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ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 1  |  Page : 43-47
 

Estimation of malondialdehyde levels in serum and saliva of children affected with sickle cell anemia


1 Department of Pedodontics and Preventive Dentistry, Sharad Pawar Dental College, Sawangi (M), Wardha, Maharashtra, India
2 Department of Oral Pathology and Microbiology, Sharad Pawar Dental College, Sawangi (M), Wardha, Maharashtra, India
3 Department of Pedodontics and Preventive Dentistry, Yenepoya Dental College, Mangalore, Karnataka, India

Date of Web Publication28-Mar-2018

Correspondence Address:
Dr. Sudhindra Baliga
Department of Pedodontics and Preventive Dentistry, Sharad Pawar Dental College, Sawangi (M), Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_87_17

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   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


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

How to cite this URL:
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 [serial online] 2018 [cited 2019 Nov 18];36:43-7. Available from: http://www.jisppd.com/text.asp?2018/36/1/43/228754





   Introduction Top


Sickle cell anemia (SCA) is an inherited disorder of hemoglobin synthesis that is associated with a 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 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 Top


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 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 Top


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]. The correlation of MDA levels in serum and saliva of children with SCA is shown in [Table 3]. Results, when compared using Pearson's correlation coefficient, were found to be statistically significant (P< 0.05). Similarly, the correlation of the MDA between serum and saliva of healthy children is shown in [Table 4]. On comparison, the MDA between saliva and serum using Pearson's 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 revealed a nonsignificant result in children with SCA while significant result in healthy controls (P< 0.05) [Table 5] and [Table 6].
Table 1: Comparative evaluation of malondialdehyde levels in serum of healthy controls and sickle cell anemic (case) children

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Table 2: Comparative evaluation of malondialdehyde levels in saliva of healthy controls and sickle cell anemic children

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Table 3: Correlation between malondialdehyde levels of serum and saliva in sickle cell anemic (case) children

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Table 4: Correlation between malondialdehyde levels of serum and saliva in healthy (control) children

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Table 5: Correlation of age with malondialdehyde levels of serum and saliva in sickle cell anemic (case) children

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Table 6: Correlation of age with malondialdehyde levels of serum and saliva in healthy (control) children

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   Discussion Top


Oxidative stress has been related to the etiopathogenesis of several chronic diseases.[6] ROS have been reported to play a very important role in cell signaling and metabolic processes [7] and also have been thought to be implicated in the pathogenesis of a variety of inflammatory disorders. Polyunsaturated fatty acids are the most commonly involved biological targets of oxidative stress providing MDA as a by-product on peroxidation.[8] MDA is able to impair several 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 as something more than a lipid peroxidation product. MDA is assessed to quantify the level of oxidative stress in vivo and in vitro using several methods. In the past 20 years, MDA has been recognized as a relevant lipid peroxidation marker, and as such, the measurement of MDA levels in biological samples from participants affected by several diseases has been widely utilized. Recent research has revealed potential applications of antioxidant/free radical manipulations in prevention or control of diseases.[9]

There have been very few studies investigating the MDA levels of saliva. Based on these preliminary observations, we hypothesize that differences in MDA exist between SCA patients and healthy controls and that increased MDA may be a feature of both local and peripheral extracellular fluids in patients with SCA. Therefore, the study evaluated both local (saliva) and peripheral (serum) levels of MDA in participants with SCA and healthy controls.

Several methods have been reported for measuring the MDA of biological fluids;[10] however, no single assay can be considered as an ideal assay even though it can be performed in an aqueous as well as in a lipophilic environment.[11] Therefore, the TBA method which is a quantitative assay was used for the determination of MDA in the present study. It has been reported that MDA is higher in unstimulated saliva as compared to stimulated saliva; therefore, in the present study, determination of MDA was done using unstimulated saliva.[12]

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.[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],[15],[16],[17],[18],[19],[20],[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 Top


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 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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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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