|Year : 2013 | Volume
| Issue : 2 | Page : 69-73
A comparative study of salivary buffering capacity, flow rate, resting pH, and salivary Immunoglobulin A in children with rampant caries and caries-resistant children
S Kuriakose1, C Sundaresan2, V Mathai3, E Khosla4, FMA Gaffoor5
1 Department of Pedodontics and Preventive Dentistry, Sri Sankara Dental College, Varkala, Kerala, India
2 Department of Pedodontics and Preventive Dentistry, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari, Kerala, India
3 Department of Conservative Dentistry and Endodontics, Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari, Kerala, India
4 Department of Pedodontics and Preventive Dentistry, Mar Baselios Dental College, Kothamangalam, Kerala, India
5 Department of Conservative Dentistry and Endodontics, N.I.College of Dental Sciences, Neyyantinkara, Trivandrum, Kerala, India
|Date of Web Publication||26-Jul-2013|
Department of Pedodontics and Preventive Dentistry, 'Mangalya', Era 160, Thottam, Manacaud, Trivandrum 695 009
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Purpose : This study was conducted to identify various factors in the development of rampant type of dental caries in South Kerala children, other than high sucrose intake and poor oral hygiene. This was done by comparing the salivary buffering capacity(BC), flow-rate(FR), resting pH and salivary immunoglobulin-A(s-IgA) levels in children who are caries resistant(CR) and who have rampant dental caries. Materials and Methods :Two study groups, a rampant caries group(RC) with more than five active caries lesions in the early stages and a CR with no caries lesions were selected based on a specific criteria. Unstimulated whole mixed saliva was collected directly from the floor of the mouth for a period of 10 min and the FR was calculated. Resting pH of saliva was measured using color coded pH paper. BC was measured by calculating the amount of citric acid of pH2.5, required to lower the initial pH of saliva down to 3. s-IgA levels were also estimated by immunoturbidometric method after forming a precipitate of s-IgA with specific anti-IgA antibodies. Result: The salivary BC, FRs, pH and s-IgA levels were significantly lower in the RC group when compared to the CR group. Conclusion : This study showed that salivary BC, flow-rate, resting pH and levels of s-IgA in saliva are risk factors in the development of RC in children.
Keywords: Buffering capacity, caries risk, flow rate, Immunoglobulin A, rampant caries, salivary parameters
|How to cite this article:|
Kuriakose S, Sundaresan C, Mathai V, Khosla E, Gaffoor F. A comparative study of salivary buffering capacity, flow rate, resting pH, and salivary Immunoglobulin A in children with rampant caries and caries-resistant children. J Indian Soc Pedod Prev Dent 2013;31:69-73
|How to cite this URL:|
Kuriakose S, Sundaresan C, Mathai V, Khosla E, Gaffoor F. A comparative study of salivary buffering capacity, flow rate, resting pH, and salivary Immunoglobulin A in children with rampant caries and caries-resistant children. J Indian Soc Pedod Prev Dent [serial online] 2013 [cited 2020 Feb 24];31:69-73. Available from: http://www.jisppd.com/text.asp?2013/31/2/69/115697
| Introduction|| |
Dental caries is a unique multifactorial, infectious disease involving internal defense factors such as saliva, tooth surface morphology and mineralization, general health, nutritional and hormonal status, and a number of external factors such as diet, microbial flora colonizing the teeth, oral hygiene, and fluoride availability.  Rampant dental caries is an extreme form of dental caries where multiple caries lesions appear suddenly, almost all teeth are affected and the disease process reaches the pulp at very rapid pace. Affected children are often in great distress due to multiple pulp exposures, do not eat properly, and become malnourished. There is no evidence that the mechanism of the decay process is different in rampant caries (RC) when compared to ordinary caries or that it occurs in teeth that are malformed or inferior in composition. It can occur in teeth that were for many years resistant to decay suggesting that there is a sudden imbalance in the oral environment. There seems to be an exaggeration of certain caries-producing factors.
The role of excess consumption of refined carbohydrates, poor oral hygiene, and accumulation of plaque with high acid concentration produced by acidogenic bacteria in the development of RC is well understood. But even in the presence of these factors, many children are found to be resistant to rampant dental caries. Some children who were resistant to caries for many years may suddenly develop RC even in the absence of the above-mentioned cariogenic factors. Presence or absence of protective host factors may be responsible for the apparent resistance or susceptibility to RC.
Many modern studies have hence introduced a new approach to dental caries from being a bacteria and sugar-induced disease to a disease that is greatly influenced by inherited salivary factors.  The dental caries process is controlled to a large extent by a natural protective mechanism inherent within the saliva. The most important caries protective functions of saliva are the flushing and neutralizing effects which is dependent on the flow rate (FR) and buffering capacity (BC) of saliva.  It has been shown that a sudden reduction in the salivary FR can lead rapid formation of caries lesions.  A clear inverse relationship between salivary BC and caries susceptibility has been clearly demonstrated.  BC is related to the FR as well as the resting pH of the saliva.  BC is one of the best indicators of caries susceptibility as it reveals the host response. Saliva acts with its BC as a regulator of plaque pH, neutralizing acids produced by cariogenic bacteria. This capacity is based on the phosphate system, the carbonic acid, and bicarbonate system. Saliva also contains a number of antibacterial compounds such as lysozyme, lactoperoxidases, lactoferrin, and various immunoglobulins which can control the growth of cariogenic oral microflora.  Many studies have shown that salivary Immunoglobulin A (s-IgA) has a role in the caries resistance shown by certain individuals and decrease in IgA can lead to increase in incidence of acute caries lesions. ,
This study was done to compare the salivary FR, BC, resting pH, and s-IgA levels in children who show an inherent resistance to dental caries and children who have rampant dental caries and thereby to identify the role of various salivary factors in the development of RC.
| Materials and Methods|| |
Forty-two children with an age range of 3-5 years reporting for dental checkup in the Department of Pedodontics and Preventive dentistry were included in this study. All the children had a comparable socio-economic background and belonged to the southern districts of Kerala and neighboring Tamil Nadu. They were divided into two study groups, a RC group and a caries-resistant (CR) group based on the following criteria.
Rampant caries group
- RC was diagnosed based on Masslers definition  of RC.
- Children with multiple (>5) cervical, proximal, or lingual white spot or brown spot lesions or shallow active caries lesions in enamel/dentine were included.
- The subjects had a reasonably good oral hygiene (plaque score: PlI 1-1.9) and did not have more than three solid sugar exposures per day based on a diet history of 3 days.
- "Baby bottle syndrome" cases were excluded.
- Children who had any infection during the past 6 months, allergic or other systemic disorders, those who who underwent tonsillectomy, and those on medications for systemic ailments were excluded.
- Subjects had no visual or radiographic signs of caries.
- Subjects with high plaque scores (PlI 2-3) and subjects having more than three solid sugar exposures per day including in-between meal sugar exposures were included.
- Children with a family history of very low dental caries prevalence were included.
| Collection of Saliva|| |
Children and their parents and caretakers were informed about the procedure and written consent was obtained before collecting saliva. Unstimulated, resting mixed saliva was collected for 10 min directly from the floor of the mouth, using a needleless aspirating syringe calibrated up to 5 ml. Each subject underwent this procedure between 8.00 A.M and 9.00 A.M in the morning. Children were advised not to eat or drink anything except water before saliva collection and were not on any sort of medication. Saliva was collected when they were totally calm. Children were asked to keep their mouths open and saliva was then collected in increments. They were allowed to close their mouths for a few seconds in-between but were instructed not to swallow any saliva.
The amount of saliva collected during a time period of 10 min was taken as a measure of the salivary flow for that individual. Resting salivary pH was estimated using an Indikrom pH paper, which was placed on the floor of the mouth with residual saliva, for 10 s. Indikrom pH papers with a pH range from 5 to 7.5 were used for estimating intraoral salivary pHs. The BC of the saliva collected was immediately estimated to prevent CO 2 gas from escaping from the sample. When the quantity of saliva during 10 min was too small for estimating the BC, saliva was collected for another 10 min after giving sufficient rest to the child.
| Procedure|| |
BC of a sample of saliva was measured as the "amount of acid needed to lower the pH of saliva through a fixed pH interval" as is described in Federation Dentaire Internationale technical report no. 31.  One milliliter of saliva collected from each subject was dispensed into a small borosil jar. After noting the initial pH of saliva with a pH paper which ranges from 5.0 to 7.5, citric acid of pH 2.5 was poured drop by drop using a small pipette (one drop = 0.05 ml) into the saliva in the borosil jar. Care was taken not to touch the sides of the borosil jar. After pouring two drops of citric acid, the pH of saliva was checked with pH paper. This continued till the pH of saliva dropped to a value of 3. For measuring lower pHs, Indikrom pH papers which range from 2 to 4.5 were used. The amount of citric acid added was calculated thereafter (no. of drops added were counted). This volume was taken as a measure of the BC of that particular sample of saliva. The initial pH of saliva was taken as such, i.e., the titration will be till the pH decreased to 3 irrespective of the initial pH of saliva. Higher initial pH was given credit for a higher BC.  The values were determined for all the subjects in both the study groups. They were tabulated and statistically analyzed.
Seventeen children from both the study groups were recalled after 1 week for an estimation of their s-IgA values. Saliva was collected again for 10 min from the floor of mouth as described earlier. The saliva samples are then transported under refrigerated conditions to the laboratory where immunological assays are performed.
s-IgA concentrations were determined using the immunoturbidometric assay. Here, the IgA samples were suitably diluted first, 2 micro aliquots of each saliva sample were diluted with 0.9% NaCl applying a dilution factor of 1:20 using a microliter dispenser, and then reacted with specific anti-IgA antibodies to form a precipitate. For this, 50 μl of the antibody reagents was taken in a labeled test tube, 500 μl of assay buffer was pipetted into this test tube, and then specific quantity of the diluted saliva (50 μl) sample, determined by protein calibration methods was incorporated into this mixture. The resultant solution was allowed to stand at room temperature for 30 min in order to promote immunoprecipitation reaction. Following immunoprecipitation reaction, each sample suspension was drawn into the sipper tube of an automatic analyzer, where a beam of light of 340 nm wavelength impinges on the reaction mixture and the reduction in transmitted light is measured using a photo spectrometer. The amount of light absorbed is converted to units of concentration using standards of known concentration.
| Results|| |
The salivary BC, FR, resting pH, and s-IgA values of both groups were compared using the unpaired t-test. When comparing the IgA values of both groups, it was seen that the mean s-IgA concentration in the RC group was 9.96 ± 2.83 mg/dl and that in the CR group was 15.15 ± 2.22 mg/dl, the difference being statistically significant (P value = 0.001 and t value = 5.95) [Table 1].
The mean unstimulated FR in the RC group was 0.81 ± 0.45 ml/10 min and 1.37 ± 0.57 ml/10 min in the CR group which was statistically significant (P value = 0.001, t value = 3.48) [Table 2].
The mean salivary resting pH value was 6.45 ± 0.50 in the RC group and 7.15 ± 0.30 in the CR group (statistically significant with a P value = 0.001 and t value + 5.43) [Table 3].
When comparing the buffering capacities of both the groups, the mean salivary BC of the RC group was 0.43 ± 0.16 units and that in the CR group was 1.18 ± 0.30 units (statistically significant t value = 9.89, P value = 0.001) [Table 4].
| Discussion|| |
In this study, RC children had a significantly lower salivary FR, pH, BC, and s-IgA levels compared to CR children. Only those RC cases where there was a diet history of < 3 solid sugar exposures per day, no bottle or breast feeding at night/day time, and where the children maintained reasonably good oral hygiene (PI-1-1.9) were included. Multiple active caries lesions (>5) in these children aged 3-5 years meant that other etiological factors were responsible for the rapid appearance and progress of Carious lesion. Children who gave a diet history of more than three solid sugar exposures daily in-between meal sugar exposures, not maintaining good oral hygiene (PI-2-3), and still not developing caries lesions were considered CR and included in the CR group. It was hypothesized that CR group had more protective salivary host factors than RC group children. The significantly higher mean values of salivary FR, pH, BC, and s-IgA, in the CR group, proved that this hypothesis was correct.
Many studies have reported that caries-free patients have higher levels of naturally induced s-IgA and that decrease in IgA levels increases caries incidence. , Some studies have shown high levels of IgA in children with caries susceptibility. Caries lesions accumulated over a long period of time lodges enormous numbers of cariogenic bacteria that can stimulate a local immune response with secondary rise in s-IgA levels.  In this study, care was taken to assess IgA levels at the initiation of rampant type of dental caries when there were only cervical, proximal, or lingual white spot lesions or shallow active caries lesions in enamel and dentine. There were no long-standing open caries lesions or root stumps such that only the IgA levels in the primary immune response were assessed. CR children had significantly higher mean IgA levels (15.15 mg/dl ± 2.22 mg/dl) when compared to (9.96 ± 2.83) RC children showing the role of IgA in resisting rampant dental caries.
Specific immune defense against Streptococcus mutans, considered to be the primary causative agent of dental caries, is provided largely by s-IgA antibodies which are generated by the common mucosal immune systems.  This system is functional right from infancy when infants develop these IgA antibodies as their mouth gets colonized by oral microorganisms. s-IgA seems to interfere with sucrose-independent and sucrose-dependent attachment of S. mutans to tooth surfaces thereby inhibiting dental caries. They may also result in agglutination of bacteria and neutralization of their toxins. 
Regarding salivary pH, significantly lower and more acidic pH values were shown by children in the RC group (6.45 ± 0.50) when compared to CR group (7.15 ± 0.30). This is in accordance with the findings of Sissons et al.  Saliva was collected for 10 min as the unstimulated saliva collected during a period of 1 min was too small for calculating the BC. The volume collected during the 10 min was taken as the FR for that individual. A mean difference of 0.56 ml/10 min was seen between the two study groups with CR group having significantly higher FR. This result was in agreement with previous studies.  Direct aspiration from the floor of the mouth ensured that the salivary flow was unstimulated. Spitting onto a tumbler could have stimulated salivary flow. Unstimulated FR seems to be more important in RC development as stimulated flow is seen during mastication only. Nocturnal flow is also unstimulated.
Jorma Tenovou  has reported a negative correlation between BC and caries activity in a population. Decrease in salivary BC leads to a drastic increase in caries susceptibility as the enamel dissolution by plaque acids is left uncontrolled.  A significantly lesser BC was shown by RC children when compared to CR children in this study. The technique described by Larsen et al.  was modified and used in this study. Simple inexpensive, indigenous equipment was used, with citric acid as the weak acid for measuring the BC of saliva. Citric acid is one of the byproducts of microbial carbohydrate metabolism. RC children with known etiological factors such as poor oral hygiene, uncontrolled sugar intake, and prolonged bottle or breast feeding were not included in this study. Hence, it is not possible to conclude that all RC children have low levels of IgA and low values of BC, FR, and resting pH. Similarly, it is not possible to conclude that all CR children have high values of BC, FR, pH, and IgA levels. Since caries is a multifactorial disease, a low-sugar diet, good oral hygiene, and low S. mutans levels can also make a child CR even if the salivary BC, FR, pH, and IgA levels are low. Other caries risk factors, such as S. mutans count and composition and mineralization of enamel, were not assessed in this study. However, this study has shown conclusively that a reduction in s-IgA, BC, FR, and resting pH values can lead to rampant dental caries formation in children. Further research is required to determine the factors that may lead to an abrupt reduction in salivary BC, FR, resting pH, and IgA levels which in turn can lead to RC formation in preschool children.
Salivary diagnostics is entering the surgery of a pedodontist, wherein a person's susceptibility to rampant type of dental caries can be assessed by measuring the salivary BC, FR, pH, and IgA levels. A SPIT-KIT, as used in this study, a kit that can be used to measure various salivary parameters such as FR, pH, and BC can be useful in identifying high-risk patients. Estimation of IgA levels in saliva requires specialized immunological laboratories and cannot be done as a chair-side procedure and currently there are no reliable techniques to increase s-IgA levels. However, IgA studies are important as they will finally help in the development of a suitable anti-caries vaccine, as increasing the levels of anti-S. mutans IgA is the basis of any caries vaccine. Streptococcal antigens like GTF, antigen 1/11 are being used to develop IgA specific to S. mutans.  In high-risk children, salivary flow and buffering effect may be increased using sugar-free chewing gums, sucking tablets, lozenges, etc. 
| Conclusion|| |
In this study
- The RC group children had significantly lower s-IgA concentration in their saliva when compared to CR children in their same age group.
- The resting salivary pH values of CR children were significantly higher than RC children.
- Salivary BC and FRs are comparatively much lower in RC children than in children who show a resistance of dental caries.
- In both RC and CR children, the BC and resting pH values of saliva showed a positive correlation with each other.
This study has shown that the salivary FR, pH, BC, and s-IgA concentrations are important risk factors in the development of rampant dental caries in preschool children aged 3-5 years. Increase in s-IgA levels, BC, FR, and resting pH can make a child resistant to dental caries while a sudden decrease in these parameters can lead to the development of RC.
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[Table 1], [Table 2], [Table 3], [Table 4]
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