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Biomedical Research

Antioxidant Status and Lipid Peroxidation in Sickle Cell Anaemia

Author(s): Prakash Hundekar, Adinath Suryakar, Aarti Karnik, Rahul Ghone, Maya Vasaikar

Vol. 21, No. 4 (2010-10 - 2010-12)

Prakash Hundekar, Adinath Suryakar٭٭, Aarti Karnik, Rahul Ghone, Maya Vasaikar٭

Department of Biochemistry, ACPM Medical College, Dhule. ٭Dept. of Pathology, Shri Bhausaheb Hire GMC, Dhule, India, ٭٭Department of Biochemistry, Dr. VM GMC, Solapur, India

Abstract

Sickle cell anemia (SCA) is an inherited disorder of hemoglobin synthesis, characterized by life-long severe hemolytic anemia and vasoocclussive crisis. Oxidative stress play important role in pathophysiology of sickle cell anaemia. Therefore, the study was undertaken to evaluate the levels of plasma lipid peroxidation product malondialdehyde (MDA), plasma total antioxidant capacity, erythrocytic activity of superoxide dismutase and catalase. We found significantly elevated plasma MDA level and activity of erythrocytic superoxide dis-mutase. While plasma total antioxidant capacity and activity of erythrocytic catalase were reduced significantly in SCA patients. These observations provide the evidence of imbalance between oxidant and antioxidant status leading to chronic oxidative stress.

Keywords: Sickle cell anaemia, malondialdehyde, superoxide dismutase, catalase, total antioxidant capacity
Accepted September 23 2010

Introduction

Sickle cell anaemia (SCA) is a congenital haemoglobinopathy characterized by deformed red blood cells, acute episodic attacks of pain and pulmonary compromise, widespread organ damage and early death [1].

The prevalence of SCA is very high in central Africa, Mediterranean region, eastern countries and in certain parts of India. In India, the prevalence of SCA is high in Dhule, Nandurbar, Jalgoan and Nasik districts of Ma-harashtra state [2].

Sickle cell anaemia results from a point mutation in the genetic code such that glutamic acid is replaced by valine at 6th position of β-globin chain of hemoglobin (Hb). This substitution transforms normal adult hemoglobin (HbA) into sickle hemoglobin (HbS). In a low oxygen tension environment, the replaced valine can bind to a comple-mentary hydrophobic site on beta subunit of another he-moglobin tetramer in a polymerization process that leads to the sickling of the red blood cells (RBCs). Polymeriza-tion of deoxygenated sickle hemoglobin (HbS) tetramers is central to the process of vasoocclusion [3]. The poly-mer make the erythrocyte rigid, distort its shape, and cause structural damage in the red-cell membrane, all of which alter the rheologic properties of cell, impair blood flow through the microvasculature and leads to hemolysis and vasoocclussive episodes [4]. Sickle cell spontane-ously generates approximately two times more amount of reactive oxygen species [5]. The reactive oxygen species can attack erythrocytic membrane directly, causing alteration in lipid and protein structure that may ultimately re-sult into hemolysis [6]. Lipid peroxidation has a major role in the pathophysiology of sickle cell anaemia. With this view, we planned to evaluate the level of lipid peroxidation product malondialdehyde (MDA), total antioxi-dant capacity (TAC), erythrocytic activity of superoxide dismutase (SOD) and catalase in homozygous (HbSS) as well as in heterozygous (HbAS) sickle cell anaemia patients.

Material and methods

The present study was carried out in Department of Bio-chemistry, ACPM medical college, Dhule, sickle cell anaemia centre, Bhausaheb Hire Govt. medical college, Dhule and Department of biochemistry, Dr. VM Govt. medical college, Solapur. Local ethical clearance obtained prior the study. A total of 69 subjects were participated in the study, including 17 homozygous SCA (HbSS), 22 heterozygous SCA (HbAS) and 30 healthy controls. Con-trol group is of age, sex matched and hematologically normal subjects. All the participants were included in the study after sickle screen test (solubility test) and HPLC analysis of blood [7]. Subjects were excluded having past 3 month history of vasoocclussive crisis, blood transfu-sion and serious illness.

5 ml of blood was collected aseptically from the antecubi-tal vein in EDTA (0.47 mol/L K3-EDTA) blood con-tainer. The blood was centrifuged at 1000g for 10min to separate the plasma. Plasma was used to analyze lipid peroxidation product malondialdehyde (MDA) according to method of Kei Satho [8] and total antioxidant capacity (TAC) of plasma by FRAP assay described by Benzie IFF [9]. The remaining erythrocytes were washed with phos-phate buffer pH 7.4 to remove buffy coat and Hb conc. was adjusted to10 gms/dl. Tsuchiasi extract was prepared by adding ethanol: chloroform in ratio of 1:0.6 to the clear hemolysate followed by addition of 3.5ml ice cold dis-tilled water. The clear extract was used to assess the activ-ity of superoxide dismutase (SOD) described by Winter-bourn et al [10] and catalase according to method of L Goth [11].

Statistics

Values are expressed as mean ± SD, significance of the mean differences between the groups was assessed by student ‘t’ test. Pearson’s correlation coefficient used for the correlation assessment.

Results

As seen in table-I, plasma MDA level and erythrocytic activity of superoxide dismutase were significantly (p< 0.001) elevated in homozygous as well as heterozygous sickle cell anaemia patients compared to the control group. In contrast, total antioxidant capacity of plasma and erythrocytic activity of catalase were significantly (p< 0.001) lowered in both homozygous and heterozygous sickle cell anaemia patients as compared to control group. We found a positive correlation (r= 0.54) between plasma MDA and erythrocytic superoxide dismutase activity.

Table I. Level of plasma malondialdehyde (MDA), total antioxidant capacity (TAC) of plasma, erythrocytic activity of superoxide dismutase (SOD) and catalase in sickle cell patients and healthy controls.

S. No. Parameters Controls (HbAA) (n=30) Mean ± SD Sickle cell Heterozygous (HbAS) (n=22) Mean ± SD Sickle cell Homozygous(HbSS) (n=17) Mean ± SD
1 MDA n moles/ml 0.84 ± 0.32 2.58 ± 0.49٭ 4.13 ± 0.48٭#
2 TAC m mol/L 2.11 ± 0.32 0.71 ± 0.16٭ 0.46 ± 0.19٭#
3 SOD U/gm Hb 1307 ± 119 2256 ± 419٭ 3403 ± 775٭#
4 Catalase KU/gm Hb 144.79 ± 17.84 82.61 ± 10.43٭ 64.79 ± 10.16٭#
Note:٭ – significance p<0.001 control Vs sickle cell heterozygous (HbAS),control Vs sickle cell homozygous (HbSS)
  1. - significance p<0.001 sickle cell heterozygous (HbAS) Vs sickle cell homozygous (HbSS)

Discussion

SCA is a hereditary disorder with higher potential for oxidative damage due to chronic redox imbalance in red cells that often results in clinical manifestation of mild to severe hemolysis in patients [12].

Hebbel and colleagues [5] have shown that, under ambi-ent oxygen tensions, sickle cells spontaneously generate O2•¯, H2O2 and OH• approximately two times more, when compared to normal RBCs. Furthermore, these workers have also demonstrated that hemichrome may facilitate OH• production in the presence of O2•¯ and H2O2.

In our study, the plasma MDA level is significantly ele-vated that agrees the previous findings [13,14]. Accumu-lation of malondialdehyde disturbs the organization of phospholipids in the human erythrocyte membrane bi-layer. Membrane damage considered as an important fac-tor contributing towards pathophysiology due to the for-mation of irreversible sickle cells (ISC) [15]. Peroxidative reactions have long been recognized as potential factors that contribute to degenerative cellular processes. RBCs are particularly susceptible to peroxidative damage be-cause they contain hemoglobin, one of the most powerful catalysts for initiation of peroxidative reaction [16]. It is also stated that excess quantity of malondialdehyde can promote erythrophagocytosis [17].

Several authors have reported about the alteration in anti-oxidant defense system in SCA. We have seen signifi-cantly elevated activity of superoxide dismutase and low-ered activity of catalase in erythrocytes which is consis-tent with previous studies [14]. The raised activity of SOD is might be due to excessive production of reactive oxygen species. It is to be noted that an increased SOD activity may build up of H2O2 in cells which subsequently removed by the peroxidases of erythrocytes. Das K and Nair [14] have observed lower glutathione peroxidase and catalase activities in erythrocytes. Thus, an excessive H2O2 in such instance may precipitate the hemoglobin as Heinz bodies and peroxidise the unsaturated lipids of erythrocyte membrane [18].

In addition, we also noted a significant (p<0.001) reduc-tion in the total antioxidant capacity of plasma in SCA patients, compared to the control group, supporting the previous studies [19,20]. TAC reflects the collective con-tribution to the reducing property of non protein individ-ual antioxidant or electron donating components. TAC values are more informative than the knowledge of indi-vidual antioxidant. More number of vasoocclussive epi-sodes are seen in patients with lowest TAC level [20]. That indicates the depletion in low molecular weight anti-oxidants, may be conducive for vasoocclussive crisis. Various studies have shown the depleted levels of non-enzyme antioxidant molecules such as carotene, vitamin E, vitamin C and trace elements contributing antioxidant activity [21,22,23]. Previous, studies have been reported low levels of zinc in SCA patients [24,25]. Deficiency of zinc is associated with impaired cell mediated immunity and growth retardation, low antioxidant status, increased rate of infection, vasoocclussive crisis and hospitalization. Thus, in our study, we observed enhanced oxidative stress in both homozygous and heterozygous SCA patients as compared controls which can increase the severity of dis-ease. Endogenous mechanism appeared to be failure to mop up reactive oxygen species. Further study needed to see the effect of combined antioxidant supplementation on oxidative stress and disease course that anyway help patients.

Conclusion

Together these finding suggest that oxidant formed by sickle erythrocytes are less likely to be removed effectively with the endogenous mechanism. Therefore, the antioxidant supplementation is required to ameliorate the oxidative stress and improve the clinical course in sickle cell patients.

References

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Correspondence:
Prakash Hundekar

Department of Biochemistry
ACPM Medical College, Dhule, India
E-mail: pshundekar(at)gmail.com

Biomedical Research 2010 Volume 21 Issue 4 461
Biomedical Research 2010; 21 (4): 461-464

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