The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 42  |  Issue : 3  |  Page : 123--127

Oxidative stress in pediatric patients with β thalassemia major


Asmaa Nafady1, Sanaa Shaker Ali2, Hosny Mohammed Ahmed El Masry3, Khaled Abdel Baseer4, Heba M Qubaisy4, Samar Gallab Mahmoud4, Hanaa A Nafady-Hego5,  
1 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
2 Clinical and Chemical Pathology, Qena Faculty of Medicine, South Valley University, Qena, Egypt
3 Department of Pediatric, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
4 Department of Pediatric, Faculty of Medicine, Assiut University, Assiut, Egypt
5 Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt

Correspondence Address:
Asmaa Nafady
Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut
Egypt

Abstract

Background β-thalassemia major (β-TM) is a common inherited hemolytic type of anemia. Repeated blood transfusions predispose β-TM patients toward peroxidative tissue injury because of secondary iron overload. Objectives This study aimed to evaluate the effects of iron overload on antioxidant enzymes and liver cell damage in β-TM patients undergoing regular blood transfusions. Patients and methods This prospective case–control cohort study included 30 pediatric patients with a confirmed diagnosis of β-TM on regular blood transfusions and 20 age-matched and sex-matched healthy children attending the Qena University Hospital, Pediatric Clinic. Blood samples were withdrawn from each patient to measure serum levels of ferritin, glutathione peroxidase (GPX), and superoxide dismutase (SOD). Results Total bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), and ferritin levels were significantly higher in the β-TM group (P<0.001, 0.001, 0.001, and 0.001, respectively), whereas GPX and SOD were significantly lower in the β-TM group (P<0.001 and 0.001). The correlation between serum ferritin level and age, bilirubin, AST, and ALT in patients group showed that, the correlation between serum ferritin level and age was (0.745) while P-value was <0.001, the correlation between serum ferritin level and bilirubin level was (0.665) while P-value was <0.001, the correlation between serum ferritin level and (AST) level was (0.727) while P-value was <0.001 and the correlation between serum ferritin level and (ALT) level was (0.737) while P-value was <0.001. The correlation between SOD and age, ferritin, bilirubin, AST, and ALT in patients group in patients group showed that, the correlation between (SOD) and age was (−0.454) while P-value was 0.012, the correlation between (SOD) and ferritin level was (−0.664) while P-value was <0.001, the correlation between (SOD) and bilirubin level was (−0.535) while P-value was 0.002, the correlation between (SOD) and (AST) level was (−0.567) while P-value was <0.001 and the correlation between (SOD) and (ALT) level was (−0.558) while P-value was <0.001. Conclusion Impaired levels of antioxidant enzymes SOD and GPX in patients with β-TM on repeated transfusion, in addition to excessive free iron concentration, iron overload may attribute to oxidative damage in these patients. Antioxidant systems that compensate for reduced lipid peroxidation to lower tissue damage are needed.



How to cite this article:
Nafady A, Ali SS, El Masry HA, Baseer KA, Qubaisy HM, Mahmoud SG, Nafady-Hego HA. Oxidative stress in pediatric patients with β thalassemia major.Egypt J Haematol 2017;42:123-127


How to cite this URL:
Nafady A, Ali SS, El Masry HA, Baseer KA, Qubaisy HM, Mahmoud SG, Nafady-Hego HA. Oxidative stress in pediatric patients with β thalassemia major. Egypt J Haematol [serial online] 2017 [cited 2020 Sep 20 ];42:123-127
Available from: http://www.ehj.eg.net/text.asp?2017/42/3/123/217882


Full Text

 Introduction



β-Thalassemia major (β-TM) is one of the most common inherited hemolytic types of anemia. β-TM manifests itself with severe anemia, which necessitates 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 an improved survival outcome in β-TM, secondary iron overload is still a major concern [1]. Secondary iron overload in the body is difficult to eliminate, and the excess iron is deposited as hemosiderin and ferritin in the liver, spleen, endocrine organs, and myocardium [2]. Iron accounts for excessive generation of free radicals that cause oxidative damage to erythrocytes. Antioxidant defenses, a complex and diverse group of molecules, protect key biological sites from oxidative damage; they scavenge free radicals and other reactive oxygen species [3]. Removal of these oxygen metabolites is the function of antioxidant enzymes such as superoxide dismutases (SOD) and glutathione peroxidase (GPX) [4]. Glutathione and its redox enzyme system are crucial elements of the erythrocyte antioxidant defense mechanism against free radical accumulations and functions by scavenging free radicals and detoxifying lipid peroxides through GPX. Superoxide radicals generated in excess following autoxidation of the isolated hemoglobin chain are an essential contributor toward the hemolytic process. It is thus necessary to measure the erythrocyte SOD activity in β-TM patients and examine its relation to the severity of the disease. SODs are the proteins cofactor with iron, zinc manganese, copper, or nickel. In humans, it exists in three forms including SOD1, SOD2, and SOD3, which are present in the cytoplasm, mitochondria, and extracellular space, respectively. Superoxide is the main reactive oxygen species that react with nitric oxide radicals and forms peroxynitrite, thereby causing oxidative stress and cellular damage. Erythrocyte SOD is responsible for erythrocyte protection from damage during oxidative stress [5].

 Patients and methods



This study was carried out on 30pediatric patients (<18 years) diagnosed with β-TM on regular blood transfusions (β-TM group) and 20 age-matched and sex-matched healthy volunteers (control group) attending the Qena University Hospital, Pediatric Clinic. The patients were blood transfusion dependent. Patients with diabetes mellitus, renal failure, or hereditary hyperlipidemia were excluded. None of the patients or control participants enrolled in this study received antioxidant supplementations. About 5 ml of venous blood was aseptically withdrawn from the anticubital vein from the study groups. The blood sample was separated into two different tubes: one contained EDTA and the other was plain. The samples in the plain tube were centrifuged at 3000 rpm for 5 min to separate serum. Serum was stored in separate vials at −20°C until analysis of GPX and SOD by a spectrophotometer. A complete blood count was performed on a Beckman Coulter Automated Hematology Analyzer MaxM (Beckman Coulter Diagnostics, Nyon, Switzerland). Liver function and serum ferritin were measured (liver function) using a Cobas c311 Automated Chemistry Analyzer (Roche Diagnostics GmbH, Mannheim,, Germany). Serum ferritin was determined by a two-site sandwich immunoassay using direct technology by a Cobas e411 Automated Chemistry Analyzer (Roche Diagnostics). Serum GPX and SOD were measured by spectrophotometer Erba them 7 Germany using the Bio Diagnostic Kit (Bio Diagnostics, Giza, Egypt) (Cat. no. GP 2524) and (Cat. no. SD 2521).

The Human Research Ethics Committee of the Faculty of Medicine, Qena University, approved this study. Informed consent was obtained for pediatric patients from their parents according to the Declaration of Helsinki [6].

Statistical analysis

All the data are presented as mean±SD. Data analysis was carried out using an independent t-test or Pearson’ s correlation coefficient as appropriate. A P value less than 0.05 was considered significant. The statistical package for the social science for Windows statistical software (SPSS for Windows, Version 16.0; SPSS Inc., Chicago, IL, 2007) was used.

 Results



Characteristics of the participants

The clinical profiles of the participants are detailed in [Table 1] and [Table 2].{Table 1}{Table 2}

β-TM patients with diabetes mellitus, renal failure, or hereditary hyperlipidemia were not included in this study. Thirty patients were included. There were 20 males and 10 females, with a mean age of 7.77 years (3–15 years). Twenty age-matched healthy volunteers were also included control group. They showed no evidence of disease. There were seven males and 13 females, with a mean age of 7.5 years (3–14 years). The age, sex, and BMI in the two groups showed no significant difference. However, weight and height were significantly lower in the β-TM group compared with the control group (P=0.007 and 0.0324, respectively).

Liver function, ferritin, GPX, and SOD levels in the study groups: liver function tests [total bilirubin, aspartate transaminase (AST), and alanine transaminase (ALT)] were significantly higher in the β-TM group compared with the control group (P<0.001, <0.001, and <0.001, respectively). In addition, the level of ferritin was significantly higher in the β-TM group compared with the control group (P<0.001); in contrast, the levels of GPX and SOD were significantly lower in the β-TM group compared with the control group (P<0.001 and <0.001) ([Table 3]).{Table 3}

The serum ferritin level correlated positively with age (R=0.745, R2=0.56, P<0.001). Total bilirubin (R=0.665, R2=0.44, P<0.001), AST (R=0.727, R2=0.53, P<0.001), and ALT (R=0.737, R2=0.54, P<0.001) ([Table 3]). However, the serum GPX level correlated negatively with age (R=−0.585, R2=0.34, P<0.001). The correlation between GPX and age, ferritin, bilirubin, AST, and ALT in patients group showed that, the correlation between (GPX) and age was (−0.585) while P-value was <0.001, the correlation between (GPX) and ferritin level was (−0.725) while P-value was <0.001, the correlation between (GPX) and bilirubin level was (−0.626) while P-value was <0.001, the correlation between (GPX) and (AST) level was (−0.588) while P-value was <0.001 and the correlation between (GPX) and (ALT) level was (−0.626) while P-value was <0.001. The correlation between GPX and age, ferritin, bilirubin, AST, and ALT in patients group showed that, the correlation between (GPX) and age was (−0.585) while P-value was <0.001, the correlation between (GPX) and ferritin level was (−0.725) while P-value was <0.001, the correlation between (GPX) and bilirubin level was (−0.626) while P-value was <0.001, the correlation between (GPX) and (AST) level was (−0.588) while P-value was <0.001 and the correlation between (GPX) and (ALT) level was (−0.626) while P-value was <0.001 ([Table 3], [Figure 1] and [Figure 2]).{Figure 1}{Figure 2}

 Discussion



We found that the serum ferritin level was significantly higher in patients with β-TM compared with the control group. Our finding is in agreement with recent reports from Abed Mahdy [7], who reported a significant increase in serum ferritin in patients younger than 18 years old, with β-TM indicating an existing iron overload.

The bilirubin level of patients with β-TM was statistically significantly higher in patients with β-TM compared with the control group, which can be attributed to the hemolysis in the patient group. In addition, liver enzymes, AST, and ALT levels were significantly elevated in patients with β-TM compared with the control group; our findings were in agreement with reports from three different centers [8],[9],[10] that reported higher levels of transaminases in β-TM patients. In the liver, lipid peroxidation is associated with malfunction of the mitochondria and lysosomes [10]. In fact, iron overload participates in impaired hepatic mitochondrial respiration primarily by reducing cytochrome c oxidase activity, which can explain hepatocellular calcium homeostasis dysfunction through damage to microsomal and mitochondrial calcium sequestration.

However, our study showed downregulation of serum GPX and serum SOD levels in pediatric patients with β-TM. Our finding is in agreement with that of others who reported that SOD and GPX were significantly lower in thalassemic patients because of iron over load compared with controls [7]. Decreased levels of this antioxidant cause inactivation by increased superoxide anion production, leading to an increase in oxidative stress [11].

Moreover, Dhawan et al. [12] found that the mean SOD enzyme activity was at least 1.5 times lower in 209 thalassemia major patients compared with healthy volunteers. Similar results were obtained by Patne et al. [13] and Del Bo et al. [14], who reported that SOD and GPX were significantly lower in thalassemic patients. Intracellular enzymes are responsible for changes in the oxidant–antioxidant balance in cells. Their function is to catalyze modifying ion free radicals, especially O2− into H2O. In patients with thalassemia, enormous free radicals build up because of the state of iron overload (resulting from transfusions and ineffective erythropoiesis). Iron can accelerate the transformation of molecular oxygen into reactive oxygen radicals, superoxide, and hydroxyl groups through the Fenton reaction; this is in agreement with our study.

In contrast to our finding of SOD, three groups reported a significant increase in its level of activity in patients with β-TM [8],[9],[15]; another group reported no difference in its level of activity in β-TM patients compared with controls [16]. These findings could be attributed to the fact that SOD scavenges superoxide radicals to form hydrogen peroxide and protects the cell membrane from damage. Increased erythrocyte SOD activity may be because of blood transfusion and an increase in the proportion of younger erythrocytes as a compensatory mechanism after increased oxidative stress.

Moreover, GPX as well as SOD activities were found to be higher in patients with β thalassemia as a compensatory mechanism accompanying iron overload [8],[9].

In our study, there was a significant correlation between ferritin level and AST and ALT levels, which can be explained by liver injury caused by iron overload. This is in agreement with a previous report that showed that aminotransferase serum activity in patients with transfusion iron overload caused by acquired anemia depends on the hepatic iron concentration and is directly related to the log-transformed urinary iron excretion. Serum ferritin was correlated positively with ALT, AST, and total bilirubin, representing cell damage as a consequence of iron overload [17].

The results of our study showed that there was a statistically significant negative correlation between GPX and SOD with ferritin, AST, ALT, and total bilirubin. This is in agreement with Baumgartener [3], who reported that serum ferritin, which determines the extent of iron loading, was found to have a significant negative correlation with the antioxidant enzymes, that is GPX and SOD, thus showing increased oxidative stress.

 Conclusion



This report provided detailed evidence that pediatric β-TM patients have impairments in antioxidant enzymes level because of iron overload. Free radicals, altered serum trace elements, and antioxidant enzymes status do not exclusively play an essential role in β-TM pathogenesis, but they can lead to complications. Therefore, novel therapeutic strategies should focus on administration of selective antioxidants along with essential trace elements and minerals. Hence, treatment with antioxidant vitamins may improve the liver functions and reduce the percentage of erythrocytes hemolysis, therefore improving the total hemoglobin concentration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Elizabeth G, Mary Ann TJA. Genotype-phenotype diversity of beta-thalassemia in Malaysia: treatment options and emerging therapies. Med J Malaysia 2010; 65:256–260.
2Milena R, Branka Z, Biljana S, Belton N, Canat B. Thalassaemia syndrome in Serbia. Hemoglobin 2010; 34:477–485.
3Baumgartener TG. Vitamins In: Van Way CW, editor. Nutrition secrets. Philadelphia: Hanley and Belfus; 1999: pp. 13–20.
4Waseem F, Khemomal KA, Sajid R. Antioxidant status in beta thalassemia major: a single-center study. Indian J Pathol Microbiol [serial online] 2011; 54:761–763. [cited 2014 Aug 10].
5Qaiser S, Zahirul Hoque M, Iqbal M, Mudin DKD. Evaluation of antioxidant status in beta thalassemia major patients in Sabah, Malaysian Borneo. Biores Comm 2015; 1:45–47.
6Burns JP. Research in children, Crit Care Med 2003; 31.
7Bazvand F, Shams S, Borji Esfahani M, Koochakzadeh L, Monajemzadeh M, Haghi Ashtiani M-T, Rezaei N. Total antioxidant status in patients with major β-thalassemia. Iran J Pediatr 2011; 21:159–165.
8Qari1 MH, Wali Y, Albagshi MH, Alshahrani M, Alzahrani A, Alhijji IA et al. Regional consensus opinion for the management of Beta thalassemia major in the Arabian Gulf area Qari et al. Orphanet J Rare Dis 2013; 8:143..
9Abed Mahdy E. Relationship between oxidative stress and antioxidant status in beta thalassemia major patients. Acta Chim Pharm Indica 2014; 4:137–145. ISSN 2277-288X.
10Attia M, Sayed A, Ibrahim F, Meaad H. Effect of antioxidant vitamins of the oxidant/antioxidant status and liver function in homozygous beta thalassemia. Roman J Biophes 2011; 21:93–106.
11Kassab-Chekir A, Laeadi S, Ferchichi S, Haj KA, Feki A, Amri F. Oxidant, antioxidant status and metabolic data in patients with β thalassemia. Clin Chem Acta 2003; 338:79–86.
12Aziz BN, Al-Kataan MA, Ali WK. Lipid peroxidation and antioxidant status in β-thalassemic patients: effect of iron overload. Iraqi J Pharm Sci 2009; 18: Antioxident.
13Dhawan V, Kumar KhR, Marwaha RK, Naravan S, Kamgar M. Antioxidant status in children with homozygous thalassemia. Indian Pediatr 2005; 42:1141–1145.
14Patne AB, Hisalkar PJ, Gaikwad SB, Pati SV. Alterations in antioxidant enzyme status with lipid peroxidation in thalassemia major patients. Int J Pharm Life Sci 2012; 3:2003–2006.
15Del Bo C, Porrini M, Campolo J, Parolini M, Lanti C, Klimis-Zacas D, Riso P. A single blueberry (Vaccinium corymbosum) portion does not affect markers of antioxidant defence and oxidative stress in healthy volunteers following cigarette smoking. Mutagenesis 2016; 31:215–224. doi: 10.1093/mutage/gev079. Epub 2015 Nov 23.
16Şimşek F, Öztürk G, Kemahl S, Erbaş D, Hasanoğlu A. Oxidant and antioxidant status in beta thalassemia major patients. Ankara Üniversitesi Tıp Fakültesi Mecmuası 2005; 58:34–38.
17Ghone RA, Kumbar KM, Suryakar AN, Katkam RV, Joshi NG. Oxidative stress and disturbance in antioxidant balance in beta thalassemia major. Indian J Clin Biochem 2008; 23:337–340.