https://doi.org/10.3889/oamjms.2020.3907 eISSN: 1857-9655
Category: B - Clinical Sciences Section: Hematology
Role of Iron Chelators, Hydroxyurea, and Splenectomy on Serum Total Antioxidant Capacity in β-Thalassemia
Nagwa Abdallah Ismail1, Ahmed A. Talaat1, Sonia Adolf Habib2, Hisham A. Orban2
1Department of Pediatrics, National Research Centre, Cairo, Egypt; 2Department of Medical Biochemistry, National Research Centre, Cairo, Egypt
Abstract
BACKGROUND: Iron overload is the main cause of oxidative stress in beta-thalassemia (βT) by the increased production of free radicals and reactive oxygen species. Antioxidants counteract the toxic effects of oxidative stress.
AIM: This study aims to evaluate the total antioxidant capacity (TAC) and the possible impact of splenectomy, iron chelators, and hydroxyurea (hydra) on serum level of TAC.
MATERIALS AND METHODS: Fifty children and adolescents with βT were studied in comparison to 25 healthy age- and sex-matched subjects. Complete medical history, clinical examination, and laboratory assessment of serum TAC, ferritin, hepcidin, and hemoglobin (Hb) were carried out.
RESULTS: There was no statistically significant difference between the three groups; thalassemia major (TM), thalassemia intermedia (TI), and controls as regard age and sex. β-TM patients had significantly higher serum ferritin, serum hepcidin, and serum TAC (p < 0.000, 0.002, and 0.000, respectively) than controls. β-TI patients had significantly higher serum ferritin and serum TAC (p < 0.000) than controls. Serum TAC was lower in children with splenectomy, but this difference was not statistically significant. In addition, we observed no statistically significant difference in serum TAC of patients under different (deferasirox or deferiprone) medication. Serum TAC concentration was significantly higher in patients taking hydroxyurea (hydra) (p < 0.010).
CONCLUSIONS: The study showed an increased level of serum TAC in patients with β-T in comparison with controls. Serum TAC was also increased in those taking hydroxyurea, however, it was low in βT patients under regular chelation therapy, while splenectomy had no significant effect on serum TAC.
Introduction
Thalassemia is a genetic disease that leads to impaired globin chains and severe oxidative stress.
In Egypt, clinical management of patients with beta- thalassemia (βT) major depends mainly on blood transfusion (BT) and chelation therapy [1], [2]. However, iron overload is the main complication after long-term treatment [3]. Iron overload leads to peroxidative damage through increased production of free radicals and reactive oxygen species and is the main cause of oxidative stress [4]. Indirect bilirubin and uric acid are the main endogenous antioxidants, while both Vitamin E and ascorbic acid are important exogenous antioxidants.
These antioxidants counteract the toxic effects of oxidative stress [5]. Different studies have evaluated oxidative stress biomarkers in various disorders, along with hemolytic anemia [5], [6], [7], [8], [9]. Moreover, genetic mutations in the Mediterranean population may result in different responses to oxidative stress [10].
Oxidative stress occurs along with a decrease in total antioxidant capacity (TAC) [11]. On the other hand, iron chelation regimen for BT patients can be helpful in the regulation of the antioxidant status in those patients [12].
The aim of this study was to evaluate TAC and the possible impact of splenectomy, different iron chelators, and hydroxyurea (hydra) on serum level of TAC.
Materials and Methods
The current case–control study was conducted in Pediatrics Clinic in Centre of Excellence in National Research Centre and El Helal Giza Children Hospital Hematology Clinic in 2019. The study included 50 patients (30 thalassemia major [TM] and 20 thalassemia intermedia [TI]). Another group of 25, age- and sex-matched healthy subjects were enrolled as a control group. The study was performed in accordance with the Helsinki Declaration for human research, and the study protocol was approved by the National Research Centre Ethics Committee. Inclusion criteria were Age >5 years <18 years, absence of cardiac, renal disease, and any chronic disease. The exclusion criteria were patients with a history of any recent infection or surgery, congestive heart failure,
Edited by: Sinisa Stojanoski Citation: Ismail NA, Talaat AA, Habib SA, Orban HA.
Role of Iron Chelators, Hydroxyurea, and Splenectomy on Serum Total Antioxidant Capacity in β-Thalassemia. Open Access Maced J Med Sci. 2020 Mar 25; 8(B):26-30.
https://doi.org/10.3889/oamjms.2020.3907.
Keywords: β-thalassemia; Hydroxyurea; Iron chelators;
Splenectomy; Total antioxidant capacity
*Correspondence: Ahmed A. Talaat, Department of Pediatrics, National Research Centre, Cairo, Egypt.
E-mail: [email protected] Received: 22-Oct-2019 Revised: 05-Jan-2020 Accepted: 15-Mar-2020 Copyright: © 2020 Nagwa Abdallah Ismail, Ahmed A. Talaat, Sonia Adolf Habib, Hisham A. Orban Funding: This research did not receive any financial support Competing Interests: The authors have declared that no competing interests exist Open Access: This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial 4.0 International License (CC BY-NC 4.0)
moderate or high liver enzymes as well as positive viral hepatitis as hepatitis C or B, and patients on antioxidant medications. Data were collected by reviewing medical records as well as patient interviewing where detailed history taking and thorough clinical examinations were carried out. Blood was collected before BT.
Laboratory investigation Sample collection
Four milliliters of venous blood were withdrawn under aseptic conditions, blood was centrifuged to get serum to measure liver function and kidney function tests. Serum samples were stored in −80°C for the assessment of serum ferritin, hepcidin, and serum TAC.
Liver function and kidney function tests were done using an autoanalyzer Olympus AU400 (Olympus America Inc., Center Valley, PA, USA). Serum ferritin and hepcidin were measured by ELISA assay (Bioneovan Co., Ltd., Beijing, China)
Serum total antioxidants capacity (TAC) was colorimetrically measured according to the methods described by Koracevic et al. [13].
Statistical analysis
Data analysis was carried out using the standard computer program Statistical Package for the Social Sciences (SPSS) for Windows, release 17.0 (SPSS Inc., USA). All numeric variables were expressed as mean ± standard deviation. The intergroup comparisons were performed using an independent -sample t-test and a one-way analysis of variance and Chi-square tests for categorical variables. Pearson’s and Spearman’s correlation tests (r = correlation coefficient) were used for correlating normal and non-parametric variables, respectively. For all tests, p < 0.05 was considered statistically significant.
Results
The mean age of the children with TM and TI was 9.1 ± 3.8 years (from 5 to 18 years) and 10.2 ± 4.4 years (from 5 to 18 years old), respectively, with no statistically significant difference. Considering gender distribution, patients consisted of 29 (58%) boys and 21 (42%) girls, there was no statistically significant difference. β-TM patients had significantly higher serum ferritin, serum hepcidin, and serum TAC (p < 0.000, 0.002, and 0.000, respectively) than controls, while hemoglobin (Hb) and hepcidin/ferritin were significantly lower in patients than controls (p < 0.000 and 0.018, respectively), as shown in Table 1. β-TI patients had significantly higher serum ferritin and serum TAC (p < 0.000) than controls, while Hb and hepcidin/ferritin were significantly lower in patients
than controls (p < 0.000 and 0.030, respectively), as shown in Table 2.
Table 1: Comparison between β-thalassemia major patients and controls
Cases and controls n Mean Std. deviation Sig. (two tailed) Age (years)
β-TM patients 30 9.23 3.80 0.06
Controls 25 10.24 4.45
Hb (g/dL)
β-TM patients 30 6.59 0.948 0.000
Controls 25 11.76 1.012
Ferritin (ng/mL)
β-TM patients 30 2099.10 574.50 0.000
Controls 25 142.92 59.137
Hepcidin (ng/mL)
β-TM patients 30 21.74 13.11 0.002
Controls 25 12.53 6.69
TAC (mmol/L)
β-TM patients 30 1.77 0.300 0.000
Controls 25 1.25 0.60
Hepcidin/ferritin
β-TM patients 30 0.026 0.0576 0.018
Controls 25 0.0916 0.037
TAC: Total antioxidant capacity; TM: Thalassemia major; TI: Thalassemia intermedia; Hb: Hemoglobin.
Table 2: Comparison between β-thalassemia intermedia patients and controls
Cases and controls n Mean Std. deviation Sig. (two tailed) Age (years)
β-TI patients 20 8.9000 3.98550 0.08
Controls 25 10.24 4.45
Hb (g/dL)
β-TI patients 20 7.8050 0.55010 0.000
Controls 25 11.76 1.012
Ferritin (ng/mL)
β-TI patients 20 958.1000 517.77468 .000
Controls 25 142.92 59.137
Hepcidin (ng/mL)
β-TI patients 20 18.2225 14.37003 0.086
Controls 25 12.53 6.69
TAC (mmol/L)
β-TI patients 20 2.0300 0.22501 0.000
Controls 25 1.25 0.60
Hepcidin/ferritin
β-TI patients 20 0.021500 0.0213431 0.030
Controls 25 0.0916 0.037
TAC: Total antioxidant capacity; TM: Thalassemia major; TI: Thalassemia intermedia; Hb: Hemoglobin.
Demographic characteristics of thalassemia patients are shown in Table 3.
Table 3: Demographic characteristics of thalassemia patients
Variable Patients (n=50)
Age (years) 9.1±3.8
Sex (male/female) 21/29
Number of patients TM and TI 30/20
Number of patients on chelation therapy (none/regular) 10/40
Hydra intake (yes/no) 15/35
Splenectomized /non-splenectomized 14/36
Iron overload (serum ferritin ≥1000 ng/ml) 35/15 TM: Thalassemia major; TI: Thalassemia intermedia.
Figures 1 show boxplot of serum TAC in all groups.
Figure 1: Boxplot of serum total antioxidant capacity in β-thalassemia major, β-thalassemia intermedia, and controls
Figures 2 shows boxplot of serum ferritin in all groups. Relation between total antioxidant capacity and ferritin in β-thalassemia major is shown in Figure 3.
Figure 2: Boxplot of serum ferritin in β-thalassemia major, β-thalassemia intermedia, and controls
Patients with β-TM had significantly higher ferritin ng/Ml (p < 0.001). While, β-TI had significantly higher level of Hb (g/dL) and TAC (mmol/L) (p < 0.001 and < 0.002, respectively). Details are included in Table 4.
Table 4: Comparison of clinical and laboratory data of patients with β-thalassemia major and TI
Β-TM and Β-TI n Mean Std. deviation Sig. (two tailed) Age (years)
β-TM 30 9.23 3.80 0.767
β-TI 20 8.90 3.98
Hb (g/dL)
β-TM 30 6.59 0.948 0.001
β-TI 20 7.80 0.550
Ferritin (ng/mL)
β-TM 30 2099.10 574.50 0.001
β-TI 20 958.10 517.77
Hepcidin (ng/mL)
β-TM 30 21.74 13.11 0.375
β-TI 20 18.22 14.37
TAC (mmol/L)
β-TM 30 1.77 0.30 0.002
β-TI 20 2.03 0.22
Hepcidin/ferritin
β-TM 30 0.026 0.057 0.702
β-TI 20 0.0215 0.021
TAC: Total antioxidant capacity; TM: Thalassemia major; TI: Thalassemia intermedia; Hb: Hemoglobin.
No correlation existed between TAC, age, and sex in thalassemia patients or controls. Interestingly, the observed reverse correlation between TAC and ferritin, Hb, hepcidin, and hepcidin-ferritin ratio in βT patients was not statistically significant. Details are included in Table 5.
Table 5: Correlations between serum concentrations of TAC and biochemical parameters in children with thalassemia and controls
Group Age, Y Ferritin,
ng/mL Hemoglobin, g/dL Hepcidin/
ferritin Hepcidin, ng/mL
r p r p r p r p r p
β-
Thalassemia M0.134 0.481 −0.113 0.554 −0.088 0.644 −0.107 0.575 −0.166 0.379 β-TI 0.255 0.278 −0.472 0.026* −0.188 0.426 −0.119 0.616 −0.205 0.121 Controls 0.081 0.749 −0.270 0.202 0.022 0.919 0.219 0.304 −0.045 0.834
*correlation is significant at P value ≤ 0.05. TI: Thalassemia intermedia.
To evaluate the possible effect of several parameters on serum TAC in thalassemia children, samples were subdivided, as shown in Table 6. Serum TAC concentration was significantly higher in patients with no iron overload and those taking hydra (p < 0.001
and 0.010), and TAC concentration was significantly lower in patients on chelating agents (p < 0.003). Serum value was lower in children with splenectomy, but this difference was not statistically significant. In addition, we observed no statistically significant difference in serum TAC of the patients under (deferasirox =DFX or deferiprone = DFP) treatment, as shown in Table 7.
Furthermore, there were no statistically significant differences in serum TAC levels between males and females in patients with thalassemia (mean 1.89 ± 0.28 and 1.86 mmol/L ± 0.31 mmol/L, respectively, p = 0.731).
Table 6: Comparison between the mean serum TAC (mmol/L) and several parameters in β-thalassemia patients
Item n TAC (mmol/L) Sig. (two tailed)
Mean Std. deviation Serum TAC (mmol/L)
No iron overload 15 2.08 0.185 0.001
Iron overload 35 1.79 0.296
Serum TAC (mmol/L)
Splenomegaly 36 1.89 0.268 0.608
Splenectomized 14 1.84 0.37151
Serum TAC (mmol/L)
Chelators 40 1.81 0.299 0.003
No 10 2.12 0.113
Serum TAC (mmol/L)
DFX 23 1.81 0.270 0.465
DFP 15 1.88 0.299
Serum TAC (mmol/L)
Hydra intake 15 2.04 0.206 0.010
No 35 1.80 0.306
Serum TAC (mmol/L) Female
Male 19
21 1.89
1.86 0.285
0.310 0.731
DFX: Deferasirox; DFP: Deferiprone; Hydra: Oral hydroxyurea; TAC: Total antioxidant capacity;
TM: Thalassemia major; TI: Thalassemia intermedia.
Table 7: Comparison between serum TAC (mmol/L) under different chelating agents in thalassemic patients
Group statistics
β-TM DFX and DFP n Mean Std. deviation Sig. (two tailed)
Serum TAC (mmol/L) DFX 18 1.7833 0.28128 0.500
DFP 10 1.8600 0.28363
β-TI DFX and DFP n Mean Std. deviation Sig. (two tailed)
Serum TAC (mmol/L) DFX 5 1.9400 0.20736 1.00
DFP 5 1.9400 0.35777
TAC: Total antioxidant capacity; TM: Thalassemia major; TI: Thalassemia intermedia; DFX: Deferasirox;
DFP: Deferiprone.
Discussion
The use of frequent, regular BTs in β-TM patient has improved the span and quality of life, but increases chronic iron overload. This leads to oxidative stress in βT patients. This activates various antioxidant systems to protect the body from its damaging effects.
In β-TI, it seems that hydra increases Hb levels and leads to transfusion independence, but it is not high enough to suppress bone marrow activity and ineffective erythropoiesis. Consequently, oxidative stress is increased.
There are controversial results about TAC in thalassemia patients. In the present study, we estimated TAC in serum, as several studies suggest that assessing TAC is more useful than measuring antioxidants individually, so their synergistic interactions could be evaluated [9], [13], [14], [15]. Our study showed
a significantly increased level of TAC in Egyptian β-TM and in β-TI patients. Bazvand et al. reported a significant increase in antioxidant status in Iranian βT patients when compared to normal controls [9]. Besides this, Karakas et al. reported that TAC was significantly increased in β-TM and β-TI [16]. It must be pointed out that another study showed decreased levels of TAC in Italian and Greek βT patients [5]. A possible explanation for this difference could be the different age ranges (41.5
± 9.5 years) and/or different medications. Furthermore, genetic mutations in different population may result in different responses to oxidative stress [11]. A probable mechanism to explain the increased antioxidant status in patients with βT is the compensating antioxidant response due to excessive oxidative stress [16].
Endogenous antioxidants such as ferritin, bilirubin, and uric acid can result in increased level of TAC in patients with β-TM [9]. However, Cakmak et al. reported no significant differences in TAC between βT patients and controls [17].
Serum TAC concentration was significantly higher in βT patients with no iron overload than those with iron overload. Iron overload leads to peroxidative damage in β-TM and antioxidant systems try to decrease tissue damage by decreasing lipid peroxidation. Glutathione peroxidase levels were lower in iron overload patients as compared to controls [18].
In the present study, βT patients not receiving chelation therapy had significantly higher TAC compared with those on regular chelation. Metwalley and El-Saied reported that βT patients not receiving or on irregular chelation therapy had significantly lower TAC compared with those on regular chelation [19]. This difference could be explained by the fact that most patients not on chelation therapy in our study belong to the β-TI group who have significantly higher TAC concentration than β-TM group, probably due to less iron load and less oxidative stress. Likewise, this could explain the significantly higher TAC concentration in those taking hydra in our study. Aggressive regular chelation and antioxidant therapy should be used to cut the progression of the adverse effects of oxidative stress and to improve carbohydrate physiology in transfusion- dependent β-TM [20]. Antioxidant therapy should be
advised for thalassemia patients to improve glucose hemostasis [19].
There was no significant relationship between TAC and splenectomy status. Same result was reported by Karakas et al. [16]. No correlation existed between TAC and age, sex in thalassemia patients, or controls.
Karakas et al. [16] reported the same results. Our results indicate that there was a negative non-significant correlation between TAC and ferritin. Different studies showed the same result [10,16].
Conclusions
The study showed an increased level of serum TAC in patients with βT in comparison with controls.
Serum TAC was also increased in those taking hydroxyurea, however, it was low in βT patients under regular chelation therapy, while splenectomy had no significant effect on serum TAC.
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