Original Paper
Caries Res 2014;48:271–275 Received: November 26, 2012
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
Key Words
Oxidative stress · Saliva · Severe early childhood caries ·
Total antioxidant capacity
Abstract
Oxidative stress may play an important role in the onset and
development of oral inflammatory and dental decay diseas-
es. The aim of this study was to compare total antioxidant
capacity (TAC) levels in the unstimulated whole saliva of chil-
dren with severe early childhood caries (S-ECC) and caries-
free 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 sa-
liva 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
© 2014 S. Karger AG, Basel
0008–6568/14/0484–0271$39.50/0
E-Mail karger@karger.com
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Accepted after revision: September 10, 2013
Published online: February 7, 2014
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
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Dir.General de Bibliotecas, UNAM
Jila Masrourroudsari, MD
Infectious Diseases and Tropical Medicine Research Center
Babol University of Medical Sciences, Babol (Iran)
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E-Mail researchbabol @ gmail.com
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 on-
set and development of inflammatory oral and dental de-
cay diseases. Saliva is the first line of defense against oxi-
dative stress through free radicals and it contains differ-
ent 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 oxi-
dants or additional oxidants in the body produced by ox-
idation 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 in-
terfere with internal antioxidant systems and if the anti-
oxidant systems do not work efficiently, oxidative dam-
age may occur [Battino et al., 2002].
Much research has been conducted about the risk fac-
tors 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 indica-
tors in S-ECC [Kumar et al., 2011]. Therefore, more com-
prehensive studies in this regard need to be done in order
to clarify the potential role of oxidative stress in the patho-
genesis 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-benzthiazoline-
6-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 caries-
active 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] exam-
ined 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 in-
creasing age of patients. Recently, Kumar et al. [2011] eval-
uated the TAC in Indian children with S-ECC by the ABTS
method. Their study showed that TAC in the saliva of
272 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, oxida-
tive stress might have an important role in the onset and
development of inflammatory oral and dental decay dis-
eases. 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 an-
tioxidant 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 rela-
tion 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 Pe-
diatric Dentistry and Biochemistry of Babol University of Medical
Sciences subsequent to its approval by the Research Ethics Com-
mittee.
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, eli-
gible children (aged 3–5 years) were selected and they were all ex-
amined 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. There-
after, dentistry examination and determination of dmfs index was
done by an experienced dentist and dental caries status was re-
ported 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 ex-
amination. After examination, unstimulated saliva was collected
for the study (with the observation of all conditions in proper sam-
pling and without pollution) in a disposable sterile laboratory con-
tainer with wide opening and lid. The samples were then trans-
ferred to Eppendorf microtubes through volume samplers and af-
ter 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.
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Mahjoub/Ghasempour/Gharage/Bijani/
Masrourroudsari
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 Experimental Methods
After all the samples were refrozen and had reached laboratory Error bars: 95% CI
0.70 p = 0.025
temperature, they were centrifuged for 10 min with 4,000 rpm at a
temperature of 4 ° C. (32R Refrigerator Universal Centrifuge,
    0.60

Salivary TAC (mmol/l)

Germany). The separated transparent solution on the top surface
0.50
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 0.40
described [Benzie and Strain, 1999]. In the FRAP method, ferric to
ferrous ion reduction at low pH causes a colored ferrous-tripyrid- 0.30
yltriazine complex to form due to the presence of antioxidants. The 0.20
maximum absorbance of this blue complex was at 593 nm. A stan- Mean = 0.647 Mean = 0.579
dard linear curve was prepared according to the absorbance of dif- 0.10 SD = 0.121 SD = 0.145
ferent concentrations of standard solution of ferrous sulfate, and 0
TAC levels of the samples were obtained by using a standard curve S-ECC Caries-free
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 Fig. 1. The mean and standard deviation of TAC levels and total
Bradford assay, Coomassie Brilliant Blue G-250 binds to proton- protein in the saliva of children with S-ECC and caries-free chil-
ated amine groups of amino acids in the polypeptide chain and dren. SD = Standard deviation.
maximum absorption wavelength is 595 nm. Total protein con-
centration 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 Error bars: 95% CI
samples were performed through spectrophotometer UV-Vis 400 p = 0.033
(JENWAY 6505, UK). 350
Salivary total protein (mg/dl)

300
Statistical Analysis
Statistical analysis was performed using SPSS 18.00 software 250
(Chicago, Ill., USA). Data are expressed as mean ± standard devia- 200
tion. To compare the mean values of TAC and total protein that
were normally distributed, the statistical independent Student 150
t test, Pearson’s correlation test and linear regression were used. 100
Mean = 323.18 Mean = 270.14
Moreover, the correlation coefficient was applied to determine the 50 SD = 128.71 SD = 86.22
relationship of TAC with dmfs and TAC with total protein. The
mean and standard deviation values of the variables were calcu- 0
lated; p < 0.05 was considered significant in the difference between S-ECC Caries-free
TAC and total protein levels in the two groups.
Fig. 2. The mean and standard deviation of total protein in the sa-
liva of children with S-ECC and caries-free children. SD = Stan-
Results dard 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 de-
viation 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 correla-
tion 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-year-
old children affected by S-ECC. Distribution between
TAC level and salivary total protein level also had a sig-
nificant 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 chil-
dren 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).
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Total Antioxidant Capacity in Saliva of Caries Res 2014;48:271–275 273

S-ECC and Caries-Free Children DOI: 10.1159/000355581
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 correlation with ECC and rampant caries. Their results
1.0 suggest that TAC of saliva is increased in caries-active
0.9 children. TAC also increased alongside the increment in
the age of the patients. Uberos et al. [2008] showed that
Salivary TAC (mmol/l)

0.8
the TAC level in the saliva of patients with dental caries
0.7 is 2.89 times more than in caries-free individuals. Recent-
0.6 ly, Kumar et al. [2011] reported that the salivary TAC
0.5
level in the study group with S-ECC in Indian children
R2 linear = 0.468
was significantly higher than the TAC level in caries-free
0.4
F = 33.48; p < 0.001 children. However, they evaluated the salivary TAC level
y = 0.269 + 0.001x
0.3 through another method (ABTS method), while the pres-
100 200 300 400 500 600 700 800 ent study used a technique known as FRAP. Despite the
Salivary total protein (mg/dl) 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
Fig. 3. Correlation of TAC with total protein levels in the saliva of
S-ECC children. 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
1.0
the only relatively similar study to the present one al-
though, in addition to the difference in methods of TAC
0.9 measurement, salivary total protein and its relationship
Salivary TAC (mmol/l)

0.8
0.7
0.6
0.5
0.4
100 200 300 400
Salivary total protein (mg/dl)
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 antioxi-
dant level of saliva in caries-active individuals was great-
er. Hegde et al. [2009] examined the TAC of saliva and its
with TAC have not been reported in their study. More-
over, 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
R2 linear = 0.072 5-year-old children with S-ECC. Increased TAC in the
F = 2.929; p = 0.095
y = 0.566 + 0.000x saliva of children with S-ECC might be a compensatory
mechanism of the antioxidant system against early dental
500 600 700 800 caries as it has been reported that this type of compensa-
tory mechanism was observed in certain systemic diseas-
es [Mahjoub et al., 2007; Gholami et al., 2012].
Some studies, including the present one, have demon-
strated higher levels of antioxidants in the saliva of caries-
active children of different ages and this suggests the ef-
fect of increased levels of salivary proteins with antioxi-
dant properties. Therefore, we can expect a positive
correlation between salivary total antioxidant levels and
total protein in saliva [Moore et al., 1994]. Our results in-
dicated that the mean amount of total protein in the sa-
liva of children with early dental caries was significantly
greater than the caries-free group and confirmed the
above-mentioned hypothesis. Dodds et al. [1997] exam-
ined the parotid salivary gland proteins in adults with se-
vere and active caries and showed that similar proteins in
the individuals might present different biological activi-
ties. 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
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274 Caries Res 2014;48:271–275 Mahjoub/Ghasempour/Gharage/Bijani/

DOI: 10.1159/000355581 Masrourroudsari
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findings, it is suggested that in early dental caries, signifi-
cant changes should occur in protein level and antioxi-
dant components in total saliva since these changes in
both biochemical indices in saliva have a significant pos-
itive 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
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 den-
tal 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 Sci-
ences (grant No. 6131228).

It seems that the present study is the first of its kind to

examine the antioxidant levels of saliva in S-ECC chil- Disclosure Statement
dren aged 3–5 years using the FRAP method. Further-
more, our findings showed a significant positive correla- The authors have no conflict of interest to disclose.

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