Copper, zinc, and magnesium status among patients with thalassemia attending pediatric hematological unit at Sohag University Hospital

Eman M Fahmy; Eman H Salama; Nesreen A Mohammed

doi:10.4103/ejh.ejh_1_19

ORIGINAL ARTICLE

Year : 2019 | Volume : 44 | Issue : 2 | Page : 98-104

Copper, zinc, and magnesium status among patients with thalassemia attending pediatric hematological unit at Sohag University Hospital

Eman M Fahmy¹, Eman H Salama², Nesreen A Mohammed³
¹ Department of Pediatric, Sohag University Hospital, Sohag, Egypt
² Department of Clinical Pathology, Sohag University Hospital, Sohag, Egypt
³ Public Heath and Community Medicine, Sohag University Hospital, Sohag, Egypt

Date of Submission	05-Jan-2019
Date of Acceptance	21-Mar-2019
Date of Web Publication	15-Nov-2019

Correspondence Address:
Nesreen A Mohammed
Lecturer of Pediatric Medicine Sohag University. 01063011409
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_1_19

Abstract

Introduction Thalassemias are a group of inherited blood-born disorders caused by abnormalities in the synthesis of hemoglobin chain (α or β chain). β-Thalassemia major is the severest form where individuals usually experience severe transfusion-dependent anemia within their first two years of life; thus, it requires regular transfusions of packed red blood cell (RBC) and desferrioxamine injections. Patients with thalassemia have ineffective erythropoiesis and faster RBC turnover owing to the short life span of RBCs and so increased body demand of energy and nutrients to keep normal erythropoiesis. Trace elements and minerals have a vital role in the appropriate functioning of the body.
Aim of the study The aim of this study is evaluation of zinc, copper, and magnesium levels in patients having β-thalassemia major and establish if these patients need copper, zinc, and magnesium supplementation or no.
Patients and methods In this study, we included 35 patients diagnosed as having β-thalassemia major attending pediatric hematological unit at Sohag University Hospital within a period of 6 months from January 2018 to June 2018. We studied the serum level of various trace elements in children with β-thalassemia major of both sexes aged below 18 years in a cross-sectional study by assessment of serum level of zinc, copper, and magnesium using classical colorimetric end-point technique by a semiautomated, programmable photometer 5010.
Results Only 6.7% of the studied patients had serum zinc levels of less than 66 µg/dl and 13.3% had copper levels of less than 63.7 µg/dl, whereas more than one-third of the participants had copper levels higher than 140.12 µg/dl. Serum magnesium levels of all the studied patients were within the normal range (1.46–2.68 µg/dl).
Conclusion Empirical zinc, copper, and magnesium supplementation should not be regularly recommended in patients with β-thalassemia major.

Keywords: β-thalassemia, copper, erythropoiesis, magnesium, serum ferritin, zinc

How to cite this article:
Fahmy EM, Salama EH, Mohammed NA. Copper, zinc, and magnesium status among patients with thalassemia attending pediatric hematological unit at Sohag University Hospital. Egypt J Haematol 2019;44:98-104

How to cite this URL:
Fahmy EM, Salama EH, Mohammed NA. Copper, zinc, and magnesium status among patients with thalassemia attending pediatric hematological unit at Sohag University Hospital. Egypt J Haematol [serial online] 2019 [cited 2019 Dec 5];44:98-104. Available from: http://www.ehj.eg.net/text.asp?2019/44/2/98/271073

Introduction

Thalassemias are a group of genetic blood-born disorders caused by abnormal hemoglobin chain biosynthesis. This may involve either α-hemoglobin or β-hemoglobin chains. The abnormal hemoglobin chain synthesis necessitates ∼200 point mutations in that particular chain of hemoglobin. The lack of β chain synthesis causes β-thalassemia, which causes deficiency in the formation of new blood cells leading to anemia. β-Thalassemia major is a severe form of this blood disorder, where transfusion is needed for survival in these patients, and a bone marrow transplant may be the only way for complete cure [1].

β-Thalassemia is one of the most common inherited single gene disorder caused by ∼200 mutations in the β-globin genes. In β-thalassemia, although there is reduced or even no synthesis of β globin chains, the α chain will continue to be produced in normal or segmented rate. This augmented synthesis of α chains makes the resultant RBCs more fragile than usual, leading to ineffective erythropoiesis, early damage, and decreased life span of RBCs, and thus leading to anemia [2].

There are three main types of β-thalassemia, which are β-thalassemia major, β-thalassemia intermedia, and β-thalassemia minor. β-Thalassemia major is the most severe form of thalassemia, where the patients have severe transfusion-dependent anemia within their first 2 years of their life, thus requiring regular and repeatable RBC transfusions with desferrioxamine injections [3]. This severe hereditary anemia necessitates lifelong blood transfusion, and according to this modality of treatment, metabolic, endocrine, and growth abnormalities occur. Approximately 150 million people carry the thalassemia gene all over the world, and they are more prevalent in the Mediterranean countries. In Egypt, β-thalassemia major is the most common type of thalassemia, with carrier rate of up to 9% and more than 1000 new cases born with β-thalassemia major per year of the total live births of ∼1.5 million according to 2007 data [4].

Patients with thalassemia major can live longer and better if they obtain proper treatment. Patients with thalassemia have ineffective erythropoiesis and faster RBC turnover owing to the short life span of RBCs, which causes increased body requirements of energy and nutrients to keep normal erythropoiesis [5],[6].

Trace elements and minerals have a very important role in the body to achieve this. These elements and minerals should be present in the human body in their normal range and must be accessible to react with other elements or minerals to form many important and vital molecules, thus participating in the various important chemical reactions and physiological processes [7]. Copper is one of these important trace element; it is present in high levels in heart, kidney, and brain. Moreover, copper has an important role in the development of connective tissues, nerves, and bones. It also participates in many enzymatic reactions like reduction of superoxide dismutase, cytochrome oxidase, lysil oxidase, dopamine hydroxylase, and other oxidases, resulting in reduction in molecular oxygen function [8]. Many nutrient products contain copper, especially meats, oysters, nuts, seeds, whole grain, and dark chocolate. Evaluation of copper levels in patients with β-thalassemia major revealed contradictory results, with some report copper excess, whereas others report copper deficiency [9].

Zinc is also another essential micronutrients in the human body, acting as a cofactor for as high as 300 enzymes, and has essential roles in human growth and development [9]. Moreover, zinc plays a central role in heme production by activating δ-aminolevulinic acid dehydratase, an enzyme that catalyzes δ-aminolevulinic acid into porphobilinogen, which represents a common precursor for heme, cytochromes, and other hemoproteins [9],[10]. Some reports stated that patients having β-thalassemia major experience zinc deficiency. These reports claimed that this zinc deficiency may be the cause of delayed maturity in patients with thalassemia [11].

Magnesium is the second most copious intracellular mineral after potassium and plays an important role in the activation of many enzymes involved in some hematologic parameters of human β-thalassemia intercellular metabolism [12]. Abnormalities of magnesium metabolism have been described in patients with β-thalassemia, and low serum magnesium has been reported in children affected by the homozygous form of this disorder. Moreover, patients with heterozygous form of β-thalassemia and β-thalassemia intermedia show low erythrocyte magnesium content when compared with either normal controls or patients with other forms of anemia (e.g. hypochromic sideroblastic anemia) [13]. There is no plain explanation for the abnormalities in magnesium metabolism seen in β-thalassemia. There were no systematic studies on the possible role of magnesium deficiency in the anemia caused by β-thalassemia major.

The aim of this study is to evaluate zinc, copper, and magnesium levels among patients with β-thalassemia major and determine if these patients need copper, zinc, and magnesium empirical supplementation or not.

Patients and methods

Study design

It is a cross-sectional study conducted among patients with β-thalassemia major attending the pediatric hematological unit at Sohag university hospital within a period of 6 months from January 2018 to June 2018.

Patients

In this study, we included 35 patients diagnosed as having β-thalassemia major attended the pediatric hematological unit at Sohag University Hospital within the study period. We studied the serum levels of various trace elements in children with β-thalassemia major of both sexes aged below 18 years in this cross-sectional study by assessment of serum level of zinc, copper, and magnesium using classical colorimetric end-point technique by a semiautomated, programmable photometer 5010 (RIETE German company). Exclusion criteria were patients with serological evidence of hepatitis B or C, HIV infections, diabetes mellitus, thyroid dysfunction, autoimmune or systemic diseases, any metabolic hereditary diseases other than β-thalassemia major, and patients whose parents refused permission for participation in the study.

Methods

We initially enrolled 35 consecutive children with β-thalassemia major in the study; clinical diagnosis of β-thalassemia major was evaluated by an experienced pediatric hematologist. At the time of enrollment, demographic data, anthropometric measures, and medical and blood transfusion history were recorded for each patient. Written informed consent was obtained from parents of all enrolled participants, and this study protocol was reviewed and approved by scientific and ethical committees of Sohag Faculty of Medicine. Overall, 3 ml of peripheral blood was collected in a sterile plain vacutainer from 35 patients for serum zinc, copper, and magnesium assay. We excluded five cases (two blood sample collections were not sufficient to complete the test, and three were hemolysis samples). Serum for sample analysis was obtained after centrifugation of the whole blood, following complete clot formation has taken place, at 3000 rpm for 5 min and stored freeze at 80° for 6 months till the time of study. Estimation of serum level of zinc, copper, magnesium, and ferritin is performed by using photometer 5010 V5+. They were estimated by classical colorimetric end-point method using Randox reagent.

Principle for copper estimation

At pH 4.7, copper which is bound to ceruloplasmin is released by a reducing agent. It then reacts with a specific color reagent 4-(3.5-dibromo-2-pyridylazo)-N-ethyl-N-(3-sulphopropyl) aniline to form a stable colored chelate. The intensity of the color is directly proportional to the quantity of copper in the sample. Overall, 60 µl of nonhemolyzed serum sample is mixed with 1000 µl of the provided reagent 1 in a test tube. In another test tube, 60 µl of the provided standard reagent is mixed with 1000 µl of the provided reagent 1. In a third test tube, 60 µl of double distilled water is mixed with the provided reagent 1. The three tubes are mixed well and allowed to stand for 60 s at 37°C. Then initial absorbance (A₁) of sample and standard against the reagent blank at 580-nm wavelength was read. Then 250 µl of chromogen (R₂) reagent provided is added to each tube and mixed well, then incubated at 37°C for 5 min, and then final absorbance (A₂) against reagent blank is read.

Calculation

Copper reference value in serum sample according to the used reagents is 63.7–140.12 µg/dl for children.

Principle for zinc estimation

Zinc present in the sample is chelated by 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)-phenol in the reagent. The formation of this complex is measured at a wavelength of 560 nm. Overall, 500 µl of nonhemolyzed serum sample is mixed with 500 µl of the provided deproteinizing reagent in a test tube. In another test tube, 500 µl of standard reagent is mixed with 500 µl of the provided deproteinizing reagent. In a third test tube, 500 µl of double distilled water is mixed with the provided deproteinizing reagent. The three tubes are mixed well and then centrifuged for 10 min at 3000 rpm. Overall, 500 µl of supernatant in the zinc assay is used within 2 h. The provided color reagents A and B are mixed in a 4 : 1 ratio for preparation of working reagent. This working reagent is stable for 2 days at 15–5°C or 1 week at 2–8°C. Overall, 500 µl of supernatant from test sample is mixed with 2500 µl of the working reagent in a test tube. In another test tube, 500 µl of supernatant from standard is mixed with 2500 µl of the working reagent. In a third test tube, 500 µl of supernatant of blank double distilled water is mixed with the 2500 µl of the working reagent. They are mixed well and then incubated at 25°C for 5 min. Absorbance of the standard and the sample against the reagent blank is measured within 60 min at wavelength of 560 nm.

Calculation

Zinc reference value in the serum sample according to the used regent is 66–110 µg/dl for children.

Principle for magnesium estimation

Magnesium ions react with xylidyl blue in an alkaline medium to form a water-soluble purple-red chelate, whose color intensity is highly proportional to magnesium concentration in the sample. Calcium is excluded from the reaction by mixing with EGTA. Overall, 20 µl of nonhemolyzed serum sample is mixed with 2000 µl of the provided color reagent in a test tube. In another test tube, 20 µl of provided standard reagent is mixed with 2000 µl of the provided color reagent. In a third test tube, 20 µl of double distilled water is mixed with the provided color reagent. The three tubes are mixed well and incubated for 5 min at room temperature. Then, final absorbance of the sample and standard is measured against the reagent blank at 520-nm wavelength. The final color is stable for 3 h at room temperature.

Calculation

Magnesium reference value in the serum sample according to the used reagent is 1.46–2.68 mg/dl.

Data analysis

Data were analyzed using IBM SPSS Statistics for Windows, version 22.0 (IBM SPSS by Stata release 15). Quantitative data were expressed as mean±SD, median, and range. Qualitative data were expressed as number and percentage. Quantitative data were tested for normality by Shapiro–Wilk test. Independent Samples t-test and one-way analysis of variance test were used for normally distributed data. Mann–Whitney U-test, Kruskal–Wallis test, and Spearman’s correlation were used for data that were not normally distributed. χ²-Test and Fisher’s exact test were used for comparison of qualitative variables as appropriate. A 5% level was chosen as a level of significance in all statistical tests used in the study.

Results

The current study included 30 patients with thalassemia, of whom 22 (73.3%) were males. The mean age of the studied patients was 7.67±4.02 years. More than three-fourths of the studied patients (76.7%) were dependent on blood transfusion. Those who received iron chelation were 33.3%, and 26.7% of the participants were hydroxyurea recipients. One-fifth (20%) of the studied patients underwent splenectomy ([Table 1]). [Table 2] shows the means of the studied nutrients among the participants. The mean zinc level was 115.37±30.01 µg/dl, the mean copper level was 130.47±58.09 µg/dl, and the mean magnesium level was 1.86±0.28 µg/dl. Only 6.7% of the studied patients had serum zinc levels of less than 66 µg/dl and 13.3% had copper levels of less than 63.7 µg/dl, whereas more than one-third of the participants had copper levels higher than 140.12 µg/dl. Serum magnesium levels of all the studied patients were within the normal range (1.46–2.68 µg/dl; [Table 3]). No significant correlation was found between serum copper and zinc levels and serum ferritin level. On the contrary, a significant negative moderate correlation between serum magnesium and ferritin levels was found ([Table 4]). As shown in [Table 5] and [Table 6], there was no significant association between the levels of the studied nutrients among the participants and their age, sex, weight, height, and disease-related data (P>0.05).

Table 1 Distribution of the studied patients by age, sex, anthropometric measures, and disease-related criteria (N=30)

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Table 2 Serum ferritin, zinc, copper, and magnesium levels in patients with thalassemia (N=30)

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Table 3 Serum zinc, copper, and magnesium levels in patients with thalassemia

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Table 4 Correlation of serum ferritin, zinc, copper, and magnesium levels among patients with thalassemia

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Table 5 Relation between age, sex, anthropometric measures, disease-related criteria, and zinc level

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Table 6 Relation between copper level and disease-related criteria

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Discussion

Thalassemia major is a severe form of the β-thalassemia disease, and the patients require frequent blood transfusions and chelation therapy to maintain their lives. Although with new therapies, the patients’ life span has increased up to the fourth or fifth decades; these patients are still more liable to a variety of complications such as abnormal growth, endocrinopathy, hypogonadism, and others [14].

Trace elements and minerals have an essential role in appropriate functioning of the body. They should be present in the body in proper amounts and must be existent for reacting with other elements to produce critical molecules as well as to share in different important chemical reactions [15]. Zinc is one of the important trace elements, which has been well recognized for its role in human health [16],[17],[18],[19],[20]. It is important to evaluate the status of zinc between the general population, preferably in children [21],[22].

In the present study, we found that zinc level was normal in 93.3% of our included patients, whereas hypozincemia was present in only two (2.6%) cases. Similar to our study, the study of Reshadat et al. [23] showed that ∼77% of patients with thalassemia had normal serum zinc level and the remainder had even more than normal levels, so medical treatment of these patients is not suitable, and the value of giving zinc should be more assessed. Another report from Pakistan by El Missiry et al. [24] stated that there was no zinc deficiency among patients with thalassemia major as proved from normal zinc activities among patients with β-thalassemia major. Moreover, Mehdizadeh et al. [25] stated that the mean serum zinc level was considerably higher in patients with thalassemia compared with controls. They also noted that zinc deficiency is very uncommon among patients with thalassemia. On the contrary, in contrast to the present study and the aforementioned studies, Tabatabei et al. [26] reported that up to 84.8% of patients with thalassemia major had zinc deficiency. They claimed that zinc deficiency in these patients may be owing to insufficient dietary zinc. Moreover, Yazdideha and Faranosh [27] reported that the mean serum concentration level of zinc among patients with thalassemia was much lower than that of the control group; with a significant difference. As a result, they recommended zinc supplement for patients with thalassemia. It was suggested by these authors that the cause of zinc deficiency is mainly malnutrition and insufficient dietary zinc, and so, they recommended zinc supplementation for these patients [28]. In addition, the study by Nidumuru et al. [29] in India showed that the mean role of serum zinc among β-thalassemia major cases (39.25±13.45) was found to be significantly lower than the control subjects (85.31±13.53). In Egypt, the study by Sherief et al. [30] reported significantly lower levels of serum zinc in thalassemic group compared with controls. They explained that the reason of zinc deficiency may be inadequate dietary zinc, chronic hemolysis, and deferoxamine and deferiprone medications [31].

Copper is a trace element that had an vital role in the development of nerve, bone, and connective tissues [8]. This trace element plays the role of cofactor for at least 30 enzymes [32]. In the present study, serum copper level was normal in 50% of the patients with β-thalassemia major, being higher than normal in 36.7% and lower in 13.3% of the cases. There are contradictory results about serum copper in patients with thalassemia. This may be partially owing to the fact that the serum copper concentration in patients with β-thalassemia major depends on multiple factors such as dietary intake and intestinal absorption of copper, iron accumulation, renal function, copper to zinc ratio, and intake of deferoxamine [33]. Changes in one or more of these factors will seriously affect the serum copper level. Some studies reported increased serum level of copper in patients with β-thalassemia major [34]. Al-Samarrai et al. [35] hypothesized that the cause of hypercupremia is hemochromatosis, which is one of the main complication of thalassemia. On the contrary, some other reports show decrease in serum level of copper, whereas Mahyar et al. [36] showed no change in serum copper concentration as the factors that affect copper level are under control. In Egypt, Sherief and colleagues showed significant low copper level in patients with β-thalassemia major compared with the control group, with the mean level of copper of 12.2 and 90.5 µg/dl, respectively, and they explained that by the factors that affect copper levels were not accurately controlled in patients with thalassemia [37]. Magnesium is the second most profuse intracellular metal after potassium which is necessary for maintaining appropriate body functions. Cell magnesium is an important modulator in the regulation of cell volume and also affects the activity of transport pathways of different membrane cation [38]. It is essential for body’s immune system, cardiovascular, and also the musculoskeletal systems. Deficiency of this element will cause hypertension, diabetes, and cardiovascular diseases. In this study, the serum magnesium level was within normal range in all included children with β-thalassemia major. The study by Arcasoy and Cavdar [39] also showed that magnesium levels were within normal levels in patients with thalassemia, whereas serum magnesium levels were significantly higher than normal control in the study by Al-Samarrai et al. [40]. Other studies revealed low serum Mg in children affected by the homozygous form of β-thalassemia [41]; therefore, they advise using magnesium supplementation to stabilize damaged red blood cell by its specific interactions with K–Cl cotransport and effects on the red blood cell membrane and improved erythrocyte survival and morphology [39].

Conclusion

We concluded from our study that there is no zinc, copper, and magnesium deficiency in our patients of β-thalassemia major, and empirical supplementation of them is not recommended. Different levels in different studies suggest variation in the factors influencing their levels in each patient, like dietary intake and the regular use of blood transfusion and chelating programs, so we recommend regular measurement of these elements in patients with β-thalassemia major to detect the needed patients of supplementation. Moreover, we recommend studying other trace elements in serum levels and various vitamins in a wider scale of patients with β-thalassemia major to improve our knowledge about the need to use empirical supplementation in treating these patients.

Financial support and sponsorship

Nil.

Conflicts of interest

Their is no need for zinc, copper or magnesium empirical supplementation in Sohag pediatric B thalaseima major patients.

References

1.	Thein SL. Molecular basis of β thalassemia and potential therapeutic targets. Blood Cells Mol Dis 2018; 70:54-65.
2.	Elizabeth G, Ann MTJA. Genotype–phenotype diversity of beta thalassemia in malaysia: treatment options and emerging therapies. Med J Malaysia 2010; 65:256–260.
3.	Naeem S, Fouzia B, Rubab N, Ghulam MS, Hafiz A, Abdul Q. Role of iron chelation therapy for beta-thalassemia major: a review. J Appl Environ Biol Sci 2014; 4:17–25.
4.	El-Beshlawy A, Kaddah N, Moustafa A, Mouktar G, Youssry I. Screening for Β-thalassaemia carriers in Egypt: significance of the osmotic fragility test. East Mediterr Health J 2007; 13:780–786.
5.	Borgna-Pignatti C. Modern treatment of thalassaemia intermedia. Br J Haematol 2007; 138:291–304.
6.	Fung EB. Nutritional deficiencies in patients with thalassemia. Ann N Y Acad Sci 2010; 1202:188–196.
7.	Claster S, Wood JC, Noetzli L, Carson SM, Hofstra TC, Khanna R, Coates TD. Nutritional deficiencies in iron overloaded patients with hemoglobinopathies. Am J Hematol 2009; 84:344–348.
8.	Fraga CG. Review relevance, essentiality and toxicity of trace elements in human health. Mol Aspects Med 2005; 26:235–244.
9.	Mahyar A. The preventive role of zinc from communicable and non-communicable diseases in children. NCD Malaysia 2005; 4:21–26.
10.	Marengo-Rowe AJ. The thalassemias and related disorders. Proc (Bayl Univ Med Cent) 2007; 20:27–31.
11.	Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis 2010; 5:11.
12.	Rayssignier Y, Gueux E, Motta C. Magnesium deficiency effects on fluidity and function of plasma and subcellular membranes. In: Lasserre B, Durlach J, editors. Magnesium: a relevant ion. New York, NY: Libbey; 1991. p. 311.
13.	De Franceschi L, Cappellini MD, Olivieri O, Graziadei G, Fiorelli F, Corrocher R et al. Modulation of erythrocyte potassium chloride cotransport, potassium content, and density by dietary magnesium intake in transgenic SAD mouse. Blood 1996; 88:2738-2744.
14.	Quirolo K, Vichinnky E. Hemoglobin disorders. In: Behraman RE, editor. Nelson textbookof pediatrics. 18th ed. Philadephia, PA: Saunders; 2007. pp. 2033–2039.
15.	Shazia Q, Mohammad ZH, Rahman T, Shekhar HU. Correlation of oxidative stress with serum trace element levels and antioxidant enzyme status in beta thalassemia major patients: a review of the literature. Anemia 2012; 2012:7.
16.	Gammoh NZ, Rink L. Zinc in infection and inflammation. Nutrients 2017; 9:624.
17.	Jarosz M, Olbert M, Wyszogrodzka G, Młyniec K, Librowski T. Antioxidant and anti-inflammatory effects of zinc. Zinc-dependent NF-κB signaling. Inflammopharmacology 2017; 25:11–24.
18.	Terrin G, Canani BR, Di Chiara M, Pietravalle A, Aleandri V, Conte F, De Curtis M. Zinc in early life: a key element in the fetus and preterm neonate. Nutrients 2015; 7:10427–10446.
19.	Prasad AS. Discovery of human zinc deficiency: its impact on human health and disease . Adv Nutr 2013; 4:176–190.
20.	Maret W. Zinc biochemistry: from a single zinc enzyme to a key element of life. Adv Nutr 2013; 4:82–91.
21.	Cantoral A, Téllez-Rojo M, Shamah-Levy T, Schnaas L, Hernández-Ávila M, Peterson KE, Ettinger AS. Prediction of serum zinc levels in Mexican children at 2 years of age using a food frequency questionnaire and different zinc bioavailability criteria. Food Nutr Bull 2015; 36:111–119.
22.	Galetti V, Mitchikpè CE, Kujinga P, Tossou F, Hounhouigan DJ, Zimmermann MB, Moretti D. Rural Beninese children are at risk of zinc deficiency according to stunting prevalence and plasma zinc concentration but not dietary zinc intakes. J Nutr 2016; 146:114–123.
23.	Reshadat S, Kiani A, Iranfar SH. Zinc level of major thalassemic patients in Kermanshah. Behbood 2006; 2:157–167.
24.	El Missiry M, Hamed Hussein M, Khalid S, Yaqub N, Khan S, Itrat F et al. Assessment of serum zinc levels of patients with thalassemia compared to their siblings. Anemia 2014; 2014:125452.
25.	Mehdizadeh M, Zamani G, Tabatabaee S. Zinc status in patients with major beta-thalassemia. Pediatr Hematol Oncol 2008; 25:49–54.
26.	Tabatabei M, Kamkar M, Habibzadeh MR. Metabolic and endocrine complications inβ-thalassemia major; a multicenter study in Tehran. Boshehr Med J 2003; 5:72–73.
27.	Yazdideha MS, Faranosh M. Evaluation of serum zinc in children affected with betathalassemic patients. Res Med 2004; 24:7–9.
28.	Hashemipoor M, Modaresi MR, Sepah Vand N, Shahabi I, Ahmadiniar A. Zinc concentration in thalassemic patient’s hair. J Med Faculty Guilan Uni Med Sci 2000; 33:74–78.
29.	Nidumuru S, Boddula V, Vadakedath S, Kolanu BR, Kandi V. Evaluating the role of zinc in beta thalassemia major: a prospective case–control study from a tertiary care teaching hospital in India. Cureus 2017; 9:1495.
30.	Sherief LM, Abd El-Salam SM, Kamal NM, El Safy O, Almalky MA, Azab SF et al. Nutritional biomarkers in children and adolescents with beta-thalassemia-major: an Egyptian center experience. BioMed Res Int 2014; 2014:261761.
31.	Mahyar A, Ayazi P, Pahlevan A-A, Mojabi H, Sehhat M-R, Javadi A. Zinc and copper status in children with beta-thalassemia major. Iran J Pediatr 2010; 20:297–302.
32.	Anderson JB. Minerals. In: Escott-Stump KL, Krause S, editors. Food, nutrition and diet therapy. 11th ed. Philadelphia, PA: Sunders; 2004. pp. 134–154.
33.	Bashir NA. Serum zinc and copper levels in sickle cell anaemia and β-thalassaemia in North Jordan. Ann Trop Paediatr 1995; 15:291–293.
34.	Claster S, Wood JC, Noetzli L, Carson SM, Hofstra TC, Khanna R, Coates TD. Nutritional deficiencies in iron overloaded patients with hemoglobinopathies. Am J Hematol 2009; 84:344–348.
35.	Eshghi P, Alavi S, Ghavami S, Rashidi A. Growth impairment in β-thalassemia major: the role of trace element deficiency and other potential factors. J Pediatr Hematol Oncol 2007; 29:5–8.
36.	Mahyar A, Ayazi P, Pahlevan AA, Mojabi H, Sehhat MR, Javadi A. Zinc and copper status in children with Beta-thalassemia major. Iran J Pediatr 2010; 20:297–302.
37.	Franceschi LD, Brugnara C, Beuzard Y. Dietary magnesium supplementation ameliorates anemia in a mouse model of beta-thalassemia. Blood 1997; 90:1283–1290.
38.	Shazia Q, Mohammad ZH, Rahman T, Shekhar HU. Correlation of oxidative stress with serum trace element levels and antioxidant enzyme status in beta thalassemia major patients: a review of the literature. Anemia 2012; 2012:270923.
39.	Arcasoy A, Cavdar AO. Changes of trace minerals (serum iron, zinc, copper and magnesium) in thalassemia. Acta Haematol 1975; 53:341–346.
40.	Al-Samarrai AH, Adaay MH, Al-Tikriti KA. Evaluation of some essential element levels in thalassemia major patients in Mosul district. Iraq Saudi Med J 2008; 29:94–97.
41.	Abbasciano V, Bader G, Graziano L, Mazzotta D, Vecchiati G, Guglielmini C et al. Serum and erythrocyte levels of magnesium in microcytosis: comparison between heterozygous beta-thalassemia and sideropenic anemia. Haematologica 1991; 76:339-341.