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Year : 2016  |  Volume : 34  |  Issue : 3  |  Page : 227-232

Evaluation of total antioxidant level of saliva in modulation of caries occurrence and progression in children

Department of Pedodontics and Preventive Dentistry, Modern Dental College and Research Centre, Indore, Madhya Pradesh, India

Date of Web Publication25-Jul-2016

Correspondence Address:
Naveen Reddy Banda
Department of Pedodontics and Preventive Dentistry, Modern Dental College and Research Centre, Airport Road, Gandhi Nagar, Indore, Madhya Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-4388.186747

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Context: Recent studies have implicated a direct relation between dental caries and salivary total antioxidant level (TAL), which can be suggested as an indicator (either harmful or protective) for susceptibility of individuals for dental caries. Aim: To evaluate the total antioxidant level (TAL) in unstimulated saliva of children, and to correlate the TAL with caries experience and BMI (body mass index). Settings and Design: 60 children, aged 6-12 yrs, were randomly selected for the study. Two groups were made (study group n=30 and control group n=30), and subdivisions based on gender were made out of both groups [male (n=15) and female (n=15)]. In control group, subjects with no dental caries and study group, subjects having decayed teeth score ≥4 were included. Statistical Analysis: Data analysis was done using Statistical Package for Social Sciences (SPSS V. 21). Shapiro – Wilks test showed that TAL (absorbance) did not follow normal distribution. Hence, for comparison between cases and control for TAL non-parametric test namely Mann- Whitney U test was applied. Results: Statistically significant strong positive correlation was seen between age and TAL (P< 0.05).

Keywords: Body mass index, dental caries, oxidative stress, total antioxidant level

How to cite this article:
Banda NR, Singh G, Markam V. Evaluation of total antioxidant level of saliva in modulation of caries occurrence and progression in children. J Indian Soc Pedod Prev Dent 2016;34:227-32

How to cite this URL:
Banda NR, Singh G, Markam V. Evaluation of total antioxidant level of saliva in modulation of caries occurrence and progression in children. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2022 Nov 29];34:227-32. Available from: http://www.jisppd.com/text.asp?2016/34/3/227/186747

   Introduction Top

Despite the advancement in treatment modalities of oral health needs among the world population, there are many developing countries facing oral health problems.[1] Good oral health is an essential part of healthy living, which can be affected by various dental diseases such as dental caries, periodontitis, gingivitis, and periapical pathology. These problems can affect the normal day to day activities such as chewing and talking. These diseases initiate in a very inconspicuous manner, and if ignored, result in a more severe damage to oral hard and soft tissues. Sometimes leading to unesthetic appearance.[2]

Among dental diseases, the most common chronic disease which has been considered as a global oral health problem is dental caries.[3] Dental caries is a multifactorial disease. According to Shafer, “Dental caries is an irreversible microbial disease of calcified tissue of the teeth, characterized by demineralization of the inorganic portion and destruction of organic substance of the tooth, which often leads to cavitation.”[4]

Numerous authors with expertise on dental caries stated that it is an infectious and transmissible disease. Its multifactorial nature influence the onset and progression, which also require host (tooth), a dietary substrate and causative organism.[5]

Saliva also plays a crucial role in dental caries. It is a heterogeneous fluid consisting of various electrolytes, glycoproteins, proteins, and compounds transported from the systemic circulation. It has various functions such as cleaning of teeth, acts as an ion reservoir, helps in lubrication, and has buffering action.[6] To a larger extent, saliva maintains a balance between remineralization and demineralization as protective mechanism and thus protects the enamel.[7]

In addition to its mentioned properties, it also provides defense against free radical (FR)-mediated oxidative stress, since most of the physiological activities such as mastication and digestion of ingested food promotes a variety of reactions including lipid peroxidation (Terao and Nagao 1991). Battino et al., 2002, stated that antioxidant property of saliva provides the first line of defense against oxidative stress. Oxidative imbalance is referred to as an imbalance between antioxidant defense system and reactive oxygen species (ROS) production. This imbalance is peculiar to many oral diseases.[6]

With the recent advances in knowledge of microbiological aspects of dental caries, it has been argued that oxidative stress plays an important role in onset and progression of dental caries.[6] Antioxidant system present in all biological species, helps in mediating the balance, and neutralizing the harmful effects of the free radial and ROS. Thus, pathological diseases can be prevented mainly by the dietary antioxidants, intercellular antioxidants, and enzymes of the antioxidant system.[8]

Activity of FR/ROS is regulated by antioxidant system. Antioxidants counteract FRs/ROS and balance the chain reaction by terminating it. The activity of antioxidants is a concerted effect rather than individual. It might be misleading to investigate and evaluate individual antioxidants.[9] Hence, evaluation of total antioxidant level (TAL) in saliva can pave way in understanding the risk of individuals to dental caries.

   Materials and Methods Top


Sixty random selected, 6–12-year-old children who reported to the Pedodontics and Preventive Dentistry Department were included as subjects of this study. They were divided into two groups; Group I study group (n = 30) and Group II control group (n = 30). Subdivision of these groups was done based on gender with 15 children in each group.

Inclusion criteria

  • For caries-active children, number of decayed teeth >4
  • For caries free (CF) children, decayed teeth = 0
  • Patients who are free from systemic conditions affecting salivary properties.

Exclusion criteria

  • Mentally and physically challenged children
  • Patients on medications
  • Children with fluorosis, previous restoration, and arrested carious lesion.

Data recording of carious teeth

Caries status was recorded based on WHO recommendations. Sterile mouth mirror and probe were used, and number of caries teeth were recorded as caries experience.

Calculation of body mass index

Height and weight measurements were recorded for all the children who participated in the study. For body mass index (BMI) calculation, following formula was used.

Saliva collection and salivary analysis of total antioxidant level

On the day of collection of saliva samples, participating children were instructed not to eat or drink anything for at least 1 h before the saliva sample was collected. To control the circadian variations, samples were collected between 9 and 10 am. Unstimulated saliva samples were collected.

The subjects were seated comfortably with eyes open, head tilted slightly forward with restricted orofacial movements and were instructed to rest for 5 min. Saliva was allowed to accumulate in the floor of the mouth, and the subjects were instructed to spit into a sterile saliva collecting vial. The collected samples were stored in a hermetically sealed case containing ice and transported to laboratory within 1 h of collection and were kept at −80°C, till analysis.

Estimation of salivary total antioxidant level

Two milliliters of each saliva sample was centrifuged, and obtained supernatant was kept at 4°C for subsequent analysis.

Phosphomolybdenum method for estimation of salivary total antioxidant level

The basic principle of this assay is based on the conversion of molybdenum (Mo VI) to molybdenum (Mo V) by reducing agents such as antioxidants, resultant molybdenum (Mo V). A reagent solution (consisting of 0.6 M sulfuric acid, 28 mM sodium phosphate, 4 mM ammonium molybdate) was used, which results in the formation of green color complex under acidic pH [Figure 1]. Reading of color intensity was done using spectrophotometer at 695 nm.[10]
Figure 1: Supernatant obtained from saliva mixed with reagent forms green-color complex

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Statistical analysis

Mean, standard deviation, median, minimum and maximum values of TAL (absorbance, nM) in caries active (CA) group and controls (CF group) were calculated [Table 1]. Shapiro–Wilk test showed that TAL (absorbance, nM) did not follow normal distribution. Hence, for comparison between case and control for TAL, nonparametric test namely Mann–Whitney U-test was applied.
Table 1: Group statistics: Comparison of case and control according to variables

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Correlations of TAL (absorbance, nM) with age, weight, height, BMI, and decayed missing filled teeth (DMFT) scores were evaluated by Spearman's rank correlation test [Table 2]. P < 0.05 was considered statistically significant. Data analysis was done using Statistical Package for Social Sciences (SPSS) v. 21 (SPSS Inc., Chicago, Illinois).
Table 2: Correlation between total antioxidant level and variable

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   Results Top

Statistically significant strong positive correlation was seen between age and TAL and between DMFT scores and TAL [Graph 1 [Additional file 1]]. There is no relationship of TAL (absorbance) with weight (kg), height (inch), and BMI in total study subjects [Graph 2 [Additional file 2]] and [Graph 3 [Additional file 3]].

   Discussion Top

Dental caries is a multifactorial microbial disease, and the documented results regarding antioxidants and proteins show an increased level in patients with dental caries. Antioxidant molecules either delay or inhibit oxidation of substrate (host/pathogen/both). Chemically, they donate one of their electron thereby ending the electron stealing reaction. However, antioxidants are stable in either form; therefore, they do not become FRs.

Based on method of action antioxidants are:[11],[12]

  • Chain breaking/scavenging. E.g.,: Vitamins E, C, A, urate, bilirubin.
  • Preventing antioxidants: Those substance contain “thiol” group. Their function largely by sequestering transitional metal ions and preventing Fenton reaction and are therefore protein in nature. E.g.: Albumin, transferrin, lactoferrin, ceruloplasmin, etc.
  • Enzyme antioxidants: Function by catalyzing the oxidation of other molecules. E.g.: Catalase, glutathione peroxide.

In living system there are various source of ROS such as by-products of cellular respiration, synthesized by enzyme systems-phagocytic cells, neutrophils, and macrophases, exposure to ionizing radiation, etc., In addition, smoking, fired foods, etc., are the sources of ROS. Recent work has demonstrated that ROS have role in cell signaling, including the following: Apoptosis, gene expression, and activation of cell signaling cascades. It should be noted that ROS can serve as both intracellular and intercellular messengers.[13]

In previous studies, increased TAL is found in subjects with caries. FRs/ROS result as normal metabolic by-products, which is controlled by antioxidant system of host. These FRs also trigger activation of antioxidant system of microorganism for self-defense.[14] Due to potential damaging effects of ROS, in bacteria, iron solubilization, and metabolism is strictly regulated. That could be a possible reason of an increase in TAL in caries group because significantly more number of pathogens are present in caries group. Hence, we think that increased TAL in saliva in caries group is a defensive action from pathogen rather than host. This fact has to be, especially by biochemical methods, which was not possible in our study.

Loss of equilibrium between antioxidant defense system and pro-oxidant mechanism may tip to tissue destruction by increased radical production, which in turn activates antioxidant system of bacteria. As has been found in previous studies, the adherent activity of some bacteria may be modified by the antioxidant activity of their environment.[15] In this sense, an increase in the TAL of saliva could produce modifications in the adherence of Streptococcus to the dental plaque and lead to greater cariogenic activity. Increased TAL could also counteract the bactericidal effect of the pro-oxidant molecules present in saliva and on the dental plaque, in turn favoring the development of caries.[16]

Cariogenic bacteria such as Streptococcus mutans, Streptococcus sobrinus, and lactobacilli produce acids following an individual's sugar consumption (Loesche, 1986). These acids, mainly lactic acid, diffuse through the dental calcified tissues and drop the local pH to below 5.5, which in turn leads to a dissolution of the mineral crystals and cause caries initiation in enamel.[17]

As the caries process reaches to dentin, degradation of dentin matrix starts. ROS activate matrix metalloproteinases (MMPs) in dentin matrix. Though activated MMPs are tightly regulated by tissue inhibitors of metalloproteinases and alfa-macromolecules in normal physiological conditions, but certain local environmental changes (pH changes and ROS) results in imbalance and the regulatory mechanism is bypassed leading to tissue destruction.[17]

In this study, we have found statistically significant strong positive correlation (P < 0.05) between age and TAL of saliva in total study subjects [Graph 2]. The age group, which was taken is 6–12 years nutrition, could be a possible reason for raise in TAL of saliva with increase in age. Younger children possibly consuming nutritious diet but lesser in volume, as compared to older age groups.[18] Age is also the well-known marker for variability of oxidative stress and it increases with age.[19],[20] The positive correlation between age and TAL is also stated in other similarly focused papers.[21],[22]

There is no relationship of TAL (absorbance, nM) with weight (kg), height (Inch) in total study subjects. Except the TAL and BMI which has a weak negative correlation (P = 0.093), but not a significant correlation. In other studies,[23] negative correlation was found between TAL and BMI, suggestive of pathogenesis of obesity. This can be further checked by a study with a large sample size.

Moreover, there is no significant difference in TAL of saliva difference in gender subgroups [Graph 3] in both CA and control groups (CF). There is no relation of antioxidant level of saliva with gender.[24],[25] Some researchers have noted no effect, where as some have reported male predominance.[26],[27]

Possible reasons for variations of TAL in subjects could be there diet, oral hygiene practices, and use of fluoridated measures, socioeconomic status, and age.

   Conclusion Top

Most common dental diseases are dental caries, gingivitis, periodontitis, pulpitis as well as periapical pathosis. The key biochemical changes occur in these diseases are loss of collagen due to degradation. There are several ways by which extracellular matrix can be degraded. Most of these are directly or indirectly interlinked with the release of enzymes by host or pathogen, generation of ROS, cytokine release or influence of inflammatory mediators, and apoptotic proteins. The elevated levels of these molecules are an indicator of inflammation; hence, they can be used as biomarkers as well as medicinal target.

Limitations of study

  • Stricter standardization protocol including same dietary pattern would give an in depth knowledge of the vagaries in antioxidant variations
  • Unable to differentiate the source of antioxidants whether it is host or pathogen in origin
  • Stricter standardized protocol to pin point the exact reason of variation in antioxidant levels, e.g., Children having same diet would help
  • Small sample size.


We thank the Biochemistry Department of Modern Dental College and Research Center, Indore, Madhya Pradesh, for helping us with this study, as well as all the children who took part in this study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Figure 1]

  [Table 1], [Table 2]

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