|Year : 2017 | Volume
| Issue : 1 | Page : 14-18
Determination of toral antioxidant capacity of saliva in sickle cell anemic patients – A cross-sectional study
Sudhindra Baliga1, Minal Chaudhary2, Sham S Bhat3, Poonam Bhatiya4, Nilima Thosar1, Pooja Bhansali1
1 Department of Pedodontics and Preventive Dentistry, Sharad Pawar Dental College, Sawangi, Wardha, Maharashtra, India
2 Department of Oral pathology, Sharad Pawar Dental College, Sawangi, Wardha, Maharashtra, India
3 Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College, Mangalore, Karnataka, India
4 Private Practitioner, Raipur, Chhattisgarh, India
|Date of Web Publication||31-Jan-2017|
Sharad Pawar Dental College and Hospital, DMIMS (DU), Wardha, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Sickle cell anemia is a congenital hemoglobinopathy characterized by deformed red blood cells. Oxidative stress plays an important role in the pathophysiology of sickle cell anaemia as it destroys free radicals, and thereby depleting the protective mechanisms such as antioxidants in serum. These antioxidants are essential to protect against harmful oxidation-reduction reactions preventing oxidative damage to the cells. Aim: To evaluate and compare the Total Antioxidant Capacity (TAC) of serum and saliva in sickle cell anemia patients. Materials and Methods: A total of 150 children aged 4-12 yrs were selected and divided into two equal groups: Children suffering from sickle cell anemia and healthy controls. Blood and saliva samples were collected aseptically from both groups and were subjected to phosphomolybdenum method. Absorbance was read spectrophotometrically at 695 nm. The concentration of total antioxidants was obtained by plotting absorbance of the test against the standard graph. Results: TAC levels in serum (0.29±0.19) and saliva (0.29±0.14) of sickle cell anaemic patients was reduced when compared with serum (0.32+ 0.18) and saliva (0.33+ 0.16) of the healthy children. The correlation between levels of TAC in saliva and serum was found to be statistically significant in sickle cell anaemic patients. Conclusion: A significant correlation of the TAC was found in saliva and serum of the patients with SCA suggests that saliva could be used as a non invasive alternative for assessing the antioxidant status in patients with SCA.
Keywords: Saliva, sickle cell anemia, total antioxidant capacity
|How to cite this article:|
Baliga S, Chaudhary M, Bhat SS, Bhatiya P, Thosar N, Bhansali P. Determination of toral antioxidant capacity of saliva in sickle cell anemic patients – A cross-sectional study. J Indian Soc Pedod Prev Dent 2017;35:14-8
|How to cite this URL:|
Baliga S, Chaudhary M, Bhat SS, Bhatiya P, Thosar N, Bhansali P. Determination of toral antioxidant capacity of saliva in sickle cell anemic patients – A cross-sectional study. J Indian Soc Pedod Prev Dent [serial online] 2017 [cited 2018 Jan 18];35:14-8. Available from: http://www.jisppd.com/text.asp?2017/35/1/14/199219
| Introduction|| |
Sickle cell anemia (SCA) is a hemoglobinopathy inherited through a mutant autosomal recessive gene present on chromosome II. Sickle cell trait develops if only one of the pair of chromosomes is affected, while involvement of both results in SCA. Due to substitution of an amino acid in the b-hemoglobin chain in the red blood cells (RBCs), an abnormal hemoglobin S develops which is less soluble than normal hemoglobin. This gives rise to the classic “sickle shape” of the cell which is more susceptible to distortion.
The clinical problems associated with SCA are mainly attributed to the deformed shape of the sickle cells. A hemolytic type of anemia, hyperplasia and widening of the marrow spaces, and a greater susceptibility to infections  have been reported. Due to increased adherence of the sickled RBCs to the vascular endothelium, vascular occlusion and impediment to blood flow may occur. This may lead to tissue anoxia, infarcts, necrosis, and pain. Oral problems such as mandibular osteomyelitis, anesthesia of the mandibular nerve, and asymptomatic pulpal necrosis have been described, however, these are relatively uncommon.,,,
In India, SCA has been reported to be more common in central and southern parts of the country. In Maharashtra, Bankar et al. reported a prevalence of the disease from 1.9% to 33.5% in different communities. In Vidarbha region of Maharashtra, a prevalence from 9.4% to 22.2% in nontribal population has been reported. The overall prevalence of SCA in Vidarbha has been reported to be 2.9%, with maximum cases reported in Samudrapur block followed by Wardha and Hinganghat block.
Reactive oxygen species (ROS) include reduced metabolites of oxygen that are toxic to the cells. Free radicals (FRs) and/or ROS can cause tissue damage by a variety of mechanisms which include DNA damage, lipid peroxidation (through activation of cyclooxygenases and lipoxygenases), protein damage, oxidation of important enzymes, for example, antiproteases such as α 1-antitrypsin, and stimulation of pro-inflammatory cytokine release by monocytes and macrophages. The body fluids and tissues are naturally protected against the endogenously formed FRs by an array of protective antioxidants  to counteract ROS and to reduce their damage. Free-radical mediated hemolysis has been reported to occur in SCA, with the sickled cells having the ability to generate approximately two times more amount of ROS.
Saliva is a natural body fluid, and any hormonal, nutritional, and metabolic disturbances that occur in serum reflect equally in saliva. Noninvasive and safe methods of salivary sample collection, the possibility of repeated sampling and longitudinal monitoring have all made salivary analysis more lucrative nowadays. There is currently no literature available on the total antioxidant capacity (TAC) of saliva, especially in patients with SCA. Therefore, the present study was carried out to evaluate and correlate the antioxidant capacity of serum and saliva in children with SCA.
| Materials and Methods|| |
A total of 150 children aged between 4 and 12 years were selected using convenient sampling method. Group I consisted of 75 children with sickle cell anemia and Group II (n = 75) consisted of healthy controls. Children with any other systemic diseases, who were immunocompromised or giving any history of vaso-occlusive crisis in the past 3 months, who had blood transfusion or any serious illness were excluded from this study. The study protocol was first approved by the Institutional Ethical Committee and written informed consent was taken from parents/guardians accompanying the children. The biochemical analysis for the study was conducted in the Central Research Laboratory of the University.
Collection of saliva and serum samples
To minimize diurnal variations, the study subjects were instructed not to eat or drink anything except water for at least 1 h before the sample collection. For a collection of the salivary sample, patients were asked to sit comfortably for 5 min with eyes open and head titled down slightly. Saliva accumulated on the floor of the mouth was then collected by asking the subject to spit it into sterile plastic plain tubes.
To obtain serum samples about 2 ml blood was drawn from the cubital vein with a 24 gauge needle. Blood was then transferred to a plain sterile bulb which was kept undisturbed for half an hour at room temperature. The supernatant was removed and centrifuged at 3000 rpm for 4–5 min. Serum obtained was then stored at −20°C for subsequent analysis.
Estimation of total antioxidant capacity of saliva and serum by phosphomolybdenum method
This quantitative assay is based on the conversion of molybdenum (Mo VI) by reducing agents like antioxidants to molybdenum (Mo V), which further reacts with phosphate under acidic pH resulting in the formation of a green colored complex, the intensity of which can be read spectrophotometrically at 695 nm.
| Results|| |
The values of TAC evaluated, in the study groups, both in saliva as well as serum is given in [Table 1] and [Table 2] respectively. When the TAC of serum was compared between children with SCA and healthy controls, using the student's unpaired 't' test, the result was non- significant. Similarly, when the TAC of saliva was compared between the two groups using the student's unpaired 't' test the results were found to be non-significant. The correlation of the TAC between serum and saliva of children with SCA is given in [Table 3]. Results, when compared using Pearson's correlation coefficient, was found to be higly significant. Similarly, the correlation of the TAC between serum and saliva of healthy children is given in [Table 4]. Comparison of the TAC between saliva and serum using Pearson's correlation coefficient showed a highly significant result [Table 4]. When the TAC of serum and saliva was correlated with age, the statistical analysis revealed a highly significant result, both in children with SCA and healthy controls [Table 5] and [Table 6] respectively].
|Table 1: Comparative evaluation of TAC levels in serum of healthy controls and sickle cell anemic (case) children|
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|Table 2: Comparative evaluation of TAC levels in saliva of healthy controls and sickle cell anemic children|
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|Table 3: Correlation between TAC levels of serum and saliva in sickle cell anemic (case) children|
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|Table 4: Correlation between TAC levels of serum and saliva in healthy (control) children|
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|Table 5: Correlation of age with TAC levels of serum and saliva in sickle cell anemic children|
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|Table 6: Correlation of age with TAC levels of serum and saliva in healthy (control)|
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| Discussion|| |
ROS have been reported to play a very important role in cell signaling and metabolic processes  and also have been thought to be implicated in the pathogenesis of a variety of inflammatory disorders. Currently, research has revealed potential applications of antioxidant/FR manipulations in prevention or control of diseases.
Congenital hemoglobin mutations may alter the delicate balance of free-radical generation and antioxidant defense by virtue of a shift in favor of the former, giving rise to an oxidative stress within the tissues. The limited availability of disease specific biomarkers of oxidative stress has made an evaluation of the oxidant-antioxidant imbalance problematic. Earlier studies have measured isolated antioxidants to know the status of the systemic condition. However, estimation of individual antioxidant activity may be misleading and less representative of the whole antioxidant status, since FRs and antioxidant systems appear to work in concert through chain-breaking reactions rather than act alone. Therefore, assessment of the TAC may provide more relevant biological information.,,
There have been very few published studies in the literature investigating the antioxidant capacity of fluids local to the oral cavity. Based on these preliminary observations, we hypothesize that differences in total antioxidant levels exist between SCA patients and healthy controls and that reduced TAC may be a feature of both local and peripheral extracellular fluids in patients with SCA. Therefore, the aim of this study was to examine both local (saliva) and peripheral (serum) TAC, in subjects with SCA and age- and gender-matched healthy controls.
Several methods have been reported for measuring the TAC of biological fluids, however, no single assay can be considered as a TAC assay even though it can be performed in an aqueous as well as in a lipophilic environment. Therefore, the phosphomolybdenum method which is a quantitative assay was used for the determination of TAC in the present study. It has been reported that TAC is higher in unstimulated saliva as compared to stimulated saliva; therefore, in the present study, determination of antioxidant capacity was done using unstimulated saliva.
Saliva is considered functionally equivalent to serum. Although the blood is the gold standard for doing many medical tests, changes in serum have been reported to be reflected equally in saliva. Therefore, the salivary evaluation could serve as an alternative. In a study done by Sayedda et al., 2012, it has been stated that both the serum and salivary TAC levels could serve as markers for assessing the severity of disease process but compared to serum the salivary analysis is noninvasive and easier to perform.
In the present study, a significant correlation was found between the TAC of serum and saliva in SCA as well as the control group. Therefore, salivary estimation of the TAC may serve as an independent predictor of vaso-occlusive episodes in SCA patients which are generally noted with a reduced level of TAC.
In the present study, the TAC in serum was decreased in children with SCA compared to healthy children. The decrease in TAC could be attributed to the enhanced ROS formation in SCA which forms a very stable structure by extracting electrons from other sources including enzymatic and nonenzymatic antioxidants. Results observed in the present study are in agreement with the study done by Hundekar and Suryakar who found reduced levels of TAC in SCA patients compared to healthy individuals. This finding further emphasizes the role of oxidative stress in the pathophysiology of SCA and any intervention aimed at increasing the antioxidant capacity of these patients may be beneficial. In contrary, studies have reported increased levels of superoxide dismutase in homozygous sickle cell anemia patients. This has been reported to be a part of a defense mechanism causing increased production of hydroxyl radicals in response to increased oxidative stress in the presence of iron.
Salivary estimation revealed depleted TAC levels in the present study, similar to the findings in serum. Since saliva possesses a wide range of antioxidants, it may form the first line of defense against FR-mediated oxidative stress in SCA. This could account for the enhanced utilization of antioxidants and could lead to a reduction of TAC levels in saliva. Decreased TAC levels have also been reported in studies done in immune-compromised states like HIV, and respiratory conditions like asthma.,
The antioxidant capacity has been stated to be related to the intake of dietary antioxidants, and may alter as a function of age. In the present study, the TAC was found to increase in older children with SCA. Even though the study involved SCA patients from low socioeconomic status; statistical tests revealed a positive correlation between the TAC values and advancing age. This may be related to a dietary shift from semisolid to solid food which may contain larger volumes of antioxidants in the form of micronutrients with increasing age. Similar findings were also seen in healthy children. In addition to the dietary changes, with advancing age absence of an infectious challenge  and good immunity may also be some of the other reasons for the increased TAC levels seen in healthy children.
| Conclusions|| |
The TAC was found to be reduced in children with SCA and a significant correlation between the TAC of serum and saliva indicates that changes in serum may be reflected equally in saliva. Therefore, assessment of antioxidant status in the saliva of SCA patients could serve as a noninvasive alternative to that in serum. In the present study, depleted antioxidant defense mechanisms may account for the reduced TAC in SCA.
Saliva, a noninvasive biomarker can be used as an alternative for assessing the antioxidant status in sickle cell anemic patients. Antioxidants supplements in the form of diet substances rich in beta-carotene, Vitamin C and Vitamin E such as carrots, corn, green peppers, broccoli, brussels sprouts, cauliflower, and turnip greens, should be recommended more and more in sickle cell anemic patients to reduce the accumulation of FRs.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Bishop K, Briggs P, Kelleher M. Sickle cell disease: A diagnostic dilemma. Int Endod J 1995;28:297-302.
Cherry-Peppers G, Davis V, Atkinson JC. Sickle-cell anemia: A case report and literature review. Clin Prev Dent 1992;14:5-9.
Kelleher M, Bishop K, Briggs P. Oral complications associated with sickle cell anemia: A review and case report. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;82:225-8.
Andrews CH, England MC Jr., Kemp WB. Sickle cell anemia: An etiological factor in pulpal necrosis. J Endod 1983;9:249-52.
Patton LL, Brahim JS, Travis WD. Mandibular osteomyelitis in a patient with sickle cell anemia: Report of case. J Am Dent Assoc 1990;121:602-4.
Shroyer JV 3rd
, Lew D, Abreo F, Unhold GP. Osteomyelitis of the mandible as a result of sickle cell disease. Report and literature review. Oral Surg Oral Med Oral Pathol 1991;72:25-8.
Hundekar P, Suryakar A. Antioxidant status and lipid peroxidation in sickle cell anaemia. Biomed Res 2010;21:461-4.
Dhumne UL, Jawade AA. Sickle cell anemia and morbidity in rural population of Chandrapur District, Maharashtra, India. Anthropologist 2011;13:61-3.
Bankar MP, Kate SL, Mokashi GD, Phadke MA. Distribution of sickle cell haemoglobin amongst different tribal groups in Maharashtra. Indian J Haematol 1984;2:4-224.
Shukla RN, Solanki BR. Sickle-cell trait in Central India. Lancet 1958;1:297-8.
Deshmukh P, Garg BS, Garg N, Prajapati NC, Bharambe MS. Prevalence of sickle cell disorders in rural Wardha. Indian J Community Med 2006;31:26-7. [Full text]
Chapple IL. Reactive oxygen species and antioxidants in inflammatory diseases. J Clin Periodontol 1997;24:287-96.
Jiya NM, Matazu KK, Bilbis LS, Shehu A. Serum levels of antioxidant vitamins in foetal haemoglobin (Hbf) persistant sickle cell anaemia children in Sokoto, Nigeria. Ann Afr Med 2005;4:168-71.
Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of Vitamin E. Anal Biochem 1999;269:337-41.
Bogdan C, Röllinghoff M, Diefenbach A. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 2000;12:64-76.
Davies KJ. Oxidative stress: The paradox of aerobic life. Biochem Soc Symp 1995;61:1-31.
Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, Lele RD. Free radicals and antioxidants in human health: Current status and future prospects. J Assoc Physicians India 2004;52:794-804.
Gopinath VK, Arzreanne AR. Saliva as a diagnostic tool for assessment of dental caries. Arch Orofac Sci 2006;1:57-9.
Uberos J, Alarcon JA, Penalver MA. Influence of the antioxidant content of saliva on dental caries in an at risk community. Br Dent J 2008;5:205.
Battino M, Ferreiro MS, Gallardo I, Newman HN, Bullon P. The antioxidant capacity of saliva. J Clin Periodontol 2002;29:189-94.
Cao G, Prior RL. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin Chem 1998;44(6 Pt 1):1309-15.
Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 2005;53:4290-302.
Pereslegina IA. The activity of antioxidant enzymes in the saliva of normal children. Lab Delo 1989:20-3.
Kumar D, Pandey RK, Agrawal D, Agrawal D. An estimation and evaluation of total antioxidant capacity of saliva in children with severe early childhood caries. Int J Paediatr Dent 2011;21:459-64.
Sayedda K, Ahmed QS, Kamal S. Comparison of serum and salivary total antioxidant activity in unstable angina pectoris patients. J Phys Pharm Adv 2012;2:195-201.
Agarwal MB, Mehta BC. Sickle-syndromes: A study of 44 cases from Bombay. Indian Pediatr 1980;17:793-6.
Das SK, Nair RC. Superoxide dismutase, glutathione peroxidase, catalase, catalase and lipid peroxide of normal and sickled erythrocytes. Br J Haematol 1980;44:87-92.
Halliwell B. Reactive oxygen species in living systems: Source, biochemistry, and role in human disease. Am J Med 1991;91:14S-22S.
Padmanabhan V, Rai K, Hegde AM, Shetty S. Total antioxidant capacity of saliva in children with HIV. J Clin Pediatr Dent 2010;34:347-50.
Hegde AM, Raj K, Shetty S. Relation of caries status on the salivary total antioxidant levels in asthmatic children. Contemp Clin Dent 2012;3:402-5.
] [Full text]
Cao G, Booth SL, Sadowski JA, Prior RL. Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables. Am J Clin Nutr 1998;68:1081-7.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]