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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2011  |  Volume : 54  |  Issue : 4  |  Page : 761-763
Antioxidant status in beta thalassemia major: A single-center study


1 Department of Biochemistry, Liaquat College of Medicine and Dentistry, Karachi, Pakistan
2 Department of Biochemistry, Basic Medical Sciences Institute, Jinnah Post Graduate Medical Center, Karachi, Pakistan
3 Department of Pathology and Microbiology, Aga Khan University, Karachi, Pakistan

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Date of Web Publication6-Jan-2012
 

   Abstract 

Background: Homozygous β thalassemia may lead to a marked reduction or absence of normal β chain production and accumulation of unpaired alpha-globin chains. A crucial component in the oxidant susceptibility of the thalassemic RBC is the release of heme and iron from the excessive, unpaired α-globin chains. This release can initiate self-amplifying redox reactions, which deplete the cellular reduction potential (e.g., GSH), oxidize additional hemoglobin and accelerate RBC destruction. Furthermore, β-thalassemia patients are under continuous blood transfusion, which, although life-saving, leads to an iron overload with a resultant increase in non-transferrin-bound iron that may cause greater tissue toxicity than iron in other forms. Iron-induced oxidative stress is known to be one of the most important factors determining cell injury in thalassemic patients. Therefore, we designed this study to obtain a comprehensive picture of the iron overload, antioxidant status and cell damage in β thalassemia major patients undergoing regular blood transfusion. Materials and Methods: A total of 48 diagnosed patients of β thalassemia major and 30 age- and sex-matched healthy subjects were included in the study. Estimation of hemoglobin, hematocrit, glutathione peroxidase (GPX), superoxide dismutase (SOD),vitamin E, serum ferritin, total and direct bilirubin, AST and ALT was carried out. Results: The levels of vitamin E, antioxidant enzymes GPX and SOD were significantly lowered in β thalassemic patients as compared with the control group (P<0.001). Serum total and direct bilirubin, AST and ALT were significantly elevated in thalassemic subjects as compared with the control group, indicating liver cell damage. Conclusion: Thus, our findings indicate that thalassemics are in a state of enhanced oxidative stress and that the administration of selective antioxidants would represent a promising approach toward counteracting oxidative damage and its deleterious effects on the disease status.

Keywords: Glutathione peroxidase, oxidative stress, superoxide dismutase, thalassemia major

How to cite this article:
Waseem F, Khemomal KA, Sajid R. Antioxidant status in beta thalassemia major: A single-center study. Indian J Pathol Microbiol 2011;54:761-3

How to cite this URL:
Waseem F, Khemomal KA, Sajid R. Antioxidant status in beta thalassemia major: A single-center study. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Apr 23];54:761-3. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/761/91489



   Introduction Top


The β thalassemia syndromes are one of the most common hemoglobinopathies prevalent in Pakistan, with an estimated more than 5,000 patients being born with thalassemia major every year in Pakistan. [1] Thalassemia major manifests itself with severe anemia and a lifelong dependence on blood transfusions to sustain life. Regular blood transfusions lead to iron overload, with its toxic effects including endocrinopathies and cardiac arrhythmias. Although iron chelation has led to improved survival in thalassemia major, secondary iron overload is still a major concern. [2]

In patients with iron overload from fully saturated transferrin, non-transferrin-bound iron (NTBI) is increased and causes tissue toxicity leading to increased lipid peroxidation processes with subsequent consumption of antioxidants. [3] This consumption of antioxidants is responsible for constant intracellular oxidative stress and release of free (non-heme) iron. [4]

Glutathione and its redox enzyme system are an essential element of erythrocyte's antioxidant defense mechanism against free radical accumulations and functions by scavenging free radicals and detoxifying lipid peroxides via glutathione peroxidase (GPX). [5],[6] Superoxide radicals generated in excess following autoxidation of the isolated hemoglobin chain is an important contributor to the hemolytic process. [7] It is thus important to measure the erythrocyte superoxide dismutase (SOD) activity in thalassemia and examine its relation to severity of the disease. Vitamin E counteracts free radical attack and appears to be a highly efficient antioxidant. [8]

This study was designed to analyze the effects of iron overload on antioxidant enzymes and liver cell damage in β thalassemia major patients undergoing regular blood transfusion.


   Materials and Methods Top


Patients with confirmed diagnosis of thalassemia major on regular blood transfusions were enrolled, consent was taken and a proforma was filled. Thalassemia intermedia and minor cases, or those with ferritin level less than 1000 ng/ml and/or taking any vitamin supplements, were excluded from the study.

Blood from thalassemic patients was collected just before transfusion. For comparison, control blood was drawn from healthy individuals not exhibiting any signs and symptoms.

Ten milliliters of venous blood was collected just before transfusion from the antecubital vein under aseptic measures. Whole blood was taken simultaneously in lithium heparin and EDTA tubes. Serum was separated within 1 h. Sample in the EDTA tube was used for automated hemoglobin and hemotocrit estimation on a Beckman Coulter automated hematology analyzer MaxM (Beckman Coulter Diagnostics, Switzerland). Whole blood sample in the lithium heparin tube was used for estimation of GPX and SOD. For rest of the parameters, aliquots were initially stored at -50°C. The data was entered into statistical package for social sciences (SPSS) version 16.0 and was analyzed using Student's independent samples t-test and Pearson's correlation coefficient.

GPX assay was performed using Ransel GPX and the results were read on a spectrophotometer, model AE-350, Erma Inc., Tokyo, Japan.

SOD was assayed using the Ransod kit and the results were read on a spectrophotometer; model AE-350, Erma Inc. Tokyo, Japan.

Serum ferritin was analyzed by the enzyme immunoassay method using the kit of Pointe Scientific. inc. 5449 research drive, canton MI 48188, USA.

Total and direct bilirubin, AST and ALT were measured in serum using Randox kits.

Serum Tocopherol was determined by the Baker and Frank method, 1968, and the absorbance was read at 520 nm.

This study was approved by the departmental research committee.


   Results Top


Forty-eight diagnosed cases of β thalassemia major, 34 males and 14 females, aged 1-23 years (mean 11.21 ± 0.68 years), were recruited with consent for this study. All the patients were on regular bimonthly transfusions of 15 ml/kg body weight at each transfusion to maintain hemoglobin levels above 10 g/dl. The mean duration of transfusion was 10.49 ± 0.69 years. Thirty control subjects (21 males and 9 females) with a mean age of 11.13 ± 0.62 years were also recruited in the study.

The patient and control data is presented in [Table 1] with mean values and standard error of mean. Vitamin E level was found to be significantly lower (P<0.001) in the β-thalassemic patients as compared with the control group. GPX (U/L) was found to be significantly low (P<0.001) in the β-thalassemic group as compared with the control group. Similarly, the mean level of enzyme SOD (U/L) was also found to be significantly low (P<0.001) in the β-thalassemic patients as compared with controls.
Table 1: Patient and control data (values are expressed as mean ± SEM)

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[Table 2] shows the correlation coefficient between serum ferritin and other biochemical parameters. Serum ferritin, which determines the extent of iron loading, was found to have a significant negative correlation with the antioxidant enzymes, i.e. GPX, SOD and vitamin E, thus showing increased oxidative stress. Serum ferritin was positively correlated with ALT, AST, total bilirubin and direct bilirubin, representing cell damage as a consequence of iron overload.
Table 2: Correlation coeffi cient between serum ferritin and other biochemical parameters in patients with thalassemia major

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[Table 3] depicts a negative correlation between vitamin E and ALT, AST, total bilirubin and direct bilirubin, suggesting that liver damage may play a major role in the extent of depletion of lipid-soluble antioxidants. Similarly, GPX and SOD were also negatively correlated with ALT, AST, total bilirubin and direct bilirubin, showing decreased activity of antioxidant enzymes in relation to increasingly disturbed signs of liver damage.
Table 3: Correlation coeffi cient between anti oxidant parameters, hemoglobin and liver function tests in patients with thalassemia major

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A strongly positive correlation was found between SOD and hemoglobin and GPX and hemoglobin. Thus, decreased activities of antioxidants enzymes reflect their role in severity of the disease.


   Discussion Top


The intracellular antioxidative mechanisms normally prevent damage due to a dangerous combination of oxygen and iron (hemoglobin or hemoglobin derived). The released heme has been shown to directly inhibit a number of cytoplasmic enzymes, further disrupting normal cellular homeostasis and predisposing the cell to additional injury. In addition to the released heme, the α-hemoglobin chain-mediated production of reactive oxygen species (O- 2 H 2 O2 ) catalyzes the production of the hydroxyl radical (OH) and reactive lipid radicals. These species are believed to mediate much of the damage seen in thalassemic erythrocytes. [9],[10] Removal of these oxygen metabolites is the function of antioxidant enzymes such as SOD and GPX. We found significantly lower mean SOD activity in children with β-thalassemia major when compared with healthy controls, and our results are in agreement with those of Dhawan et al., who found that the mean SOD enzyme activity was at least 1.5-times lower in the thalassemics than in controls. [10]

The findings, pertaining to erythrocytic SOD enzyme activity, reported by other investigators are varied. They ranged from high SOD activity to no difference in patients and controls. [11],[12]

In our study, significantly reduced levels of GPX were observed in patients with β thalassemia major as compared with controls. Our findings are in confirmation with the study of Garelnabi et al. [13] Low level of GPX seems to result from the enzyme inhibition or reduced activity due to excessive production of hydrogen peroxide. This study showed significantly lower levels of all the antioxidants-vitamin E, GPX and SOD - in thalassaemic children compared with the matched healthy controls. Our results of significantly low level of vitamin E in thalassaemic patients agree with those of Simsek et al. and Nasr et al.[11],[14] The decreased serum vitamin E in our patients indicated an overconsumption to protect against oxidative hemolysis caused by NTBI. [3]

In contrast to these findings, we found a significant elevation of signs of iron overload and cell damage (serum ferritin, AST, ALT and total and direct bilirubin) in patients with β thalassemia major when compared with controls. Our results agree with those of Filosa et al. and Laksmitawati et al.[8],[15] Increase in bilirubin in thalassemia may be related to hemolytic process and existing hepatic damage. Elevated transaminasaemia in β thalassemic children is indicative of liver dysfunction and leakage of liver metabolites into the plasma. [6]

In conclusion, our observations (depletion of vitamin E, decreased activities of SOD and GPX and the increase of ferritinaemia) indicate that thalassemics are in a state of enhanced oxidative stress, and understanding the clinical manifestations of thalassemia major from the viewpoint of the enzymatic antioxidant defense system in diseased erythrocytes is of great significance for the future management of these patients.


   Acknowledgment Top


The authors would like to thank Dr. Walid Bin Azhar, Chief Operating Officer, Fatimid Foundation, Karachi.

 
   References Top

1.Ahmed S, Saleem M, Modell B, Petrou M. Screening extended families for genetic hemoglobin disorders in Pakistan. N Engl J Med 2002;347:1162-8.  Back to cited text no. 1
    
2.Olivieri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. Blood 1997;89:739-61.  Back to cited text no. 2
    
3.Reller K, Dresow B, Collell M, Fischer R, Engelhardt R, Nielsen P, et al. Iron overload and antioxidant status in patients with â-thalassemia major. Ann N Y Acad Sci 1998;850:463-5.  Back to cited text no. 3
    
4.Kattamis C, Kattamis AC. Oxidative stress disturbances in erythrocytes of â-thalassemia. Pediatr Hematol Oncol 2000;8:85-8.  Back to cited text no. 4
    
5.Chakraborty D, Bhattacharyya M. Oxidant defense status of red blood cells of patients with â-thalassemia and E â thalassemia. Clin Chim Acta 2001;305:123-9.  Back to cited text no. 5
    
6.Walter PB, Fung EB, Killilea DW, Jiang Q, Hudes M, Madden J, et al. Oxidative stress and inflammation in iron-overloaded patients with â-thalassemia or sickle cell disease. Br J Haematol 2006;135:254-63.  Back to cited text no. 6
    
7.Traber MG, Packer L. Vitamin E: Function beyond antioxidant. Am J Clin Nutr 1995;62:1501S- 9S.  Back to cited text no. 7
    
8.Filosa A, Valgimigli L, Pedulli GF, Sapone A, Maggio A, Renda D, et al. Quantitative evaluation of oxidative stress status on peripheral blood in â-thalassemic patients by means of electron paramagnetic resonance spectroscopy. Br J Hematol 2005;131:135-40.  Back to cited text no. 8
    
9.Scott MD, Repka T, Hebbel RP, van den Berg JM, Wagner TC, Lubin BH et al. Membrane deposition of heme and non-heme iron in model thalassemic erythrocytes. Blood 1991;78:771.  Back to cited text no. 9
    
10.Dhawan V, Kumar KhR, Marwaha RK, Ganguly NK. Antioxidant status in children with homozygous thalassemia. Indian Pediatr 2005;42:1141-5.  Back to cited text no. 10
    
11.Simsek, Ozturk G, Kemahli S, Erbas D, Hasanoglu A. A Oxidant and antioxidant status in â thalassemia major patients. J Ankara Univ Fac Med 2005;58:34-8.  Back to cited text no. 11
    
12.Gerli GC, Beretta L, Bianchi M, Pellegatta A, Agostoni A. Erythrocyte superoxide dismutase, catalase and glutathione peroxidase activities in â-thalassemia (major and minor). Scand J Haematol 1980;25:87-92.  Back to cited text no. 12
    
13.Garelnabi M, Paradhan P. Splenectomy may not influence glutathione metabolism in children with â thalassemia major. Turk J Haematol 2005;22:25-30.  Back to cited text no. 13
    
14.Nasr MR, Ali S, Shaker M and Elgabry E. Antioxidant micronutrient in children with thalassemia in Egypt. East Mediterr Health J 2002;8:1-5.  Back to cited text no. 14
    
15.Laksmitawati DR, Handayani S, Udyaningsih-Freisleben SK, Kurniati V, Adhiyanto C, Hidayat J, et al. Iron status and oxidative stress in â-thalassemia patients in Jakarta. Biofactors 2003;19:53-62.  Back to cited text no. 15
    

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Correspondence Address:
Raihan Sajid
Department of Pathology and Microbiology, Aga Khan University, Stadium Road, Karachi - 74800
Pakistan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.91489

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