantages of the method. The results of this study indicated that salivary CA VI concentrations were significantly higher in individuals with poor oral hygiene compared to individuals with a good or moderate oral hygiene status. The results showed a positive relationship between higher PI and GI scores and salivary CA VI levels in patients with poor oral hygiene, and supported the findings of Kivela et al.14 In a neglected oral environment, the salivary pH is reduced to acidic levels, and the salivary buffering system is immediately activated in order to neutralize the pH. Salivary CA is an important component of this buffering system, and its salivary levels would be expected to be elevated in individuals with poor oral hygiene. Our findings also confirmed that there were significant positive correlations between CA VI activity and OHI scores in patients with poor oral hygiene. Previously, it was suggested that higher CA levels increased the salivary pH and the tendency to precipitate calcium salts.10,16 Thus, it is reasonable to consider that the level of CA VI in saliva of patients with poor oral hygiene would increase to prevent caries processes. As mentioned above, several studies reported that some salivary enzymes facilitate the accumulation of inorganic salts in saliva with a basic pH.17,40 The
T. Arabacı et al hypothesis of this study suggested a potential relationship between higher levels of salivary CA and the amount of supragingival dental calculus. Consequently, significant positive correlations were found between CA VI levels and VMI scores in patients with supragingival dental calculus. Furthermore, the correlation was more significant in heavy calculus formers. Since there are few investigations of the relationship between these parameters, data of this study could not be compared with the previous literature. Although the correlation between increased salivary CA activity and the amount of supragingival dental calculus was statistically significant, especially in a neglected oral hygiene status, many factors including individual variations and the methodology of this study might have influenced this finding. However, if these parameters were correlated with the other components and the pH of saliva samples, we believe that the findings would be more accurate. Therefore, further investigations are needed to define the exact role of salivary CA in calculus formation, including an analysis of ion transport between the saliva and deposits on tooth surfaces.
Funding statement This study was self-funded by our institution.
References 1. Parkkila S, Parkkila A- K. Carbonic anhydrase in the alimentary tract. Roles of the different isozymes and salivary factors in the maintenance of optimal conditions in the gastrointestinal canal. Scand J Gastroenterol 1996;31:305e17. 2. Bardow A, Moe D, Nyvad B, Nauntofte B. The buffer capacity and buffer systems of human whole saliva measured without loss of CO2. Arch Oral Biol 2000;45:1e12. 3. Supuran CT, Scozzafava A. Applications of carbonic anhydrase inhibitors and activators in therapy. Exp Opin Ther Patents 2002;12:217e42. 4. Supuran CT, Scozzafava A, Casini A. Carbonic anhydrase inhibitors. Med Res Rev 2003;23:146e89. 5. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors and their therapeutic potential. Exp Opin Ther Patents 2000;10: 575e600. 6. Kimoto M, Kishino M, Yura Y, Ogawa Y. A role of salivary carbonic anhydrase VI in dental plaque. Arch Oral Biol 2006;51: 117e22. 7. Ogawa Y, Matsumoto K, Maeda T, Tamai R, Suzuki T, Sasano H, et al. Characterization of lacrimal gland carbonic anhydrase VI. J Histochem Cytochem 2002;50:821e7. 8. Kimoto M, Iwai S, Maeda T, Yura Y, Fernley RT, Ogawa Y. Carbonic anhydrase VI in the mouse nasal gland. J Histochem Cytochem 2004;52:1057e62. 9. Fleming RE, Parkkila S, Parkkila A-K, Rajaniemi H, Waheed A, Sly WS. Carbonic anhydrase IV in rat and human gastrointestinal tract: regional cellular and subcellular localization. J Clin Invest 1995;96:2907e13. 10. Rapp GW. The biochemistry of oral calculus. II. The presence of carbonic anhydrase in human saliva. J Am Dental Assoc 1946; 33:191. 11. McConnell D, Frajola WJ, Deamer DW. Relation between the inorganic chemistry and biochemistry of bone mineralization. Science 1960;133:281e2. 12. Parkkila S, Parkkila AK, Vierjoki T, Sta ˚hlberg T, Rajaniemi H. Competitive time-resolved immunofluorometric assay for
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014
+
MODEL
Carbonic anhydrase VI and dental calculus
13.
14.
15. 16.
17.
18.
19.
20. 21. 22.
23.
24.
25. 26.
27.
28.
quantifying carbonic anhydrase VI in saliva. Clin Chem 1993;39: 2154e7. Kivela J, Parkkila S, Metteri J, Parkkila A-K, Toivanen A, Rajaniemi H. Salivary carbonic anhydrase VI concentrations and its relation to basic characteristics of saliva in young men. Acta Physiol Scand 1997;161:221e5. Kivela J, Parkkila S, Parkkila AK, Rajaniemi H. A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence. Caries Res 1999;33:178e84. Szabo I. Carbonic anhydrase activity in the saliva of children and its relation to caries activity. Caries Res 1974;8:187e91. Lang NP, Mombelli A, Attstro ¨m R. Dental plaque and calculus. In: Lindhe J, ed. Clinical Periodontology and Implant Dentistry, 9th ed. Philadelphia: W.B. Saunders Company, 2002:106e49. Watanabe T, Toda K, Morishita M, Iwamoto Y. Correlations between salivary protease and supragingival or subgingival calculus index. J Dent Res 1982;61:1048e51. Yamalık N, Ozer N, Caglayan F, Caglayan G. Salyadaki alkalen fosfataz du ¨zeyi ve supragingival dis‚tas‚ı olus‚umu arasındaki ilis‚kinin incelenmesi. Hacettepe Dis Hek Fak Derg 1990;14:65e7. Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:121e35. Loe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand 1963;21:533e51. Greene JC, Vermillion JR. The simplified oral hygiene index. J Am Dental Assoc 1964;68:7e13. Volpe AR, Manhold JH, Hazen SP. In vivo calculus assessment: Part I. A method and its examiner reproducibility. J Periodontol 1965;36:32e44. Volpe AR, Kupczak LJ, King WJ. In vivo calculus assessment: Part III: scoring techniques, rate of calculus formation, partial mouth exams vs. full mouth exams, and intra-examiner reproducibility. Periodontics 1967;5:184e93. Beiswanger BB, Segreto VA, Mallatt ME, Pfeiffer HJ. The prevalence and incidence of dental calculus in adults. J Clin Dent 1989;1:55e8. Navazesh M. Methods for collecting saliva. Ann NY Acad Sci 1993;694:72e7. Gulcin I, Beydemir S, Buyukokuroglu ME. In vitro and in vivo effects of dantrolene on carbonic anhydrase enzyme activities. Biol Pharm Bull 2004;27:613e6. Tashian RE. The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioassays 1989;10: 186e92. Tashian RE. Genetics of the mammalian carbonic anhydrases. Adv Genet 1992;30:321e56.
5 29. Parkkila S, Parkkila A-K, Rajaniemi H. Circadian periodicity in salivary carbonic anhydrase VI concentration. Acta Physiol Scand 1995;154:205e11. 30. Thatcher BJ, Doherty AE, Orvisky E, Martin BM, Henkin RI. Gustin from human parotid saliva is carbonic anhydrase VI. Biochem Biophys Res Commun 1998;250:635e41. 31. Kivela J, Parkkila S, Parkkila A-K, Leinonen J, Rajaniemi H. Salivary carbonic anhydrase isoenzyme VI. Topical review. J Physiol 1999;520:315e20. 32. Fernley RT, Wright RD, Coghlan JP. Radioimmunoassay of carbonic anhydrase VI in saliva and sheep tissues. Biochem J 1991; 274:313e6. 33. Fernley RT, Farthing J, Cooper EJ. Radioimmunoassay for salivary carbonic anhydrase in human parotid saliva. Arch Oral Biol 1995;40:567e9. 34. Parkkila S, Kaunisto K, Rajaniemi L, Kumpulainen T, Jokinen K, Rajaniemi H. Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II and I in human parotid and submandibular glands. J Histochem Cytochem 1990;38: 941e7. 35. Parkkila S, Parkkila A-K, Lehtola J, Reinila ¨ A, So ¨dervik HJ, Rannisto M, et al. Salivary carbonic anhydrase protects gastroesophageal mucosa from acid injury. Dig Dis Sci 1997;42: 1013e9. 36. Yildiz M, Nalbantoglu B. The comparison of saliva carbonic anhydrase activities in carrious and noncarrious subjects. J Dent Fac Atatu¨rk Univ 2000;10:1e3. 37. Leinonen J, Kivela J, Parkkila S, Parkkila AK, Rajaniemi H. Salivary carbonic anhydrase isoenzyme VI is located in the human enamel pellicle. Caries Res 1999;33:185e90. 38. Hannig C, Hannig M, Attin T. Enzymes in the acquired enamel pellicle. Eur J Oral Sci 2005;113:2e13. 39. Sharma NC, Galustians HC, Qaqish J, Cugini M, Warren PR. The effect of two power toothbrushes on calculus and stain formation. Am J Dent 2002;15:71e6. 40. Morita M, Watanabe T. Relation between the presence of supragingival calculus and protease activity in dental plaque. J Dent Res 1986;65:703e5. 41. Friskopp J, Hammarstrom L. An enzyme histochemical study of dental plaque and calculus. Acta Odontol Scand 1982;40: 459e66. 42. Isacsson G, Hammarstrom L. An enzyme histochemical study of human salivary duct calculi. J Oral Pathol 1983;12:217e22. 43. Draus FJ, Tarbet WJ, Miklos FL. Salivary enzymes and calculus formation. J Periodontal Res 1968;3:232e5. 44. Jin Y, Yip H- K. Supragingival calculus: formation and control. Crit Rev Oral Biol Med 2002;13:426e41.
Please cite this article in press as: Arabacı T, et al., Are increased salivary carbonic anhydrase VI levels related to the amount of supragingival dental calculus formation and clinical periodontal scores?, Journal of Dental Sciences (2013), http://dx.doi.org/10.1016/ j.jds.2012.12.014