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ORIGINAL ARTICLE  
Year : 2014  |  Volume : 57  |  Issue : 1  |  Page : 39-42
What regulates hepcidin in poly-transfused β-Thalassemia Major: Erythroid drive or store drive?


1 Department of Pathology, Lady Hardinge Medical College and Kalawati Saran Children's Hospital,New Delhi, India
2 Department of Pediatrics, Lady Hardinge Medical College and Kalawati Saran Children's Hospital,New Delhi, India

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Date of Web Publication17-Apr-2014
 

   Abstract 

Background: Hepcidin, a key regulator of iron homeostasis, is increased by iron overload and inflammation while suppressed by hypoxia. In spite of iron overload in β-Thalassemia Major (β-TM), a paradoxical decrease in hepcidin is observed. Aim: To assess the opposing effects of enhanced erythropoiesis due to anemia and iron overloading on hepcidin in β-TM patients. Setting and Design: This prospective observational study was done at our tertiary care hospital. Materials and Methods: Eighty-three pediatric polytransfused (> 20 transfusions) patients of β-TM were compared with 70 children who served as controls. Serum assays for ferritin, transferrin receptors (sTfR) and hepcidin were performed. Statistical analysis: Independent Student t test was used to compare variables between both the groups. A Pearson correlation coefficient was used to find any correlation between ferritin, sTfR and hepcidin. Results: The mean value of hepcidin in β-TM children was 13.88±10.68 ng/ml (range, 0.9-60 ng/ml) and showed significant negative correlation with sTfR (r = -0.296, P < 0.0066). However, there was no correlation of hepcidin with ferritin. Ferritin and sTfR were significantly elevated in β-TM children compared to controls (P < 0.001). The mean serum hepcidin/ferritin index in the study group (0.00552) was significantly lower (P value < 0.001) than the controls (0.378) thus indicating inappropriate levels of hepcidin to iron overload. Conclusion: In polytransfused β-TM children increased iron demand dominates over iron overload in regulating hepcidin. In spite of excessive iron load, the inappropriate hepcidin levels may further contribute to iron overload enhancing iron toxicity.

Keywords: β-Thalassemia, hepcidin, iron overload

How to cite this article:
Chauhan R, Sharma S, Chandra J. What regulates hepcidin in poly-transfused β-Thalassemia Major: Erythroid drive or store drive?. Indian J Pathol Microbiol 2014;57:39-42

How to cite this URL:
Chauhan R, Sharma S, Chandra J. What regulates hepcidin in poly-transfused β-Thalassemia Major: Erythroid drive or store drive?. Indian J Pathol Microbiol [serial online] 2014 [cited 2019 Dec 9];57:39-42. Available from: http://www.ijpmonline.org/text.asp?2014/57/1/39/130891



   Introduction Top


Hepcidin is a newer molecule in iron homeostasis. It is a 25 amino-acid cationic peptide hormone produced in the liver .[1] It is an important key regulator of iron homeostasis by regulating its absorption from intestine, recycling by macrophages, and mobilization from hepatic stores. Iron overload and inflammation increase hepcidin synthesis while anemia and hypoxia suppress hepcidin expression. [2],[3],[4]

βeta-Thalassemia Major (β-TM) is a hereditary hemolytic anemia caused by deficient synthesis of the β-chain leading to ineffective erythropoiesis and hemolysis and therefore, marked anemia. Erythropoietin (EPO) level is increased in TM causing massive erythroid hyperplasia. There is excessive dietary iron absorption in these patients. This accompanied by iron accumulation due to repeated blood transfusions leads to a state of severe iron overload. In contrast, the patients with Thalassemia Intermedia (TI), who have a milder form of anemia and remain for the most part transfusion independent, nevertheless also experience iron overload because of increased iron absorption.

Iron overload state in these patients should lead to increase hepcidin levels. However, a paradoxical decrease in hepcidin is observed in thalassemia patients.

In patients of TM, "erythroid" drive exerts a dominant influence over "store" signals. Tissue hypoxia triggers EPO production causing massive erythroid proliferation accompanied by increased serum transferrin receptor (sTfR) levels. EPO, through indirect links and also by Growth Differentiating Factor 15 (GDF15) over-expression by expanded erythroid compartment, leads to suppression of the iron regulatory protein hepcidin. [5] Hepcidin seems to play an important role in the pathogenesis of iron overload in patients with thalassemia. Its inappropriate low levels may further contribute to iron overload and thus lead to iron toxicity. To assess the opposing effects of enhanced erythropoiesis due to anemia and iron overloading on hepcidin as their common target, hepcidin levels in thalassemia syndrome are being analyzed.


   Materials and Methods Top


The study included 248 β-TM patients registered in Thalassemia Day Care Centre. A detailed clinical history and relevant physical examination was done. Patients with history of ≤ 20 transfusions, fever or clinical evidence of infection, serum C-reactive protein (CRP) level > 8 mg/l, deranged liver function, increased serum urea or serum creatinine, and sero-positivity for HBsAg, anti-HCV antibody and HIV were excluded from the study. After applying the exclusion criteria out of 248 patients, 83 regularly chelated cases were selected for the study (Group A). The control group comprised of 70 age- and sex-matched individuals (Group B).

Sampling was done pre-blood transfusion. Two milliliters of blood, from a clean venipuncture was collected each in a dipotassium EDTA coated and in a plain vacutainer. Complete blood count was performed on Sysmex KX-21 hematology analyzer. Serum was separated from the sample collected in plain vacutainer after centrifugation at 1000 rpm for 5 minutes and stored at -40°C in four aliquots.

Serum hepcidin was estimated by Elisa kit-DRG International, Germany, with a reference range of 13.3-54.4 ng/ml. Serum ferritin was determined by ELISA kit from Calbiotech with a reference range of 10-160 ng/ml. The sTfR was measured using Human sTfR ELISA kit-DRG international, Germany, with a reference range of 1.0-2.9 μg/ml. CRP was determined by Biolatex CRP-EKO.

The data were expressed as mean ± standard deviation. The independent Student 't' test was done for comparison between the two groups. Pearson correlation coefficient (r) was used to test the correlation between continuous variables. A P value of <0.05 was considered to be significant.


   Results Top


The age of the patients ranged from 1.5 to 18 years (mean 9.59 ± 3.96 years) with a male:female ratio of 0.97:1. The maximum number of patients was in the age group of 8.1-12 years (33/83 cases).

The mean hemoglobin in Group A was significantly low as compared to control, i.e., 9.17 ± 1.03 g/dl versus 12.97 ± 1.18 g/dl, respectively (P < 0.001). Number of transfusions received by Group A patients ranged from 22 to 186 (mean 87.3).

The serum hepcidin levels were comparable in both the groups. The range of serum hepcidin was 0.9-60 ng/ml; mean 13.88 ± 10.68 ng/ml in Group A which was similar to that of Group B (0.9-60 ng/ml; mean 14.47 ± 11.68 ng/ml; (P = 0.768). Serum ferritin was significantly increased in Group A as compared to Group B (900 to 8800 ng/ml; mean 3428.80 ± 1741.65 ng/ml vs 10.4 to 494.3 ng/ml; mean 107.0 ± 121.31 ng/ml; P value <.001).

The value of sTfR in Group A ranged from 0.45 to 10 μg/ml with a mean of 5.18 ± 2.58 μg/ml, and was significantly higher in Group B (0.8 to 10 μg/ml with a mean of 3.60 ± 2.26 μg/ml; P value <0.001) [Table 1]. The hepcidin levels in Group A showed no correlation with serum ferritin levels (r = -0.009, P = 0.408) [Figure 1]. However, there was a significant negative correlation between sTfR and serum hepcidin levels (r = -0.296, P = 0.0066) [Figure 2].
Figure 1: Correlation between serum hepcidin and serum ferritin in Group A (r = – 0.009, P = 0.408)

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Figure 2: Correlation between serum hepcidin and serum transferrinreceptors in Group A ( r = – 0.296, P=0.0066)

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The serum hepcidin/ferritin index ranged from 0.000127 to 0.0272 in Group A. The mean was 0.00552 for the study group. Group B showed a statistically significant higher range for this ratio, i.e. 0.0023 to 1.893 with an average value of 0.378 (P value <0.001) [Table 1].
Table 1: Serum ferritin, sTfR, and hepcidin in study and control groups

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   Discussion Top


Hepcidin, a recently identified iron regulatory hormone, was initially identified as an antimicrobial peptide [6] and its role in iron-overloading states was unravelled until 2001. [2] While most of the earlier studies were done on mouse models, the evolution of techniques to assay hepcidin levels in human body has lead to upsurge of human experimentation of hepcidin in regulation of iron homoeostasis in various physiological as well as pathological states.

The role of hepcidin in iron overload states especially thalassemia has been a subject of interest because of its paradoxically low level inspite of iron overload. To understand the regulation of hepcidin in thalassemia, research has been going on upon the complex feedback mechanisms involving iron overload, inflammation and degree of erythropoiesis. Earlier studies done on mouse models of β-TM showed variable results. Hepcidin was initially measured in the form of hepatic hepcidin mRNA expression in β-TI and β-TM mice using RT-PCR. [7],[8] They found a 16-fold decrease of hepcidin mRNA level in β-TM mouse models as compared to wild-type mouse models. Franceschi et al. studied serum hepcidin in 4 months and 10 month old mice. They found higher hepcidin levels in younger mice as compared to controls (P < 0.05) while comparable levels in 10-month-old mice. So they hypothesized that severe anemia in older mice than 4 month old animals might contribute to upper hand of erythroid drive over store drive. The ability of increasing hepcidin synthesis in 4 month old animals in β- TM that faced with modest iron overload, was lost in older mice. [9]

Papanikolaou et al. used the immunodot assay to determine urinary hepcidin in 8 human β-TM subjects. Six out of eight had undetectable or low hepcidin levels as compared to controls. [10] While Nemeth et al. and Origa et al. found increased urinary hepcidin levels in β-TM patients as compared to controls. [3],[11]

However, contrary to this, in the present series the serum hepcidin levels were not significantly different from that of controls (P = 0.786). Similarly, the level of hepatic hepcidin mRNA was found to be comparable with controls in a series of 10 β-TM patients by Camberlein et al. [12] Kroot et al. studied serum hepcidin levels in 23 adult sickle cell disease patients and found very low levels in only 5 patients , rest were in the normal range. [15]

Among the human studies, a few authors found a significant positive correlation of indices of iron overload with hepcidin. Origa et al. reported a significant positive correlation of serum ferritin and hepatic mRNA levels, i.e, r 2 = 0.68, P < 0.001. [11] However, the current study showed no correlation of serum ferritin and serum hepcidin. (r = -0.009, P = 0.408). Similarly, Kattamis et al. too found a lack of correlation between the two variables; r = 0.001, P = 0.97. [13]

The regulation by increased erythropoeitic activity has been demonstrated by studying variables such as TfR1 expression and sTfR. In the present study, there was a significant increase of sTfR in β-TM as compared to controls (P < 0.001) indicating erythroid expansion because of marked ineffective erythropoiesis. This was negatively corelated with hepcidin levels (r = -0.296, P = 0.0066). Orly Weizer-Stern et al. found a 3.6-fold increase in TfR1 levels along with a 16-fold decrease in hepcdin mRNA in mouse models inspite of iron overload suggesting an upper hand of erthroid drive over store drive. [8] Kattamis et al. had found a statistically significant negative correlation between sTfR and hepcidin; rho = -0.59, P < 0.01. [13] Similar results were also obtained by Origa et al. in a series of 11 βTM patients; r 2 = 0.83, P < 0.001. [11] Although Camberlein et al. reported significantly higher levels of sTfR in β-TM patients, but they did not observe a significant decrease in hepatic hepcidin mRNA. [12]

Hepcidin-to-ferritin ratio, an index of appropriateness of hepcidin expression relative to the degree of iron loading, was found to be low in thalassemia major(normal hepcidin/ferritin ratio close to 1). Urinary hepcidin to ferritin index in β-TM patients ranged from 0.004-0.21 (median 0.054), as calculated by Kattamis et al. [13] Origa et al., Kearney et al., and Kroot et al., also found this index to be significantly low in β-TM. [11],[14],[15] The ratio of serum hepcidin to ferritin in this series was found to be significantly low in β-TM patients as compared to the control group (P < 0.001). Thus, in spite of iron overload in β-TM patients, hepcidin was not increased proportionately to the degree of iron overload. But its levels were inappropriately low.

HFE gene mutation results in inappropriately reduced level of hepcidin in herediatry hemachromatosis which contributes to enhanced iron absorption and iron overload. Besides multiple blood transfusions and increased gut absorption of iron, contribution of HFE gene mutation has been investigated in thalassemia patients. Longo et al. found 18 cases with HFE gene mutation among 71 transfusion-dependent β-TM patients. They did not find any signicant effect of single mutation of HFE gene on iron overload, however, they observed unusually severe iron overload in a single patient with homozygous H63D mutation of the HFE gene. [16] Contrary to this, an Indian study found higher chances of developing iron overload in thalassemia intermedia patients with co-existent HFE mutation, thus requiring early therapeutic intervention. [17] However, muation analysis for the HFE gene could not be performed in the present series.


   Conclusion Top


This study suggests that erythroid drive takes an upper hand over store drive in controlling hepcidin levels in β-TM patients and thus enhancing iron absorption in spite of iron overload. Also, low hepcidin levels further increase iron absorption, contributing to iron toxicity in β-TM patients. Thus, concentration of hepcidin in these patients might help to pick those who are most susceptible to iron overload and its complications. [18] Hepcidin can be used as a therapeutic target to allay side effects of transfusion therapy in thalassemia major patients. [19] The monitoring of hepcidin-ferritin index might be used as a sensitive indicator of anemia correction or non- compliance with iron chelation therapy in β-TM patients. Therefore, chelation therapy should be adjusted for the individual patient. Also, further studies need to be conducted to demonstrate the clinical utility of this index.

 
   References Top

1.Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 2001;276:7806-10.  Back to cited text no. 1
    
2.Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P et al. A new mouse liver specific gene encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 2001;276:7811-9.  Back to cited text no. 2
    
3.Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute phase protein. Blood 2003;101:2461-3.  Back to cited text no. 3
    
4.Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn A, Vaulont S. Hepcidin, A new iron regulatory peptide. Blood Cell Mol Dis 2002;29:327-35.  Back to cited text no. 4
    
5.Tanno T, Bhanu NV, Oneal PA, Goh SH, Staker P, Lee YT et al. High levels of GDF-15 in thalassemia suppresses expression of the iron regulatory protein hepcidin. Nat Med 2007;13:1096-101.  Back to cited text no. 5
    
6.Krause A, Neitz S, Magret HJ, Schulz A, Forssmann WG, Schulz-Knappe P, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 2000;480:147-50.  Back to cited text no. 6
    
7.Adamsky K, Weizer O, Amariglio N, Breda L, Harmelin A, Rivella S, et al. Decreased hepcidin mRNA expression in thalassemic mice. Br J Haematol 2004;124:123-4.  Back to cited text no. 7
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8.Orly Weizer-Stern, Adamsky K, Amariglio N, Rachmilewitz E, Breda L, Rivella S, et al. mRNA expression of regulatory genes in β-Thalassemia major mouse models. Am J Hematol 2006;81:479-83.  Back to cited text no. 8
    
9.Franceschi LD, Daraio F, Filippini A, Carturan S, Muchitsch EM, Roetto A, et al. Liver expression of hepcidin and other iron genes in two mouse models of β- Thalassemia. Haematologica 2006;91:1336-42.  Back to cited text no. 9
    
10.Papanikolaou G, Tzilianos M, Christakis JI, Bogdano D, Tsimirika K, MacFarlane J, et al. Hepcidin in iron overload disorders. Blood 2005;105:4103-5.  Back to cited text no. 10
    
11.Origa R, Galanello R, Ganz T, Giagu N, Maccioni L, Faa G, Nemeth E. Liver iron concentrations and urinary hepcidin in beta-thalassemia. Haematologica 2007;92:583-8.  Back to cited text no. 11
    
12.Camberlein E, Zanninelli G, Detivaud L, Lizzi AR, Sorrentino F, Vacquer S, et al. Anemia in β-thalassemia patients targets hepatic hepcidin transcript levels independently of iron metabolism genes controlling hepcidin expression. Haematologica 2008;93:111-5.  Back to cited text no. 12
    
13.Kattamis A, Papassotiriou I, Palaiologou D, Apostolakou F, Galani A, Ladis V, et al. The effects of erythropoeitic activity and iron burden on hepcidin expression in patients with thalassemia major. Haematologica 2006;91:809-12.  Back to cited text no. 13
    
14.Kearney SL, Nemeth E, Neufeld EJ, Thapa D, Ganz T, Weinstein DA, et al. Urinary hepcidin in congenital chronic anemias. Pediatr Blood Cancer 2007;48:57-63.  Back to cited text no. 14
    
15.Kroot JJ, Laarakkers CM, Kemna EH, Biemond BJ, Swinkels DW. Regulation of serum hepcidin levels in sickle cell disease. Haematologica 2009;94:885-7.  Back to cited text no. 15
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16.Longo F, Zecchina G, Sbaiz L, Fischer R, Piga A, Camaschella C. The influence of hemochromatosis mutations on iron overload of thalassemia major. Haematologica 1999;84:799-803.  Back to cited text no. 16
    
17.Sharma V, Panigrahi I, Dutta P, Tyagi S, Choudhry VP, Saxena R. HFE mutation H63D predicts risk of iron overload in thalassemia intermedia irrespective of blood transfusions. Indian J Pathol Microbiol 2007;50:82-5.  Back to cited text no. 17
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18.Kroot JJ, Tjalsma H, Fleming RE, Swinkels DW. Hepcidin in human Iron disorders: Diagnostic implications. Clin Chem 2011;57:1650-69.  Back to cited text no. 18
    
19.Bartnikas TB, Fleming MD. A tincture of hepcidin cures all: The potential for hepcidin therapeutics. J Clin Invest 2010;120:4187-90.  Back to cited text no. 19
    

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Richa Chauhan
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DOI: 10.4103/0377-4929.130891

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