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American Journal of Clinical Nutrition, Vol. 88, No. 4, 1026-1031, October 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Iron metabolism in heterozygotes for hemoglobin E (HbE), {alpha}-thalassemia 1, or β-thalassemia and in compound heterozygotes for HbE/β-thalassemia1,2,3

Michael B Zimmermann, Suthat Fucharoen, Pattanee Winichagoon, Pornpan Sirankapracha, Christophe Zeder, Sueppong Gowachirapant, Kunchit Judprasong, Toshihiko Tanno, Jeffery L Miller and Richard F Hurrell

1 From the Laboratory for Human Nutrition, Swiss Federal Institute of Technology Zürich, Zürich, Switzerland (MBZ, CZ, and RFH); the Thalassemia Research Center, Institute of Science and Technology for Research and Development (SF and PS) and the Institute of Nutrition (PW, SG, and KJ), Mahidol University, Salaya, Nakon Pathom, Thailand; and the Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD (TT and JLM)

2 Supported by SUSTAIN (Washington, DC), The Global Alliance for Improved Nutrition (Geneva, Switzerland), the International Atomic Energy Agency (Vienna, Austria), and the Swiss Federal Institute of Technology (ETH) Zürich (Zürich, Switzerland). SF is a senior research scholar of The Thailand Research Fund.

3 Reprints not available. Address correspondence to MB Zimmermann, Laboratory for Human Nutrition, Swiss Federal Institute of Technology (ETH) Zürich, Schmelzbergstrasse 7, LFV E19, CH-8092 Zürich, Switzerland. E-mail: michael.zimmermann{at}ilw.agrl.ethz.ch.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Despite large populations carrying traits for thalassemia in countries implementing universal iron fortification, there are few data on the absorption and utilization of iron in these persons.

Objective: We aimed to determine whether iron absorption or utilization (or both) in women heterozygous for β-thalassemia, {alpha}-thalassemia 1, or hemoglobin E (HbE) differed from that in control subjects and compound HbE/β-thalassemia heterozygotes.

Design: In Thai women (n = 103), red blood cell indexes, iron status, non-transferrin-bound iron, and growth differentiation factor 15 were measured, and body iron was calculated. Fractional iron absorption was measured from meals fortified with isotopically labeled (57Fe) Fe sulfate, and iron utilization was measured by the infusion of (58Fe) Fe citrate.

Results: Iron utilization was {approx}15% lower in {alpha}-thalassemia 1 or β-thalassemia heterozygotes than in controls. When corrected for differences in serum ferritin, absorption was significantly higher in the {alpha}- and β-thalassemia groups, but not the HbE heterozygotes, than in controls. HbE/β-thalassemia compound heterozygotes had lower iron utilization and higher iron absorption and body iron than did controls. Nontransferrin-bound iron and growth differentiation factor 15 were higher in the compound heterozygotes, but not in the other groups, than in the controls.

Conclusions: In {alpha}-thalassemia 1 and β-thalassemia heterozygotes with ineffective erythropoesis, dietary iron absorption is not adequately down-regulated, despite a modest increase in body iron stores. In populations with a high prevalence of these traits, a program of iron fortification could include monitoring for possible iron excess and for iron deficiency.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Globally, iron excess occurs mainly in persons with geographically specific genetic mutations that permit the absorption from the diet of more iron than is physiologically needed. Two main types of hereditary iron overload are well recognized: 1) hereditary hemochromatosis, which is seen in populations derived from northern Europe, and 2) the thalassemias and related hemoglobinopathies of South and Southeast Asia, the Middle East, and the Mediterranean (1). Thalassemia mutations are extremely common (2): up to 25% of Thai people are carriers of {alpha}-thalassemia, and, in regions of Thailand, Laos, and Cambodia, up to 60% of people are carriers of hemoglobin E (HbE), a hemoglobinopathy caused by a mutation of the β-globin gene. In southern China, which has a population of >350 million, 5% of people are carriers for {alpha}-thalassemia and 4% are carriers for β-thalassemia or HbE (3).

Heterozygotes for {alpha}-thalassemia 1, β-thalassemia, and HbE typically are asymptomatic and have mild microcytic, hypochromic anemia. In contrast, in thalassemia homozygotes and compound heterozygotes such as HbE/β-thalassemia compound heterozygotes, ineffective erythropoiesis in an expanded marrow stimulates iron absorption even if iron stores are adequate, and this stimulation increases the risk of iron excess when transfusions are given (4, 5). If heterozygotes have some degree of ineffective erythropoesis and absorb more dietary iron, they may, to a lesser degree than homozygotes and compound heterozygotes, also be at risk of iron excess. In heterozygotes for hemochromatosis, one study reported that iron absorption from a meal with added iron was 3-fold that in controls (6); another study found no differences in absorption (7).

Despite large populations that are heterozygous for thalassemia in countries implementing iron fortification, there are few data on the absorption and utilization of iron in these persons. Earlier studies in β-thalassemia heterozygotes are difficult to interpret because they used oral solutions of 59Fe given with ascorbic acid that overestimate dietary iron absorption and made comparisons without adjustment for differences in iron status (4, 8–11). In countries where thalassemias are common, public health officials are hesitant to adopt iron fortification because of concerns about possible iron excess in thalassemia carriers. Therefore, our study aim was to determine whether iron metabolism—absorption and utilization (incorporation into erythrocytes)—in heterozygotes for β-thalassemia, {alpha}-thalassemia 1, or HbE differs from that in controls with normal hemoglobin A (HbA) or in symptomatic compound HbE/β-thalassemia heterozygotes.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
The study was carried out in Thai women recruited from the western suburbs of Bangkok. Inclusion criteria were age 18–50 y; premenopausal status; body weight < 65 kg; self-reported nonpregnancy and no plans for pregnancy; no chronic medical illnesses; and desired Hb type: heterozygotes for β-thalassemia; heterozygotes for {alpha}-thalassemia 1; HbE heterozygotes; compound HbE/β-thalassemia heterozygotes; and normal HbA. None of the subjects were taking medicinal iron at entry into the study. Twelve percent of the women, distributed through the 5 groups, were taking oral contraceptives. Sample size calculations indicated that 24 women should be included in the control group and the groups heterozygous for β-thalassemia, {alpha}-thalassemia 1, and HbE. This number was based on 80% power to detect a 50% difference in iron absorption compared with the control subjects, an SD of 8.2% for log-transformed absorption data from previous absorption studies with the same meal and iron compound in a similar population of Thai women, and a type I error rate of 5%. We also studied 9 compound heterozygotes for HbE/β-thalassemia, a disorder with a clinical phenotype; these persons exhibit clearly ineffective erythropoesis and were therefore studied as positive controls (4, 5).

Written informed consent was obtained from all subjects. Ethical approval for the study was given by ethical review committees at Mahidol University (Salaya, Nakon Pathom, Thailand) and the Swiss Federal Institute of Technology (ETH, Zürich, Switzerland).

Iron absorption and utilization study
Iron absorption was estimated by using stable-isotope techniques in which the incorporation of 57Fe and 58Fe into erythrocytes is measured 14 d after administration (12, 13). If the subjects were taking vitamin or mineral supplements or any other medications, administration was stopped for 2 wk before the study and until the final venipuncture. On day 1, a baseline venous blood sample was drawn after an overnight fast for determination of isotopic composition and confirmation of iron status. The subjects then received a test meal (rice with vegetable soup; see below) labeled with 57Fe as ferrous sulfate in fish sauce, which was fed under standardized conditions and close supervision. Each test meal contained 3 mg labeled 57Fe. One hour later, 2 mL of an aqueous solution containing 100 µg 58Fe as iron citrate was taken into a syringe containing 10 mL of 0.9% saline solution and, via a 100-mL infusion bag leading into a 0.9% saline drip, slowly infused over 50 min (7, 14). The rate of intravenous infusion of iron was based on the estimated 2 µg/min plasma appearance of iron normally absorbed from the gastrointestinal tract (7). No intake of food and fluids was allowed for 4 h after the test meal intake. Fourteen days later, a second venous blood sample was drawn.

Preparation of isotopically labeled iron
57Fe was prepared from isotopically enriched 57Fe by dilution in 0.1 mol H2SO4/L (12). The solution was kept refrigerated in plastic containers, under argon atmosphere. Iron citrate, enriched with 58Fe, was prepared for intravenous infusion from elemental 58Fe according to the method previously described (14). The solution was divided in ampoules containing 100 µg Fe, sterilized, and checked for pyrogens. Enrichment of isotopic labels was 95.5% for 57Fe and 93.1% for 58Fe. The isotopic composition of the stable-isotope labels was measured by using negative thermal ionization–mass spectrometry (13). Iron-isotope solutions were divided into aliquots and sterilized at the Zürich University Hospital Pharmacy (Zürich, Switzerland).

Test meal composition
The test meal was composed of steamed white rice (50 g dry weight; Jasmine perfume rice; Dragon Phoenix Brand, Bangkok, Thailand), which was served with a vegetable soup containing white cabbage, Chinese cabbage, Thai mushrooms, and steamed carrots in 120 mL of water and seasoned with fish sauce. All ingredients were purchased in bulk and used for the entire study. The food portions were kept frozen until use, and each portion was microwaved on the day of feeding.

Laboratory analysis
Whole blood was transported on ice to the thalassemia laboratory at Mahidol University. Complete blood count and reticulocyte count was done by using the Advia 120 Hematology System with 3-level Advia 120 TESTpoint hematology controls (Bayer, Singapore). Hemoglobin typing for β-globin abnormality was done by using HPLC (Variant Hemoglobin Testing System; BioRad, Hercules. CA) with calibrators and controls provided by the manufacturer. DNA analysis for {alpha}-globin abnormalities was done by using a GeneAmp PCR System (Applied Biosystem, Foster City, CA) and a Gel Doc 2000 Gel Documentation System (BioRad, Hercules, CA). Serum ferritin (SF) and serum transferrin receptor (TfR) measurements were made by using an enzyme-linked immunosorbent assay (ELISA; Ramco Laboratories Inc, Stafford, TX) on microplates (CODATM Open Microplate System; BioRad); normal ranges: SF, 12–300 µg/L; TfR, <8.2 mg/L. Serum erythropoietin was measured by using an ELISA (R&D Systems, Minneapolis, MN) with controls provided by the manufacturer; the normal range is 3.3–16.6 IU/L. Serum iron (SI), total iron–binding capacity, and transferrin saturation were measured by using colorimetric spectrophotometry (UV-Vis Spectrophotometer 8453; Hewlett-Packard, Palo Alto, CA); the normal range is 9–29 µmol/L for SI, 45–70 µmol/L for total iron–binding capacity, and 30–50% for transferrin saturation. High-sensitivity C-reactive protein was measured by using latex nephelometry (BNProSpec System; Dade Behring Limited, Tokyo, Japan); the normal range is <3 mg/L. SF data from one subject with a CRP concentration > 10 (a HbE/β-thalassemia compound heterozygote) were excluded from the data analysis. For measurement of plasma NTBI, plasma (450 µL) was incubated with 800 mmol nitrilotriacetate/L, pH 7.0 solution (50 µL), at room temperature for 30 min. Plasma proteins were then removed by centrifugation of the treated plasma with the use of an ultracentrifugation filtration device (30-kDa cutoff, polysulfone type, NanoSep; Pall Life Sciences, Ann Arbor, MI) at 12 000 rpm (10620 x g; Hettich Centrifugation, Bach, Germany) and 15 °C for 45 min. The ultrafiltrate was then analyzed by HPLC (15). Nontransferrin-bound iron (NTBI) concentrations in healthy subjects using this method are <0 µmol/L. Serum growth differentiation factor 15 (GDF-15) was measured in blinded samples by using a DuoSet ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. Whole blood was mineralized by microwave digestion, and iron was separated by anion-exchange chromatography and a subsequent solvent-solvent extraction step into diethylether. Iron was analyzed by negative thermal ionization–mass spectrometry with a magnetic sector field mass spectrometer (Finnigan MAT 262; Thermo Finnigan, Bremen, Germany) equipped with a multicollector system for simultaneous ion beam detection; isotopic dilution calculations were done as described previously (13).

Statistical analysis
Data were analyzed by using PRISM (version 3; GraphPad, San Diego, CA) and EXCEL (XP 2002; Microsoft, Redmond, WA) software. Body iron stores (BFe) were calculated from the ratio of TfR to SF (TfR:SF) (16). The amount of 57Fe and 58Fe label present in the blood was calculated from isotope dilution. Circulating iron was calculated from the blood volume and hemoglobin concentration. The amount of stable isotope administered was used to calculate the fractional iron incorporation into erythrocytes (13). The absorption of the oral iron was calculated by dividing the percentage of erythrocyte incorporation of the oral dose by the fractional erythrocyte incorporation of the intravenous dose (7, 14). Results were presented as geometric means ± SDs. Analysis of variance with unpaired t tests and a Bonferroni correction were used to compare the incorporation and absorption data between the groups, and values for iron incorporation and absorption were logarithmically transformed before statistical analysis. In the overall analysis of variance and in the Bonferroni correction, P < 0.05 and P < 0.005, respectively, were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Age, anthropometric data, and hematologic indexes by group are shown in Table 1Go. There were no significant between-group differences in age, height, or weight, except that the HbE group was significantly heavier than the normal hemoglobin and HbE/ β-thalassemia groups (P < 0.05). Compared with controls, the {alpha}- and β-thalassemia and HbE heterozygote groups had higher RBC concentrations and lower mean corpuscular volume and mean corpuscular hemoglobin (P < 0.05). The {alpha}- and β-thalassemia groups had significantly lower hemoglobin than did the control subjects (P < 0.02), and most of these heterozygotes were mildly anemic. The HbE/β-thalassemia group had significantly lower RBC, hemoglobin, and mean corpuscular hemoglobin and significantly greater RBC distribution width than did the other groups (P < 0.01 for all).


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  		TABLE 1 Age, anthropometric variables, and hematologic indexes in Thai women by hemoglobin (Hb) type1

 
The iron status indicators are shown in Table 2Go. The HbE heterozygotes did not differ from the control subjects in any of the iron indexes. The {alpha}-thalassemia 1 heterozygotes had significantly (P < 0.05) higher SF, BFe, and erythropoietin (but not NTBI) than did control subjects. The β-thalassemia heterozygotes had significantly (P < 0.05) higher SF, TfR, and BFe (but not NTBI) than did control subjects. The β-thalassemia heterozygotes had slightly higher NTBI than did control subjects, but this difference was not significant. The HbE/β-thalassemia group had significantly higher serum iron, transferrin saturation, SF, TfR, BFe, erythropoietin, NTBI, and GDF-15 concentrations than did the other groups (P < 0.01 for all).


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  		TABLE 2 Iron indexes and biochemical variables in Thai women by hemoglobin (Hb) type1

 
As shown in Table 3Go, the incorporation of iron into erythrocytes (utilization) from the intravenous iron dose was significantly (P < 0.05) lower in the {alpha}- and β-thalassemia groups (but not the HbE group) than in control subjects. Uncorrected fractional iron absorption from the test meal was slightly but significantly (P < 0.05) lower in the β-thalassemia heterozygotes than in the other 4 groups. When corrected for differences in SF, absorption was significantly (P < 0.05) higher in the {alpha}- and β-thalassemia groups (but not the HbE heterozygotes) than in control subjects. The HbE/β-thalassemia group had significantly higher absorption and lower incorporation than did the other groups (P < 0.02 for both). The relation between GDF-15 and erythrocyte incorporation of 58Fe is shown in Figure 1Go.


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  		TABLE 3 Iron absorption and erythrocyte iron utilization in Thai women by hemoglobin (Hb) type1

 

Figure 1
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  		FIGURE 1. The relation between erythrocyte incorporation of 58Fe and log growth differentiation factor 15 (GDF-15) in Thai women (n = 103) who had normal hemoglobin A; who were heterozygous for β-thalassemia, {alpha}-thalassemia 1, or hemoglobin E (HbE); or who were compound heterozygous for HbE/β-thalassemia.

 
In a stepwise regression of all the hematologic variables in Tables 1Go and 2Go on erythrocyte incorporation of 58Fe, only hemoglobin was a significant positive predictor, and SF and GDF-15 were negative predictors (Table 4Go). In a stepwise regression of these variables on fractional absorption of 57Fe, significant negative and positive predictors were BFe and the red blood cell distribution width, respectively (Table 4Go).


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  		TABLE 4 Stepwise regressions of the hematologic variables in 1 and 2 on the dependent variables of erythrocyte incorporation of 58Fe and fractional absorption of 57Fe1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Early ferrokinetic studies in β-thalassemia homozygotes reported that erythrocyte incorporation of iron was as low as 15%, whereas it was 75–90% in control subjects (4,8–11). In subjects with β-thalassemia intermedia, iron absorption from labeled meals varied from 26% to 54%, a range that is sharply higher than values in control subjects (4). These studies and others (17) suggested that greater iron absorption and lower utilization in β-thalassemia homozygotes resulted in large increases in body iron that exceeded iron-binding capacity (18, 19). At the same time, there appeared to be a certain component of functional iron deficiency in subjects with thalassemia, because excess iron stores were not entirely available for erythropoiesis (20–22).

If thalassemia heterozygotes partially express the homozygote phenotype (ineffective erythropoiesis and higher plasma iron turnover) and absorb significantly more dietary iron, they may accumulate iron and be susceptible to damage from excess body iron. In previous studies reporting SF or serum iron concentrations (or both) in persons who were heterozygous for β-thalassemia, most found that SF concentrations were similar to or modestly higher than those in healthy persons (23–29). Heterozygotes for β-thalassemia also have higher erythropoietin and TfR concentrations (30, 31) than do control subjects, which indicates a modest increase in erythropoietic drive. Whereas TfR concentrations are higher than those in control subjects, they are lower than those in subjects with iron-deficiency anemia, which suggests ineffective erythropoiesis (32). Previous studies of iron absorption in thalassemia heterozygotes from the 1960s and 1970s were done by using oral solutions of 59Fe given with ascorbic acid (4, 8–11), a method that significantly overestimates true dietary iron absorption (33, 34). Those studies produced mixed results, and a review (8) concluded that mild erythropoetic hyperplasia in heterozygotes for β-thalassemia does not influence iron absorption, and, if iron absorption increases, that change is due to concurrent iron deficiency. The findings of the present study differ. In women heterozygous for {alpha}- or β-thalassemia, erythrocyte iron incorporation was {approx}15% lower, whereas serum TfR or erythropoietin concentrations (or both) were significantly higher, which indicated ineffective erythropoesis and increased erythropoetic drive. At the same time, iron absorption was less well regulated by the increase in iron stores and was approximately twice that expected for the level of iron stores in persons with normal hemoglobin. This higher iron absorption resulted in modest increases in storage iron as reflected by significantly higher concentrations of SF and BFe.

Both heterozygotes and homozygotes for HbE, the most common form of β-thalassemia, are asymptomatic and minimally anemic, and they have microcytic and hypochromic red blood cells. However, when the βE allele interacts with a β-thalassemia mutation in the compound heterozygous state, a variable and often severe anemia is produced (35) that has evidence of ineffective erythropoiesis and shortened red blood cell survival (5, 36). In the women in the present study who were heterozygous for HbE, erythrocyte incorporation of 58Fe and dietary iron absorption were similar to those in control subjects. However, the HbE/β-thalassemia compound heterozygotes showed sharply lower erythrocyte iron incorporation and significantly higher iron absorption, body iron, and plasma NTBI.

Hepcidin concentrations should be high in iron-loaded persons with β-thalassemia (37); however, hepcidin concentrations are low in these persons, unless they have recently received a transfusion (30, 38, 39). Production of GDF-15 by the expanded erythroid compartment contributes to iron overload in thalassemia by inhibiting hepcidin gene expression (40). Compared with the concentration in control subjects, The concentrations of serum GDF-15 in persons with β-thalassemia syndromes is >100-fold those in control subjects, but they are not significantly higher in heterozygotes for {alpha}- and β-thalassemia (40). Our data support these findings in that, in the HbE/β-thalassemia compound heterozygotes, serum GDF-15 was sharply higher ({approx}40-fold) than in control subjects, whereas GDF-15 in heterozygotes for HbE or {alpha}- or β-thalassemia was not higher than that in control subjects. Circulating GDF-15 was a significant negative predictor of erythrocyte incorporation of iron, which suggests that ineffective incorporation of iron may stimulate the synthesis of GDF-15 by the developing erythron.

The present study has several limitations. First, we did not measure hepcidin concentrations in our subjects. Also, we studied only young Thai women, and our results may not be generalizable to other population groups, such as older men or groups with differing ethnicities or diets (or both). Finally, estimates of body iron (16) in persons with ineffective erythropoesis may not be valid; however, if we had used SF alone, we would have obtained similar results and relations, and SF concentrations were used to correct the iron absorption values in Table 3Go.

Although the prevention of iron deficiency through fortification of foods is a recommended strategy (41, 42), there may be potential health risks associated with an overabundant iron supply (43). Because of fears over possible iron overload, iron fortification of flour has been discontinued in Sweden and Denmark (44, 45). Moreover, in developing countries with a high prevalence of hemoglobinopathies (eg, Egypt and Cambodia), public health officials are reluctant to adopt iron fortification (P Winichagoon, R Hurrell, personal communication, 2008). In this context, what is the significance of our findings? In HbE heterozygotes, the most common hemoglobinopathy in Thailand, iron absorption and utilization do not differ significantly from those in control subjects, which suggests that additional dietary iron from universal fortification may be beneficial in populations with low dietary iron intakes. In HbE/β-thalassemia compound heterozygotes, iron absorption and body iron are sharply higher and utilization is impaired, which suggests that additional dietary iron would not be beneficial. But these persons typically are followed clinically, and potential iron excess is monitored and treated. In heterozygotes for {alpha}- or β-thalassemia, iron utilization is lower and absorption is not appropriately down-regulated, despite modestly higher concentrations of SF and storage iron. In regions with a high prevalence of these traits, iron should be targeted to groups vulnerable to iron deficiency, such as women and children. If universal iron fortification is implemented, it may be useful to monitor iron stores in groups with lower iron turnover, such as adult males and postmenopausal women.


   ACKNOWLEDGMENTS
 
We thank the subjects for their participation in the study and the nursing staff at Mahidol University; we also thank Napaporn Reabroy (Nakon Pathom, Thailand) and Adam Krzystek (Zürich, Switzerland) for laboratory assistance.

The authors’ responsibilities were as follows—MBZ, SF, PW, CZ, and RFH: designed the research; all authors: performed the research and participated in the editing of the manuscript; MBZ, SF, and PW: analyzed the data; and MBZ: wrote the first draft of the manuscript. None of the authors had a personal or financial conflict of interest.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication May 14, 2008. Accepted for publication July 3, 2008.





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