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Original Article
Estimation of malondialdehyde levels in serum and
saliva of children affected with sickle cell anemia
Sudhindra Baliga, Minal Chaudhary1, Sham Bhat2, Pooja Bhansali, Akshat Agrawal, Satwik Gundawar
Departments of Pedodontics and Preventive Dentistry and 1Oral Pathology and Microbiology, Sharad Pawar Dental College,
Sawangi (M), Wardha, Maharashtra, 2Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College, Mangalore,
Karnataka, India
ABSTRACT
Background: Sickle cell anemia (SCA) is an inherited
disorder of hemoglobin synthesis characterized by
deformed erythrocytes. Hemoglobin S present in
sickle‑shaped erythrocytes exhibits an enhanced
rate of auto‑oxidation compared with normal
hemoglobin A. It produces more of reactive oxygen
species  (ROS) which promotes oxidatively stressed
environment. ROS degrade the membranes of sickle
cell erythrocytes composed of polyunsaturated
lipids and form malondialdehyde  (MDA) as a
by‑product. Aim: The aim of the study is to evaluate
and compare the MDA levels of serum and saliva
in SCA patients. Design: A  total of 150 children
aged 4–12  years were divided into two groups:
Group  A  (n  =  75) consisting of children suffering
from SCA and Group B (n = 75) consisting of healthy
children. Blood and saliva samples were collected
aseptically from both the groups, and they were
subjected to thiobarbituric acid assay. Absorbance
was evaluated spectrophotometrically at 531 nm, and
the values of concentration of MDA were derived.
Results: The mean MDA levels in serum and saliva
were 8.9825  ±  1.04 and 0.5152  ±  0.28, respectively,
in Group  A and they were found to be higher
than mean MDA levels of serum  (5.87  ±  0.92) and
saliva (0.2929 ± 0.06) of Group B and the difference
of their mean was found to be statistically significant.
Conclusion: A significant correlation of the MDA was
found in saliva and serum of the patients with SCA.
This finding suggests that saliva can be effectively
used as a noninvasive alternative for assessing the
oxidative stress in patients with SCA.
KEYWORDS: Malondialdehyde, saliva, serum, sickle
cell anemia
Introduction
Sickle cell anemia  (SCA) is an inherited disorder
of hemoglobin synthesis that is associated with a
© 2018 Journal of Indian Society of Pedodontics and Preventive Dentistry | Published by Wolters Kluwer - Medknow
Address for correspondence:
Dr. Sudhindra Baliga,
Department of Pedodontics and Preventive Dentistry,
Sharad Pawar Dental College, Sawangi (M),
Wardha, Maharashtra, India.
E‑mail: baligams@hotmail.com
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DOI:
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significant morbidity and mortality due to its sequelae
leading to episodic vaso‑occlusive events, pain crises,
and multi‑organ damage. It has been traced to a single
point mutation that substitutes valine for glutamic
acid in the β‑globin subunit. Normal erythrocytes
incorporate hemoglobin A  (HbA) and are biconcave,
in contrast to this, the erythrocytes of SCA patients
incorporate hemoglobin S and are sickle shaped.
HbS exhibits an enhanced rate of auto‑oxidation
in comparison to HbA in the presence of reactive
oxygen species  (ROS), namely, superoxide, peroxide,
and hydroxyl radical. Under normal physiological
conditions, antioxidant enzymes and oxygen radical
scavengers inhibit basal fluxes of ROS. However, when
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For reprints contact: reprints@medknow.com
How to cite this article: Baliga S, Chaudhary M, Bhat S,
Bhansali P, Agrawal A, Gundawar S. Estimation of malondialdehyde
levels in serum and saliva of children affected with sickle cell
anemia. J Indian Soc Pedod Prev Dent 2018;36:43-7.
43
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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
antioxidant defense mechanism, it results in an
oxidatively stressed environment. Children affected
with SCA show an impaired antioxidant status due to
reduced antioxidant defenses.
Oxidative stress has been related to the etiopathogenesis
of several chronic diseases.[1]
It can damage specific molecular targets such as
lipids, proteins, and carbohydrates resulting in cell
dysfunction and/or cell death. However, lipids are
the most commonly affected class of biomolecules
and its oxidation gives rise to a number of secondary
products. Membranes of sickle‑shaped erythrocytes
are high in polyunsaturated fatty acids which are
more susceptible to endogenous free radical‑mediated
oxidative damage. Thus, it affects the hemostatic
environment. ROS degrade polyunsaturated lipids,
forming malondialdehyde  (MDA) as a by‑product
which is said to be the biomarker of increased oxidative
stress.
MDA is an end product of the radical‑initiated
oxidative decomposition of polyunsaturated fatty
acids, and therefore, it is a frequently measured
biomarker of oxidative stress.[2] Saliva is a natural body
fluid, which is acclaimed as the first defense system of
the body and any hormonal, nutritional, and metabolic
disturbances that occur in serum is equally reflected
in saliva.[3] Noninvasive and safe methods of salivary
sample collection, the possibility of repeated sampling,
and longitudinal monitoring have all made salivary
analysis more lucrative.[4] Estimation of oxidative stress
is an essential part of routine blood investigations,
which are employed for monitoring health and disease.
There is a lack of literature which estimates oxidative
stress using salivary MDA, especially in patients with
SCA. Therefore, the present study was carried out to
evaluate and correlate the MDA levels in serum and
saliva in children with SCA.
Materials and Methods
A total of 150 children in the age group of 4–12 years
participated in the study which consisted of two
groups. Group  A  (n  =  75) included children who
were randomly selected from the patients attending
Sickle Anemia Clinic in the Department of Pediatrics
at Acharya Vinoba Bhave Rural Hospital, Sawangi,
whereas Group  B  (n  =  75) consisted of healthy
Table 1: Comparative evaluation of malondialdehyde levels in serum of healthy controls and sickle cell
anemic (case) children
Groups n Mean±SD
Saliva MDA
Case 75 0.5152±0.28195
Control 75 0.2929±0.06166
*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 |
controls. Children with any other systemic diseases,
immunocompromised states, or having any history of
vaso‑occlusive crisis in the past 3 months, who had a
blood transfusion or any serious illness, were excluded
from the study. The study protocol first approved by
the Institutional Ethical Committee, Datta Meghe
Institute of Medical Sciences, Sawangi, Wardha, India.
A  written informed consent was obtained from the
parents/guardians of all the children. The biochemical
analysis for the study was conducted in the Central
Research Laboratory, Datta Meghe Institute of Medical
Sciences.
Collection of saliva and serum samples
To minimize diurnal variations, all the samples were
collected in the morning. The study participants were
instructed not to eat or drink anything except water
for at least 2 h before the sample collection. For the
collection of the salivary sample, patients were asked
to sit comfortably with head tilted down, and the
2  ml of saliva pooled on the floor of the mouth was
then collected by asking the patient to spit into sterile
plastic tubes.
To obtain serum samples, 2 ml of blood was drawn from
the cubital vein of the left arm with a 24‑gauge needle.
Blood was then transferred to a plain sterile bulb which
was immediate. 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 MDA levels of saliva and serum was done
using thiobarbituric acid (TBA) assay method as given
by Satoh.[5] The TBA reacts with MDA giving rise to a
high absorptivity adduct which can be easily assessed
with a spectrophotometer at 531 nm. A standard graph
was plotted, and concentration of MDA was expressed
as nmol/ml. Mean and standard deviation of the
measurements obtained was calculated.
Results
The values of MDA evaluated in both the study groups
in saliva as well as serum are shown in Table 1. While
the levels of MDA in serum were compared between
children with SCA and healthy controls using the
Student’s unpaired t‑test, the result was statistically
significant (P  <  0.05). However, when the levels of
MDA in saliva were compared between the two
groups using the Student’s unpaired t‑test, the results
were found to be nonsignificant  (P  <  0.05)  [Table  2].
SEM Mean difference t P
0.03256 0.22227 6.669 0.001*
0.00712
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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
alternative.[11]
In the present study, the MDA levels in serum were
found to be increased in children with SCA compared
to healthy children. The increase in levels of MDA
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.[13] Increased serum
levels of MDA have been reported in various systemic
conditions such as diabetes mellitus, malignancies
of the stomach, breast, cervix, and premalignant
lesions, and conditions such as leukoplakia and
oral submucous fibrosis[14-21] including SCA.[22]
The results of the present study are in accordance
with the observation of above studies. 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.[1]
Estimation of MDA levels in saliva of children with
SCA showed its elevated levels while it was within
the normal range in the healthy controls, and these
findings were in accordance to the serum MDA levels
obtained in the present study. Since saliva possesses
a wide range of antioxidants,[23] it may form the first
line of defense against free radical‑mediated oxidative
stress in SCA. The presence of MDA levels higher than
normal in SCA patients may be due to oxidatively
stressed environment which also suggests enhanced
utilization of antioxidants and leading to a reduction
of total antioxidant capacity in saliva.
The antioxidant capacity has been stated to be related
to the intake of dietary antioxidants[14] and may alter
as a function of age.[23] In the present study, the MDA
was found to decrease in older children with SCA.
Even though the study involved SCA patients from
low socioeconomic status, statistical tests revealed
a positive correlation between the MDA values and
advancing age.[24,25] 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, the absence of an infectious
challenge[14] and good immunity may also be some of
the other reasons for the decreased MDA levels seen in
healthy children.[26,27]
Conclusion
The MDA was found to be increased in children with
SCA, and a significant correlation between the MDA of
serum and saliva indicates that changes in serum may
be reflected equally in saliva. Therefore, assessment
of MDA in the saliva of SCA patients could serve as a
noninvasive alternative to that in serum. In the present
46 Journal of Indian Society of Pedodontics and Preventive Dentistry | Volume 36 | Issue 1 | January-March 2018 |
study, increased oxidative stress may account for
raised MDA level which serves as a biomarker.
Recommendations
Saliva, a noninvasive biomarker, can be used as an
alternative for assessing the oxidative stress in SCA
patients. Antioxidants’ supplements in the form of
dietary 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 SCA
patients to reduce the accumulation of free radicals.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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