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Salivary Proteins May Be Useful for Determining Caries Susceptibility
REVIEW ANALYSIS & EVALUATION ARTICLE TITLE AND BIBLIOGRAPHIC INFORMATION Salivary proteins as a biomarker for dental caries-A systematic review. Martins C, Buczynski AK, Maia LC, Siqueira WL, Castro GF. J Dent 2013; 41(1): 2-8.
REVIEWER Martin Levine, BDS, BSc, PhD
PURPOSE/QUESTION What is the evidence for an association between individual salivary protein composition or content and dental caries experience?
SOURCE OF FUNDING Canadian Government Institutes of Health Research
TYPE OF STUDY/DESIGN Systematic review
Salivary Proteins May Be Useful for Determining Caries Susceptibility SUMMARY Selection Criteria An electronic search was performed in the PubMed Medline, Ovid Medline, ISI Web of Science, Medline, Cochrane Library, Lilacs, Scielo, BBO, Paho, and Wholis databases applying the following MeSH terms: ‘‘dental caries’’ OR ‘‘tooth demineralization’’ OR ‘‘dental caries susceptibility’’ OR ‘‘dental enamel solubility’’ AND ‘‘salivary proteins and peptides’’ OR ‘‘saliva’’ AND ‘‘proteins.’’ All searches were limited to human studies published between 1950 and 2011. From the electronically selected papers, a hand search of the references and a ‘related articles’ link search was also performed.
Key Study Factor A study was only included in this review if it was an original investigation in which groups of individuals with and without caries were compared with a measurement of salivary proteins or peptides. The importance of each study was assessed from ‘risk of bias,’ defined by the following criteria: (1) no medically compromised subjects or an unrelated aim; (2) no fluoride exposure; (3) clinical diagnosis; (4) radiographic assessment; (5) experienced examiner(s); (6) calibrated examiner(s); (7) saliva collection method given; (8) saliva analytical method given; (9) statistical analysis done; and (10) paired groups. None of the included studies were blinded or randomized controlled trials.
LEVEL OF EVIDENCE
Main Outcome Measure
Level 2: Limited-quality, patientoriented evidence
The statistical significance of the protein composition or content of saliva from subjects with caries experience compared with that of subjects without caries experience.
STRENGTH OF RECOMMENDATION GRADE Grade B: Inconsistent or limitedquality patient-oriented evidence
J Evid Base Dent Pract 2013;13:91-93 1532-3382/$36.00 Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jebdp.2013.07.005
Main Results The electronic search found 186 articles. An additional paper was found from the hand search and another from a ‘related articles’ link. Of these 188 articles, only 7 (4%) met inclusion criteria with a ‘low or moderate risk of bias; ’ i.e., they met 3 or more of the 10 criteria. All 7 included studies compared data from subjects with no caries with that of subjects who had caries irrespective of whether the caries had been treated. Two of the 7 studies reported a significant difference in types of salivary proteins between subjects with caries compared to those without caries: a lack of large basic proline-rich proteins in parotid saliva1 or a greater content of an uncharacterized 17 kDa salivary protein in unstimulated whole saliva from men.2 A third study found that subjects with caries had a greater content of proteins in unstimulated whole saliva than those without caries.3 The remaining 4 studies found no association of salivary protein type or content with caries.4–7
JOURNAL OF EVIDENCE-BASED DENTAL PRACTICE
Conclusions Only three of the seven studies found a statistically significant difference between individuals with and without caries experience in terms of salivary proteins and dental caries. Although these studies found a relationship regarding protein phenotypes, total protein concentration, or protein molecular weight, there was insufficient evidence to establish salivary proteins as a biomarker for dental caries.3
COMMENTARY AND ANALYSIS The search omitted two important papers by Vitorino et al8,9 that fulfill the inclusion criteria and support the finding of the Ayad paper.1 In first paper, whole saliva was collected from 20 males, each characterized by an experienced dentist as caries-free (DMFT = 0) or having treated caries (DMFT > 0), although the number in each group was not recorded. Multivariate analysis showed large amounts of phosphopeptides (PRP1/3, histatin 1, and statherin) in the whole saliva from the caries-free group, but their replacement by many smaller peptide fragments in the saliva from the group with caries. In the second paper,9 32 male subjects were divided into 16 caries-free and 16 caries-treated subjects (DMFT = 3 through 12). In this second study, greater quantities of lipocalin and cystatins S and SN were present in the whole saliva samples from caries-free subjects, suggesting that these natural protease inhibitors may provide saliva with greater protection from caries. This finding agrees with a reported study by Tulunoglu et al3 The biuret method they used detects small peptides better than large proteins and glycoproteins. Their finding of more ‘protein’ in saliva from caries active children may be due to more small peptides than in the saliva from caries-free children. In the absence of the Vitorino papers, the reviewers queried the Tulunoglu et al result because it was not confirmed by Shimotoyodome et al6 and Roa et al2 However, the salivary protein concentration was measured differently in both of those studies and was likely less influenced by the content of small peptides in whole saliva. Thus, contrary to the reviewers’ conclusion, the presence of caries likely associates with a reduced content of secreted protease inhibitors, greater protease activity, and a corresponding lack of PRPs, histatins, and statherin from whole and parotid saliva. There is even a rationale for this conclusion that could be investigated. In addition to a greater content of acidic PRPs, histatins, and statherin binding to enamel and slowing its acid dissolution, large basic PRPs from parotid saliva attach to mutans streptococci10,11 by their cell surface antigen I/II, homologs of which are present in many other streptococci.12 Basic PRPs may therefore be incorporated by many oral viridans streptococci into biofilms where they would prevent the pH fall after carbohydrate inges92
tion. Indeed, a paper excluded from the review because it did not specifically examine salivary protein composition or content demonstrates that saliva from cariesresistant individuals modifies the biofilm pH fall.13 When the biofilm was denied access to saliva, the pH minima reached by 10 caries-free subjects (DMFT = 0) and 10 subjects with caries (DMFT > 20) following exposure to sucrose were comparable. When access to saliva was allowed, the pH minima were higher (less acidic) in the caries-resistant subjects compared with the subjects with caries. Although only 3 of 7 studies selected for review presented evidence for a relationship between salivary proteins and dental caries, a more complete survey would have provided evidence for an association between certain salivary proteins, protease activity, and caries. Moreover, the studies considered not to support a role for salivary proteins4–7 did not employ methods that detected small peptides and proteases, or differences in the content of subgroups of highly variable mixtures of proteins such as statherin, histatins, and PRPs. As echoed in this review, the conventional wisdom is that genetic variability across many different genes14 explains the well-established effect of heritability on caries susceptibility,15,16 along with variable diets, fluoride exposure, and microbial environment. By omitting the Vitorino studies, strong scientific4 evidence for an association of salivary proteins and proteases with caries susceptibility was not considered. In conclusion, this review failed to include all the evidence for an association of salivary proteins with caries susceptibility.16 Along with diet and fluoride exposure, these protein differences may affect microbial colonization of the oral cavity.17 A more detailed review of the contribution of the PRPs to caries susceptibility and an explanation of why variants of the basic PRPs cannot be detected by genome-wide association scans or direct Illumina sequencing is published elsewhere.18
REFERENCES 1. Ayad M, Van Wuyckhuyse BC, Minaguchi K, et al. The association of basic proline-rich peptides from human parotid gland secretions with caries experience. J Dent Res 2000;79(4):976-982. 2. Roa NS, Chaves M, Gomez M, Jaramillo LM. Association of salivary proteins with dental caries in a Colombian population. Acta Odontol Latinoam 2008;21(1):69-75. 3. Tulunoglu O, Demirtas S, Tulunoglu I. Total antioxidant levels of saliva in children related to caries, age, and gender. Int J Paediatr Dent 2006;16(3):186-191. 4. Anderson LC, Lamberts BL, Bruton WF. Salivary protein polymorphisms in caries-free and caries-active adults. J Dent Res 1982;61(2):393-396. 5. Dodds MW, Johnson DA, Mobley CC, Hattaway KM. Parotid saliva protein profiles in caries-free and caries-active adults. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;83(2):244-251. 6. Shimotoyodome A, Kobayashi H, Tokimitsu I, Matsukubo T, Takaesu Y. Statherin and histatin 1 reduce parotid saliva-promoted
September 2013
JOURNAL OF EVIDENCE-BASED DENTAL PRACTICE
7.
8.
9.
10.
11.
12.
13.
Streptococcus mutans strain MT8148 adhesion to hydroxyapatite surfaces. Caries Res 2006;40(5):403-411. Stuchell RN, Mandel ID. A comparative study of salivary lysozyme in caries-resistant and caries-susceptible adults. J Dent Res 1983;62 (5):552-554. Vitorino R, Lobo MJ, Duarte JR, Ferrer-Correia AJ, Domingues PM, Amado FM. The role of salivary peptides in dental caries. Biomed Chromatogr 2005;19(3):214-222. Vitorino R, de Morais GS, Ferreira R, et al. Two-dimensional electrophoresis study of in vitro pellicle formation and dental caries susceptibility. Eur J Oral Sci 2006;114(2):147-153. Matsumoto-Nakano M, Tsuji M, Amano A, Ooshima T. Molecular interactions of alanine-rich and proline-rich regions of cell surface protein antigen c in Streptococcus mutans. Oral Microbiol Immunol 2008;23(4):265-270. Russell MW, Mansson-Rahemtulla B. Interaction between surface protein antigens of Streptococcus mutans and human salivary components. Oral Microbiol Immunol 1989;4(2):106-111. Brady LJ, Maddocks SE, Larson MR, et al. The changing faces of Streptococcus antigen I/II polypeptide family adhesins. Mol Microbiol 2010;77(2):276-286. Abelson DC, Mandel ID. The effect of saliva on plaque pH in vivo. J Dent Res 1981;60(9):1634-1638.
Volume 13, Number 3
14. Shaffer JR, Wang X, Feingold E, et al. Genome-wide association scan for childhood caries implicates novel genes. J Dent Res 2011;90 (12):1457-1462. 15. Bretz WA, Corby PM, Schork NJ, et al. Longitudinal analysis of heritability for dental caries traits. J Dent Res 2005;84(11):1047-1051. 16. Werneck RI, Mira MT, Trevilatto PC. A critical review: an overview of genetic influence on dental caries. Oral Dis 2010;16(7):613-623. 17. Kanasi E, Dewhirst FE, Chalmers NI, et al. Clonal analysis of the microbiota of severe early childhood caries. Caries Res 2010;44 (5):485-497. 18. Levine M. Susceptibility to dental caries and the salivary proline-rich proteins. Int J Dent 2011;2011:953412.
REVIEWER Martin Levine, BDS, BSc, PhD Dept of Biochemistry & Molecular Biology, University of Oklahoma HSC, 940 S.L. Young Blvd, Oklahoma City, OK 73104, USA. Tel.: þ1 405 271 2227x61238, 405 990 42832 (Cell) [email protected]
93
REVIEWER Martin Levine, BDS, BSc, PhD
PURPOSE/QUESTION What is the evidence for an association between individual salivary protein composition or content and dental caries experience?
SOURCE OF FUNDING Canadian Government Institutes of Health Research
TYPE OF STUDY/DESIGN Systematic review
Salivary Proteins May Be Useful for Determining Caries Susceptibility SUMMARY Selection Criteria An electronic search was performed in the PubMed Medline, Ovid Medline, ISI Web of Science, Medline, Cochrane Library, Lilacs, Scielo, BBO, Paho, and Wholis databases applying the following MeSH terms: ‘‘dental caries’’ OR ‘‘tooth demineralization’’ OR ‘‘dental caries susceptibility’’ OR ‘‘dental enamel solubility’’ AND ‘‘salivary proteins and peptides’’ OR ‘‘saliva’’ AND ‘‘proteins.’’ All searches were limited to human studies published between 1950 and 2011. From the electronically selected papers, a hand search of the references and a ‘related articles’ link search was also performed.
Key Study Factor A study was only included in this review if it was an original investigation in which groups of individuals with and without caries were compared with a measurement of salivary proteins or peptides. The importance of each study was assessed from ‘risk of bias,’ defined by the following criteria: (1) no medically compromised subjects or an unrelated aim; (2) no fluoride exposure; (3) clinical diagnosis; (4) radiographic assessment; (5) experienced examiner(s); (6) calibrated examiner(s); (7) saliva collection method given; (8) saliva analytical method given; (9) statistical analysis done; and (10) paired groups. None of the included studies were blinded or randomized controlled trials.
LEVEL OF EVIDENCE
Main Outcome Measure
Level 2: Limited-quality, patientoriented evidence
The statistical significance of the protein composition or content of saliva from subjects with caries experience compared with that of subjects without caries experience.
STRENGTH OF RECOMMENDATION GRADE Grade B: Inconsistent or limitedquality patient-oriented evidence
J Evid Base Dent Pract 2013;13:91-93 1532-3382/$36.00 Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jebdp.2013.07.005
Main Results The electronic search found 186 articles. An additional paper was found from the hand search and another from a ‘related articles’ link. Of these 188 articles, only 7 (4%) met inclusion criteria with a ‘low or moderate risk of bias; ’ i.e., they met 3 or more of the 10 criteria. All 7 included studies compared data from subjects with no caries with that of subjects who had caries irrespective of whether the caries had been treated. Two of the 7 studies reported a significant difference in types of salivary proteins between subjects with caries compared to those without caries: a lack of large basic proline-rich proteins in parotid saliva1 or a greater content of an uncharacterized 17 kDa salivary protein in unstimulated whole saliva from men.2 A third study found that subjects with caries had a greater content of proteins in unstimulated whole saliva than those without caries.3 The remaining 4 studies found no association of salivary protein type or content with caries.4–7
JOURNAL OF EVIDENCE-BASED DENTAL PRACTICE
Conclusions Only three of the seven studies found a statistically significant difference between individuals with and without caries experience in terms of salivary proteins and dental caries. Although these studies found a relationship regarding protein phenotypes, total protein concentration, or protein molecular weight, there was insufficient evidence to establish salivary proteins as a biomarker for dental caries.3
COMMENTARY AND ANALYSIS The search omitted two important papers by Vitorino et al8,9 that fulfill the inclusion criteria and support the finding of the Ayad paper.1 In first paper, whole saliva was collected from 20 males, each characterized by an experienced dentist as caries-free (DMFT = 0) or having treated caries (DMFT > 0), although the number in each group was not recorded. Multivariate analysis showed large amounts of phosphopeptides (PRP1/3, histatin 1, and statherin) in the whole saliva from the caries-free group, but their replacement by many smaller peptide fragments in the saliva from the group with caries. In the second paper,9 32 male subjects were divided into 16 caries-free and 16 caries-treated subjects (DMFT = 3 through 12). In this second study, greater quantities of lipocalin and cystatins S and SN were present in the whole saliva samples from caries-free subjects, suggesting that these natural protease inhibitors may provide saliva with greater protection from caries. This finding agrees with a reported study by Tulunoglu et al3 The biuret method they used detects small peptides better than large proteins and glycoproteins. Their finding of more ‘protein’ in saliva from caries active children may be due to more small peptides than in the saliva from caries-free children. In the absence of the Vitorino papers, the reviewers queried the Tulunoglu et al result because it was not confirmed by Shimotoyodome et al6 and Roa et al2 However, the salivary protein concentration was measured differently in both of those studies and was likely less influenced by the content of small peptides in whole saliva. Thus, contrary to the reviewers’ conclusion, the presence of caries likely associates with a reduced content of secreted protease inhibitors, greater protease activity, and a corresponding lack of PRPs, histatins, and statherin from whole and parotid saliva. There is even a rationale for this conclusion that could be investigated. In addition to a greater content of acidic PRPs, histatins, and statherin binding to enamel and slowing its acid dissolution, large basic PRPs from parotid saliva attach to mutans streptococci10,11 by their cell surface antigen I/II, homologs of which are present in many other streptococci.12 Basic PRPs may therefore be incorporated by many oral viridans streptococci into biofilms where they would prevent the pH fall after carbohydrate inges92
tion. Indeed, a paper excluded from the review because it did not specifically examine salivary protein composition or content demonstrates that saliva from cariesresistant individuals modifies the biofilm pH fall.13 When the biofilm was denied access to saliva, the pH minima reached by 10 caries-free subjects (DMFT = 0) and 10 subjects with caries (DMFT > 20) following exposure to sucrose were comparable. When access to saliva was allowed, the pH minima were higher (less acidic) in the caries-resistant subjects compared with the subjects with caries. Although only 3 of 7 studies selected for review presented evidence for a relationship between salivary proteins and dental caries, a more complete survey would have provided evidence for an association between certain salivary proteins, protease activity, and caries. Moreover, the studies considered not to support a role for salivary proteins4–7 did not employ methods that detected small peptides and proteases, or differences in the content of subgroups of highly variable mixtures of proteins such as statherin, histatins, and PRPs. As echoed in this review, the conventional wisdom is that genetic variability across many different genes14 explains the well-established effect of heritability on caries susceptibility,15,16 along with variable diets, fluoride exposure, and microbial environment. By omitting the Vitorino studies, strong scientific4 evidence for an association of salivary proteins and proteases with caries susceptibility was not considered. In conclusion, this review failed to include all the evidence for an association of salivary proteins with caries susceptibility.16 Along with diet and fluoride exposure, these protein differences may affect microbial colonization of the oral cavity.17 A more detailed review of the contribution of the PRPs to caries susceptibility and an explanation of why variants of the basic PRPs cannot be detected by genome-wide association scans or direct Illumina sequencing is published elsewhere.18
REFERENCES 1. Ayad M, Van Wuyckhuyse BC, Minaguchi K, et al. The association of basic proline-rich peptides from human parotid gland secretions with caries experience. J Dent Res 2000;79(4):976-982. 2. Roa NS, Chaves M, Gomez M, Jaramillo LM. Association of salivary proteins with dental caries in a Colombian population. Acta Odontol Latinoam 2008;21(1):69-75. 3. Tulunoglu O, Demirtas S, Tulunoglu I. Total antioxidant levels of saliva in children related to caries, age, and gender. Int J Paediatr Dent 2006;16(3):186-191. 4. Anderson LC, Lamberts BL, Bruton WF. Salivary protein polymorphisms in caries-free and caries-active adults. J Dent Res 1982;61(2):393-396. 5. Dodds MW, Johnson DA, Mobley CC, Hattaway KM. Parotid saliva protein profiles in caries-free and caries-active adults. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;83(2):244-251. 6. Shimotoyodome A, Kobayashi H, Tokimitsu I, Matsukubo T, Takaesu Y. Statherin and histatin 1 reduce parotid saliva-promoted
September 2013
JOURNAL OF EVIDENCE-BASED DENTAL PRACTICE
7.
8.
9.
10.
11.
12.
13.
Streptococcus mutans strain MT8148 adhesion to hydroxyapatite surfaces. Caries Res 2006;40(5):403-411. Stuchell RN, Mandel ID. A comparative study of salivary lysozyme in caries-resistant and caries-susceptible adults. J Dent Res 1983;62 (5):552-554. Vitorino R, Lobo MJ, Duarte JR, Ferrer-Correia AJ, Domingues PM, Amado FM. The role of salivary peptides in dental caries. Biomed Chromatogr 2005;19(3):214-222. Vitorino R, de Morais GS, Ferreira R, et al. Two-dimensional electrophoresis study of in vitro pellicle formation and dental caries susceptibility. Eur J Oral Sci 2006;114(2):147-153. Matsumoto-Nakano M, Tsuji M, Amano A, Ooshima T. Molecular interactions of alanine-rich and proline-rich regions of cell surface protein antigen c in Streptococcus mutans. Oral Microbiol Immunol 2008;23(4):265-270. Russell MW, Mansson-Rahemtulla B. Interaction between surface protein antigens of Streptococcus mutans and human salivary components. Oral Microbiol Immunol 1989;4(2):106-111. Brady LJ, Maddocks SE, Larson MR, et al. The changing faces of Streptococcus antigen I/II polypeptide family adhesins. Mol Microbiol 2010;77(2):276-286. Abelson DC, Mandel ID. The effect of saliva on plaque pH in vivo. J Dent Res 1981;60(9):1634-1638.
Volume 13, Number 3
14. Shaffer JR, Wang X, Feingold E, et al. Genome-wide association scan for childhood caries implicates novel genes. J Dent Res 2011;90 (12):1457-1462. 15. Bretz WA, Corby PM, Schork NJ, et al. Longitudinal analysis of heritability for dental caries traits. J Dent Res 2005;84(11):1047-1051. 16. Werneck RI, Mira MT, Trevilatto PC. A critical review: an overview of genetic influence on dental caries. Oral Dis 2010;16(7):613-623. 17. Kanasi E, Dewhirst FE, Chalmers NI, et al. Clonal analysis of the microbiota of severe early childhood caries. Caries Res 2010;44 (5):485-497. 18. Levine M. Susceptibility to dental caries and the salivary proline-rich proteins. Int J Dent 2011;2011:953412.
REVIEWER Martin Levine, BDS, BSc, PhD Dept of Biochemistry & Molecular Biology, University of Oklahoma HSC, 940 S.L. Young Blvd, Oklahoma City, OK 73104, USA. Tel.: þ1 405 271 2227x61238, 405 990 42832 (Cell) [email protected]
93