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Original Article
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Baliga, et al.: MDA levels in serum and saliva of sickle cell anemic children
the production of ROS overwhelms the endogenous controls. Children with any other systemic diseases,
antioxidant defense mechanism, it results in an immunocompromised states, or having any history of
oxidatively stressed environment. Children affected vaso‑occlusive crisis in the past 3 months, who had a
with SCA show an impaired antioxidant status due to blood transfusion or any serious illness, were excluded
reduced antioxidant defenses. from the study. The study protocol first approved by
the Institutional Ethical Committee, Datta Meghe
Oxidative stress has been related to the etiopathogenesis Institute of Medical Sciences, Sawangi, Wardha, India.
of several chronic diseases.[1] A written informed consent was obtained from the
parents/guardians of all the children. The biochemical
It can damage specific molecular targets such as analysis for the study was conducted in the Central
lipids, proteins, and carbohydrates resulting in cell Research Laboratory, Datta Meghe Institute of Medical
dysfunction and/or cell death. However, lipids are Sciences.
the most commonly affected class of biomolecules
and its oxidation gives rise to a number of secondary Collection of saliva and serum samples
products. Membranes of sickle‑shaped erythrocytes To minimize diurnal variations, all the samples were
are high in polyunsaturated fatty acids which are collected in the morning. The study participants were
more susceptible to endogenous free radical‑mediated instructed not to eat or drink anything except water
oxidative damage. Thus, it affects the hemostatic for at least 2 h before the sample collection. For the
environment. ROS degrade polyunsaturated lipids, collection of the salivary sample, patients were asked
forming malondialdehyde (MDA) as a by‑product to sit comfortably with head tilted down, and the
which is said to be the biomarker of increased oxidative 2 ml of saliva pooled on the floor of the mouth was
stress. then collected by asking the patient to spit into sterile
plastic tubes.
MDA is an end product of the radical‑initiated
oxidative decomposition of polyunsaturated fatty To obtain serum samples, 2 ml of blood was drawn from
acids, and therefore, it is a frequently measured the cubital vein of the left arm with a 24‑gauge needle.
biomarker of oxidative stress.[2] Saliva is a natural body Blood was then transferred to a plain sterile bulb which
fluid, which is acclaimed as the first defense system of was immediate. The supernatant was removed and
the body and any hormonal, nutritional, and metabolic centrifuged at 3000 rpm for 4–5 min. Serum obtained
disturbances that occur in serum is equally reflected was then stored at − 20°C for subsequent analysis.
in saliva.[3] Noninvasive and safe methods of salivary
sample collection, the possibility of repeated sampling, Estimation of MDA levels of saliva and serum was done
and longitudinal monitoring have all made salivary using thiobarbituric acid (TBA) assay method as given
analysis more lucrative.[4] Estimation of oxidative stress by Satoh.[5] The TBA reacts with MDA giving rise to a
is an essential part of routine blood investigations, high absorptivity adduct which can be easily assessed
which are employed for monitoring health and disease. with a spectrophotometer at 531 nm. A standard graph
There is a lack of literature which estimates oxidative was plotted, and concentration of MDA was expressed
stress using salivary MDA, especially in patients with as nmol/ml. Mean and standard deviation of the
SCA. Therefore, the present study was carried out to measurements obtained was calculated.
evaluate and correlate the MDA levels in serum and
saliva in children with SCA. Results
Materials and Methods The values of MDA evaluated in both the study groups
in saliva as well as serum are shown in Table 1. While
A total of 150 children in the age group of 4–12 years the levels of MDA in serum were compared between
participated in the study which consisted of two children with SCA and healthy controls using the
groups. Group A (n = 75) included children who Student’s unpaired t‑test, the result was statistically
were randomly selected from the patients attending significant (P < 0.05). However, when the levels of
Sickle Anemia Clinic in the Department of Pediatrics MDA in saliva were compared between the two
at Acharya Vinoba Bhave Rural Hospital, Sawangi, groups using the Student’s unpaired t‑test, the results
whereas Group B (n = 75) consisted of healthy were found to be nonsignificant (P < 0.05) [Table 2].
Table 1: Comparative evaluation of malondialdehyde levels in serum of healthy controls and sickle cell
anemic (case) children
Groups n Mean±SD SEM Mean difference t P
Saliva MDA
Case 75 0.5152±0.28195 0.03256 0.22227 6.669 0.001*
Control 75 0.2929±0.06166 0.00712
*P<0.05, significant. MDA=Malondialdehyde; SD=Standard deviation; SEM=Standard error of mean
44 Journal of Indian Society of Pedodontics and Preventive Dentistry | Volume 36 | Issue 1 | January-March 2018 |
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Baliga, et al.: MDA levels in serum and saliva of sickle cell anemic children
Table 2: Comparative evaluation of malondialdehyde levels in saliva of healthy controls and sickle cell
anemic children
Groups n Mean ± SD SEM Mean difference t P
Serum MDA
Case 75 8.9825±1.04329 0.12047 3.10587 19.288 0.001*
Control 75 5.8767±0.92536 0.10685
*P<0.05, significant. MDA=Malondialdehyde; SD=Standard deviation; SEM=Standard error of mean
The correlation of MDA levels in serum and saliva of Table 3: Correlation between malondialdehyde
children with SCA is shown in Table 3. Results, when levels of serum and saliva in sickle cell
compared using Pearson’s correlation coefficient, were anemic (case) children
found to be statistically significant (P < 0.05). Similarly, Mean±SD n Correlation (r) P
the correlation of the MDA between serum and saliva
Serum level 8.9825±1.04329 75 −0.002 0.98
of healthy children is shown in Table 4. On comparison,
Saliva level 0.5152±0.28195 75
the MDA between saliva and serum using Pearson’s
SD=Standard deviation
correlation coefficient showed a highly significant
result (P < 0.05). When the levels of MDA in serum and
saliva were correlated with age, the statistical analysis Table 4: Correlation between malondialdehyde
revealed a nonsignificant result in children with SCA levels of serum and saliva in healthy (control)
while significant result in healthy controls (P < 0.05) children
[Tables 5 and 6]. Mean±SD n Correlation (r) P
Serum level 5.8767±0.92536 75 0.323 0.005*
Discussion Saliva level 0.2929±0.06166 75
*P<0.05, significant. SD=Standard deviation
Oxidative stress has been related to the etiopathogenesis
of several chronic diseases.[6] ROS have been reported Table 5: Correlation of age with malondialdehyde
to play a very important role in cell signaling and levels of serum and saliva in sickle cell
metabolic processes[7] and also have been thought anemic (case) children
to be implicated in the pathogenesis of a variety of
Mean±SD n Correlation (r) P
inflammatory disorders. Polyunsaturated fatty acids
Age (years) 8.3±3.27 75
are the most commonly involved biological targets
of oxidative stress providing MDA as a by‑product Serum level 8.9825±1.04329 75 −0.018 0.877
on peroxidation.[8] MDA is able to impair several Saliva level 0.5152±0.28195 75 0.080 0.494
SD=Standard deviation
physiological mechanisms of the human body through
its ability to react with molecules such as DNA and
proteins. It is, therefore, useful to consider this molecule Table 6: Correlation of age with malondialdehyde
as something more than a lipid peroxidation product. levels of serum and saliva in healthy (control)
MDA is assessed to quantify the level of oxidative stress children
in vivo and in vitro using several methods. In the past Mean±SD n Correlation (r) P
20 years, MDA has been recognized as a relevant lipid Age (years) 8.17±3.05 75
peroxidation marker, and as such, the measurement
Serum level 5.8767±0.92536 75 0.619 0.001*
of MDA levels in biological samples from participants
Saliva level 0.2929±0.06166 75 0.313 0.006*
affected by several diseases has been widely utilized.
*P<0.05, significant. SD=Standard deviation
Recent research has revealed potential applications of
antioxidant/free radical manipulations in prevention
or control of diseases.[9] can be considered as an ideal assay even though it can
be performed in an aqueous as well as in a lipophilic
There have been very few studies investigating the environment.[11] Therefore, the TBA method which is a
MDA levels of saliva. Based on these preliminary quantitative assay was used for the determination of
observations, we hypothesize that differences in MDA MDA in the present study. It has been reported that
exist between SCA patients and healthy controls and MDA is higher in unstimulated saliva as compared
that increased MDA may be a feature of both local and to stimulated saliva; therefore, in the present study,
peripheral extracellular fluids in patients with SCA. determination of MDA was done using unstimulated
Therefore, the study evaluated both local (saliva) and saliva.[12]
peripheral (serum) levels of MDA in participants with
SCA and healthy controls. Saliva is considered functionally equivalent to
serum. Although the blood is the gold standard
Several methods have been reported for measuring the for doing many medical tests, changes in serum
MDA of biological fluids;[10] however, no single assay have been reported to be reflected equally in saliva.
Journal of Indian Society of Pedodontics and Preventive Dentistry | Volume 36 | Issue 1 | January-March 2018 | 45
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Baliga, et al.: MDA levels in serum and saliva of sickle cell anemic children
Therefore, the salivary evaluation could serve as an study, increased oxidative stress may account for
alternative.[11] raised MDA level which serves as a biomarker.
46 Journal of Indian Society of Pedodontics and Preventive Dentistry | Volume 36 | Issue 1 | January-March 2018 |
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Baliga, et al.: MDA levels in serum and saliva of sickle cell anemic children
and its relationship with antioxidant enzymes in saliva of serum malondialdehyde levels in patients of cerebrovascular
periodontitis patients. Eur J Dent 2009;3:100‑6. accident. Indian Acad Clin Med 2005;6:229-31.
12. Elias DB, Freitas RM, Gonçalves RP, Magalhães HY, 20. Bitla AR, Reddy P, Sambasivaih K, Suchitra MM, Reddy SV,
Sousa JH, Magalhães SM. Evaluation of the concentration Rao S. Evaluation of plasma malondialdehyde as a biomarker in
of malondialdehyde and nitrite in patients with sickle cell patients with carcinoma of stomach. Biomed Res 2011;22:63‑8.
anemia treated or not with hydroxyurea. Einstein (Sao Paulo) 21. Kaur J, Politis C, Jacobs R. Salivary
2010;8:414‑8. 8‑hydroxy‑2‑deoxyguanosine, malondialdehyde, Vitamin
13. Khalili J, Biloklytska HF. Salivary malondialdehyde levels in C, and Vitamin E in oral pre‑cancer and cancer: Diagnostic
clinically healthy and periodontal diseased individuals. Oral value and free radical mechanism of action. Clin Oral Investig
Dis 2008;14:754‑60. 2016;20:315‑9.
14. Gerritsen WB, van Boven WJ, Boss DS, Haas FJ, van 22. Gupta VK, Singh N, Nigam P , Shah J, Patil SKB. Status of
Dongen EP, Aarts LP. Malondialdehyde in plasma, a biomarker antioxidants vitamin and plasma malondialdehyde (MDA)
of global oxidative stress during mini‑CABG compared to in sickle cell anemia patients of Chhattisgarh region. Int J
on‑ and off‑pump CABG surgery: A pilot study. Interact
Contemp Med 2014; 2: 62-5.
Cardiovasc Thorac Surg 2006;5:27‑31.
23. Rai B, Kharb S, Jain R, Anand SC. Salivary lipid peroxidation
15. Celec P, Hodosy J, Celecová V, Vodrázka J, Cervenka T,
product malonaldehyde in various dental diseases. World J
Halcák L, et al. Salivary thiobarbituric acid reacting substances
Med Sci 2006;1:100‑1.
and malondialdehyde – Their relationship to reported smoking
24. Nielsen F, Mikkelsen BB, Nielsen JB, Andersen HR, Grandjean
and to parodontal status described by the papillary bleeding
index. Dis Markers 2005;21:133‑7. P. Plasma malondialdehyde as a biomarker for oxidative stress:
16. Trivedi S, Lal N, Mahdi AA, Mittal M, Singh B, Reference interval and effects of life‑style factors. Clin Chem
Pandey S. Evaluation of antioxidant enzymes activity and 1997;43:1209‑14.
malondialdehyde levels in patients with chronic periodontitis 25. Tukozkan NA, Erdamar H, Seven I. Measurement of total
and diabetes mellitus. J Periodontol 2014;85:713‑20. malondialdehyde in plasma and tissues by high‑performance
17. Tonguç MÖ, Öztürk O, Sütçü R, Ceyhan BM, Kilinç G, liquid chromatography and thiobarbituric acid assay. Fırat Tıp
Sönmez Y, et al. The impact of smoking status on antioxidant Derg 2006;11:88‑92.
enzyme activity and malondialdehyde levels in chronic 26. Ghallab NA, Hamdy E, Shaker OG. Malondialdehyde,
periodontitis. J Periodontol 2011;82:1320‑8. superoxide dismutase and melatonin levels in GCF of
18. Turki A, Naji M. Measurement of serum malondialdehyde aggressive and chronic periodontitis patients. Aust Dent J
(MDA) levels as a marker of lipid peroxidation in neonatal 2016;61:53-61.
sepsis. Med J 2011;5:9‑17. 27. Weiss SL, Deutschman CS. Elevated malondialdehyde levels
19. Beg M, Ahmad S, Gandhi S, Akhtar N, Ahmad Z. A study of in sepsis – Something to ‘stress’ about? Crit Care 2014;18:125.
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