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ROLE OF HEPCIDIN HORMONE IN PATIENTS OF β -THALASSEMIA MAJOR
A thesis
Submitted for Partial Fulfillment of the Requirements
of Master Degree in clinical Pathology
By Abeer Elsayed Ahmed Badawy
M.B.B.CH
Faculty of Medicine-Cairo University
Under Supervision Of
Prof. Dr. Nagelaa Ali Khalifa
Professor of clinical &chemical pathology Faculty of Medicine Zagazig University
Prof. Dr.
Mervat Abdallah Hesham
Professor of Pediatrics Faculty of Medicine Zagazig University
Dr. Ibtessam Ibrahim Ahmed Assist. Professor of Clinical and
Chemical Pathology Faculty of Medicine Zagazig University
Faculty of Medicine Zagazig University
2010
ROLE OF HEPCIDIN HORMONE IN PATIENTS OF β -
THALASSEMIA MAJOR
A thesis
Submitted for Partial Fulfillment of the Requirements of Master Degree in clinical Pathology
By
Abeer Elsayed Ahmed Badawy M. B. B. CH.
Faculty of Medicine - Cairo University
Under Supervision Of
Prof. Dr. Nagelaa Ali Khalifa
Professor of clinical & chemical pathology Faculty of Medicine Zagazig University
Prof. Dr.
Mervat Abdallah Hesham Professor of Pediatrics Faculty of Medicine Zagazig University
Dr. Ibtessam Ibrahim Ahmed
Assist. Professor of Clinical and Chemical Pathology Faculty of Medicine Zagazig University
Faculty of Medicine Zagazig University
2010
ii
االرحمن االرحيیم بسم هللا
إإنـك أأنـت االعليیـم قالواا سبحانك ال علم لنا إإال ما علمتنـا﴿
﴾االحكيیـم
االعظيیم هللاصدقق
)32( آآيیةسوررةة االبقرةة:
ACKNOWLEDGMENT
iii
First and foremost, thanks to Allah who helped me to finish this work.
I am honered to express my deepest appreciation and profound gratitude
to Prof. Dr.Nagelaa Ali Khalifa Professor of clinical & chemical pathology,
Faculty of Medicine, Zagazig University, for her kind supervision,
encouragement and constant guidance.
My deepest thanks and gratefulness to Prof. Dr. Mervat Abdallah
Hesham, Professor of Pediatrics, Faculty of Medicine, Zagazig University,
for her continuous support and advice.
I would like to express my deepest sense of gratitude and obligations to
Prof. Dr. Ibtessam Ibrahim Ahmed, Assist.Professor at department of
Clinical and Chemical Pathology, Faculty of Medicine, Zagazig University.
Lastly, I would like to express my deepest thanks to all my colleges in
clinical & chemical pathology department for their help and encouragement.
Dr. Abeer Badawy
2010
LIST OF ABBREVIATIONS
BMP: Bone morphogenetic protein.
iv
BMT:
Bp:
Dcytb:
DFO:
DMT1:
FEP:
GVHD:
HFE:
HIF:
HIV:
HJV:
HLA:
LCR:
MOD:
NGAL:
NMD:
Nrmap2:
NTBI:
PASP:
PHT:
sTfR:
TfR:
UGTIA:
VSS:
Bone marrow transplantation.
Base pair.
Duodenal cytochrome b.
Desferrioxamine.
Divalent Metal ion Transporter 1.
Free Erythrocyte Porphyrin.
Chronic graft-Versus-Host Disease.
High iron Fe.
Hypoxia-inducible factor.
Human Immune deficiency virus.
Hemojuvelin.
Human leukocyte antigen.
Locus control region.
Multi - organ dysfunctions.
Neutrophil gelatinase-associated lipocalin.
Nonsense- mediated mRNA decay.
Natural resistance macrophage-associated protein 2.
Non-transferrin bound iron.
Pulmonary artery systolic pressure.
Pulmonary hypertension.
Soluble Transferrin receptors.
Transferrin receptors.
Uridine diphosphate-glucoronyl transferase IA.
Volume of distribution at steady state.
LIST OF TABLES
Table 1 Main characteristics of genetic iron overload disorders
v
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
Table 8
Table 9
(Deugnier et al., 2008)………………………………………...
The clinical data of the studied groups…………….......
Liver and kidney functions results of the studied groups.........
Complete blood count results of the studied groups…………..
Results of iron study of the studied groups……………………
Hemoglobin Electrophoresis data……………………………...
Hepcidin concentration levels………………………………....
Ratio between hepcidin and Serum Ferritin…………………...
Correlation between hepcidin and other parameters……….....
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LIST OF FIGURES
vi
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
The β-Globin Gene Cluster on the Short Arm of
Chromosome 11 (Nancy & Livieri, 1999)…………………..
The Normal Structure of the β-Globin Gene and the
Locations and Types of Mutations Resulting in β-
Thalassemia (Nancy & Livieri, 1999)……………………….
Effects of Excess Production of Free α-Globin Chains
(Nancy & Livieri, 1999)……………………………………..
Complications of beta thalassemia…………………………..
Peripheral blood film in Cooley anemia…………………….
Essential roles of iron (Taketani, 2005)……………………..
Normal distribution and storage of body iron (Andrews,
1999)……….………………………………………………..
Major pathways of iron transfer between cells and tissues
(Andrews, 2000)……………………………………………..
Iron absorption (Andrews, 2000)……………………………
Hepatic Iron Burden over Time and the Effect of Various
Hepatic Iron Concentrations in Patients with Thalassemia
Major, Homozygous Hemochromatosis, and Heterozygous
Hemochromatosis (Nancy & Livieri, 1999)………………...
Amino acid sequence and a model of the major form of
human hepcidin. The amino and carboxy termini are labeled
as N and C, The pattern of disulfide linkages between the 8
cysteines is also shown in the amino acid sequence (Ganz,
2003)……………………………………………….………..
Physiology of hepcidin-ferroportin interaction (Rivera et al.,
2005b)……………………………………………………….
10
16
21
23
26
36
37
39
42
47
52
55
vii
Fig. 13
Fig. 14
Fig. 15
Fig. 16
Fig. 17
Fig. 18
Fig. 19
Fig. 20
Fig. 21
Normal iron homeostasis mediated by an iron-sensing
feedback loop (Wrighting & Andrews, 2008)………………
Hepcidin mRNA expression (Nicolas et al., 2002)………….
Concentration of hepcidine of patients (in circles) and
concentration of hecidin of control (in squares)…………….
The variations of RBCs, HB, MCV, HCT (PCV) for 40
patients (30 patients and 10 controls)……………………….
Hepcidin and Serum Ferritin for 40 patients (30 patients and
10 controls)……………………………………………..
Correlation between hepcidin and Hb………………………
Correlation between hepcidin and HCT…………………….
Correlation between hepcidin and MCV……………………
Correlation between hepcidin and Serium Ferritin………….
56
60
72
76
78
80
80
81
81
viii
CONTENTS
INTRODUCTION……………………………………………………
AIM OF THE WORK………………………………………………..
LITERATURE REVIEW…………….………………………………
THALASSEMIA………………….………………………………
IRON METABOLISM…………...……………………………….
HEPCIDIN……..…………………………………………………
SUBJECTS AND METHODS………….……………………………
RESULTS………………………………...………………………….
DISCUSSION………………………………………………………..
CONCLUSIONS……………………………….…………………….
RECOMMENDATION…………………………….………………..
SUMMARY…………………………………………….……………
REFERENCES………………………..…………………………...…
.........................................................................................االملخص االعربى
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Introduction
1
INTRODUCTION
The thalassemias are a heterogeneous group of genetic disorders of
haemoglobin synthesis, occurring more frequently in the Mediterranean
region, the Indian subcontinent, Southeast Asia, and West Africa .The
thalassemias are divided according to their severity into major which is
severe and transfusion dependent, intermediate and minor forms of illness.
The β-thalassemias are the most important types of thalassemia because
they are so common and usually produce severe anemia in their
homozygous and compound heterozygous states (Hillman et al., 2005).
In β-thalassemia major, the neonate is well at birth but develops severe
anemia, bone abnormalities, failure to thrive, and life-threatening
complications. In many cases, the first signs are pallor, yellow skin and
scleras in infants ages 3 to 6 months. Later clinical features, in addition to
severe anemia, include splenomegaly or hepatomegaly, with abdominal
enlargement, frequent infections, bleeding tendencies (especially toward
epistaxis), and anorexia (Fucharoen et al., 2000).
Transfusional iron overload is the most important complication of β-
thalassemia and is a major focus of management, which can be prevented
by adequate iron chelation. Extensive iron deposits are associated with
cardiac hypertrophy and dilatation, degeneration of myocardial fibers
(Aessopos et al., 1995; Du et al., 1997).
Hepcidin is a 25-amino-acid iron peptide hormone. Initially identified in
human plasma and urine as an anti-microbial molecule. Hepcidin is the key
regulator of systemic iron homeostasis and a pathogenic factor in anemia of
inflammation and hereditary hemochromatosis. Hepcidin inhibits iron
influx into plasma from duodenal enterocytes that absorb dietary iron, from
Introduction
2
macrophages that recycle iron from senescent erythrocytes and from
hepatocytes that store iron (Park et al., 2001).
Iron–Loading anemias are characterized by ineffective erythropoiesis
and increased intestinal iron absorption. Erythrocyte transfusions further
exacerbate the iron overload. The development of hepcidin- based
diagnostics and therapies for iron-loading anemias may offer more effective
approaches to prevent the toxicity associated with iron overload. The most
common iron- loading anemias are major forms of β-thalassemia
(Papanikolaou et al., 2005).
In the presence of systemic iron overload Patients with thalassemia
major in whom iron overload was more severe and anemia was partially
relieved by transfusions, had urinary hepcidin concentrations that were
higher than in thalassemia intermedia. These findings were interpreted as
supporting the dominant erythropoietic effect of exogenous hepcidin could
prevent the iron overload in iron–Loading anemias (Loreal et al., 2005).
Adamsky and his co-authors (2004) have found that iron overload is
less dominant than anaemia in regulating hepcidin expression in the setting
of the β-thalassemia major mouse model. The decreased expression of
hepcidin may explain the increased absorption of iron in thalassemia.
Aim of THE work
3
AIM OF THE WORK
The aim of the present work is to measure hepcidin concentration in
patients of β thalassemia major to explain its role in iron metabolism for
those patients who have iron overload.
Literature review
4
LITERATURE REVIEW
THALASSEMIA
Definition The thalassemias are a heterogeneous group of genetic disorders of haemoglobin
synthesis, all of which result from a reduced rate of production of one or more of the
globin chains of haemoglobin. The thalassemias are among the most common genetic
disorders worldwide, occurring more frequently in the Mediterranean region, the Indian
subcontinent, Southeast Asia, and West Africa (Weatherall & Clegg, 2001). Brief historical review
Thalassemia was first defined in 1925 when Dr-Thomas B.Cooley
described five young children with severe anemia, splenomegally, and
unusual bone abnormalities and called the disorder erythroblastic or
Mediterranean anemia because of circulating nucleated red blood cells and
because all of his patients were of Italian or Greek ethnicity (Hillman et al.,
2005).
In Europe, Riette 1925 described Italian children with unexplained mild
hypochromic and microcytic anemia in the same year Cooley reported the
severe form of anemia later named after him. In addition, Wintrobe and
coworkers in the United States reported a mild anemia in both parents of a
child with Cooley anemia. This anemia was similar to the one that Riette
described in Italy. Only then was Cooley's severe anemia recognized as the
homozygous form of the mild hypochromic and microcytic anemia that
Riette and Wintrobe described. This severe form was then labeled as
thalassemia major and the mild form as thalassemia minor. The word
thalassemia is a Greek term derived from thalassa, which means "the sea"
(referring to the Mediterranean), and emia, which means "related to blood"
(Yaish, 2007).
Literature review
5
Classification of the Thalassemias:
First, a clinical classification, which describes the degree of severity.
Second, the thalssemia can be defined by the particular globin chain that is
synthesized at a reduced rate. Finally, it is now often possible to subclassify
them according to defect in the globin chain synthesis (Cohen et al., 2004).
I. Clinical classification of the Thalassemias
The thalassemias are divided according to their severity into
major,intermediate and minor forms of illness, thalassemia major which is
severe and transfusion dependent , and the symptomless minor forms,
which usually represent the carrier state, or trait. Thalassemia intermedia
describes conditions that associated with a more severe degree of anemia
and splenomegaly than the trait but, not as severe as the major forms to
require regular transfusions (Weatherall & Clegg, 2001).
(a) Thalassemia major
Thalassemia major, the severe form of Thalassemia occurs when a child
inherits two mutated genes, one from each parent. Children born with
Thalassemia major usually develop the symptoms of severe anemia within
the first year of life. They lack the ability to produce normal adult
hemoglobin (HbA, α2β2). Children with Thalassemia major are so
chronically fatigued they fail to thrive and do not grow normally. Left
untreated, this disorder will cause bone deformities and eventually will lead
to death within the first decade of the child’s life (Talwar & Srivastava,
2004).
Literature review
6
(b) Thalassemia Intermedia
Thalassemia Intermedia is a mild form of thalassemia that is caused by
the one of the more severe thalassemic genes and one of the milder
thalassemic genes. Children with Thalassemia intermedia start to develop
symptoms later in life than those with Thalassemia major. They are
moderately anemic but a large number of the patients survive without
regular blood transfusions. The severity of Thalassemia intermedia isn't
determined by hemoglobin levels alone; it also depends on how the
individual's feelings,their growth rate and development (Eldor &
Rachmilewitz, 2002).
(c) Thalassemia minor
Thalassemia minor, people with a Thalassemia trait in one gene are
known as carriers or are said to have Thalassemia minor. The only way to
know if they carry the Thalassemia trait is to have a special blood
hemoglobin electrophoresis which can identify the gene. The carriers of
Thalassemia minor show mild hypochromic microcytic anemia (Talwar &
Srivastava, 2004).
Normal Human Hemoglobin:
Function
Hemoglobin main function is carrying oxygen through the body to all of
the organs (Moreno et al., 2004).
Hemoglobin synthesis and structure
Hemoglobin synthesis requires the coordinated production of heme and
globin. Heme is the prosthetic group that mediates reversible binding of
Literature review
7
oxygen by hemoglobin. Globin is the protein that surrounds and protects the
heme molecule (Handin et al., 2003).
The combination of two alpha chains and two gamma chains form
"fetal" hemoglobin (α2γ2 ), termed "hemoglobin F". With the exception of
the first 10 to 12 weeks after conception, fetal hemoglobin is the primary
hemoglobin in the developing fetus. The combination of two alpha chains
and two beta chains form "adult" hemoglobin (α2β2), also called
"hemoglobin A". Although hemoglobin A is called "adult", it becomes the
predominate hemoglobin within about 18 to 24 weeks of birth (Handin et
al., 2003).
The genes that encode the alpha globin chains are on chromosome 16.
Those that encode the non-alpha globin chains are on chromosome 11. The
alpha complex is called the "alpha globin locus", while the non-alpha
complex is called the "beta globin locus" (Handin et al., 2003).
In the first trimester of intrauterine life, ζ, ε, α, and γ chains attain
significant levels and in various combinations form Hb Gower I (ζ2ε2), Hb
Gower II (α2ε2), Hb Portland (ζ2γ2), and fetal hemoglobin (HbF) (α2γ2 136-
G and α2γ2 136-A) .Whereas Hb Gower and Hb Portland soon disappear,
HbF persists and forms the predominant respiratory pigment during
intrauterine life. Before birth, gamma-chain production begins to wane so
that after the age of 6 months postpartum, only small amounts of HbF (<
2%) can be detected in the blood (Wood et al ., 2001).
In early intrauterine life, beta-chain synthesis is maintained at a low
level but gradually increases to significant concentrations by the end of the
third trimester and continues into neonatal and adult life. The synthesis of
delta chains remains at a low level throughout adult life (< 3%). Hence
during normal development, the synthesis of the embryonic hemoglobins
Literature review
8
Gower and Portland is succeeded by the synthesis of HbF, which in turn is
replaced by the adult hemoglobins, HbA and HbA2 (Hardison , 2001).
Alpha Globin Locus
Each chromosome 16 has two alpha globin genes that are aligned one
after the other on the chromosome. For practical purposes, the two alph
globin genes (termed α1 and α2) are identical. Since each cell has two
chromosomes 16, a total of four alpha globin genes exist in each cell. Each
of the four genes produces about one-quarter of the alpha globin chains
needed for hemoglobin synthesis. The mechanism of this coordination is
unknown. Promoter elements exist 5' to each alpha globin gene. In addition,
a powerful enhancer region called the locus control region (LCR) is
required for optimal gene expression. (Hoff brand, 2006).
Beta Globin Locus
The genes in the beta globin locus are arranged sequentially from 5' to 3'
beginning with the gene expressed in embryonic development (the first 12
weeks after conception; called episolon). The beta globin locus ends with
the adult beta globin gene. The sequence of the genes is: epsilon, gamma,
delta, and beta. There are two copies of the gamma gene on each
chromosome 11. The others are present in single copies. Therefore, each
cell has two beta globin genes, one on each of the two chromosomes 11 in
the cell. These two beta globin genes express their globin protein in a
quantity that precisely matches that of the four alpha globin genes. The
mechanism of this balanced expression is unknown (Hoffbrand, 2006).
Upstream of the entire β globin complex is the locus control region
(LCR), which is essential for the expression of all the genes in the complex
Literature review
9
(Fig. 1). The general structure of the β globin gene is typical of the other
globin loci. The genomic sequence spans 1600 bp and codes for 146 amino
acids; the transcribed region is contained in three exons separated by two
introns or intervening sequences.The first exon encodes amino acid 1 to 29
together with the first two bases for codon 30, exon 2 encodes part of
residue 30 together with amino acids 31 to 104, and exon 3, amino acids
105 to 146 (Hardison, 2001).
Exon 2 encodes the residues involved in heme binding and αβ dimer
formation, while exons 1 and 3 encode for the non- heme- binding regions
of the β globin chain.The β globin gene promoter includes 3 positive cis-
acting elements: TATA box, CCAAT box and duplicated CACCC motifs.
In addition to these motifs, the region upstream of the β globin promoter
contains two binding motifs for the erythroid transcription factor GATA
1(Marini et al., 2004).
Literature review
10
Fig. 1 The β-Globin Gene Cluster on the Short Arm of Chromosome 11
(Nancy & Livieri, 1999).
In Panel A, the β-globin–like genes are arranged in the order during
development. Panel B shows the timing of the normal developmental switching of
human hemoglobin.
Conserved sequences important for gene function are found in the
5’promoter region, at the exon-intron junction, and in the 3 untranslated
regions (3-UTR) at the end of the mRNA sequences. The 5’
untranslated region (UTR) occupies a region of 50 nucleotides between
the CAP site, the start of transcription, and the initiation (ATG) codon.
There are two prominently conserved sequences in the 5’ UTR of the
various globin genes (both α and β). The 3’ UTR constitutes the region
between the termination codon (TAA) and the poly (A) tail. It consists
of 132 nucleotides with one conserved sequence, AATAAA, located 20
nucleotides upstream of the poly (A) tail (Forget et al., 2001).
Literature review
11
II. Genetic Classification of the Thalassemias a) Alpha-thalassemia
Alpha thalassemia syndromes can be expressed as α0 and α+. In the α0,
no alpha chains are produced. In the α+, the output of one of the linked pair
of alpha-globin genes is defective, and only some alpha chains are
produced. Within these general categories of the alpha-thalassemia
syndromes, there is considerable genetic and clinical heterogeneity due to
the interaction of the many possible mutations directing globin chain
synthesis (Rimoin et al., 2006).
Silent Carrier (α+-thalassemia carriers):
The loss of one gene diminishes the production of the alpha protein
slightly. This condition is so close to normal that it can be detected only by
specialized laboratory techniques which, until recently, were confined to
research laboratories. A person with this condition is called a "silent carrier"
because of the difficulty of detection (Sabella & Cunningham, 2006).
α0-Thalassemia minor or trait:
The loss of two genes produces a condition with small red blood cells,
and at most a mild hypochromic anemia. People who have this condition
look and feel normal, but thecondition can’t be detected certainly except by
DNA analysis (Sabella & Cunningham, 2006).
Hemoglobin H disease:
Deficiency of 3 α chains leads to production of excess β chains, forms
β4-tetramer and produces a serious hematological problem. Patients with
Literature review
12
this condition have a severe anemia, and often require blood transfusions to
survive (Sabella & Cunningham, 2006).
Hemoglobin Bart’s hydrops syndrome:
The loss of all four alpha genes during intrauterine life results in γ4-
tetramers, produces a condition that is incompatible with life. Foetus with
four-gene deletion alpha thalassemia die in utero or shortly after birth.
Rarely, four-gene deletion alpha thalassemia has been detected in utero,
usually in a family where the disorder occurred in an earlier child. In utero
blood transfusions have saved some of these children. These patients
require life-long transfusions and other medical support (Sabella &
Cunningham, 2006).
b) Delta-Thalassemia A hereditary disorder characterized by reduced or absent delta-globin
thus effecting the level of hemoglobin A2 (Bouva et al., 2006).
c) Delta-beta-thalassemia In δβ+ thalassemia, mutational basis is due to extensive deletions of
delta and beta globin structural genes. Abnormal hemoglobin (Hb Lepore)
is produced due to unequal crossing over between mispaired δ and β globin
genes leading to δ and β fusion with segments of δ, β lost (Weatheral &
Clegg, 2000).
d) Beta-thalassemia
Definition:
The β-thalassemias are the most important types of thalassemia because
they are so common and usually produce severe anemia in their
Literature review
13
homozygous and compound heterozygous states. The fact that there are
only two genes for the beta chain of hemoglobin makes beta thalassemia a
bit simpler to understand than alpha thalassemia. (Hillman et al., 2005).
Local Prevalence and geographic distribution of β –thalassemia (in
Egypt)
El-Beshlawy stated that β-thalassemia is the most common chronic
hemolytic anemia in Egypt (85.1%). A carrier rate of 9-10.2% has been
estimated in 1000 normal random subjects from different geographical areas
of Egypt (El-Beshlawy, 1999).
International Prevalence and geographic distribution of β –thalassemia
Worldwide, 15 million people have clinically apparent thalassemic
disorders. People who carry thalassemia in India alone number
approximately 30 million. These facts confirm that thalassemias are among
the most common genetic disorders in humans. β thalassemia is much more
common in Mediterranean countries such as Greece, Italy, and Spain. Many
Mediterranean islands, including Cyprus, Sardinia, and Malta, have a
significantly high incidence of severe β thalassemia, constituting a major
public health problem. For instance, in Cyprus, 1 in 7 individuals carries the
gene, which translates into 1 in 49 marriages between carriers and 1 in 158
newborns expected to have β thalassemia major. As a result, preventive
measures established and enforced by public health authorities have been
very effective in decreasing the incidence among their populations. β
thalassemia is also common in North Africa, the Middle East, India, and
Eastern Europe. Conversely, β thalassemia is more common in Southeast
Asia, India, the Middle East, and Africa (Yaish, 2007).
Literature review
14
Classification of β- thalassemia:
It occurs in three clinical forms: major, intermediate, and minor. The
resulting anemia’s severity depends on whether the patient is homozygous
or heterozygous for the thalassemic trait (Rund & Rachmilewitz, 2005).
Molecular basis of β thalassemia
There are two main varieties of β thalassemia alleles; β 0 thalassemia in
which no β globin is produced, and β+ thalassemia in which some β globin
is produced, but less than normal. In contrast to the α thalassemias, the β
thalassemias are rarely caused by deletions. One group of deletions affects
only the β globin gene and ranges in size from 290 bp to > 60 Kb. Of these,
only the 619 bp deletion at the 3’ end of the β gene is common (Thein,
1998).
The other deletions, although extremely rare, are of particular functional
and phenotypic interest because they are associated with unusually high
levels of Hb A2 in heterozygotes. These deletions differ widely in size, but
remove in common a region from positions –125 to +78 relative to the
mRNA cap site in the β promoter which includes the CACCC, CCAAT and
TATA elements (Forget et al., 2001).
The mechanism underlying the markedly elevated levels of Hb A2
appears to be related to the removal of the 5’ promoter region of the β gene.
This may remove competition for the upstream LCR leading to its increased
interaction with the γ and δ genes in cis, enhancing their expression (Wood
et al ., 2001).
There is a disproportionate increase of Hb A2 (α2 δ2) derived from the δ
globin gene cis to the β globin gene deletion. This mechanism may also
Literature review
15
explain the moderate increases in Hb F which characterize this group of
deletions and those due to point mutations affecting the promoter region.
Although the increases in Hb F are variable, and modest in heterozygotes,
they are adequate to compensate for the complete absence of β globin in
homozygotes. Two homozygotes for different deletions of this kind have a
mild disease despite the complete absence of Hb A2 (α2 β2) (Craig et al .,
1992).
The vast majority of β thalassemias are caused by point mutations
within the gene or its immediate flanking sequences (Fig. 2). These single
base substitutions, minor insertions or deletions of a few bases are classified
according to the mechanism by which they affect gene regulation:
transcription, RNA processing or RNA translation. Mutations affecting
transcription can involve either the conserved DNA sequences that form the
β globin promoter or the stretch of 50 nucleotides in the 5’UTR. Generally
they result in a mild to minimal deficit of β globin output and can be silent
in carriers (Thein et al ., 2001).
Literature review
16
Fig. 2 The Normal Structure of the β-Globin Gene and the Locations
and Types of Mutations Resulting in β-Thalassemia (Nancy & Livieri,
1999).
All β-globin–like genes contain three exons and two introns between
codons 30 and 31 and 104 and 105, respectively. Approximately half of the
β thalassemia alleles affect the different stages of RNA translation and in all
instances, no β globin is produced resulting in β0 thalassemia. Most of these
defects result from the introduction of premature termination codons due to
frameshifts or nonsense mutations and nearly all terminate within exon 1
and 2. Mutations that result in premature termination early in the sequence
(in exons 1 and 2) are associated with minimal steady-state levels of β
mRNA in erythroid cells, due to an accelerated decay of the abnormal
mRNA referred to as nonsense- mediated mRNA decay (NMD)
(Maquat,1995) .
Literature review
17
Variants of β thalassemia
Dominantly inherited β thalassemia
In contrast to the common β thalassemia alleles that are prevalent in
malarial regions and inherited typically as Mendelian recessives, some
forms of β thalassemia are dominantly inherited, in that inheritance of a
single β thalassemia allele results in a clinically detectable disease despite a
normal α globin genotype. Heterozygotes have a thalassemia intermedia
phenotype with moderate anemia, splenomegaly and a thalassemic blood
picture. Apart from the usual features of heterozygous β thalassemia, such
as increased levels of HbA2 and the imbalanced α/β globin biosynthesis,
large inclusion bodies similar to those seen in thalassemia major are often
observed in the red cell precursors, hence the original term of inclusion
body thalassemia (Fei et al ., 1989).
Normal Hb A2 β thalassemias
The diagnostic feature of β thalassemia is the hypochromic microcytic
red cells and an elevated level of Hb A2 in heterozygotes, whether β+ or β0.
Normal Hb A2 β thalassemias refer to the forms in which the blood picture
is typical of heterozygous β thalassemia except for the normal levels of Hb
A2. Most cases result from co-inheritance of δ thalassemia (δ0 or δ+) in cis
or trans to the β thalassemia gene, which can be of the β0 or β+ type. One
relatively common form of normal Hb A2 thalassemia is that associated
with Hb Knossos. The mutation activates an alternative splice site reducing
the amount of normal transcript that contains the variant (Trifillis et al .,
1991).
Literature review
18
Another fairly common cause of normal Hb A2 β thalassemia phenotype
in the Greek population is the Corfu form of δ β thalassemia. The phenotype
of normal Hb A2 β thalassemia is also seen in heterozygotes for ε γ δ β
thalassemia and overlaps the phenotypes encountered in carriers of α
thalassemia (Trifillis et al ., 1991).
Silent β thalassaemia
The silent β thalassemias cause only a minimal deficit of β globin
production. Heterozygotes do not have any evident hematologic phenotype;
the only abnormality being a mild imbalance of globin chain synthesis.
These mutations have been identified in homozygotes who have a typical β
thalassemia trait phenotype or in the compound heterozygous state with a
severe β thalassemia allele where they cause thalassemia intermedia
(Gonzalez-Redondo et al ., 1989).
β thalassemia trait with unusually high Hb A2
Despite the vast heterogeneity of mutations, the increased levels of Hb
A2 observed in heterozygotes for the different β thalassemia alleles in
different ethnic groups are remarkably uniform, usually 3.5-5.5% and rarely
exceeding 6%. Unusually high levels of Hb A2 over 6.5% seem to
characterize the sub-group of β thalassemias caused by deletions that
remove the regulatory elements in the β promoter (Codrington et al .,
1990).
β thalassemia due to insertion of a transposable element
Transposable elements may occasionally disrupt human genes and result
in their inactivation. The insertion of such an element, a retrotransposon of
the family called L1 has been reported with the phenotype of β +
thalassemia (Divoky et al ., 1996).
Literature review
19
β thalassemia due to trans-acting determinants
Population studies have shown that ~1% of the β thalassemias remain
uncharacterized despite extensive sequence analysis, including the flanking
regions of the β globin genes. In several families, linkage studies
demonstrated that the β thalassemia phenotype aggregates independently of
the β globin complex implying that the genetic determinant is trans-acting
(Giordano et al ., 1998).
Somatic deletion of β globin gene
This novel mechanism was recently described in an individual who had
moderately severe thalassemia intermedia despite being constitutionally
heterozygous for β0 thalassemia with a normal genotype. Subsequent
investigations revealed that he had a somatic deletion of a region of
chromosome 11p15 including the β globin complex giving rise to a mosaic
of cells, 50% with one and 50% without any β globin gene. The sum total of
the β globin product is ~25% less than the normally asymptomatic β
thalassemia trait (Badens et al ., 2002).
Clinical features of severe β-thalassemic syndromes
Infants and children affected with β thalassemia have pallor, poor
development, and abdominal enlargement. Hemoglobin electrophoretic
patterns show a variable quantity of HbA2 (0% – 6%) depending on the
genotype of the patient. The anemia is due to a combination of ineffective
erythropoiesis, excessive peripheral red blood cell hemolysis, and
progressive splenomegaly (Weatherall & Clegg, 2000). The latter causes
an increase in plasma volume and a decrease in total red cell mass. The red
Literature review
20
cells are microcytic (mean corpuscular volume <70 fL) with marked
anisochromasia. The bone marrow shows marked erythroid hyperplasia, and
the serum ferritin level is elevated (Wonke, 2001).
In children and young adults, radiologic abnormalities include thinning
of the long bones with sun-ray appearance and dilatation of the marrow
cavities. The skull has a “hair-on-end” appearance because of widening in
the diploic space. Patients with thalassemia have enlarged maxillary sinuses
and tend to have a maxillary overbite. The face gradually assumes a
“mongoloid” appearance. Such changes promote infections in the ears,
nose, and throat. Because of chronic anemia and iron overload,
endocrinopathies such as hypopituitarism, hypothyroidism,
hypoparathyroidism, diabetes mellitus, cardiomyopathy, and testicular or
ovarian failure become common as the child with thalassemia grows older
(Cunningham et al., 2004; Rund & Rachmilewitz, 2005).
Thalassemia can be regarded as a chronic hypercoagulable state.
Venous and arterial thromboembolic phenomena tend to occur more
frequently in thalassemic patients who have undergone splenectomy.
Furthermore, such patients may develop progressive pulmonary arterial
disease due to platelet thrombi in the pulmonary circulation (Eldor &
Rachmilewitz, 2002).
Pathophysiology
Mechanisms of Anemia
Normal hemoglobin, hemoglobin A, is composed of 2 beta and 2 alpha
subunits. In beta thalassemia major, more than 200 mutations have been
described in the beta-globin genes, cause loss of both beta-globin subunits.
Literature review
21
This leaves the normally paired alpha subunits unpaired. Unpaired subunits
are catatonic (Handin et al., 2003).
Normally, compensatory mechanisms are present to protect the cell
from the small amounts of unpaired alpha subunits, which may regularly be
present. This significant excess of free α chains caused by the deficiency
of β chains causes destruction of the RBC precursors in the bone marrow
(i.e., ineffective erythropoiesis) (Fig. 3). This ineffective erythropoiesis and
profound hemolysis result in a severe anemia that is usually manifest in
affected individuals by age 6 months (Rimoin et al., 2006).
Fig. 3 Effects of Excess Production of Free α-Globin Chains (Nancy &
Livieri, 1999).
The physiologic response is to attempt to increase red cell production by
expanding the bone marrow space up to 30-fold and/or increase production
of non-beta hemoglobin chains such as A2 (δ) and fetal (γ) hemoglobin.
However, despite these mechanisms, erythropoiesis remains ineffective and
Literature review
22
these patients become transfusion-dependent early in life. In fact, the
presence or absence of adequate transfusions significantly impacts the
appearance of these patients and the course of the disease (Sabella &
Cunningham, 2006).
Excess unbound α-globin chains and their degradation products
precipitate in red-cell precursors, causing defective maturation and
ineffective erythropoiesis. Hemolysis Anemia stimulates the synthesis of
erythropoietin, leading to an intense proliferation of the ineffective marrow,
which in turn causes skeletal deformities and a variety of growth and
metabolic abnormalities (Cunningham et al., 2004).
Clinical features of β-thalassemia major
In β-thalassemia major, the neonate is well at birth but develops severe
anemia, bone abnormalities, failure to thrive, and life-threatening
complications. In many cases, the first signs are pallor and yellow skin and
scleras in infants ages 3 to 6 months. Later clinical features, in addition to
severe anemia, include splenomegaly or hepatomegaly, with abdominal
enlargement, frequent infections, bleeding tendencies (especially toward
epistaxis), and anorexia (Fucharoen et al., 2000).
Children with thalassemia major typically have small bodies and large
heads and may also be mentally retarded. Infants may have mongoloid
features because bone marrow hyperactivity has thickened the bone at the
base of the nose. As these children grow older, they become susceptible to
pathologic fractures as a result of expansion of the marrow cavities with
thinning of the long bones. They’re also subject to cardiac arrhythmias,
heart failure, and other complications that result from iron deposits in the
Literature review
23
heart and in other tissues from repeated blood transfusions (Fucharoen et
al., 2000).
Complications of β-thalassemia major
Iron overload of tissue is the most important complication of β-
thalassemia and is a major focus of management. Thalassemia major can be
complicated with CCF, hepatic failure, aplastic crisis, intercurrent infection,
growth retardation, delayed puberty, hemosiderosis and hemochromatosis.
Transfusion related infection (HIV, HB, HC), complications related to iron-
chelation therapy, endocrinopathies (diabetes mellitus, hypothyroidism,
hypogonadism), skeletal complications and multiorgan dysfunctions (MOD)
also may found (Fig. 4) (Deugnier et al., 2008).
Fig. 4 Complications of beta thalassemia.
Management of thalassemia and treatment-related complications
(Cunningham et al., 2004).
Literature review
24
Hyperbilirubinemia and a propensity to gallstone formation is a
common complication of β thalassemia and is attributed to the rapid turn-
over of the red blood cells, bilirubin being a break-down product of
hemoglobin. Studies have shown that the levels of bilirubin and the
incidence of gallstones in β thalassemia, from trait to major, is related to a
polymorphic variant (seven TA repeats) in the promoter of the uridine
diphosphate-glucoronyltransferase IA (UGTIA) gene, also referred to as
Gilbert’s syndrome (Sampietro et al., 1997).
Cardiac iron overload is the most frequent cause of death from chronic
transfusion therapy. Recurrent pericarditis may be the initial manifestation
of myocardial iron deposition. Ventricular tachycardia and fibrillation or
severe congestive heart failure often proves fatal . Cardiac complications,
such as pulmonary hypertension (PHT), are the leading cause of death in
beta-thalassemia patients. L-Carnitine, due to its role in fatty acid oxidation,
might help control the elevation in pulmonary artery systolic pressure
(PASP) (El-Beshlawy et al., 2008).
Causes of death in patients with Beta - thalassemia major:
The prognosis of patients with homozygous β-thalassemia major has
been improved by transfusion and iron-chelation therapy. Prognosis for
survival without cardiac disease is excellent for patients who receive regular
transfusions and whose serum ferritin concentrations remain below 2500ng
per milliliter with chelation therapy (Borgna-Pignatti et al., 2004).
The most common cause of death in patients with beta thalassemia is
heart failure followed by infection, liver cirrhosis, thrombosis, cancer, and
diabetus (Borgna-pignatti et al., 2004).
Literature review
25
Laboratory diagnosis of β-thalassemia major
The CBC count and peripheral blood film examination results are
usually sufficient to suspect the diagnosis. Hb evaluation confirms the
diagnosis in β thalassemia, Hb H disease, and Hb E/β thalassemia.
• In the severe forms of thalassemia, the Hb level ranges from 2-8
g/dL.
• MCV and MCH are significantly low, but, unlike thalassemia trait,
thalassemia major is associated with a markedly elevated RDW,
reflecting the extreme anisocytosis.
• The WBC count is usually elevated in β thalassemia major; this is
due, in part, to miscounting the many nucleated RBCs as leukocytes.
Leukocytosis is usually present, even after excluding the nucleated
RBCs. A shift to the left is also encountered, reflecting the hemolytic
process.
• Elevated reticulocytic count.
• Platelets count is usually normal, unless the spleen is markedly
enlarged.
• Peripheral blood film examination reveals marked hypochromasia
and microcytosis, hypochromic macrocytes that represent the
polychromatophilic cells, nucleated RBCs, basophilic stippling, and
occasional immature leukocytes shown in (Fig. 5)
• In thalassemia major, laboratory results show elevated bilirubin,
urinary and fecal urobilinogen levels (Wonke, 2001).
• Hb electrophoresis and alkali denaturation test reveal an elevated Hb
F fraction, which is distributed heterogeneously in the RBCs of
patients with β thalassemia, Hb H in patients with Hb H disease, and
Literature review
26
Hb Bart in newborns with β thalassemia trait. In β -0 β, no Hb A is
usually present; only Hb A2 and Hb F are found (Wonke, 2001).
• Free erythrocyte porphyrin (FEP) tests may be useful in situations in
which the diagnosis of beta thalassemia minor is unclear. FEP level is
normal in patients with the beta thalassemia trait, but it is elevated in
patients with iron deficiency or lead poisoning (Wonke, 2001).
• Decreased hepcidin level in patients with β thalassemia major.
Fig. 5 Peripheral blood film in Cooley anemia.
Iron studies are as follow:
Serum iron level is elevated, with saturation reaching as high as 80%
with decreased total iron binding capacity. The serum ferritin level, which is
frequently used to monitor the status of iron overload, is also elevated.
However, an assessment using serum ferritin levels may underestimate the
iron concentration in the liver of a transfusion-independent patient with
thalassemia. Increased levels of transferrin receptors (TfR) and soluble TfR
(sTfR). Complete RBC phenotype, hepatitis screen, folic acid level, and
Literature review
27
human leukocyte antigen (HLA) typing are recommended before initiation
of blood transfusion therapy (Hillman et al., 2005).
Management of B-Thalassemia Major
1- Regular blood transfusion and chelation therapy:
Regular transfusion therapy to maintain hemoglobin levels of at least 9
to 10 g per deciliter allows for improved growth and development and also
reduces hepatosplenomegaly due to extramedullary hematopoiesis as well
as bone deformities (Cunningham et al., 2004 & Old et al., 2001).
Choice of the scheme for blood transfusion in thalassemia major:
(a) Intermediate schemes: mean Hb 9-10 gm/dl are acceptable in terms
of daily living.
(b) Hypertransfusion schemes: mean Hb 10 gm/dl or greater, improve
the quality of life without accelerating the lethal complication of iron
overload
(c) Supertransfusion program: Maintaince of mean hemoglobin level at
11-12 gm/dl Hb level is not allowed to drop below 12 gm/dl and is
raised regularly to 14 by transfusion every 2-3 weeks
supertransfusion permits an excellent quality of life (Cohen et al.,
2004).
Prevention of Secondary Complications
The most common secondary complications are those related to
transfusional iron overload, which can be prevented by adequate iron
chelation. After ten to 12 transfusions, chelation therapy is initiated with
desferrioxamine B (DFO) administered five to seven days a week by 12-
hour continuous subcutaneous infusion via a portable pump. Recommended
Literature review
28
dosage depends on the individual's age and the serum ferritin concentration.
Young children start with 20-30 mg/kg/day, increasing up to 40 mg/kg/day
after age five to six years. The maximum dose is 50 mg/kg/day after growth
is completed. The dose may be reduced if serum ferritin concentration is
low. By maintaining the total body iron stores below critical values (i.e.,
hepatic iron concentration <7.0 mg per gram of dry weight liver tissue),
desferrioxamine B therapy prevents the secondary effects of iron overload,
resulting in a consistent decrease in morbidity and mortality (Borgna-
Pignatti et al., 2004).
The survival of individuals who have been well transfused and treated
with appropriate chelation extends beyond age 30 years. Offbrand et al
(2003); said that iron-chelation therapy is largely responsible for doubling
the life expectancy of patients with thalassemia major but Deferoxamine
continues to be the most common iron-chelating agent in use, but it has
several limitations: the need for parenteral administration (which is painful
and reduces compliance), side effects, and cost (which is prohibitive in
underdeveloped countries) (Borgna-Pignatti et al., 2004).
Much effort has been invested in the development of new orally active
chelators. Deferiprone, an orally administered chelator, was initially thought
to be an inadequate chelator that might worsen hepatic fibrosis. However,
cumulative worldwide experience indicates that the drug is safe and
effective. Long-term administration of deferiprone does not appear to be
associated with liver damage (Wanless et al., 2003). Adverse effects of
deferiprone include arthralgia, nausea and other gastrointestinal symptoms,
fluctuating liver enzyme levels, leukopenia, and rarely agranulocytosis and
zinc deficiency. Most of these effects can be monitored and controlled
(Ceci et al., 2002)..
Literature review
29
Overly vigorous chelation is associated with deferoxamine-induced
bone dysplasia, which can slow growth velocity in children and may be
only partially reversible (Rund & Rachmilewitz, 2005).
Deferasirox recently became available for clinical use in patients with
thalassemia. It is effective in adults and children and has a defined safety
profile that is clinically manageable with appropriate monitoring. The most
common treatment-related adverse events are gastrointestinal disorders,
skin rash, and a mild, non-progressive increase in serum creatinine
concentration. Post-marketing experience and several phase IV studies will
further evaluate the safety and efficacy of deferasirox. New strategies of
chelation using a combination of desferrioxamine and deferiprone have
been effective in individuals with severe iron overload; toxicity was
manageable (Wu et al., 2004; Tanner et al., 2007).
2- Bone marrow transplantation
Bone marrow transplantation (BMT) from an HLA-identical sib
represents an alternative to traditional transfusion and chelation therapy. If
BMT is successful, iron overload may be reduced by repeated phlebotomy,
thus eliminating the need for iron chelation. The outcome of BMT is related
to the pretransplantation clinical conditions, specifically the presence of
hepatomegaly, extent of liver fibrosis, and magnitude of iron accumulation.
In children who lack the above risk factors, disease-free survival is over
90% (Gaziev & Lucarelli, 2003).
A lower survival rate of approximately 60% is reported in individuals
with all three risk factors. Chronic graft-versus-host disease (GVHD) of
variable severity may occur in 5%-8% of individuals. BMT from unrelated
donors has been carried out on a limited number of individuals with β-
thalassemia. Provided that selection of the donor is based on stringent
Literature review
30
criteria of HLA compatibility and that individuals have limited iron
overload, results are comparable to those obtained when the donor is a
compatible sib. However, because of the limited number of individuals
enrolled, further studies are needed to confirm these preliminary findings
(La Nasa et al., 2005).
3- Cord blood transplantation.
Cord blood transplantation from a related donor offers a good
probability of a successful cure and is associated with a low risk of GVHD
(Locatelli et al., 2003; Walters et al., 2005). For couples who have already
had a child with thalassemia and who undertake prenatal diagnosis in a
subsequent pregnancy, prenatal identification of HLA compatibility
between the affected child and an unaffected fetus allows collection of
placental blood at delivery and the option of cord blood transplantation to
cure the affected child. On the other hand, in case of an affected fetus and a
previous normal child, the couple may decide to continue the pregnancy and
pursue BMT later, using the normal child as the donor (Orofino et al.,
2003).
4- Hematopoietic Stem-Cell Transplantation
Although hematopoietic stem-cell transplantation is the only available
curative approach for thalassemia, it has been limited by the high cost and
the scarcity of HLA-matched, related donors. The past several years have
brought progress in the realms of conditioning regimens, donor
identification and selection, and the development of alternative sources of
hematopoietic stem cells (Talwar & Srivastava, 2004).
Literature review
31
5- Splenectomy
Splenectomy is usually not needed if regular transfusion therapy is
followed. If the child already has a big spleen ( his transfusion requirement
increases to more than times of normal or more than 200 ml packed red
cells or over 400 ml of whole blood per kg), splenectomy is indicated .
Portal vein thrombosis is a recognized complication after splenectomy due
to hypercoagulable state in thalassemia (Wonke, 2001).
6- Diet and vitamins:
No strict regulations regarding diet can be recommended. However,
food rich in iron e.g., meat, liver, kidney and green leafy vegetables should
be avoided.Diet should include food high in phosphorus or phytates e.g.,
cereals bread, milk, soya beans, roasted peas, etc. to inhibit iron absorption.
Similarly tea can be taken along within an hour after meals to reduce iron
absorption (Wonke, 2001).
7- Vitamin C
Ascorbate repletion (daily dose not to exceed 100-150 mg) increases the
amount of iron removed after DFO administration. Vitamin C facilitates
iron chelation with DFO and should be supplemented in patients receiving
DFO (5 mg/kg/d maximum of 200 mg/d). However, in unchelated patients a
low vitamin C status is beneficial (Wonke, 2001).
8- Folic acid
Folic acid (5 mg per week) should be given to patients receiving on or
irregular transfusion. This is because of relative folate deficiency due to
increasesd folate consumption. However,patients receiving regular blood
transfusions ordinarily do not require folic acid unless actual deficiency
state exist (Wonke, 2001).
Literature review
32
Other therapies
1- Combination therapy and Induction of fetal hemoglobin synthesis
New chelation strategies, including the combination or alternate
treatment with the available chelators, are under investigation. Induction of
fetal hemoglobin synthesis can reduce the severity of β-thalassemia by
improving the imbalance between alpha and non-alpha globin chains.
Several pharmacologic compounds including 5-azacytidine, decytabine, and
butyrate derivatives have had encouraging results in clinical trials. These
agents induce Hb F by different mechanisms that are not yet well defined.
Their potential in the management of β-thalassemia syndromes is under
investigation (Pace & Zein, 2006).
2- Hydroxyurea treatment
The efficacy of hydroxyurea treatment in individuals with thalassemia is
still unclear. Hydroxyurea is used in persons with thalassemia intermedia to
reduce extramedullary masses, to increase hemoglobin levels, and, in some
cases, to improve leg ulcers. A good response, correlated with particular
polymorphisms in the beta-globin cluster (i.e., C > T at -158 G gamma), has
been reported in individuals with transfusion dependence. However,
controlled and randomized studies are warranted to establish the role of
hydroxyurea in the management of thalassemia syndromes (Bradai et al.,
2003 ).
3- Correction of molecular defects
The possibility of correction of the molecular defect in hematopoietic
stem cells by transfer of a normal gene via a suitable vector or by
homologous recombination is being actively investigated (Sorrentino &
Niehuis, 2001).
Literature review
33
Initial efforts at gene therapy were directed against diseases of the β -
globin gene. This therapeutic strategy involves the insertion of a normally
functioning β -globin into the patient's autologous hematopoietic stem cells
(Puthenveetil & Malik 2004).
The major problems with this type of gene therapy have been related to
vector construction. The genetic elements of the vector that are necessary
for appropriate regulation of the inserted gene have been defined .However,
the therapeutic gene must be inserted into a hematopoietic stem cell and
must be expressed at high levels, over an extended period, in an erythroid-
specific manner. In addition, the vector must be safe from recombination or
mutagenesis. Oncoretroviral and adenoviral vectors have been found to be
unsuitable for various reasons) (Puthenveetil &Malik 2004).
Recombinant human erythropoietin was shown to provide the benefit of
increasing "thalassemic erythropoiesis" without raising fetal hemoglobin.
The effect appeared to be dose-dependent and was observed primarily in
patients with thalassemia intermedia who had undergone splenectomy
(Persons et al., 2003).
Recently, long-acting darbepoetin alfa was shown to increase
hemoglobin levels substantially in patients with hemoglobin E β–
thalassemia disease. Two important obstacles to the use of recombinant
human erythropoietin are its relatively high cost and its subcutaneous
administration route, which restrict its use in developing countries.
Appropriate clinical protocols are needed to delineate the role of
recombinant human erythropoietin (alone or in combination with the
aforementioned drugs) in the treatment of thalassemia (Persons et al.,
2003).
Literature review
34
IRON METABOLISM
Iron is an essential nutrient that is required for the oxygen-carrying
capacity of hemoglobin. Failure to incorporate adequate iron into heme
results in impaired erythrocyte maturation, leading to microcytic,
hypochromic anemia. Therefore, circulating factors that modulate iron
availability are of major importance in erythropoiesis. Normally, the total
body iron endowment is maintained within a tight range between 3 and 5 g
(Andrews, 2000).
Systemic iron is distributed among erythrocyte precursors in the bone
marrow, tissue macrophages, liver, and all other tissues, with the largest
amount found in circulating erythrocytes. Homeostasis is maintained by
regulating the levels of plasma iron. Hepcidin, a circulating peptide
hormone, has recently emerged as a key modulator of plasma iron
concentration and thus, a central regulator of iron homeostasis (Nemeth,
2004).
The cross-talk which has taken place in recent years between clinicians
and scientists has resulted in a greater understanding of iron metabolism
with the discovery of new iron-related genes including the hepcidin gene
which plays a critical role in regulating systemic iron homeostasis.
Consequently, the distinction between (a) genetic iron-overload disorders
including haemochromatosis related to mutations in the HFE, hemojuvelin,
transferrin receptor 2 and hepcidin genes and (b) non-haemochromatotic
conditions related to mutations in the ferroportin, ceruloplasmin, transferrin
and di-metal transporter 1 genes, and (c) acquired iron-overload syndromes
has become easier (Loreal et al., 2005).
Iron is one of the most common elements in nature and a transition
metal , iron is involved in electron transport and maintenance of the
Literature review
35
respiratory chain , it is required for the functioning of proteins involved in (
oxidative energy production, oxygen transport, mitochondrial respiration
and inactivation of harmful oxygen radicals) , it is essential for the synthesis
of hemoglobin and myoglobin , it plays an important role in detoxification
of the reactive species and it is rate limiting in DNA synthesis (Fig. 6)
(Taketani , 2005).
In a normal balanced state, 1–2 mg of iron enters and leaves the body
every day. Dietary iron is absorbed by duodenal enterocytes and circulates
in the plasma bound to transferrin, the main iron transport protein. Most of
the circulating iron is used by the bone marrow to generate hemoglobin for
red blood cells, while around 10–15% is utilized by muscle fibers to
generate myoglobin. Iron released by tissue breakdown is absorbed and
recycled. Excess iron is stored by parenchymal cells in the liver and
reticuloendothelial macrophages. Traces of iron are lost each day by
sloughing of mucosal cells, loss of epithelial cells and any blood loss. Since
the human body has not evolved a mechanism to clear excess iron, disorders
of iron balance, such as iron overload, are among the most common
diseases in humans (Andrews, 1999).
Literature review
36
Fig. 6 Essential roles of iron (Taketani, 2005).
In a normal state, once iron has been absorbed it is complexed with
transferrin . Because of the important role of iron in metabolism, the human
body has many mechanisms to absorb, transfer and store iron, but none to
excrete excess iron Although serum ferritin levels are indicative of body
iron levels, a number of conditions can alter the correlation between serum
ferritin levels and body iron stores. Acute and chronic inflammation and
infections can greatly influence levels; ascorbate levels and increased
erythropoiesis can also affect circulating ferritin levels (Fig. 7) (Fleming
and Bacon, 2005).
Literature review
37
Fig. 7 Normal distribution and storage of body iron (Andrews, 1999).
Iron Metabolism and Homeostasis
Iron metabolism
Nearly all circulating iron is bound by the abundant serum glycoprotein
transferrin. Transferrin can carry one (monoferric transferrin) or two
(holotransferrin) atoms of iron per protein molecule. Erythroid precursors
are the primary consumers of circulating transferrin-bound iron. During
differentiation, changing rates of hemoglobin production correlate with
variations in the cell surface complement of transferrin receptor-1 (TFR1).
Fluctuations in TFR1 expression control the amount of transferrin-bound
iron entering into erythroid cells (Chan &Gerhardt, 1992).
The process by which transferrin delivers iron to these cells is called the
transferrin cycle. Upon binding to TFR1 at the cell surface, transferrin and
its iron are endocytosed. These endosomal compartments are actively
Literature review
38
acidified by proton pumps. Acidification facilitates iron release from
transferrin because low pH decreases the affinity of the protein for iron.
Iron then leaves the endosome through divalent metal ion transporter 1
(DMT1, also known as Nramp2 and SLC11A2) to become available for
heme biosynthesis (Canonne-Hergaux et al., 2001).
The insertion of iron into protoporphyrin IX, the final step in heme
biosynthesis, occurs in the mitochondrion. Mitoferrin, a protein mutated in
anemic zebra fish, was recently shown to act as a mitochondrial iron
importer necessary for heme biosynthesis . However, another group has
postulated that iron is transferred from endosomes directly into
mitochondria through direct membrane contacts between the organelles.
The fates of TFR1 and apotransferrin are more certain—they are recycled to
the cell surface and circulation, respectively, where they repeat the cycle
(Sheftel, 2007).
The amount of circulating, transferrin-bound iron is determined by three
coordinated processes: macrophage iron recycling, duodenal iron
absorption, and hepatic iron storage. When iron is administered
therapeutically, it is assimilated by one or more of these three tissues, which
play critical roles in iron metabolism and the maintenance of iron
homeostasis (Fig. 8) (Andrews, 2000).
1. Macrophage iron recycling Normal human erythrocytes have a finite life span of approximately 4
months. Tissue macrophages remove senescent and damaged erythrocytes
from circulation and breakdown hemoglobin to recycle iron, supplying most
of the requirement for new erythropoiesis. The process by which
macrophages distinguish aged and damaged erythrocytes is not fully
Literature review
39
understood, but it is likely that morphological changes in the erythrocyte
membrane facilitate recognition and uptake by macrophages (Knutson &
Wessling-Resnick, 2003).
Binding of erythrocytes to the macrophage cell surface initiates
phagocytosis and lysosome-mediated degradation of the erythrocyte
membrane. Heme oxygenases catalyze the oxidation of heme to biliverdin,
free iron, and carbon monoxide (Yachie, 1999).
Fig. 8 Major pathways of iron transfer between cells and tissues
(Andrews, 2000).
Similar to the transferrin cycle, liberated iron may be pumped from the
phagosome into the cytoplasm by DMT1, though this has not been
definitively established. Heme-derived iron can be utilized by the cell,
sequestered within the multimeric iron storage protein ferritin or exported
into the plasma (Jabado et al., 2002).
Literature review
40
The transmembrane transporter ferroportin is activated in macrophages
after erythrophagocytosis (Canonne-Hergaux et al., 2006). Ferroportin is
the only cellular iron exporter that has been identified in vertebrates
(Abboud & Haile, 2000). It is expressed in cells of the extraembryonic
visceral endoderm that provide nourishment to the developing embryo, in
the intestinal epithelium and in spleen and liver macrophages that recycle
iron (Donovan et al., 2005).
Tissue macrophages contain large amounts of iron, apparently because
they are unable to export it to the circulation. These results support the idea
that most iron liberated from heme in macrophages is mobilized through
ferroportin-mediated iron export to be utilized for erythropoiesis. Because
ferroportin transports ferrous iron, iron must be oxidized to its ferric form in
order to bind circulating transferrin (Harris, 1999).
A circulating ferroxidase, ceruloplasmin, is thought to carry out the
oxidation of iron exported from macrophages also ceruloplasmin appears to
be necessary to keep ferroportin on the cell surface. For both of these
reasons, it is not surprising that ceruloplasmin deficiency (a
ceruloplasminemia) leads to tissue iron loading with low transferrin
saturation and, often, mild anemia (De Domenico, 2007).
2. Duodenal iron absorption
In contrast to some other metals, there is no regulated mechanism for
iron excretion through the liver or kidneys. Macrophage-mediated iron
recycling alone cannot sustain erythropoiesis over the long term. Early in
life, the overall iron endowment must be increased to support growth. Later,
small obligatory iron losses from bleeding and exfoliation of skin and
Literature review
41
mucosal cells would lead to negative iron balance if not offset by
continuous iron intake. Thus, iron balance is achieved through regulated
dietary iron absorption (Andrews, 2000).
Dietary iron absorption occurs in the most proximal part of the
duodenum, the first section of the small intestine (Fig. 9). There, acidity
from stomach acid aids in the absorption of both the heme iron, primarily
derived from hemoglobin and myoglobin in meats and the inorganic iron,
from other food sources (Qiu et al., 2007).
Inorganic iron in the intestinal lumen is primarily in its ferric, oxidized
form. In order for iron to be absorbed, it must be reduced to the ferrous
form. Reduction of iron can be carried out by an enterocyte apical
membrane protein duodenal cytochrome b (Dcytb) (McKie et al., 2001).
The fact that the same iron transporter is used for cellular iron uptake
both in transferrin cycle endosomes and at the apical surface of the
intestinal epithelium is somewhat surprising. These two membranes are
quite different and must be reached through distinct targeting signals.
However, both sites are within a low-pH milieu, important because DMT1
uses cotransport of protons to move iron across the membrane (Sacher et
al., 2001; Xu et al., 2004).
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42
Fig. 9 Iron absorption (Andrews, 2000).
Depending on body iron needs, intracellular iron can be stored within
the enterocyte or exported into the plasma. Net absorption requires transfer
across the basolateral surface of the epithelium—iron retained within
enterocytes is lost from the body when those cells senesce and are shed into
the gut lumen. It appears that the transmembrane transporter ferroportin is
responsible for most if not all basolateral iron transfer (Donovan et al.,
2005).
A ferroxidase activity acts in concert with ferroportin-mediated export.
This can be supplied by the enterocyte-associated multicopper oxidase,
hephaestin, or by its circulating homologue, ceruloplasmin (Vulpe et al.,
1999).
3. Hepatocyte iron storage
Hepatocytes serve many important functions including detoxification of
the blood production of proteins that aid in host defense and storage of
essential nutrients such as glucose and iron. Excess circulating iron in both
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43
transferrin-bound and nontransferrin-bound forms can be taken up by
hepatocytes (Trenor et al., 2000).
Similarly, DMT1 cannot account for all hepatocyte iron uptake because
DMT1 knockout mice and human patients carrying loss-of-function
mutations in DMT1 accumulate iron in the liver (Iolascon et al., 2006).
Other hepatic iron importers must exist to participate in efficient iron
storage. Hepatocyte membrane proteins such as megalin, TFR2, CD163,
and L-type calcium channels are candidate iron importers that employ a
variety of molecular mechanisms to bring iron into cells (Borregaard &
Cowland, 2006).
Although hepatocyte iron uptake is not fully understood, it is clear that
once inside the cell, iron can be utilized in cellular processes or sequestered
in ferritin. When iron loss or demand is too great to rely solely on recycling
and absorption, iron is mobilized from hepatic storage to sustain
erythropoiesis. Ferroportin and ceruloplasmin are believed to be involved in
the process of iron export from hepatocytes, but this has not been shown
directly (Kozyraki et al., 2001; Kristiansen et al., 2001).
4. Systemic iron homeostasis
Systemic iron homeostasis maintains body iron content within tolerable
limits and dictates iron distribution. As the largest consumer of iron, the
erythron is particularly sensitive to iron insufficiency. When body iron
stores are depleted, iron deficiency anemia ensues. Rarely, iron deficiency
anemia can be caused by genetic lesions that interfere with intestinal iron
absorption, erythroid iron assimilation, or both. The best characterized of
these are mutations preventing the production of transferrin or inactivating
DMT1 (Iolascon et al., 2006; Mims et al., 2004).
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44
Far more commonly, deficiency results from an imbalance between
increased iron requirements associated with growth and blood loss and iron
acquisition from the diet. In this case, iron deficiency anemia is
characterized by low plasma iron, decreased iron stores, and the
accumulation of iron-free protoporphyrins. Iron-deficient erythrocytes are
small (microcytic), pale (hypochromic), and relatively fragile. Oxygen-
carrying capability caused by iron deficiency has measurable effects on
quality of life, associated with symptoms including fatigue and tachycardia
(Beutler et al., 2000).
Iron replacement therapy, through dietary supplementation, intravenous
iron, or transfusion of iron-rich erythrocytes, is the only treatment for iron
deficiency anemia. Although essential for several important cellular
functions, iron’s capacity to donate and accept electrons makes the metal
toxic. Several proteins such as transferrin, ferritin, lactoferrin, lipocalin, and
myoglobin exist to bind iron in a variety of contexts. Binding to these
proteins renders iron less able to react with its environment (Britton et al.,
2002).
Thus, a safe upper limit of body iron for each individual is set by the
capacity of iron-binding proteins to sequester iron. Once this capacity is
reached, free iron accumulates within the plasma and cells. The
accumulation of excess iron is undesirable because ferrous iron reacts with
hydrogen peroxide to produce hydroxyl radicals. These radicals damage
macromolecules resulting in cellular and tissue dysfunction and organ
failure associated with iron overload disorders (Britton et al., 2002).
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45
Iron Overload
The human body can only excrete one to two milligrams of iron. With
every unit of blood, about 200 to 250 milligrams of iron are transfused,
therefore iron overload will occur. Generally, iron overload can be
classified as primary (hereditary) or secondary (acquired), depending on
whether it results from a primary defect in the regulation of iron balance or
secondary to other genetic or acquired disorders or their treatment:
Primary iron overload results from abnormally regulated iron
absorption: Hereditary hemochromatosis is an example; caused by a
missense mutation on the C282Y gene. This mutation results in the
incorrect processing of regulatory receptors in the intestine (Porter, 2001).
Secondary iron overload can result as the indirect effect of a condition
or occur as a result of its treatment:
Ineffective erythropoiesis and anemias requiring repeated blood
transfusions such as (beta-thalassemia major, thalassemia intermediate,
myelodysplastic syndromes and sickle cell disease) (Porter, 2001).
When iron homeostasis is in balance, iron is absorbed from the diet
(gut) at a rate equivalent to 1–2 mg/day. After absorption from the
duodenum, iron enters the plasma where it is complexed with transferrin.
Transferrin-bound iron in the plasma is the main pool supplying iron to the
erythron, which cycles 20–30 mg of iron each day, as well as to hepatocytes
and other parenchyma, which cycle around 10% of this amount (Anderson
et al., 2007).
When transferrin becomes completely saturated during conditions of
iron overload, iron circulates in the bloodstream as extracellular non-
transferrin-bound iron (NTBI). In the case of transfusion the
reticuloendothelial system loses the capacity or gets filled up and has no
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46
ability to store more iron. It is bound to transferrin and when the capacity of
transferrin is saturated, it becomes the non-transferrin bound iron (NTBI)
(Anderson et al., 2007).
As iron loading progresses, the capacity of serum transferrin, the main
transport protein of iron, to bind and detoxify iron may be exceeded and a
non–transferrin- bound fraction of plasma iron may promote the generation
of free hydroxyl radicals, propagators of oxygen-related damage. The body
maintains a number of antioxidant mechanisms against damage induced by
free radicals, including superoxide dismutases, catalase, and glutathione
(Hershko et al., 1998).
In the absence of chelating therapy the accumulation of iron results in
progressive dysfunction of the heart, liver, and endocrine glands. Within the
heart, changes associated with chronic anemia are usually present in
patients who are not receiving transfusions and are aggravated by iron
deposition. In response to iron loading, human myocytes in vitro increase
the transport of non–transferrin-bound iron possibly thereby aggravating
cardiac iron loading (Parkes et al., 1993).
Extensive iron deposits are associated with cardiac hypertrophy and
dilatation, degeneration of myocardial fibers. In patients who are receiving
transfusions but not chelating therapy, symptomatic cardiac disease has
been reported within 10 years after the start of transfusions and may be
aggravated by myocarditis and pulmonary hypertension (Aessopos et al.,
1995; Du et al., 1997).
The survival of patients with β-thalassemia is determined by the
magnitude of iron loading within the heart. Iron-induced liver disease is a
common cause of death in older patients and is often aggravated by
infection with hepatitis C virus. Within two years after the start of
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47
transfusions, collagen formation and portal fibrosis have been reported; in
the absence of chelating therapy, cirrhosis may develop in the first decade
of life. The risk of hepatic fibrosis is augmented at body iron burdens
corresponding to hepatic iron concentrations of more than 7 mg per gram of
liver, dry weight (Fig. 10) (Niederau et al., 1996).
Fig. 10 Hepatic Iron Burden over Time and the Effect of Various
Hepatic Iron Concentrations in Patients with Thalassemia Major,
Homozygous Hemochromatosis, and Heterozygous Hemochromatosis
(Nancy & Livieri, 1999).
The transport of non–transferrin-bound iron is increased, possibly
aggravating iron loading in vivo. Iron loading within the anterior pituitary is
the primary cause of disturbed sexual maturation, early secondary
amenorrhea occurs in approximately one quarter of female patients over the
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48
age of 15 years. Even in the modern era of iron-chelating therapy, diabetes
mellitus is observed in about 5 percent of adults (Olivieri et al., 1998).
As the iron burden increases and iron-related liver dysfunction
progresses, hyperinsulinemia occurs as a result of reduced extraction of
insulin by the liver, leading to exhaustion of beta cells and reduced
circulating insulin concentrations. Studies reporting reduced serum
concentrations of trypsin and lipase suggest that the exocrine pancreas is
also damaged by iron loading. Over the long term, iron deposition also
damages the thyroid, parathyroid, adrenal glands and may provoke
pulmonary hypertension, right ventricular dilatation, and restrictive lung
disease. Bone density is markedly reduced in patients with β-thalassemia
(Tai et al., 1996; Olivieri et al., 1998).
Iron Chelation Therapy
Indications for chelation therapy
(i) Transfusions of 2 units/month persisting for at least one year.
(ii) Ferritin level of 1000 ng/ml.
(iii) Patients in which transplant is imminent.
(iv) Consider earlier chelation therapy in patients with compromised
organ function who experience increased transfusion burden
(Franchini & Veneri 2004).
Treatment Options
Three products are available worldwide:
1) Deferoxamine: Deferoxamine is indicated for the treatment of acute iron intoxication
and of chronic iron overload due to transfusion-dependent anemias.
Deferoxamine chelates iron by forming a stable complex that prevents the
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49
iron from entering into further chemical reactions. It readily chelates iron
from ferritin and hemosiderin but not readily from transferrin; it does not
combine with the iron from cytochromes and hemoglobin. Deferoxamine
does not cause any demonstrable increase in the excretion of electrolytes or
trace metals. Theoretically, 100 parts by weight of Deferoxamine is capable
of binding approximately 8.5 parts by weight of ferric iron (Borgna-
Pignatti et al, 2004).
Deferoxamine is metabolized principally by plasma enzymes, but the
pathways have not yet been defined. The chelate is readily soluble in water
and passes easily through the kidney, giving the urine a characteristic
reddish color. Some is also excreted in the feces via the bile. Long-term
therapy with deferoxamine slows accumulation of hepatic iron and retards
or eliminates progression of hepatic fibrosis. Iron mobilization with
deferoxamine is relatively poor in patients under the age of 3 years with
relatively little iron overload. The drug should ordinarily not be given to
such patients unless significant iron mobilization (e.g., 1 mg or more of iron
per day) can be demonstrated (Borgna-Pignatti et al, 2004).
2) Deferiprone: Promising chelating compounds are the 3-hydroxypyrid- 4-ones which
form strong, highly stable and water soluble 3:1 complexes with the Fe3+-
ion at physiological pH both in vitro and in vivo. The binding constants are
high in comparison with those of desferrioxamine and transferrin, the
physiological transport protein for Iron. They are capable of mobilizing iron
from transferrin, ferritin, hemosiderin, hepatocytes and macrophages .The
affinity for divalent metal ions is low (Franchini & Veneri 2004).
The first representative of this group which has been tested in humans is
deferiprone. This compound has shown very little toxicity in animal studies.
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50
After oral ingestion, deferiprone is rapidly absorbed, metabolised in the
liver and excreted in the urine as glucuronide (at least 90% of the absorbed
dose), as iron complex or as unchanged drug .Relatively little is known
about the pharmacodynamics of deferiprone (Franchini & Veneri 2004).
3) Deferasirox: Exjade (deferasirox) is an orally active chelator that is selective for iron
(as Fe3+). It is a tridentate ligand that binds iron with high affinity in a 2:1
ratio. Although deferasirox has very low affinity for zinc and copper there
are variable decreases in the serum concentration of these trace metals after
the administration of deferasirox. The clinical significance of these
decreases is uncertain. Deferasirox is highly (~99%) protein bound almost
exclusively to serum albumin. The percentage of deferasirox confined to the
blood cells was 5% in humans. The volume of distribution at steady state
(Vss) of deferasirox is 14.37 ± 2.69 L in adults. Deferasirox and metabolites
are primarily (84% of the dose) excreted in the feces. Renal excretion of
deferasirox and metabolites is minimal (8% of the administered dose). The
mean elimination half-life (t1/2) ranged from 8-16 hours following oral
administration (Cappellini et al., 2006).
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51
HEPCIDIN
Hepcidin is the key regulator of systemic iron homeostasis and a
pathogenic factor in anemia of inflammation and hereditary
hemochromatosis. Hepcidin inhibits iron influx into plasma from duodenal
enterocytes that absorb dietary iron, from macrophages that recycle iron
from senescent erythrocytes and from hepatocytes that store iron. Hepcidin
acts by binding to the cellular iron exporter ferroportin and causing its
internalization and degradation. Hepcidin production is increased by iron
excess and inflammation and decreased by anemia and hypoxia, however,
the molecular mechanisms of hepcidin regulation by iron, oxygen and
anemia are still unclear. Iron-loading anemias are disorders in which
hepcidin is regulated by opposing influences of ineffective erythropoiesis
and concomitant iron overload (Pigeon et al., 2001).
Hepcidin peptide:
Hepcidin is a 25-amino-acid iron peptide hormone. Initially identified in
human plasma and urine as an anti-microbial molecule. located on
chromosome 19q13.1, encodes a precursor protein preprohepcidin of 84
amino acids (aa). During its export from the cytoplasm, this full-length pre-
prohepcidin undergoes enzymatic cleavage, resulting in the export of a 64
aa prohepcidin peptide into the ER lumen. Serum prohepcidin levels have
been widely used to evaluate iron overload in clinical and preclinical studies
(Park et al., 2001).
Bioactive hepcidin indeed bears structural similarity to disulfide-rich
antimicrobial peptides. Hepcidin is synthesized in the liver as a propeptide
and has a characteristic furin cleavage site immediately N-terminal to the
25-amino-acid peptide (Park et al., 2001).
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52
The molecule is a simple hairpin whose 2 arms are linked across by
disulfide bridges in a ladderlike configuration. One highly unusual feature
of the molecule is the presence of disulfide linkage between 2 adjacent
cysteines near the turn of the hairpin. Compared with most disulfide bonds,
disulfide bonds formed between adjacent cysteines are stressed and could
have a greater chemical reactivity. Like other antimicrobial peptides,
hepcidin displays spatial separation of its positively charged hydrophilic
side chains from the hydrophobic ones, a characteristic of peptides that
disrupt bacterial membranes (Fig. 11) (Detivaud et al., 2005).
Fig. 11 Amino acid sequence and a model of the major form of human hepcidin.
The amino and carboxy termini are labeled as N and C, The pattern of disulfide
linkages between the 8 cysteines is also shown in the amino acid sequence (Ganz,
2003).
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53
Molecular regulation of hepcidin
The expression of hepcidin is regulated by many genes as HFE,
Hemojuvelin (HJV) and growth differentiation factor 15 (GDF15)
(Papanikolaou et al., 2004) .
HFE, Human hemochromatosis protein an MHC-class 1-like molecule,
is highly expressed in crypt cells (enterocyte, macrophage and hepatocyte).
The HFE gene is located on short arm of chromosome 6 at location 6p21.3.
It seems to enhance transferrin-bound iron uptake from the plasma into
crypt cells via TfR1, and may also inhibit the release of iron from the cell
via ferroportin. It regulates hepcidin expression, mechanism is uncertain but
it may participate in a signaling complex with TfR2 and interacts with TfR1
& β-2-microglobulin (Munoz et al.,2009).
Hemojuvelin (HJV) is a membrane-bound and soluble protein in
mammals that is responsible for the iron overload condition known as
juvenile hemochromatosis in humans, a severe form of hemochromatosis.
The hemojuvelin protein is encoded by the HFE2 gene. Mutations in HJV
are responsible for the vast majority of juvenile hemochromatosis patients.
Hemojuvelin is highly expressed in skeletal muscle and heart, and to a
lesser extent in the liver. One insight into the pathogenesis of juvenile
hemochromatosis is that patients have low to undetectable urinary hepcidin
levels, suggesting that hemojuvelin is a positive regulator of hepcidin, the
central iron regulatory hormone (Papanikolaou et al., 2004).
For many years the signal transduction pathways that regulate systemic
iron homeostasis have been unknown. However, a study by Babitt et al
2006 suggested that hemojuvelin interacts with bone morphogenetic protein
(BMP), possibly as a co-receptor, and may signal via the SMAD pathway to
regulate hepcidin expression (Zhang et al., 2008).
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54
Recently, the iron and erythropoiesis-controlled growth differentiation
factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in
β-thalassaemia patients, thereby increasing iron absorption despite iron
overload. (Tamary et al., 2008).
Mechanism of hepcidin action
Hepcidin causes a decrease in serum iron. Injection of hepcidin agonist
into mice results in hypoferremia already within 1 hour, and a similar effect
was seen with acute induction of hepcidin expression in tetracycline-
inducible transgenic mice. The hypoferremia develops because hepcidin
blocks the supply of iron into plasma while the relatively small plasma iron
pool is rapidly used up by erythrocyte precursors. Hepcidin blocks iron
flows from macrophages recycling iron, from stores in the liver and from
enterocytes absorbing dietary iron (Viatte et al., 2005).
The molecular mechanism is based on hepcidin's interaction with
ferroportin. Ferroportin is the only known cellular iron exporter in
vertebrates, and is expressed in all the tissues handling major iron flows as
reticulo-endothelial macrophages, hepatocytes and duodenal enterocytes.
Hepcidin binds to ferroportin and causes its internalization and degradation
in lysosomes, thus effectively blocking the export of iron from the cells
(Nemeth et al., 2004).
In vitro, the internalization of ferroportin occurs less than 1 hour after
addition of hepcidin, consistent with the kinetics of hypoferremia observed
in vivo. Likewise, injection of radiolabeled hepcidin in mice resulted in
equally rapid accumulation of radioactive hepcidin in ferroportin-rich
organs (spleen, duodenum and liver), providing further support for the key
role of hepcidin-ferroportin interactions in the regulation of iron transport
(Fig. 12) (Rivera et al., 2005b).
Literature review
55
Fig. 12 Physiology of hepcidin-ferroportin interaction (Rivera et al.,
2005b).
• Ferroportin = iron export protein.
• Circulating hepcidin.
• Hepcidin binds to ferroportin.
• Internalization, then ferroportin degradation.
• Degraded ferroportin.
• Decreased iron release due to decreased ferroportin.
Hepcidin maintains iron homeostasis through a physical interaction with
ferroportin has led to a plausible model for the normal maintenance of iron
homeostasis (Fig. 13) and the disruption of homeostasis in human disease
(Zoller &Cox 2005).
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56
Fig. 13 Normal iron homeostasis mediated by an iron-sensing feedback
loop (Wrighting & Andrews, 2008).
Hepcidin is the principal regulator of extracellular iron concentration
Hepcidin is increased by iron loading and this provides the homeostatic
loop to maintain normal extracellular concentrations of iron. A rise in
plasma iron (e.g. after a meal or an iron supplement) leads to increased
hepcidin production. In turn, elevated hepcidin reduces the concentration of
ferroportin molecules on the cell surface and inhibits the entry of iron into
plasma, thus allowing the iron concentration to return to normal levels.
Conversely, in iron deficiency, hepcidin production decreases, allowing a
greater export of iron through ferroportin into plasma; this results in an
appropriate rise in circulating iron (Delaby et al.,2005).
Chronic alterations of hepcidin expression result in systemic disorders
of iron metabolism and maldistribution of iron in the body. Homozygous
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57
disruption of the hepcidin gene in humans or mice leads to severe iron
overload. Conversely, overexpression of hepcidin in transgenic mice
resulted in severe microcytic, hypochromic anemia. Mice with tumor
xenografts engineered to overexpress hepcidin also developed hypoferremia
and anemia, with iron sequestration in the stores (Rivera et al., 2005a).
Similarly, overproduction of hepcidin by liver tumors in patients with
type 1a glycogen storage disease caused iron-refractory anemia which
resolved only after resection of the tumor, or after liver transplantation.
Altogether, these studies confirm the role of hepcidin as the negative
regulator of iron absorption, recycling and release from stores (Weinstein et
al.,2002).
Regulation of hepcidin synthesis; implications for the disorders of iron
metabolism
As an iron-regulatory hormone, hepcidin synthesis is increased by iron
loading and inflammation and is decreased by anemia and hypoxia. Except
for inflammation, the molecular pathways underlying regulation of hepcidin
are not well understood. Dysregulation of hepcidin synthesis, however,
appears to be the key factor in the pathogenesis of a spectrum of iron
disorders, with hepcidin deficiency causing iron overload and elevated
hepcidin mediating anemia of inflammation (Loreal et al.,2005).
Regulation by inflammation
Hepcidin synthesis is markedly induced by infection and inflammation.
In animal models, injection of turpentine, lipopolysaccharide, or Freund's
adjuvant increased hepatic hepcidin mRNA expression, and in humans,
infusion of lipopolysaccharide resulted in a rapid increase in urinary
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58
hepcidin. These effects are mediated by inflammatory cytokines including
interleukin (IL)-6 and IL-1. IL-6 is sufficient for hepcidin induction since
direct treatment of primary hepatocytes with IL-6 resulted in rapid
upregulation of hepcidin mRNA and infusion of human volunteers with IL-
6 resulted in increased urinary hepcidin excretion within just 2 hours after
infusion (Nemeth et al.,2004 ; Nemeth et al.,2003).
Hepcidin as a mediator of anemia of inflammation
Hepcidin increase was associated with hypoferremia in all the
inflammatory models. Increased hepcidin appears to be the key factor in the
development of anemia of inflammation. Hypoferremia and anemia of
inflammation have likely developed during evolution as a host defense
strategy against infection, limiting the growth of invading microbes.
However, the same strategy has become maladaptive with the increasing
incidence of non-infectious diseases associated with excessive cytokine
production, including rheumatologic diseases, inflammatory bowel disease,
multiple myeloma and other malignancies (Rivera et al., 2005a).
Anemia of inflammation is characterized by decreased serum iron and
impaired mobilization of iron from stores, evident from the presence of iron
in bone-marrow macrophages and increased ferritin levels. These are the
very features observed in mouse models with increased hepcidin.
Intraperitoneal injection of synthetic hepcidin resulted in hypoferremia
within 1 hour, and chronic over expression of hepcidin in tumors resulted in
anemia and hypoferremia despite increased liver iron stores (Rivera et al.,
2005b).
Furthermore, patients with infection or inflammatory disorders have
elevated urinary excretion of hepcidin compared to healthy controls. Thus,
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59
the molecular pathway from inflammation to anemia centers on the elevated
plasma hepcidin which causes the internalization and degradation of
ferroportin in macrophages, hepatocytes and duodenal enterocytes,
sequestering iron in these cells and blocking iron flows into plasma
(Nemeth et al.,2003).
As the bone marrow continues to utilize iron for hemoglobin synthesis,
the small plasma iron compartment becomes rapidly depleted causing
hypoferremia. Persistent hypoferremia, as in chronic inflammation, leads to
iron-restricted erythropoiesis and anemia. However, it still remains to be
established whether the increase in hepcidin is the essential factor in the
development of this disorder, since inflammation may contribute to anemia
by alternative hepcidin-independent mechanisms including decreased
erythropoietin production, blunted response to erythropoietin and shortened
erythrocyte lifespan (Nemeth et al., 2003).
Regulation of hepcidin by anemia and hypoxia
Inadequate delivery of oxygen to tissues, which occurs in anemia or
hypoxemia, would be expected to result in homeostatic decrease in hepcidin
synthesis. The decrease in hepcidin levels would in turn allow increased
iron mobilization from macrophages and hepatocytes, and increased iron
absorption from the diet, making more iron available for erythrocyte
production. Indeed, hepcidin was shown to be suppressed by anemia and
hypoxia; however, the molecular pathways that regulate this response are
still unclear. Though anemia may act by causing liver hypoxia, it is also
possible that the pathways of hepcidin regulation by oxygen and by
anemia/erythropoiesis are independent (Nicolas et al., 2002).
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60
Fig. 14 Hepcidin mRNA expression (Nicolas et al., 2002).
Exposure to hypoxia decreased hepcidin mRNA expression (Fig. 14). In
general, cellular oxygen sensing and the related transcriptional control are
largely mediated by the hypoxia-inducible factor (HIF). However, unlike
most of the target genes that are transcriptionally activated by HIF, hepcidin
expression is negatively regulated by hypoxia. In addition, except for
human hepcidin promoter, the promoters in other mammals do not contain
the consensus binding sites for HIF, and direct involvement of HIF in
transcriptional regulation of hepcidin remains to be explored (Nicolas et al.,
2002).
Hepcidin mRNA increased by dietary iron and mice injected with
bacterial LPS (indirect effect mediated by IL-6). Decreased by Hypoxemia
and increased erythroid iron demand (phlebotomy or hemolysis).
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61
Hepcidin and anemia of chronic disease
Hepcidin appears to block iron uptake in the duodenum and release
from RE macrophages, thereby decreasing delivery of iron to RBC
precursors. A hepcidin antagonist might benefit patients with ACD but
might be contraindicated in patients with infections . Elevated urinary
hepcidin levels might be useful to diagnose ACD (Nemeth et al., 2003).
Role of Hepcidin in Iron –Loading Anemias
Iron–Loading anemias are characterized by ineffective erythropoiesis
and increased intestinal iron absorption. Erythrocyte transfusions further
exacerbate the iron overload, the development of hepcidin- based
diagnostics and therapies for iron-loading anemias may offer more effective
approaches to prevent the toxicity associated with iron overload. The most
common iron- loading anemias are the intermediate and major forms of β-
thalassemia, but other rare anemias also complicated by iron loading,
including congenital dyserythropoietic anemia, X- Linked sideroblastic
anemia and anemia associated with divalent metal ion transporter 1
(DMT1) mutations (Papanikolaou et al., 2005) .
Role of Hepcidin in β-thalassemia major
In the presence of systemic iron overload, patients with thalassemia
major in whom iron overload was more severe and anemia was partially
relieved by transfusions, had urinary hepcidin concentrations that were
higher than in thalassemia intermedia. These findings were interpreted as
supporting the dominant erythropoietic effect of exogenous hepcidin could
prevent the iron overload in iron–Loading anemias (Loreal et al., 2005).
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62
Iron overload of tissue is the most important complication of β –
thalassemia major and is a major focus of management. In patients who are
not receiving transfusions, abnormally regulated iron absorption results in
increases in body iron burden ranging from 2 to 5 g per year, depending on
the severity of erythroid expansion. Regular transfusions may double this
rate of iron accumulation (Loreal et al., 2005).
Elevated levels of hepcidin in the bloodstream effectively shut off iron
absorption by disabling the iron exporter ferroportin. Conversely, low levels
of circulating hepcidin allow ferroportin to export iron into the bloodstream.
Aberrations in hepcidin expression result in disorders of iron deficiency and
iron overload. It is clear that erythroid precursors communicate their iron
needs to the liver to influence the production of hepcidin and thus the
amount of iron (Loreal et al., 2005).
In a mouse model of beta-thalassaemia, Weizer-Stern and his co-
authors (2006) observed that the liver expressed relatively low levels of
hepcidin, which is a key factor in the regulation of iron absorption by the
gut and of iron recycling by the reticuloendothelial system. It was
hypothesised that, despite the overt iron overload, a putative plasma factor
found in beta-thalassaemia might suppress liver hepcidin expression. Sera
from beta-thalassaemia patients were compared with those of healthy
individuals regarding their capacity to induce changes in the expression of
key genes of iron metabolism in human HepG2 hepatoma cells. Sera from
beta-thalassaemia major patients induced a major decrease in hepcidin
(HAMP) expression. A significant correlation was found between the
degree of downregulation of HAMP induced by beta-thalassaemia major
sera.
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63
Adamsky and his co-authors (2004) have found that iron overload is
less dominant than anaemia in regulating hepcidin expression in the setting
of the β-thalassemia major mouse model. The decreased expression of
hepcidin may explain the increased absorption of iron in thalassemia.
Recently, decreased expression of hepcidin was found in hereditary
haemochromatosis in association with elevated levels of nontransferrin
bound iron. The elevated expression of NGAL, an alternative iron delivery
vehicle, supports the role of nontranferrin bound iron in the abnormal iron
regulation in thalassemia. The decreased HFE expression level is similar to
the finding in hereditary haemochromatosis (Bridle et al., 2003).
Regulation of hepcidin by iron and the lessons from hereditary
hemochromatosis
The only clues about molecules involved in the pathway of hepcidin
regulation by iron come from mutations causing hereditary
hemochromatosis. In addition to juvenile hemochromatosis caused by
inactivating mutations in the hepcidin gene itself, it appears that hepcidin
deficiency is the unifying cause of most types of hereditary
hemochromatosis. Measurements of urinary hepcidin excretion or hepatic
mRNA expression showed that patients and animal models with
homozygous disruption of HFE, transferrin receptor 2 (TfR2) and
hemojuvelin (HJV) all had hepcidin levels inappropriately low for the
systemic iron load (Muckenthaler et al., 2003; Ahmad et al., 2002).
While the precise function of the three molecules is not known, they
likely participate in the sensing of iron or the consequent signal transduction
that regulates hepcidin synthesis and release. Importantly, the degree of
hepcidin deficiency appears to correlate with the severity of the disease. The
Literature review
64
most severe form, juvenile hemochromatosis, is caused by mutations in
either the hepcidin or HJV gene and these are phenotypically
undistinguishable. Patients with HJV mutations have very low or
undetectable urinary hepcidin suggesting that HJV is the key regulator of
hepcidin (Papanikolaou et al., 2004).
Genetic iron-overload disorders may be divided into haemochromatotic
and non-haemochromatotic forms according to patho-physiological and
phenotypic criteria (Table 1) (Pietrangelo, 2007). Haemochromatosis refers
to hereditary iron-overload disorders characterized by normal
erythropoiesis, increased transferrin saturation and parenchymal distribution
of iron deposition, and related to an inaccurate production and/or regulation
and/or activity of hepcidin (Pietrangelo, 2007).
Hepcidin and Erythropoeisis
Hepcidin expression is regulated in response to bone marrow needs
Iron absorption is increased in patients with congenital anemias
characterized by ineffective erythropoiesis. Clinically, increased intestinal
iron absorption compounds the effects of transfusional iron overload in
patients with thalassemia syndromes, sideroblastic anemia, or congenital
dyserythropoietic anemias (Adamsky et al., 2004).
Finch (1994) proposed the existence of an erythroid regulator of
systemic iron homeostasis. The erythron, composed of developing erythroid
cells in the bone marrow and circulating erythrocytes, utilizes about 80% of
the iron found in the plasma. Anemia results from the inability of the
erythroid compartment to receive its full complement of iron. The putative
erythroid regulator communicates the iron needs of the erythron to
influence changes in intestinal iron absorption (Breda et al., 2005).
Literature review
65
Table 1 Main characteristics of genetic iron overload disorders
(Deugnier et al., 2008).
Genetic iron
overload disease Gene
Chromos
ome
Transmis
sion
Onse
t
Clinical
expressio
n
Haemochromatotic
HFE HFE 6p21.3 Recessive Late
Articular
and
hepatic
Hemojuvelin HJV 1p21 Recessive Early
Cardiac
and
endocrine
Hepcidin HAMP 19q13.1 Recessive Early
Cardiac
and
endocrine
Transferrin
receptor 2 TfR2 7q22 Recessive Late Hepatic
Ferroportin disease
type B
SLC40A
1 2q32 Dominant Late
Articular
and
hepatic
Nonhaemochromat
otic Ferroportin
disease type A
SLC40A
1 2q32 Dominant Late Rare
A (hypo)
ceruloplasminemia
Cerulopl
asmin 3q23-q25 Recessive Late
Neurolog
ical
A (hypo)
transferrinemia
Transfer
rin 3q21 Recessive Early
Hematolo
gical
Literature review
66
Hepcidin is an effective inhibitor of iron absorption, the erythroid
regulator includes a mechanism to decrease hepcidin production.
Accordingly, low hepcidin levels have been reported with thalassemia and
other disorders with ineffective erythropoiesis. In these disorders, decreased
hepcidin expression leads to relief of inhibition of ferroportin, resulting in
increased iron release from recycling macrophages and absorptive
enterocytes, increasing availability of iron for erythropoiesis. However, the
iron cannot be effectively utilized by the erythron, leading to accumulation
and tissue iron overload in the face of anemia (Gardenghi et al., 2007;
Jenkins et al., 2007).
Stimulation of erythropoiesis with phenylhydrazine resulted in hepcidin
suppression as expected, but the simultaneous inhibition of erythropoiesis
by irradiation prevented hepcidin suppression despite severe anemia. In
addition, irradiation prevented hepcidin suppression after erythropoietin
administration, ruling out the direct effect of erythropoietin on hepcidin
synthesis (Breda et al., 2005).
Subjects and methods
67
SUBJECTS AND METHODS
This study was carried out in pediatric and clinical pathology
departments of Zagazig University Hospitals in the period from March 2009
to April 2010. The study included 40 children divided into two groups:
I- Group (I): healthy controls group
This group included (10) apparently healthy subjects aged between 4-11
years with a male to female ratio of 1.5:1 (6 males and 4 females).
II- Group (II): patients group
The studied group included 30 β-thalassemic major children patients
aged between 4-10 years with a male to female ratio of 1.5:1 (18 males and
12 females). They were selected from patients attending pediatric
hematology unit and already diagnosed as β-thalassemia major.
All study members were subjected to the following:
a) Full history taking to collect data from children or mothers: Personal
history, diagnosis, blood transfusion, iron chelation and history of
complications (hepatic, renal, cardiac and endocrinal complications).
b) Clinical examination included:
1) General examination
2) Local examination
• Head and neck examination for mongoloid face, earthy face of poorly
chelated patient.
• Cardiac examination to assess cardiac complications.
• Abdominal examination to assess spleen and liver state
(splenomegally, splenectomy or hepatomegally).
Subjects and methods
68
• Abnormalities of long bones and skull.
Laboratory investigations:
A-Routine:
(1) C.B.C by (SYSMEX SF 3000).
(2) Serum iron (colorimetric) (Huebers et al., 1987).
(3) Serum ferritin (Cobas E 411).
(4) TIBC( colorimetric ) (Finch & Huebers, 1986)
(5) Hemoglobin electrophoresis (Valeri et al., 1965).
(6) Liver function tests, kidney function tests and serum Alkaline
phosphatase test were done by (Selectra XL).
B- Special investigations
Hepcidin hormone:
Hepcidin was measured by Enzyme Linked Immunosorbant Assay in
the serum (Park et al., 2001). The Kit was supplied from DRG
International,Inc.,USA.
The principle:
The DRG Hepcidin ELISA Kit is a solid phase enzyme-linked
immunosorbent assay, based on the principle of competitive binding. The
microtiter wells are coated with a monoclonal antibody directed towards the
antigenic site of the bioactive Hepcidin 25 molecule. Endogenous Hepcidin
of a patient sample competes with the added Hepcidin-biotin conjugate for
binding to the coated antibody. After incubation the unbound conjugate is
washed off. An incubation with a streptavidin-peroxidase enzyme complex
Subjects and methods
69
and a second wash step follows. The addition of substrate solution results in
a colour development which is stopped after a short incubation The
intensity of colour developed is reverse proportional to the concentration of
Hepcidin in the patient sample.
The reagents:
The kit reagents include:
(a) Standard: concentrations of synthetic pepide Hepcidin.
(b) Control low and high.
(c) Assay Buffer.
(d) Enzyme Complex contains : Streptavidin peroxidase.
(e) Substrate Solution contains:Tetramethylbenzidine(TMB).
(f) Stop Solution contains: H2SO4.
(g) Wash Solution (40X concentrated).
Reagent Preparation:
• All reagents and required number of strips were brought to room
temperature prior to use.
• The lyophilized contents of the standard vials were reconstituted with
0.5 mL Aqua dest.
• The lyophilized content of the control was reconstituted with 0.5 mL
Aqua dest. And was left to stand for 10 minutes in minimum.
• Deionized water was added to the 40X concentrated Wash Solution.
30 mL of concentrated Wash Solution was diluted with 1170 mL
deionized water to a final volume of 1200 mL.
Subjects and methods
70
Specimen collection
Specimens have been prepared by collecting blood by clean
venipuncture, allowed to clot, and serum has been separated by
centrifugation at 2500 x g for 10 min at 4oC.Specimens were frozen until
use at -20oC.
Assay Procedure
• The desired numbers of micro-titer wells were secured in the holder.
• 10 µl of Sample Buffer was dispensed into each well.
• 20 µl of each Standard, Control and Sample with new disposable tips
were dispensed into appropriate wells.
• Incubation for 30 minutes at room temperature on a plate shaker at ≈
500 rpm was done.
• 150 µL of Assay Buffer and 100 µL of Enzyme Conjugate were
added to each well.
• Incubation for 180 minutes at room temperature on a plate shaker at ≈
500 rpm was done.
• The contents of the wells were briskly shaked out then the wells
were Rinsed 5 times with diluted wash solution (400 µL per well)
and were Striked sharply on absorbent paper to remove residual
droplets .
• 100 µL of Enzyme Complex was dispensed into each well.
• Incubation for 45 minutes at room temperature was done.
• The contents of the wells were briskly shaked out then the wells were
Rinsed 5 times with diluted wash solution (400 µL per well) and
were Striked sharply on absorbent paper to remove residual droplets.
• 100 µL of substrate solution was added to each well.
Subjects and methods
71
• Incubate for 30 minutes at room temperature was done.
• The enzymatic reaction was stopped by adding 100 µl of Stop
Solution to each well.
After adding the Stop Solution, the OD at 450±10 nm was red with a micro-
titer plate reader within 10 minutes.
Calculation of Results
Calculations were done as follows: The average absorbance values for
each set of standards, controls and patient samples were calculated. A
standard curve was constructed by plotting the mean absorbance obtained
from each standard against its concentration. The mean absorbance value
for each sample was used to determine the corresponding concentration .
As the value of iron absorbance increases, the concentration of hepcidin
in serum decreases and vice versa (i.e., reverse proportion relationship). The
concentration of hepcidin in serum was calculated using the curve fitting
equation and shown in Fig. 15.
Subjects and methods
72
Fig. 15 Concentration of hepcidin of patients (in circles) and
concentration of hecidin of control (in squares).
Statistical analysis Data were entered checked and analyzed Epi-Info version 6 and SPP for
windows version 11 (Dean et al., 1994). SPSS windows version 11 was
used for data analysis as follows:
A- Descriptive statistics:
In which data were summarized using:
1) The arithmetic mean ( X ) as an average describing the central
tendency of observations was applied.
2) The standard deviation (SD) as a measure of dispersion of the results around the
mean was calculated. B- Comparison of means:
The comparison was done using the student "t" test for comparison of
means of two independent groups.
Subjects and methods
73
C- Correlation study:
Correlation between variables was done using correlation coefficient
"r".
This test detects if the change in one variable was accompanied by a
corresponding change in the other variable or not. The value of r usually
lies between -1 and +1, where positive indicate a tendency for X and Y to
increase together while negative values indicate a tendency for X to
increase with decrease of Y and vice versa. The significance of ‘r’ was
obtained from the ‘t’ distribution with (n-2) degrees of freedom where n is
number of observation in each group .
D- Level of significance:
For all above mentioned statistical tests done, the threshold of
significance is fixed at 5% level (P value), where:
a) P-value > 0.05 indicates non- significant results.
b) P-value < 0.05 indicates significant results.
c) P-value < 0.001 indicates highly significant results.
Results
74
RESULTS
This study included 40 subjects divided into 10 normal control subjects (Group
I) and 30 β thalassemia major patients (Group II). The age of the normal subjects
in Group I ranged from 5 to 11 years and that of the patients in Group II ranged
from 4 to 10 years as represented in Table 2:
Table 2: Clinical data of the studied groups.
I
N = 10
II
N = 30 t p
Age (years)
Range
X ± sD
5– 11
7.8 ± 1.87
4 – 10
6.97 ± 1.9
1.2 0.23
Gender N % N % X2 P
Male 6 60.0 18 60.0 0.0 1.0
Female 4 40.0 12 40.0
Results
75
Table 3: Liver and kidney functions results of the studied groups.
I II
t P X ± SD (Range) X ± SD (Range)
Total serum Bilirubin (mg/dl)
0.61± 0.15 (0.4- 0.9)
1.7± 0.27 (1.3- 2.5) 12.0 0.001**
Direct Bilirubin (mg/dl)
0.15± 0.07 (0.1- 0.3)
0.55± 0.15 (0.3- 0.9)
7.9 0.001**
Total protein (g/dl) 7.1± 0.35 (6.2- 7.6)
6.8± 0.62 (5.4- 7.9) 1.33 0.18
S. Albumin (g/dl) 4.2± 0.3 (3.9- 4.8) 3.8± 0.9 (2.5- 5.1) 1.27 0.2 SGPT (U/L) 36.5± 3.6 (30- 40) 77.8± 6.3 (60- 89) 19.6 0.001** SGOT (U/L) 29.4± 3.6 (23- 34) 67.7± 5.5 (60- 80) 20.3 0.001**
ALP (U/L) 41.6 ± 7.8 (30- 50) 89.36 ± 6.2 (80- 99)
19.65 0.001**
Bl. Urea (mg/dl) 31.4 ± 3.1 (25-
35.5) 28.6 ± 5.5 (18- 36) 1.48 0.14
S. Creatinine (mg/dl)
0.78 ± 0.29 (0.1- 1.2)
0.86 ± 0.27 (0.2- 1.3)
0.79 0.56
BUN (mg/dl)
10.0 ± 1.16 (8- 11.5) 9.2 ± 1.3 (6.5- 12) 1.72 0.08
Table (3) showed that there was a highly significant elevation of Total
Bilirubin , Direct Bilirubinin , SGPT, SGOT and Alkaline phosphatase in patient
group compared to control group (P < 0.001). However there was no significant
difference for Total protein , S. Albumin , blood urea, S. Creatinine and BUN
between the two groups.
Results
76
Table 4 Complete blood count results of the studied groups.
I II
t P X ± SD (Range) X ± SD (Range)
WBC (103/cm) 5.2 ± 0.95 (4.5 - 6) 7.8 ± 3.3 (4 – 12.5) 2.48 0.016* RBCs (106/cm) 5.0 ± 0.13 (4.9 – 5.3) 2.9 ± 0.4 (2.3 – 3.8) 16.2 0.001**
HCT (%) 42.8 ± 1.98 (39.2 – 44.5) 18.7 ± 2.5 (15.2 – 24.6) 27.4 0.001** HB (g/dl) 10.8±0.4 (10.0 – 11.2) 7.4±0.6 (6.4 – 8.7) 16.4 0.001** MCV(fl) 84.9±3.1 (80 – 89) 63.7±2.4 (60.2 – 69.1) 22.49 0.001** MCH (Pg) 21.4 ± 0.6 (20 – 22.2) 25.6±4.2 (19.1 – 36.5) 3.09 0.003* MCHC(g/dl) 25.3 ± 1.3 (23.1 – 27.8) 40.2±6.5 (30.2 – 55.3) 7.12 0.001** RDW(%) 13.29 ± 0.5 (12.5- 14) 13.79± 1.6 (11.5- 16.9) 0.96 0.65 PLT(103/cm 290 ± 21.6 (250- 320) 245.3± 59.6 (180- 526) 1.3 0.25 PDW(%) 15.2 ± 0.96 (14- 16.6) 12.85± 1.0 (11.2- 14.7) 6.24 0.001** MPV(fl) 9.2 ± 0.9 (8- 10.5) 9.11± 1.2 (7.9- 13.9) 0.31 0.75
Table (4) and Fig. 16 showed that there was a highly significant reduction in
group II compared to group I as regard RBCs, HCT, HB, MCV, MCHC and PDW
(P < 0.001). However there was a significant elevation for WBC in group II
compared to group I (P < 0.003) . No significant difference for RDW, PLT and
MPV tests.
Fig. 16 Variations of RBCs, HB, MCV, HCT (PCV) for 40 patients (30 patients and 10 controls).
Results
77
Table 5: Results of iron study of the studied groups.
I II Mann –
Whitney U test X ± sD( Range) X ± sD( Range) Serum Iron (µg/dL)
100.5 ± 19.643 (80-150)
257.033 ± 16.866 (200-278)
p = 2.737×10-6 < 0.001**
Serum Ferritin (ng/ml)
157.0 ± 51.001 (100-250)
1442.9 ± 522.185 (597-2500)
p = 2.996×10-6 < 0.001**
TIBC(µg/dL) 274.4± 61.925 (110-330)
139.5±16.706 (120-180)
p = 1.8537×10-4 < 0.001**
Table (5) showed that there was a highly significant difference between group I
and group II for Serum Iron, Serum Ferritin and TIBC, (P < 0.001).
Table 6: Hemoglobin Electrophoresis data of the studied groups.
I II
t P X ± SD (Range) Median
X ± SD (Range) Median
HBA (%) 96.95± 0.55 (96.3- 97.9) 96.95
50.1± 26.6 (0.0- 86) 57
8.63
0.001**
HBA2
(%)
2.6± 0.4 (2- 3.2) 2.65
3.45± 1.0 (1- 6) 3
5.52 0.003**
HBF (%) 0.45± 0.2 (0.1- 0.8) 0.45
46.1± 2.7 (11- 99) 40
22.04 0.001**
Results
78
Table (6) showed that there was a highly significant difference between the two
groups for HBA, HBA2 and HBF, (P < 0.001).
Table 7: Hepcidin concentration levels of the studied groups.
I II M P Hepcidin (ng/ml) X ± SD
210.9 ± 12.8 66.2 ± 95.6 0.001**
Range (194.347 – 231.65) (-390.657– 147.47) 8.98 Median 209.89 80.351 21.98
Table (7) and Fig.17 showed that there was a highly significant reduction of
hepcidin in Group (II) compared to Group (I) (p < 0.001)
Fig. 17 Hepcidin and Serum Ferritin for 40 patients (30 patients and 10
controls).
Results
79
Table 8: Ratio between hepcidin and Serum Ferritin of the studied groups..
I II Mann–Whitney U test
X ± SD 1.455 ± 0.396 0.063 ± 0.061 P = 3.015×10-6 <
0.001** Range 1.017 0.379 median 1.444 0.074
Table (8) showed that there was highly significant increase as regard hepcidin /
Serum Ferritin ratio in Group (I) compared to Group (II). The hepcidin / Serum
Ferritin ratio in the patients group was markedly reduced.
Table 9: Correlation between hepcidin and other parameters of the studied
groups.
r P Significance Hb (g/dl) 0.68 < 0.001** HS HCT(%) 0.56 < 0.001** HS Mcv (fl) 0.61 < 0.001** HS Fe (ng/ml) -0.72 < 0.001** HS S.Iron (µg/dL) -0.63 < 0.001** HS TIBC(µg/dL) 0.21 < 0.001** HS
Table (9) and Fig. 18-21 showed that there was a positive correlation between
hepcidin in one side and Hb, Hct and Mcv in the other side (p<0.001), (Figs. 18-
20). While there was a negative (i.e., inverse) correlation between hepcidin and
(ferritin &Serum Iron) (P< 0.001) (see Fig. 21). There was a positive correlation
correlation between hepcidin in one side and TIBC (p<0.001) in the other side.
Results
80
Fig. 18 Correlation between hepcidin and Hb.
Fig. 19 Correlation between hepcidin and HCT.
Results
81
Fig. 20 Correlation between hepcidin and MCV.
Fig. 21 Correlation between hepcidin and Serium Ferritin.
Discussion
82
DISCUSSION
β-thalassemia is the most common chronic hemolytic anemia in Egypt
(85.1%). A carrier rate of 9-10.2% has been estimated in 1000 normal
random subjects from different geographical areas of Egypt. β-thalassemia
is much more common in Mediterranean countries constituting a major
public health problem (El-Beshlawy, 1999).
Finch (1994) proposed the existence of an erythroid regulator of
systemic iron homeostasis. The erythron, composed of developing erythroid
cells in the bone marrow and circulating erythrocytes, utilizes about 80% of
the iron found in the plasma. Anemia results from the inability of the
erythroid compartment to receive its full complement of iron. The putative
erythroid regulator communicates the iron needs of the erythron to
influence changes in intestinal iron absorption (Breda et al., 2005).
Iron absorption is increased in patients with congenital anemias
characterized by ineffective erythropoiesis. Clinically, increased intestinal
iron absorption compounds the effects of transfusional iron overload in
patients with thalassemia syndromes (Adamsky et al., 2004).
The most common secondary complications are those related to
transfusional iron overload, which can be prevented by adequate iron
chelation. Iron-loading anemias are disorders in which hepcidin is regulated
by opposing influences of ineffective erythropoiesis and concomitant iron
overload (Pigeon et al., 2001).
Hepcidin is an effective inhibitor of iron absorption; the erythroid
regulator includes a mechanism to decrease hepcidin production.
Accordingly, low hepcidin levels have been reported with thalassemia and
other disorders with ineffective erythropoiesis. In these disorders, decreased
hepcidin expression leads to relief of inhibition of ferroportin, resulting in
Discussion
83
increased iron release from recycling macrophages and absorptive
enterocytes, increasing availability of iron for erythropoiesis. However, the
iron cannot be effectively utilized by the erythron, leading to accumulation
and tissue iron overload in the face of anemia (Gardenghi et al., 2007 and
Jenkins et al., 2007).
This study aim was to measure hepcidin concentration in patients of β
thalassemic major to explain its role in iron metabolism for these patients
who have iron overload.
This study revealed a statistically significant elevation of total Bilirubin
and Direct Bilirubin (TB-DB) in the patient group compared to the control
group, on the other hand there was no significant difference in the Total
protein and S. Albumin. There was a statistically significant elevation in the
patient group as regard SGPT, SGOT and Alkaline phosphatase, compared
to the control group.
Hyperbillirubinemia is a result of chronic Hemolysis and ineffective
erythropoiesis. Which together cause the anemia that occurs in thalassemia.
The relative contributions of these two pathologic processes differ in
various forms of thalassemia. The bone marrow of patients with thalassemia
contains five to six times the number of erythroid precursors as does the
bone marrow of healthy controls with 15 times the number of apoptotic
cells in the polychromatophilic and orthochromic stages (Centis et al.,
2000;Mathias et al., 2000).
Ineffective erythropoiesis the major cause of accelerated apoptosis, is
caused by excess -chain deposition in erythroid precursors. Although the
exact mechanism is not known, a death-receptor–mediated pathway seems
to be involved with Fas–Fas ligand interactions (De Maria et al., 1999).
Discussion
84
In normal erythropoiesis, apoptotic mechanisms seem to play a
regulatory role and are required for normal erythroid maturation (Testa,
2004). Accelerated apoptosis is associated with a rise in extracellular
exposure of phosphatidylserine, an important signal for removal by
activated macrophages, whose numbers are increased in thalassemic bone
marrow erythrocytes , resulting in their accelerated peripheral destruction (
Angelucci et al., 2002).
In this work there was significant reduction in the patient group as
regard RBCs, HCT, HB, MCH, , MCV and MCHC. On the other hand, the
WBC count was elevated in β thalassemia major patient group.
Leukocytosis is usually present, even after excluding the nucleated
RBCs. A shift to the left is also encountered, reflecting the hemolytic
process. The anemia is due to a combination of ineffective erythropoiesis,
excessive peripheral red blood cell hemolysis, and progressive
splenomegaly. The latter causes an increase in plasma volume and a
decrease in total red cell mass. The red cells are microcytic (mean
corpuscular volume <70 fL) with marked anisochromasia. Hypochromic
microcytic anemia lead to MCV and MCH are significantly low, low
MCHc, low HCT and low Hb (Wonke, 2001).
In this study there was a highly significant difference between the two
groups for HBA, HBA2 and HBF, as HbA is the major hemoglobin found
in adults and children. Hb A2 and HbF are found in small quantities in
adult life but in β thalassemia major there was elevated HbF and Hb A2 but
HbA is in small quantities (Rachmilewitz &Schrier et al., 2001).
This study showed that there was a highly significant elevation of
Serum Iron and Serum Ferritin in the patient group compared to the control
Discussion
85
group. This was accompanied by subsequent reduction of TIBC in the
patient group.
Oxidation of α-globin subunits leads to the formation of hemichromes,
it bind to or modify various components of the mature red-cell membrane,
such as protein band 3, protein 4.1, ankyrin, and spectrin. After
precipitation of hemichromes, heme disintegrates, and toxic non–
transferrin-bound iron species are released. The resulting free iron catalyzes
the formation of reactive oxygen species (Rachmilewitz &Schrier et al.,
2001).
Iron-dependent oxidation of membrane proteins and formation of red-
cell "senescence" antigens such as phosphatidylserine (Kuypers & Jong.
2004) cause thalassemic red cells to be rigid and deformed and to aggregate,
resulting in premature cell removal (Tavazzi et al., 2001).
Ineffective erythropoiesis and hepatosplenomegaly together result in
Hypochromic microcytic anemia which in turn increases iron absorption
plus transfusional iron overload both lead to increased levels of iron, ferritin
and decreased TIBC (Porter, 2001).
The most common secondary complications are those related to
transfusional iron overload, which can be prevented by adequate iron
chelation. Iron-loading anemias are disorders in which hepcidin is regulated
by opposing influences of ineffective erythropoiesis and concomitant iron
overload (Pigeon et al., 2001).
The results of the present study revealed reduction of Hepcidin level in
patient group compared to the control group this reduction was statistically
significant. On the other hand, the ratio between hepcidin concentration and
Serum Ferritin was highly reduced in patient group compared to the control
group. This reduction was statistically significant.
Discussion
86
The results showed that there was a positive correlation between
hepcidin and Hb, PCV and MCV (i.e., as the value of hepcidin increases,
the values of the Hb, PCV and MCV increase and vice versa) while there
was a negative (i.e., inverse) correlation between hepcidin and (ferritin
&Serum Iron) (i.e., as the values of hepcidin increase, the values of ferritin
and Serum Iron decrease and vice versa).
Patients of β thalassemia major have decreased concentrations of
hepcidin due to opposing influences of ineffective erythropoiesis and
concomitant iron overload. This agreed with Wrighting and Andrews
2008 who reported that the erythroid regulator includes a mechanism to
decrease hepcidin production. Accordingly, low hepcidin levels have been
reported in patients with thalassemia and other disorders with ineffective
erythropoiesis.
Wrighting and Andrews 2008 reported also that hepcidin expression
was downregulated in a hepatocytic cell line after treatment with
thalassemic sera.
This result was in agreement with Rund and Rachmilewitz 2005 who
reported that Hepcidin levels were found to be low in patients with
thalassemia intermedia and thalassemia major. Furthermore, serum from
patients with thalassemia inhibited hepcidin messenger RNA expression in
the HepG2 cell line, which suggests the presence of a humoral factor that
downregulates hepcidin.
Zimmermann et al 2008 reported that hepcidin concentrations should
be high in iron-loaded persons with β -thalassemia; however, hepcidin
concentrations are low in these persons, unless they have recently received a
transfusion. Production of growth differentiation factor 15 (GDF-15) by the
Discussion
87
expanded erythroid compartment contributes to iron overload in thalassemia
by inhibiting hepcidin gene expression.
Nemeth and Ganz 2006 reported that when the ratio of urinary
hepcidin to serum ferritin was analyzed as an index of appropriateness of
hepcidin response to iron load, this ratio was still greatly decreased in
thalassemia major patients when compared to normal subjects, indicating
the continued regulation of hepcidin by a suppressive factor.
Pak et al 2006 reported that patients with β -thalassemia would be
expected to have high hepcidin levels. To the contrary, patients with β-
thalassemia have almost uniformly low urinary hepcidin. These and other
clinical observations in iron-loading anemias would argue that
erythropoiesis is able to suppress hepcidin production even in the face of
severe iron overload.
Papanikolaou et al 2005 reported that hepcidin was measured in 8
patients with thalassemia major and 7 with thalassemia intermedia. Patients
with thalassemia had very low urinary hepcidin levels, despite high serum
ferritin levels that reflected systemic iron overload. Several patients with
thalassemia had no detectable hepcidin.
The current study didn't agree with Origa et al 2007 who reported that
hepcidin levels were elevated in thalassemia major, due to transfusions that
reduce erythropoietic drive and deliver a large iron load, resulting in
relatively higher hepcidin levels. In the presence of higher hepcidin levels,
dietary iron absorption is moderated and macrophages retain iron,
contributing to higher serum ferritin.
This result agreed with Brissot et al 2008 who reported that hepcidin
deficiency in thalassemia major due to ineffective erythropoiesis leads to
growth differentiation factor15 (GDF15) overexpression by the
Discussion
88
erythroblasts, which inhibits hepcidin expression. This could explain why
(hepatocytic) iron overload can develop in thalassaemia in the absence of
transfusions, and why hepcidin expression is relatively low in this disease
despite transfusional iron excess (which should, by itself, lead to marked
increased hepcidin expression).
Kemna et al 2008 reported that erythropoietin that stimulates
erythropoietic activity has been shown to down-regulate liver hepcidin
expression. However, in the absence of erythropoietic activity, hepcidin
expression is no longer suppressed. The strong inverse association between
erythropoietic drive and hepcidin production was also observed in several
patients with congenital chronic anemias, which are characterized by low
urinary hepcidin levels. The dotblot method was used to observe
low/normal hepcidin levels for the degree of iron load in thalassemic
patients.
Kattamis et al 2006 reported that urinary hepcidin was found to be
suppressed in patients with thalassemia major. Tissue hypoxia triggers the
production of EPO, which results in pronounced erythroid proliferation
accompanied by increased sTfR levels. Hypoxia and yet-undefined signals
from the robust erythroid activity down-regulate hepcidin production.
Nemeth and Ganz 2006 reported that Patients with chronic anemias
with hemolysis or dyserythropoiesis, such as thalassemia syndromes suffer
from iron overload. Measurements of urinary hepcidin in these patients
indicated that hepcidin levels were severely decreased, despite systemic iron
overload reflected by the patients’ elevated serum ferritin levels. Even in
regularly transfused thalassemia patients, hepcidin levels were
inappropriately low given the patients’ iron load, as indicated by the
Discussion
89
decreased ratio of urinary hepcidin to serum ferritin, used as an index of
appropriateness of hepcidin response to iron load.
Toledano et al 2008 reported that Several diseases with chronic iron
overload such as hereditary hemochromatosis and β-thalassemia major are
characterized by low hepcidin expression in the liver. The low hepatic
hepcidin in these patients is probably responsible for the intestinal
absorption of iron.
Tanno et al 2007 also reported that Serum from thalassemia patients
suppressed hepcidin mRNA expression in primary human hepatocytes, and
depletion of GDF15 reversed hepcidin suppression. These results suggest
that GDF15 overexpression arising from an expanded erythroid
compartment contributes to iron overload in thalassemia syndromes by
inhibiting hepcidin expression.
Conclusions
90
CONCLUSIONS
• Transfusional iron overload is the most common secondary
complications in β thalassemia major.
• Hepcidin is a central regulator of iron homeostasis.
• Hepcidin concentration was decreased in the thalassemic group
although elevated iron levels.
• Patients of β thalassemia major have decreased concentrations of
hepcidin due to opposing influences of ineffective erythropoiesis and
concomitant iron overload.
Recommendation
91
RECOMMENDATION
● Further study is recommended on large scale including the
development of hepcidin- based diagnostics and therapies for iron-loading
anemias that may offer more effective approaches to prevent the toxicity
associated with iron overload.
● Further study is recommended to screen patients with β thalassemia
who develop secondary iron overload to detect hepcidin level as in the
future, therapeutic use of hepcidin and hepcidin agonists may help to
restore normal iron homeostasis.
● Further study is recommended for evaluating and measuring hepcidin
regulators as growth differentiation factor15 (GDF15) , Hemojuvelin (HJV)
and others in cases of ineffective erythropoiesis and their effects on
hepcidin expression.
Summary
92
SUMMARY
The thalassemias are a heterogeneous group of genetic disorders of
haemoglobin synthesis , all of which result from a reduced rate of
production of one or more of the globin chains of haemoglobin. The
thalassemias are among the most common genetic disorders worldwide,
occurring more frequently in the Mediterranean region, the Indian
subcontinent, Southeast Asia, and West Africa.
The most common secondary complications are those related to
transfusional iron overload, which can be prevented by adequate iron
chelation.
The survival of individuals who have been well transfused and treated
with appropriate chelation extends beyond age 30 years. Iron-chelation
therapy is largely responsible for doubling the life expectancy of patients
with thalassemia major.
Systemic iron is distributed among erythrocyte precursors in the bone
marrow, tissue macrophages, liver, and all other tissues, with the largest
amount found in circulating erythrocytes. Homeostasis is maintained by
regulating the levels of plasma iron. Hepcidin, a circulating peptide
hormone, has recently emerged as a key modulator of plasma iron
concentration, and, thus, a central regulator of iron homeostasis.
Hepcidin binds to ferroportin and causes its internalization and
degradation in lysosomes, thus effectively blocking the export of iron from
the cells.
This study was conducted to measure hepcidin concentration in patients
of β thalassemic major to explain its rule in iron metabolism for these
patients who have iron overload.
Summary
93
This study was carried out in pediatric and clinical pathology
department of Zagazig University Hospitals and included 30 β thalassemic
major children and 10 apparently healthy children as a control group.
All the studied groups were subjected to:
• Full history taking and thorough clinical examinations .
• Laboratory investigations: C.B.C, Serum iron, Serum ferritin, TIBC,
Hemoglobin electrophoresis, Liver function tests, kidney function
tests and Hepcidin hormone was measured by Enzyme Linked
Immunosorbant Assay in the serum.
From the results, it was found that:
• There was a highly significant difference between the two groups for
Total Bilirubin and Direct Bilirubin .
• There was high significant difference between the two groups as
regard SGPT, SGOT and Alkaline phosphatase.
• There was a highly significant difference between the two groups as
regards RBCs, HCT, HB, MCV, MCHC and PDW. Also there was a
significant difference for WBC and MCH.
• There was a highly significant difference between the two groups for
Serum Iron, Serum Ferritin and TIBC.
• There was a highly significant difference between the two groups for
HBA, HBA2 and HBF
• There was a highly significant increase in the control group
compared to patient group for Hepcidin . Hepcidin concentration was
decreased in the patients group although elevated iron levels
Summary
94
compared to the control group who have normal iron levels and
increased hepcidin concentration .
• The ratio between hepcidin and Serum Ferritin in the patients group
was lower than that in the control group.
• There was a positive correlation between hepcidin in one side and
Hb, Hct, and Mcv in the other side. Also there was a negative
correlation between hepcidin and serum ferritin.
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االملخص االعربى
االثالسيیميیا( االتي تسبب تكسر كريیاتت االدمم االحمرااء) من أأهھھھم أأمرااضض االدمم االوررااثيیة ااالنحالليیة
االشائعة على مستوىى االعالم بشكل عامم ووعلى مستوىى منطقة االبحر ااألبيیض االمتوسط بشكل خاصص
-لمتوسط بيیتايیميیا هھھھو مرضض اانيیميیا االبحر ااالبيیض ااس.ااهھھھم نوعع من االثال االكبرىى االتى تؤثر على اانتاجج
حيیث اانن االحديید وواالبرووتيیناتت االتى تعد ااساسيیة لحيیاةة ااالنسانن,ووبالتالى تنتج ااالنيیميیا من نقص االحديید.
تنظيیم ووااستقراارر مستوىى االحديید وواالمحافظة على هھھھذاا ااالستقراارر مهھم لصحة ااالنسانن.
-أأعرااضض اانيیميیا االبحر ااالبيیض االمتوسط بيیتا : فقر االدمم٬، االشحوبب, االخمولل ٬،قلة االكبرىى هھھھي
االحركة٬، االعصبيیة االشديیدةة٬، فقداانن االشهھيیة وو تأخر االنمو٬، قد يیعاني االطفل من بعض هھھھذهه ااألعرااضض أأوو
كلهھا. ووعند االفحص االسريیريي يیالحظ االطبيیب شحوبب االوجهھ ووتضخم بعض ااالعضاء كالطحالل
فالل فيیحدثث برووزز في عظامم وواالكبد٬، ووفي االسنوااتت االعشر ااألوولى للطفل تتغيیر مالمح بعض ااألطط
االوجهھ وواالفك ووكبر حجم االبطن ووبعض االتغيیيیر في االهھيیكل االعظمي.
يیحتاجج االمصابب إإلى عمليیاتت نقل ددمم للمحافظة على هھھھيیموجلوبيین االدمم بدررجة تسمح للمصابب
بمماررسة حيیاتهھ االطبيیعيیهھ٬، وولكن بسبب نقل االدمم االمتكررر تترسب كميیاتت كبيیرةة من االحديید في جسم
بة االتلف بكل عضو يیتركز بهھ االحديید٬، فعلى سبيیل االمثالل يیتسبب االحديید االزاائد بمرضض االمصابب مسب
االسكريي في سن صغيیرةة ووفشل عضلة االقلب ووتضخم االكبد وواالطحالل وواالعقم ووفشل االنمو وواالبلوغغ لذلك
يیحتاجج االمريیض ووبسن صغيیرةة إإلى أأددوويیة لتغسل االدمم من االحديید االمترسب مثل االديیسفراالل.
وويیتم االتحكم فى ااستقراارر مستوىى االحديید بشكل ااساسى بوااسطة االهھيیبسيیديین االذىى يیصنع فى االكبد.
حيیث تعد االخاليیا االكبديیة االمصدرر االخلوىى ااالساسى للهھيیبسيیديین.وويیتم ااخرااجج االحديید االخلوىى االى
االدمم٬، ووذذلك عبر االفيیرووبرووتيین. وواالخاليیا االمصدررةة االبالززما فى حالة اانخفاضض تركيیز االهھيیبسيیديین فى
لهھذاا االحديید هھھھى االخاليیا االطالئيیة لالثنى عشر٬، وواالخاليیا االبلعميیة وواالخاليیا االكبديیة.
ااما عند ااررتفاعع تركيیز االهھيیبسيیديین فانة يیرتبط بالفيیرووبرووتيین٬، حيیث يیتم ااستبطانن االفيیرووبرووتيین
االى ددااخل االخليیة ووتدميیرةة. ووكنتيیجة لذلك فانن ااخرااجج االحديید االخلوىى االى االبالززما يیقل بيینما يیترااكم
االحديید ددااخل االخليیة فى صوررةة االفريیتيین.
االملخص االعربى
113
ستوىى االحديید وويیقل نتيیجة لفقر االدمم وونقص ااألكسجيین. وويیزدداادد تصنيیع االهھيیبسيیديین نتيیجة لزيیاددةة م
االدمم االمصاحب لاللتهھابب يیقل مستوىى االحديید فى االبالززما مما يیحد من عمليیة تصنيیع رووفى حالة فق
كريیاتت االدمم االحمرااء.
تم ااجرااء هھھھذهه االدررااسهھ في االعيیاددةة االخاررجيیة لوحدةة االدمم بقسم ااألططفالل بمستشفى االزقاززيیق
-ن مريیض بانيیميیا االبحر ااالبيیض االمتوسط بيیتااالجامعي علي ثالثيی االكبرىى ووعشرةة أأططفالل ااصحاء
كمجموعة ضابطة.
معرفة ددووررهھھھرمونن االهھيیبسيیديین فى مرضى اانيیميیا االبحر ااالبيیض االمتوسط هھھھو هھھھدفف االدررااسة
-بيیتا االكبرىى حيیت ااززدديیادد نسبة االحديید.
ووااالصحاء بالنسبة لعد كرااتت ووقد ااظظهھرتت االنتائج ووجودد تبايین ملحوظظ بيین مجموعتى االمرضى
االدمم االحمرااء٬، نسبة االهھيیموجلوبيین وو متوسط حجم كرااتت االدمم االحمرااء. وولقد ووجد اايیضا ووجودد تبايین
ملحوظظ بيین االمجموعتيین بالنسبة لمستوىى االحديید وواالفريیتيین فى االدمم وواالقدررةة االكليیة التحادد االحديید.
فى هھھھؤالء االمرضى ووعلى االعكس ااظظهھرتت االنتائج اايیضا اانن االهھيیبسيیديین يیوجد بتركيیز ااقل
بتركيیز ااكبر فى ااالصحاء حيیث نسبة االحديید ااالقل.
اايیضا ووجد اانن نسبة االهھيیبسيیديین االى االفريیتيین فى االمرضى ااقل من نسبتهھا فى ااالصحاء.
ووهھھھذاا يیتفق مع اانن االهھيیبسيیديین مثبط فعالل المتصاصص االحديید٬، وواانن منظم االمولدةة للكريیاتت
تاجج االهھيیبسيیديین.ووبالتالى ووجدتت االتركيیزااتت االقليیلة مع االثالسيیميیا وواامرااضض االحمرااء لديیة االيیة لنقص اان
ااخرىى مصاحبة بالتكويین االغيیر فعالل لكريیاتت االدمم االحمرااء.فى مثل هھھھذةة ااالمرااضض فانن نقص
االهھيیبسيیديین يیؤددىى االى ااتاحة االفيیرووبرووتيین٬،فيیزيید ااخرااجج االحديید من االخاليیا االطالئيیة لالثنى عشر٬،
مما يیتيیح فرصة ااكبر من تصنيیع كريیاتت االدمم االحمرااء.وواالخاليیا االبلعميیة٬،
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