Original Paper Received: November 26, 2012 Accepted after revision: September 10, 2013 Published online: February 7, 2014

Caries Res 2014;48:271–275 DOI: 10.1159/000355581

Comparison of Total Antioxidant Capacity in Saliva of Children with Severe Early Childhood Caries and Caries-Free Children S. Mahjoub a M. Ghasempour b A. Gharage b A. Bijani c J. Masrourroudsari d   

 

 

 

 

a Department of Biochemistry and Biophysics, Faculty of Medicine, b Faculty of Dentistry, c Noncommunicable Pediatrics Diseases Research Center and d Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran  

 

 

 

Abstract Oxidative stress may play an important role in the onset and development of oral inflammatory and dental decay diseases. The aim of this study was to compare total antioxidant capacity (TAC) levels in the unstimulated whole saliva of children with severe early childhood caries (S-ECC) and cariesfree children. In this case-control study, 80 children aged 3–5 years from nursery schools in Babol, northern Iran were the subjects of the study. The S-ECC group contained 40 children with dmfs ≥4 (age 3), ≥5 (age 4) or ≥6 (age 5) and the control group contained 40 caries-free children (dmfs = 0). Out of consideration for growth pattern and general health, the clinical examinations of the chosen children were conducted by a physician. These two groups were age and sex matched. TAC was measured by the FRAP (ferric-reducing antioxidant power) method and total protein in unstimulated whole saliva was evaluated spectrophotometrically. According to the normal distribution of data, statistical tests including the t test and Pearson’s correlation test were used; p < 0.05 was considered significant in the difference between the two groups. TAC levels and salivary total protein increased in

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children with S-ECC compared with caries-free children (p = 0.025 and p = 0.033, respectively). Moreover, salivary TAC showed a significant positive correlation with total protein concentration and dmfs in the S-ECC group (p < 0.001, r = 0.685 and r = 0.902, respectively). The significant increment of salivary TAC in S-ECC may represent a compensatory mechanism against oxidative stress in S-ECC. © 2014 S. Karger AG, Basel

Early childhood caries (ECC) is the most common chronic disease of children and is characterized by at least 1 decayed, extracted (due to caries) or filled tooth surface in any of the primary teeth in children aged under 71 months [Nunn et al., 2009]. In children aged less than 3 years, any sign of decay in smooth surface accounts for ECC. Severe early childhood caries (S-ECC) in children aged 3–5 years was characterized by the presence of even 1 surface of decay in anterior maxillary teeth and dmfs ≥4 in 3-year-olds , dmfs ≥5 in 4-year-olds and dmfs ≥6 in 5-year-olds [Ismail and Sohn, 1999]. In spite of dental caries patency in children in the age range of 3–5 years, data collection for epidemiological studies is difficult due to the limitation of accessibility in this group [Tinanoff et al., 2002]. In 2009, 41% of children in the USA were affected by ECC [Marrs et al., 2011]. In Jila Masrourroudsari, MD Infectious Diseases and Tropical Medicine Research Center Babol University of Medical Sciences, Babol (Iran) E-Mail researchbabol @ gmail.com

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Key Words Oxidative stress · Saliva · Severe early childhood caries · Total antioxidant capacity

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Caries Res 2014;48:271–275 DOI: 10.1159/000355581

children with early caries was significantly more than the amount seen in the caries-free children. With regard to the studies that were carried out, oxidative stress might have an important role in the onset and development of inflammatory oral and dental decay diseases. Saliva can be the first line of defense against oxidative stress-mediated free radicals; that is the reason for TAC in saliva which is the outcome of the whole enzymatic and nonenzymatic salivary compounds, as has recently been considered. No study has been conducted yet with regard to salivary TAC evaluation in children aged 3–5 years with S-ECC through the standard method of ferric-reducing antioxidant power (FRAP) [Benzie and Strain, 1999]. It seems that the present study is the first concerning TAC status in S-ECC and caries-free children in the aforementioned ages using the FRAP method. The relation of TAC with dmfs and total protein amount in the saliva of children aged 3–5 years has also been studied. Methods In this case-control study, 80 children aged 3–5 years from the nursery schools in Babol, northern Iran were investigated. The study was carried out in cooperation with the Departments of Pediatric Dentistry and Biochemistry of Babol University of Medical Sciences subsequent to its approval by the Research Ethics Committee. After obtaining permission from Babol University of Medical Sciences, the study was explained to the parents and the authorities of the nursery schools. Following their consent, a questionnaire consisting of demographic questions such as nutritional habits, history of disease, fluoride therapy and consumption of drugs and vitamin supplements was given to the parents. The next day, eligible children (aged 3–5 years) were selected and they were all examined by a physician for normal growth pattern and general health. Children with a disease history, fluoride therapy or drug and supplement consumption and subjects who were physically and medically compromised were excluded from the study. Thereafter, dentistry examination and determination of dmfs index was done by an experienced dentist and dental caries status was reported on the basis of WHO recommendation [WHO, 1997]. The children were divided into two groups, which were age and gender matched. The S-ECC group contained 40 children with dmfs ≥4 (age 3), ≥5 (age 4) or ≥6 (age 5) and the control group contained 40 caries-free children (dmfs = 0). It is worth mentioning that the children did not have water and food intake for 1 h before the examination. After examination, unstimulated saliva was collected for the study (with the observation of all conditions in proper sampling and without pollution) in a disposable sterile laboratory container with wide opening and lid. The samples were then transferred to Eppendorf microtubes through volume samplers and after coding they were all transferred to the Biochemistry Research Laboratory of the university in dry ice containers. The containers were maintained in the refrigerator at –20 ° C until the execution of the experiment.  

 

Mahjoub/Ghasempour/Gharage/Bijani/ Masrourroudsari

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Tehran, the same research was carried out in children aged 3–7 years in 2006 and the dmft index was reported to be 2.56 ± 2.32 [Hematyar and Masnavi, 2009]. Oxidative stress may play an important role in the onset and development of inflammatory oral and dental decay diseases. Saliva is the first line of defense against oxidative stress through free radicals and it contains different biochemical compounds including antioxidants. Antioxidants are the various enzymatic or nonenzymatic compounds present in the tissues and biological fluids of our body and prevent the potential complications of oxidants or additional oxidants in the body produced by oxidation reaction [Battino et al., 2002; Greabu et al., 2007]. Reactive oxygen species and free radicals in the mouth originate in polymorphonuclear neutrophils, principally so that they can assist the control of bacterial growth by ‘respiratory burst’ reaction. These procedures usually interfere with internal antioxidant systems and if the antioxidant systems do not work efficiently, oxidative damage may occur [Battino et al., 2002]. Much research has been conducted about the risk factors of dental caries [de Farias and Bezerra, 2003; Mahjoub et al., 2007; Chankanka et al., 2011]. However, the studies were carried out in the field of some oxidative stress indicators in S-ECC [Kumar et al., 2011]. Therefore, more comprehensive studies in this regard need to be done in order to clarify the potential role of oxidative stress in the pathogenesis of this disease. In the study done by Tulunoglu et al. [2006], the total antioxidant level in the saliva of children aged 7–15 years was examined. They divided the children according to gender, age (7–10 and 11–15 years) and caries activity (caries-active and caries-free) into smaller groups. Total antioxidant capacity (TAC) in saliva was measured through the ABTS [2,2′-azino-di (3-ethyl-benzthiazoline6-sulphonate)] method in unstimulated saliva samples. The results of this study revealed that the total protein and total antioxidant levels showed further degree in the cariesactive group. Uberos et al. [2008] conducted a study which showed the influence of salivary antioxidant materials on dental caries in a high-risk population. In their study, TAC in the saliva of patients with dental caries was 2.89 times more than in caries-free subjects. Hegde et al. [2009] examined the TAC in the saliva of children aged less than 71 months with early caries and children aged 6–12 years with disseminated caries. The results of this study demonstrated that TAC increases in children’s saliva affected by caries. There was also an increment in TAC associated with increasing age of patients. Recently, Kumar et al. [2011] evaluated the TAC in Indian children with S-ECC by the ABTS method. Their study showed that TAC in the saliva of

Experimental Methods After all the samples were refrozen and had reached laboratory temperature, they were centrifuged for 10 min with 4,000 rpm at a temperature of 4 ° C. (32R Refrigerator Universal Centrifuge, Germany). The separated transparent solution on the top surface was used to measure the total protein and TAC. The evaluation of TAC level in saliva was carried out by the FRAP method as already described [Benzie and Strain, 1999]. In the FRAP method, ferric to ferrous ion reduction at low pH causes a colored ferrous-tripyridyltriazine complex to form due to the presence of antioxidants. The maximum absorbance of this blue complex was at 593 nm. A standard linear curve was prepared according to the absorbance of different concentrations of standard solution of ferrous sulfate, and TAC levels of the samples were obtained by using a standard curve on the basis of the FRAP index. The results were reported in terms of millimoles per liter. Total protein was measured by the Bradford method. In the Bradford assay, Coomassie Brilliant Blue G-250 binds to protonated amine groups of amino acids in the polypeptide chain and maximum absorption wavelength is 595 nm. Total protein concentration level in samples was obtained by the standard curves of albumin and the result was reported in terms of milligrams per deciliter. All materials used in the laboratory were purchased from Merck & Co. (Germany) and optical absorption measurements of samples were performed through spectrophotometer UV-Vis (JENWAY 6505, UK).

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Statistical Analysis Statistical analysis was performed using SPSS 18.00 software (Chicago, Ill., USA). Data are expressed as mean ± standard deviation. To compare the mean values of TAC and total protein that were normally distributed, the statistical independent Student t test, Pearson’s correlation test and linear regression were used. Moreover, the correlation coefficient was applied to determine the relationship of TAC with dmfs and TAC with total protein. The mean and standard deviation values of the variables were calculated; p < 0.05 was considered significant in the difference between TAC and total protein levels in the two groups.

Salivary TAC (mmol/l)

 

0.50 0.40 0.30 0.20 0.10 0

Mean = 0.647 SD = 0.121

Mean = 0.579 SD = 0.145

S-ECC

Caries-free

Fig. 1. The mean and standard deviation of TAC levels and total

protein in the saliva of children with S-ECC and caries-free children. SD = Standard deviation.

Error bars: 95% CI p = 0.033

400 Salivary total protein (mg/dl)

 

Error bars: 95% CI p = 0.025

0.70

350 300 250 200 150 100 50 0

Mean = 323.18 SD = 128.71

Mean = 270.14 SD = 86.22

S-ECC

Caries-free

Fig. 2. The mean and standard deviation of total protein in the sa-

liva of children with S-ECC and caries-free children. SD = Standard deviation.

From the 80 children aged 3–5 years participating in this study, the number of girls and boys in both the S-ECC and caries-free groups were the same (20 girls and 20 boys in each group, respectively). The mean and standard deviation values of TAC (mmol/l) and total protein (mg/dl) in the saliva of children are shown in figures 1 and 2. The mean TAC in saliva samples from children with S-ECC was significantly greater than in the group without caries (p = 0.025). Furthermore, the mean total protein in the saliva of children with S-ECC was significantly greater than in the caries-free group (p = 0.033). There was a positive correlation between salivary TAC and dmfs scores in S-ECC children by Pearson’s correlation test (r = 0.725, p < 0.001).

Figure 3 indicates a significant correlation between TAC and total protein level in the saliva of 3- to 5-yearold children affected by S-ECC. Distribution between TAC level and salivary total protein level also had a significant positive correlation in the whole saliva of the study subjects (r = 0.685, p < 0.001). Although a positive association was observed in the total protein levels in saliva and TAC in caries-free children aged 3–5 years, this was not significant (r = 0.268, p = 0.09; fig. 4). The results showed a significant positive correlation between TAC and total protein concentration and dmfs in the S-ECC group (p < 0.001, r = 0.685 and r = 0.902, respectively).

Total Antioxidant Capacity in Saliva of S-ECC and Caries-Free Children

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Results

Salivary TAC (mmol/l)

0.9 0.8 0.7 0.6 0.5 R2 linear = 0.468 F = 33.48; p < 0.001 y = 0.269 + 0.001x

0.4 0.3 100

200

300 400 500 600 700 Salivary total protein (mg/dl)

800

Fig. 3. Correlation of TAC with total protein levels in the saliva of

S-ECC children.

Salivary TAC (mmol/l)

1.0 0.9 0.8 0.7 0.6 R2 linear = 0.072 F = 2.929; p = 0.095 y = 0.566 + 0.000x

0.5 0.4 100

200

300 400 500 600 700 Salivary total protein (mg/dl)

800

Fig. 4. Correlation of salivary TAC with total protein levels in the saliva of caries-free children.

Discussion

The present study is the first of its kind to examine the antioxidant levels of saliva in S-ECC children aged 3–5 years using the FRAP method. Based on the results of this study, the mean TAC in the saliva samples of children with early dental caries was significantly greater than the caries-free group. The present findings and the results of the studies conducted in the field of dental caries in other age groups have been consistent. Tulunoglu et al. [2006] demonstrated that the antioxidant level of saliva in caries-active individuals was greater. Hegde et al. [2009] examined the TAC of saliva and its 274

Caries Res 2014;48:271–275 DOI: 10.1159/000355581

correlation with ECC and rampant caries. Their results suggest that TAC of saliva is increased in caries-active children. TAC also increased alongside the increment in the age of the patients. Uberos et al. [2008] showed that the TAC level in the saliva of patients with dental caries is 2.89 times more than in caries-free individuals. Recently, Kumar et al. [2011] reported that the salivary TAC level in the study group with S-ECC in Indian children was significantly higher than the TAC level in caries-free children. However, they evaluated the salivary TAC level through another method (ABTS method), while the present study used a technique known as FRAP. Despite the two different methods on different populations in the measurement of TAC in saliva, the results are consistent and this represents a significant change in TAC levels in the saliva of S-ECC. Perhaps in the near future, through longitudinal studies, a TAC index will be introduced as a marker of S-ECC in the determination of susceptibility in children. The study conducted by Kumar et al. [2011] is the only relatively similar study to the present one although, in addition to the difference in methods of TAC measurement, salivary total protein and its relationship with TAC have not been reported in their study. Moreover, they used the dmft index in their research while in our study the dmfs index was determined according to reliable sources and WHO recommendations and the correlation between salivary TAC and dmfs levels in 3- to 5-year-old children with S-ECC. Increased TAC in the saliva of children with S-ECC might be a compensatory mechanism of the antioxidant system against early dental caries as it has been reported that this type of compensatory mechanism was observed in certain systemic diseases [Mahjoub et al., 2007; Gholami et al., 2012]. Some studies, including the present one, have demonstrated higher levels of antioxidants in the saliva of cariesactive children of different ages and this suggests the effect of increased levels of salivary proteins with antioxidant properties. Therefore, we can expect a positive correlation between salivary total antioxidant levels and total protein in saliva [Moore et al., 1994]. Our results indicated that the mean amount of total protein in the saliva of children with early dental caries was significantly greater than the caries-free group and confirmed the above-mentioned hypothesis. Dodds et al. [1997] examined the parotid salivary gland proteins in adults with severe and active caries and showed that similar proteins in the individuals might present different biological activities. The present study also demonstrates a correlation between the salivary total protein level and salivary TAC in 3- to 5-year-old children with S-ECC. Based on these Mahjoub/Ghasempour/Gharage/Bijani/ Masrourroudsari

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findings, it is suggested that in early dental caries, significant changes should occur in protein level and antioxidant components in total saliva since these changes in both biochemical indices in saliva have a significant positive correlation with each other. The limitation of our study was that, ethically, we could not completely match the nutritional program the day before sampling and also the fasting period of more than 1 h before saliva sampling, because of the low age of the children in the two groups.

Conclusion

It seems that the present study is the first of its kind to examine the antioxidant levels of saliva in S-ECC children aged 3–5 years using the FRAP method. Furthermore, our findings showed a significant positive correla-

tion between TAC levels, dmfs levels and salivary total protein in children with S-ECC. Increased TAC in the saliva of children with S-ECC may be a compensatory mechanism of the antioxidant system against early dental caries.

Acknowledgments The authors wish to express their gratitude to Dr. Evangeline Foronda for her proofreading of the manuscript. This project was financially supported by the Vice Chancellery of Research and Technology of Babol University of Medical Sciences (grant No. 6131228).

Disclosure Statement The authors have no conflict of interest to disclose.

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Comparison of total antioxidant capacity in saliva of children with severe early childhood caries and caries-free children.

Oxidative stress may play an important role in the onset and development of oral inflammatory and dental decay diseases. The aim of this study was to ...
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