Dental caries is the result of disintegration of hard tissues due to the microflora and other factors in the biofilm contributing to the cariogenicity. These elements influence the induction and advancement of the carious process.
saliva which is an alkaline secretion of water containing enzymes, proteins, and inorganic substances adversely affects the oral conditions including the tooth surfaces when produced in minimum quantities compared to normal.[ ] The first line of protection against free radical (FR) oxidative stress is assumed to be 1 saliva which is primarily done by the phosphate and bicarbonate systems.[ ] Oxidative stress is defined as a disruption in the balance between the synthesis and accumulation of reactive oxygen species (ROS) in cells and tissues, as well as the body's ability to remove these reactive products.[ 2 ] 3
Oxidative stress biological markers are balanced in
saliva so levels of these will indicate certain caries-specific oxidation pathways.[ ] 4
This salivary buffering potential is a very important factor for caries protection. The salivary isoenzyme carbonic anhydrase VI (CAVI) formed by parotid and submandibular serous acinar cells catalyzes the reversible reaction of carbon dioxide, thus increasing the buffering potential causing acid neutralization:-
2 + H 2O ↔ H + + HCO 3.[ ] 5
Acids produced by plaque bacteria are buffered by the bicarbonates inhibiting the demineralization where the CAVI accumulates on the surface of hydroxyapatite crystals enabling acid neutralization.
The principal cause of FRs is physiological metabolism.
Saliva and its antioxidants are being investigated as redox status regulators of the oral cavity under normal and diseased conditions. Peroxidase, catalase, superoxide dismutase (SOD), and glutathione peroxidase form the major part of the antioxidant system of saliva with minor constituents as uric acid and Vitamin E, C.[ ] Earlier studies have shown that with increasing caries conditions, the total antioxidant levels also increased instantaneously.[ 6 ] These antioxidants can be enzymatic and nonenzymatic compounds which avoid complications caused by additional oxidants generated by oxidation reactions in the body. 7
The disproportionation of the superoxide anion FR (O
2-) into molecular oxygen and hydrogen peroxide (H 2O 2) is caused by SOD. It also reduces O 2 - level which destroys the cells at excessive concentration.[ ] 8
Saliva also has several proteins that act on plaque and bacteria by many strategies, decreasing tooth predisposition to dental caries. Lysozyme, lactoferrin, and salivary peroxidase are found to be contributing to restoring soft tissue and inhibiting oral infections. Tulunoglu et al. found a positive association in which total protein coupled with total antioxidant potential increased with the progression of caries.[ ] 9
Although many studies have shown the major properties of
saliva and their role in the pathogenesis of caries, very few have specifically shown its effectiveness in the caries risk assessment and diagnosis in children. Therefore, the assessment of salivary factors that may enhance the risk of tooth decay will surface the way for making recommendations that would serve the needs of an individual in particular. Existing literature has also not shown the variation of individual antioxidant components, correlating their functions with isoenzymes and proteins in carious lesions. Taking into consideration the complex interplay described above, this study was done to compare and analyze the levels of enzyme carbonic anhydrase VI, total protein, and antioxidants (SOD, catalase, lipid peroxidase and uric acid) in caries-free and caries-active children. The formulated hypothesis for the study is that there exists a definite relationship of enzyme carbonic anhydrase VI, total proteins, and antioxidants (SOD, catalase, lipid peroxidase and uric acid) levels with the caries activity. Materials and Methods
The present study was undertaken in the Department of Pedodontics and Preventive Dentistry, Manipal College of Dental Sciences, Manipal, Karnataka, in collaboration with the Department of Biochemistry, Kasturba Medical College, Manipal, Karnataka.
The study duration was 2 years which was approved by Kasturba Hospital Ethics Committee (IEC Number 826/2018) and registered with Clinical Trials Registry-India (CTRI registration number: CTRI/2019/01/017135).
The estimation of sample size was completed with the help of Department of Biostatistics, Manipal Academy of Higher Education, and using the formula:
α =5% is the level of significance
Z 1-α/2 = 1.96
Z 1-β = 0.84 σ = 0.545
d (clinically significant difference) = 0.4
Assuming the design effect of the two groups in this case–control study, the required total sample size was 60, with 30 participants each, Group 1 – caries-active group and Group 2 – caries-free group, respectively.
Children aged 6–12 years having enamel caries with decayed missing filled teeth (DMFT)/dmft >5 were included in Group 1 and caries-free children with DMFT/dmft = 0 were included in Group 2. Healthy children with a good record of general health and those who were not taking any medications were selected for the study after obtaining informed consent.
Children having deep dentinal caries and pulpally involved teeth, children with abscess due to dental caries, children with salivary gland disorders, those with special health care needs, recent antibiotics exposure (last 3 months), poor oral hygiene, and presence of periodontal disease were excluded from participating in the study.
Consent forms were issued to be signed along with patient information sheets by the parents of the children who were willing to participate in the research. After obtaining written consent from parents, a clinical examination was carried out.
The DMFT/dmft index given by Henry Klein, Carole Palmer, and Knutson JW was used to estimate the prevalence of caries.[
] After rinsing and drying the teeth with gauze, the diagnosis of dental caries was conducted by visual and tactile examination technique using mouth mirror, No. 23 explorer, and a flashlight. Current nation's recommendations and standards were followed for both infection control and waste disposal.[ 10 ] Disposable masks and gloves were used. 11
Following clinical examination, proformas with indices were filled and the children were categorized into 2 groups: Group 1 caries-active and Group 2 caries-free population.
Samples were obtained between 10 and 11:30am during the morning hours. The participants were instructed not to eat or drink anything 1 h earlier to sample collection. Each child was asked to chew a paraffin wax block of 1 cm diameter for 6 min. The initial pooled
saliva was discarded and the child was asked to expectorate 6 ml of the pooled saliva into collection funnel placed in a sterile graduated jar during the next 5 min which was then shifted to sterile Eppendorf tubes, which were transported to the laboratory within 30 min of collection. Estimation of carbonic anhydrase VI enzyme
The assessment of the CAVI concentration in
saliva was done using an ELISA kit for CAVI (ELISA kit E-EL-H0474 96 tests, Elabscience Biotechnology Inc., USA) in accordance with the manufacturer's guidelines [Figures 1 and 2]. Figure 1:
ELISA microplate reader
Standard graph for human carbonic anhydrase VI concentration (pg/ml)
Estimation of catalase
Three milliliter of H
2O 2 PBS solution was mixed with 50 μL of saliva and reading at zero time (absorbance at 240 nm) using cuvette was taken. Estimation of superoxide dismutase
One thousand eight hundred and fifty microliter of sodium carbonate buffer was mixed with 50 uL of
saliva and 100 uL of Adrenaline was included directly in the cuvette after putting in ultraviolet mode. The absorbance at 480 nm (A 0–A 60) (kinetic method) was taken. Estimation of lipid peroxidase
A mixture of 0.5 ml of
saliva, 2.5 ml of thiobarbituric acid, trichloroacetic acid, and butylated hydroxyl toluene was heated at 90°C for 10 min. The mixture was centrifuged for 5 min at 2000 rpm and absorbance was taken at 525 nm. Lipid peroxidation was expressed as nanomoles (nM) of malonaldehyde form per ml of saliva. Estimation of uric acid
The uric acid concentration test in
saliva was performed using the uric acid reagent kit following the instructions from the manufacturer.
Uricase generated allantoin and H
2O 2 from uric acid. Through the catalytic action of peroxidase, the H 2O 2 produced further reacted with a phenolic compound and 4 aminoantipyrine to form a red colored quinoneimine dye complex. The color intensity was directly proportional to the amount of uric acid contained in the sample. Estimation of total protein
Proteins along with copper ions formed an alkaline solution of the violet–blue color complex. The absorbance of the color was directly proportional to the overall protein content in the specimen.
Data were analyzed using the statistical package SPSS 22.0 (SPSS Inc., Chicago, IL, USA) and the level of significance was set at
P < 0.05. Descriptive statistics was performed to assess the mean and standard deviation of the respective groups. Normality of the data was assessed using Shapiro–Wilkinson test. Inferential statistics to find out the difference between the groups was done using independent -test. Results
This study was conducted to compare and evaluate the levels of enzyme carbonic anhydrase VI, total protein, and antioxidants (SOD, catalase, lipid peroxidase, and uric acid) in caries-free and caries-active children.
Carbonic anhydrase VI enzyme concentration in
The concentration of CAVI isoenzyme was higher in the caries active group (1925.54 ± 1398.57) compared to caries free group (1444.17 ± 1039.81). This difference was not statistically significant (
P = 0.135) [ Table 1]. Table 1:
Intergroup comparison of carbonic anhydrase VI enzyme concentration
Concentration of total protein in
The concentration of total protein was higher in the caries active group (1.723 ± 1.943) compared to caries free group (1.429 ± 1.284). This difference was not statistically significant (
P = 0.492) [ Table 2]. Table 2:
Intergroup comparison of total protein concentration
Lipid peroxidase activity in
The activity of lipid peroxidase was higher in the caries active group (0.0089 ± 0.0171) compared to caries free group (0.01423 ± 0.01344). This difference was not statistically significant (
P = 0.184) [ Table 3]. Table 3:
Intergroup comparison of lipid peroxidase activity
Concentration of uric acid in
The concentration of uric acid was higher in the caries active group (1.498 ± 0.664) compared to caries free group (0.97 ± 0.164). This difference was statistically significant (
P = 0.001) [ Table 4]. Table 4:
Intergroup comparison of uric acid concentration
Superoxide dismutase activity in
The activity of SOD was higher in the caries active group (0.027 ± 0.021) compared to caries free group (0.015 ± 0.029). This difference was not statistically significant (
P = 0.07) [ Table 5]. Table 5:
Intergroup comparison of superoxide dismutase activity
Catalase activity in
The activity of Catalase was significantly higher in the caries-free group (−0.0071 ± 0.0162) compared to caries-active group (−0.0214 ± 0.0073). This difference was statistically significant (
P = 0.001) [ Table 6]. Table 6:
Intergroup comparison of catalase activity
The present study was conducted taking into consideration the lack of evidence-based studies on evaluation of levels of oxidative stress biomarkers and bicarbonate buffering isoenzyme of
saliva in carious activity. The acidity of beverages and foods is neutralized by bacterial activity by bicarbonate, phosphate, and protein buffer systems, thus preventing the colonization of pathogenic microorganisms.[ ] In our sample, it was found that the CAVI levels in the caries-active group were higher than in the caries-free group, although the difference was not statistically significant. Concerning the interaction between CAVI concentration and dental caries, conflicting outcomes were reported by previous investigations. No substantial difference in CAVI concentrations was observed in a report by Oztürk 12 et al.[ ] when comparing caries and young adults without caries. The research conducted by Kivelä 13 et al.,[ ] on the other hand, shows that a decreased concentration of CAVI is correlated with an elevated index of caries. In accordance with this study, Szabó[ 14 ] found that 15 saliva in 7–14-year-old caries-free children had a greater CAVI concentration than in children with active caries.
et al.[ ] concluded that carbonic anhydrase VI is seen to be more active in biofilm matrix of schoolchildren with caries to lead to biofilm acid neutralization. The levels of CAVI in dental biofilm in caries-active children in our current study suggest that their biofilm pH decrease tends to activate the bicarbonate buffer since these individuals are also susceptible to cariogenic challenges, especially sucrose. Our results support this finding because earlier studies have shown that salivary pH in caries-active children is slightly lower than in children without caries. Oztürk 16 et al. (2008)[ ] showed that the rise in isoenzyme concentration after a decline in pH may suggest that during cariogenic episodes, more CAVI is available to be triggered. 13
Salivary protein has been identified as having a double-edged function that, depending on position and activity, that the microorganism colonizes, plays a protective mechanism and leads to mucin development that acts against the formation of caries and protects the tooth from desiccation.[
et al.[ ] stated that by neutralizing the acid production by Streptococci, the proline-rich protein reduces the occurrence of caries. Salivary albumin serves as a marker of the nature of the underlying disease and has an inhibitory effect on dental caries by entering enamel pores by preventing enamel demineralization.[ 18 ] 19
When we compared the total protein content in both the groups, we found that there was increased total protein in caries-active group relative to the caries-free group which was not statistically significant. This is in accordance with some previous studies done by de Farias and Bezerra[
] and Bhalla 20 et al.[ ] where no substantial difference was found between the two groups in the total protein content. Statistically significant rise in total protein levels has been seen in caries-active group in studies conducted by Pyati 21 et al.,[ ] Silva 12 et al.,[ ] Pandey 22 et al.,[ ] Preethi 23 et al.,[ ] Prabhakar 24 et al.,[ ] and Tulunoglu 25 et al.[ ] 9
Increased total salivary protein levels in children with caries may be due to a rise in many antimicrobial components such as lysozyme, lactoferrin, antioxidants such as salivary peroxidase, SOD, glutathione, and many biological factors that are all protein in nature involved in soft tissue repair.[
] The conflicting outcomes of few studies may be due to the fact that total salivary proteins may have both protective and harmful properties. 24
Chronic illnesses are characterized by the development of oxidative stress induced by FRs or ROS. To restrict disease progression, salivary antioxidants can interact with levels of FR/ROS. The onset of the ailment is in phase if imbalances between antioxidants and FR/ROS are certain.[
When the antioxidant parameters were individually analyzed and compared between the two groups, SOD, lipid peroxidase, and uric acid were increased in the caries-active group in which only uric acid showed a statistically significant increase. Another interesting finding was that catalase showed a statistically significant increase in the caries-free group.
et al.,[ ] indicated that the rise in 27 saliva uric acid levels may affect total antioxidant levels as uric acid accounts for about 70% of salivary antioxidant capacity, explaining the substantial rise in uric acid levels observed in the present study.
The results of the study conducted by Jurczak
et al.[ ] indicated high levels of antioxidants in 28 saliva which have shown to increase substantially in children in line with salivary cariogenic bacterial profiles and development of caries.
et al.[ ] concluded from their study that total antioxidant potential of 29 saliva in children with extensive dental caries substantially decreases after treatment of carious lesions.
et al.[ ] also demonstrated that the enzymatic antioxidant activity of SOD and nonenzymatic antioxidant activity of Uric acid boosted with the severity of caries, thus lowering salivary oxidative harm. It was presumed that the greater severity of caries enhances the function of the salivary antioxidant system, with a subsequent decrease in salivary oxidative stress. 30
The increase in levels of lipid peroxidase in caries-active group in the current study is in accordance with a former study conducted by Pyati
et al.[ ] which showed a statistically significant increase in malondialdehyde (MDA) levels in caries-active children. 12
The phagocytic activity is triggered by dental caries which leads to increased generation of FRs and consequent lipid peroxidation and MDA formation. These reactive oxidants aim at destroying invading microorganisms. Neutrophils and other phagocytes develop O
2-(superoxide) at the expense of NADPH by one-electron oxygen reduction. Almost all of the oxygen reacts with itself to form H 2O 2.[ ] 30
Although previous researches have studied similar parameters, there were contradicting results regarding the levels of CAVI enzymes in caries activity and also there is a lack of evidence-based studies on evaluation of levels of oxidative stress biomarkers and bicarbonate buffering isoenzyme of
saliva in carious activity.
From our results, it can be interpreted that among the antioxidants, catalase does not have an adaptive response to defense against excessive reactive species in pathogenesis of dental caries. Very few studies have been done correlating individual antioxidant parameters with dental caries.[
Further investigation is needed to evaluate the function of catalase specifically in dental caries as our groups were not analyzed according to gender which might have caused the variation which was evident in the previous study.
Previous studies have concluded that, as a result of the improved non-enzymatic and enzymatic antioxidant systems, oxidative damage to
saliva in children with extreme caries is minimized.[ ] 22
Based on the findings of the current research, the alternate hypothesis that “There exists a definite relationship of enzyme Carbonic anhydrase VI, Total proteins and antioxidants (SOD, catalase, lipid peroxidase, and uric acid) levels with the caries activity” was accepted.
For the screening and control of oral infections, including dental caries, salivary indicators of oxidative stress may be used. The assessment of these
saliva markers might constitute an adjuvant tool for recognizing patients with low adherence to dental appointments. In addition, the analysis of these biomarkers may provide useful evidence to develop new remedies for the prevention and/or management of dental caries. However, such markers should be validated in forthcoming studies conducted with a higher wide range of subjects and taking into account differing levels of caries. Conclusion
The following conclusions were drawn from the study:
There is an increased concentration of CAVI enzyme in caries-active group
Total protein showed a linear relation with caries activity
Antioxidant parameters such as SOD and lipid peroxidase were increased with caries activity. Uric acid was significantly higher in the caries-active group, whereas catalase showed an indirect relation with dental caries.
Significant variations in the levels of CAVI enzyme, total proteins, and antioxidant parameters in caries-active children imply that the levels of these components of
saliva can act as strong markers of caries status in children Financial support and sponsorship
This study was funded by the Indian Council of Medical Research, New Delhi.
Conflicts of interest
There are no conflicts of interest.
1. Ship JA. Diabetes and oral health: An overview J Am Dent Assoc. 2003;134(Spec No):4S–10S
2. Kirschvink N, De Moffarts B, Lekeux P. The oxidant/antioxidant equilibrium in horses Vet J. 2008;177:178–91
3. Pizzino G, Irrera N, Cucinotta M, Pallio G, Mannino F, Arcoraci V, et al Oxidative stress: Harms and benefits for human health Oxid Med Cell Longev 2017. 2017 8416763
4. Tóthová L, Kamodyová N, Červenka T, Celec P. Salivary markers of oxidative stress in oral diseases Front Cell Infect Microbiol. 2015;5:73
5. Kimoto M, Kishino M, Yura Y, Ogawa Y. A role of salivary carbonic anhydrase VI in dental plaque Arch Oral Biol. 2006;51:117–22
6. Panjamurthy K, Manoharan S, Ramachandran CR. Lipid peroxidation and antioxidant status in patients with periodontitis Cell Mol Biol Lett. 2005;10:255–64
7. Hegde AM, Rai K, Padmanabhan V. Total antioxidant capacity of
and its relation with early childhood caries and rampant caries J Clin Pediatr Dent. 2009;33:231–4
8. Yasui K, Baba A. Therapeutic potential of superoxide dismutase (SOD) for resolution of inflammation Inflamm Res. 2006;55:359–63
9. Tulunoglu O, Demirtas S, Tulunoglu I. Total antioxidant levels of
in children related to caries, age, and gender Int J Paediatr Dent. 2006;16:186–91
10. Peter S. Indices in dental epidemiology Essentials of Public Health Dentistry. 20135th ed(Ch. 18) New Delhi Arya Medi Publishing House Private Limited:412–74
11. Last accessed on 2021 Jan 20 Available from:
12. Pyati SA, Naveen Kumar R, Kumar V, Praveen Kumar NH, Parveen Reddy KM. Salivary flow rate, pH, buffering capacity, total protein, oxidative stress and antioxidant capacity in children with and without dental caries J Clin Pediatr Dent. 2018;42:445–9
13. Oztürk LK, Furuncuoglu H, Atala MH, Uluköylü O, Akyüz S, Yarat A. Association between dental-oral health in young adults and salivary glutathione, lipid peroxidation and Sialic acid levels and carbonic anhydrase activity Braz J Med Biol Res. 2008;41:956–9
14. Kivelä J, Parkkila S, Parkkila AK, Rajaniemi H. A low concentration of carbonic anhydrase isoenzyme VI in whole
is associated with caries prevalence Caries Res. 1999;33:178–84
15. Szabó I. Carbonic anhydrase activity in the
of children and its relation to caries activity Caries Res. 1974;8:187–91
16. Picco DC, Marangoni Lopes L, Parisotto TM, Mattos Graner R, Nobre Dos Santos M. Activity of carbonic anhydrase VI is higher in dental biofilm of children with caries Int J Mol Sci. 2019;20:2673
17. Gabryel-Porowska H, Gornowicz A, Bielawska A, Wójcicka A, Maciorkowska E, Grabowska SZ, et al Mucin levels in
of adolescents with dental caries Med Sci Monit. 2014;20:72–7
18. Shetty N, Hegde MN, Kumari S, Ullal H, Kedilaya V. Salivary proteins as biomarkers in dental caries:
study Dent Oral Craniofac Res. 2017;3:1–7
19. Monica M, Valad R, Stoica A. Analysis of salivary level of alpha-amylase as a risk factor for dental caries Acta Med Transilvanica. 2018;23:93–5
20. de Farias DG, Bezerra AC. Salivary antibodies, amylase and protein from children with early childhood caries Clin Oral Investig. 2003;7:154–7
21. Bhalla S, Tandon S, Satyamoorthy K. Salivary proteins and early childhood caries: A gel electrophoretic analysis Contemp Clin Dent. 2010;1:17–22
22. Silva PV, Troiano JA, Nakamune AC, Pessan JP, Antoniali C. Increased activity of the antioxidants systems modulate the oxidative stress in
of toddlers with early childhood caries Arch Oral Biol. 2016;70:62–6
23. Pandey P, Reddy NV, Rao VA, Saxena A, Chaudhary CP. Estimation of salivary flow rate, pH, buffer capacity, calcium, total protein content and total antioxidant capacity in relation to dental caries severity, age and gender Contemp Clin Dent. 2015;6:S65–71
24. Preethi BP, Reshma D, Anand P. Evaluation of flow rate, pH, buffering capacity, calcium, total proteins and total antioxidant capacity levels of
in caries free and caries active children: An
study Indian J Clin Biochem. 2010;25:425–8
25. Prabhakar A, Dodawad R, Os R. Evaluation of flow rate, pH, buffering capacity, calcium, total protein and total antioxidant levels of
in caries free and caries active children – An
study Int J Clin Pediatr Dent. 2009;2:9–12
26. Muchandi S, Walimbe H, Bijle MN, Nankar M, Chaturvedi S, Karekar P. Comparative evaluation and correlation of salivary total antioxidant capacity and salivary pH in caries-free and severe early childhood caries children J Contemp Dent Pract. 2015;16:234–7
27. Kumar D, Pandey RK, Agrawal D. An estimation and evaluation of total antioxidant capacity of
in children with severe early childhood caries Int J Pediatr Dent. 2011;21:459–64
28. Jurczak A, Kościelniak D, Skalniak A, Papież M, Vyhouskaya P, Krzyściak W. The role of the
antioxidant barrier to reactive oxygen species with regard to caries development Redox Rep. 2017;22:524–33
29. AlAnazi GS, Pani SC, AlKabbaz HJ. Salivary antioxidant capacity of children with severe early childhood caries before and after complete dental rehabilitation Arch Oral Biol. 2018;95:165–9
30. Araujo HC, Nakamune AC, Garcia WG, Pessan JP, Antoniali C. Carious lesion severity induces higher antioxidant system activity and consequently reduces oxidative damage in children's
Oxid Med Cell Longev. 2020;2020:3695683