New Insights into Erythropoiesis: The Roles of Folate, Vitamin B12, and Iron

Abstract

Erythropoiesis is the process in which new erythrocytes are produced. These new erythrocytes replace the oldest erythrocytes (normally about one percent) that are phagocytosed and destroyed each day. Folate, vitamin B12, and iron have crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B12 for proliferation during their differentiation. Deficiency of folate or vitamin B12 inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes erythroblast apoptosis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily with hemoglobin breakdown from destroyed old erythrocytes. Many recently identified proteins are involved in absorption, storage, and cellular export of nonheme iron and in erythroblast uptake and utilization of iron. Erythroblast heme levels regulate uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded production rates with smaller, less hemoglobinized erythrocytes.

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(Some corrections may occur before final publication online and in print)
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Annu. Rev. Nutr. 2004. 24:105–31
doi: 10.1146/annurev.nutr.24.012003.132306
NEW INSIGHTS INTO ERYTHROPOIESIS: The Roles
of Folate, Vitamin B
12
, and Iron
∗
1
Mark J. Koury
Department of Medicine, Vanderbilt University School of Medicine and
Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee 37232;
email: mark.koury@vanderbilt.edu
Prem Ponka
Departments of Physiology and Medicine, Lady Davis Institute for Medical Research of
the Jewish General Hospital, McGill University, Montreal, Quebec, H3T 1E2, Canada;
email: prem.ponka@mcgill.ca
Key Words apoptosis, heme, erythrocytes, iron deficiency anemia, megaloblastic
anemia
■ Abstract Erythropoiesis is the process in which new erythrocytes are produced.
These new erythrocytes replace the oldest erythrocytes (normally about one percent)
that are phagocytosed and destroyed each day. Folate, vitamin B
12
, and iron have
crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B
12
for prolif-
eration during their differentiation. Deficiency of folate or vitamin B
12
inhibits purine
and thymidylate syntheses, impairs DNA synthesis, and causes erythroblast apopto-
sis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large
amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily
with hemoglobin breakdown from destroyed old erythrocytes. Many recently identi-
fied proteins are involved in absorption, storage, and cellular export of nonheme iron
and in erythroblast uptake and utilization of iron. Erythroblast heme levels regulate
uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded
production rates with smaller, less hemoglobinized erythrocytes.
*The US Government has the right to retain a nonexclusive, royalty-free license in and to
any copyright covering this paper.
1
ABBREVIATIONS ALA-S2/eALA-S, erythroid-specific 5-aminolevulinic-acid syn-
thase; BFU-E, burst-forming unit-erythroid; CFU-E, colony-forming unit-erythroid;
Dcytb, duodenal cytochrome b; eIF-2, eukaryotic initiation factor 2; EPO, erythropoi-
etin; FBP, folate-binding protein; GI, gastrointestinal; HO-1, heme oxygenase 1; HRI,
heme-regulated inhibitor; IRE, iron-responsive element; Ireg1/MTP1, ferroportin 1; IRP,
iron regulatory protein; LIP, labile iron pool; Nramp/DMT1, divalent metal transporter 1;
NTBI, nontransferrin-bound iron; RFC, reduced folate carrier; THF, tetrahydrofolate;
UTR, untranslated region.
105
First published online as a Review in Advance on March 10, 2004

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CONTENTS
INTRODUCTION .....................................................106
STAGES AND REGULATION OF ERYTHROPOIESIS ......................107
FOLATE AND VITAMIN B
12
AND THEIR DEFICIENCY STATES ............108
ROLES OF FOLATE AND VITAMIN B
12
IN ERYTHROPOIESIS ..............111
THE RELATIONSHIP BETWEEN IMPAIRED DNA
SYNTHESIS AND ERYTHROID CELL APOPTOSIS .......................112
IRON METABOLISM AND THE IRON-DEFICIENCY STATE ................114
IRON EXPORT FROM CELLS TO TRANSFERRIN: A NECESSARY
PREREQUISITE FOR ERYTHROPOIESIS ...............................117
IRON ACQUISITION FROM TRANSFERRIN
BY DEVELOPING ERYTHROID CELLS ................................121
DISTINCT CONTROL OF IRON METABOLISM IN ERYTHROID
CELLS .............................................................121
THE AVAILABILITY OF IRON CONTROLS
HEMOGLOBIN SYNTHESIS ..........................................122
CONCLUSION: ERYTHROPOIESIS UNDER NORMAL,
FOLATE-DEFICIENT, AND IRON-DEFICIENT CONDITIONS ..............124
INTRODUCTION
Erythropoiesis is the process by which the hematopoietic tissue of the bone mar-
row produces red blood cells (erythrocytes). The mean lifespan of a normal human
erythrocyte is about 120 days. Erythrocytes are involved in transporting carbon
dioxide and nitric oxide, but their principal function is to deliver oxygen from the
lungs to the other tissues of the body. The amount of oxygen delivered to the tissues
is a function of the number of circulating erythrocytes. In normal adults, approx-
imately 200 billion of the oldest erythrocytes (about 1% of the total number) are
replaced every day by an equal number of newly formed erythrocytes. In situations
in which the erythrocytes are abnormally lost from the circulation by bleeding or by
increased destruction (hemolysis), the rate of new erythrocyte production can ex-
ceed one trillion per day. Thus, erythropoiesis is a dynamic process that can respond
promptly to the need for more oxygen delivery. Among the numerous requirements
for active erythropoiesis are adequate supplies of three nutrients—folate, cobal-
amin (vitamin B
12
), and iron. Deficiency of each of these three nutrients can lead to
decreased erythrocyte production and subsequently to decreased numbers of cir-
culating erythrocytes (anemia). Advances in erythropoiesis research have helped
to explain the roles of these nutrients in the production of erythrocytes and how
their respective deficiency states cause anemia. Recently reported findings related
to the development of nutrition-related anemias that will be reviewed here include:
(a) the uptake and intracellular effects of folate, vitamin B
12
, and iron; (b) the in-
duction of programmed death (apoptosis) of erythroid progenitor cells in folate or
vitamin B
12
deficiency; and (c) the cellular mechanisms in iron-deficient erythrob-
lasts that avoid apoptosis, but nonetheless decrease erythrocyte production.

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NUTRITION AND ERYTHROPOIESIS 107
Figure 1 Stages of erythroid differentiation. Specific stages of erythroid differenti-
ation beginning with the burst-forming unit-erythroid (BFU-E) and ending with the
mature erythrocyte (RBC) are shown. Multiple cell divisions occur between stages
prior to the polychromatophilic erythroblast (polyEB) stage after which the cells do
not divide. Periods of high cellular proliferation, erythropoietin (EPO) dependence, and
hemoglobin synthesis are demarcated. Other abbreviations: CFU-E, colony-forming
unit-erythroid; ProEB, proerythroblasts; BasoEB, basophilic erythroblast; OrthoEB,
orthochromatic erythroblast; and Retic, reticulocyte.
STAGES AND REGULATION OF ERYTHROPOIESIS
All types of blood cells have a finite life span and must be constantly replaced
by new cells formed in the hematopoietic tissue. Erythropoiesis is a continuous
process of proliferation and differentiation beginning with the hematopoietic stem
cell and ending with the erythrocyte (56, 60) (Figure 1). Hematopoietic stem cells
are rare, less than one in ten thousand nucleated cells of the bone marrow, and they
can self-renew or differentiate into all of the cells in the blood and the immune
system. Their commitment to differentiation and the subsequent commitment of
their progeny to the erythroid lineage appear to be stochastic events, but may be
related to the prevalence and association of specific DNA transcription factors
(18). The earliest stage of progenitor cell differentiation that is committed to the
erythroid lineage is the burst-forming unit-erythroid (BFU-E; Figure 1). Human
BFU-Es are defined by their ability to form large ‘bursts’ of erythroblast colonies
or one very large colony of erythroblasts, after two to three weeks in semisolid
tissue culture. Erythroid bursts can contain more than one thousand erythroblasts
and, thus, a single BFU-E and its progeny can have ten or more rounds of cell
division before reaching the terminal postmitotic stages of differentiation. The
next defined stage is the colony-forming unit-erythroid (CFU-E; Figure 1). Human
CFU-Es require one week to form single colonies of up to 64 erythroblasts in
tissue culture. Thus, CFU-E and their progeny have six or fewer rounds of cell
division. The erythropoietic stages subsequent to CFU-E are defined by their light
microscopic appearance in stained preparations. Cellular proliferation is not shown
in Figure 1, but the percentage of cells in active cell cycle is greatest in the CFU-
E and proerythroblast stages, and cell division ceases at the polychromatophilic
stage.
Erythropoietin (EPO) is the principal regulator of erythropoiesis (56, 63). EPO
is a glycoprotein hormone produced by a subset of peritubular, interstitial cells
in the renal cortex (61, 64). A few of these cells produce EPO under normal

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circumstances. In response to decreased oxygen delivery as occurs with anemia,
the number of these interstitial cells that produce EPO increases exponentially
(62). In the bone marrow, EPO acts upon erythroid progenitors in the stages from
CFU-E to the earliest of basophilic erythroblasts. This period of EPO dependence
precedes and does not overlap the period of hemoglobin synthesis (Figure 1).
As shown in Figure 1, progenitor cells in these stages are dependent upon EPO to
prevent apoptosis (55, 119), but they display widely varying degrees of dependence
(54). To survive this period of dependence, most erythroid progenitor cells require
greater EPO concentrations than those concentrations normally found in the blood.
Thus, the normal daily production rate of 200 billion erythrocytes requires the
survival of only a minority of the maximal number of EPO-dependent erythroid
progenitor cells.
Anemia occurs when the number of circulating erythrocytes is decreased. If
the anemia is due to transient blood loss or hemolysis and the kidneys and bone
marrow are normal, the erythropoietic system corrects the anemia. Specifically,
the decreased erythrocytes in anemia reduce oxygen delivery, and the kidneys
respond by increasing EPO production. The increased EPO results in the survival
of more erythroid progenitor cells in the EPO-dependent stages and subsequently
increased erythrocyte production. The increased erythrocytes in the circulation
deliver more oxygen, lowering the elevated EPO levels, and ultimately returning
the erythrocyte production rate and the number of circulating erythrocytes to their
normal, steady-state levels prior to the onset of anemia. In many anemias, however,
the kidneys are not normal, resulting in deficiency of EPO, or the hematopoietic
tissue of the bone marrow is not normal, resulting in an inability to respond to
the EPO. Among the numerous causes of an inability to respond to erythropoietic
demand are deficiencies of folate, vitamin B
12
, and iron. The majority of nutrition-
related anemias can be attributed to deficiency of one of these nutrients (48).
Folate and vitamin B
12
are both required for the extensive DNA synthesis that
accompanies the production of hundreds of billions of new erythrocytes each day.
All proliferating cells require iron, but the iron requirements of erythroid cells in
the late basophilic erythroblast through reticulocyte stages, when hemoglobin is
synthesized and accumulates (Figure 1), are much greater than all other cell types.
FOLATE AND VITAMIN B
12
AND THEIR
DEFICIENCY STATES
Folate, an essential nutrient found in the tissues of plants and animals, consists
of a pteridine (2-amino-4-hydroxy-pteridine) ring attached to para-aminobenzoate
with a polyglutamyl tail. The reduced tetrahydrofolate (THF) form acts as a co-
factor in multiple biochemical reactions by donating or accepting one-carbon
units (4, 108). Folate is present in both plant and animal tissues, most commonly
in the form of 5-methyl-THF. Reduced folates are absorbed in the jejunum af-
ter enzymatic cleavage to the monoglutamic form (40, 42). The absorbed folate

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NUTRITION AND ERYTHROPOIESIS 109
enters the blood and circulates in the body as 5-methyl-THF monoglutamate. Once
folate is transported from the blood into a cell, it is retained there through the ac-
tion of folylpolyglutamate synthetase that converts the folate to the polyglutamyl
form (108). Folate is transported into cells by several mechanisms, but the endo-
cytotic mechanism involving two specific glucosylphosphatidylinositol-anchored,
cell-surface folate-binding proteins (FPBs) and the bidirectional membrane trans-
porter termed the reduced folate carrier (RFC) are the best characterized (72).
Both of these transport mechanisms have been examined in erythropoietic cells.
In vitro studies with antibodies to the FBPs showed that FBPs are expressed on
early stage hematopoietic cells, but they do not transport significant amounts of fo-
late (98). Similar in vitro studies show some morphological changes in the progeny
of BFU-E and CFU-E when antibodies to FBPs are added to the culture medium,
but surprisingly the growth of these erythroid progenitors are enhanced by the
antibodies (5, 6). Mice that are rendered null for one of the FBPs by homologous
recombination have embryonic lethality due to neural defects, but no hematopoi-
etic defect has been described (83). Mice rendered null for the RFC also have
embryonic lethality, but the defect appears to involve the hematopoietic system
(123). The prenatal mice can be rescued by loading the mothers with high doses
of folic acid, but the pups die from hematopoietic failure a few weeks after birth
(123), indicating that the RFC is necessary for folate transport in erythroid cells.
Clinical folate deficiency in the developed countries has been associated with
those who have poor nutrition, such as the elderly or those with alcoholism. To
reduce the incidence of neural tube defects that develop during the first trimester of
pregnancy, grain products in the United States have been fortified with folic acid for
the last seven years. Considering serum folate concentrations of less than 3 ng/ml
as folate deficiency, one study found a reduction in folate-deficient individuals
among a middle-aged/older population from 22% prior to fortification to 1.7%
after fortification (52). Despite this dramatic decrease in the incidence of folate
deficiency in the general population, others remain at increased risk for developing
deficiency due to specific medical conditions. Among the individuals at increased
risk are those with intestinal malabsorption; general malnutrition; high erythrocyte
turnover rates such as in chronic hemolytic anemias; anticonvulsant medications
that interfere with folate absorption or utilization such as phenytoin; and antibiotic
medications with antifolate actions such as trimethoprim/sulfamethoxazole.
Vitamin B
12
(cobalamin), an essential nutrient consisting of a tetrapyrrole (cor-
rin) ring containing cobalt that is attached to 5,6-dimethylbenzimidazolyl ribonu-
cleotide, is produced in microorganisms and is found in animal tissues. Vitamin
B
12
is a coenzyme in two biochemical reactions in humans. One of these re-
actions is the transfer of a methyl group from 5-methyl-THF to homocysteine
via methylcobalamin, thereby regenerating methionine (4, 108) (Figure 2). This
reaction represents the link between folate and vitamin B
12
coenzymes and ap-
pears to account for the requirement of both vitamins in normal erythropoiesis
(106, 114). The absorption of vitamin B
12
is a relatively complex process (4, 106,
107). Protein-bound vitamin B
12
in food is released by stomach acid and binds to

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Figure 2 DNA synthesis pathways that require folate or vitamin B
12
coenzymes.
Abbreviations: THF, tetrahydrofolate; 5,10-CH
2
-THF, methylenetetrahydrofolate; 10-
CHO-THF, formyltetrahydrofolate; 5-CH
3
-THF, methyltetrahydrofolate; DHF, dihy-
drofolate; DNA-CH
3
, methylated DNA; dUMP, deoxyuridylate; dTMP, thymidylate;
dATP, deoxyadenosine triphosphate; dGTP, deoxyguanosine triphosphate; and dTTP,
thymidine triphosphate.
specific vitamin B
12
–binding glycoproteins termed haptocorrins that are present in
the secretions of the salivary glands and stomach. In the duodenum, the haptocor-
rins are digested, and the vitamin B
12
binds to intrinsic factor, another glycoprotein
secreted by the stomach. The vitamin B
12
–intrinsic factor complex subsequently
binds to specific receptors in the terminal ileum. These receptors consist of cubilin,
which binds the vitamin B
12
–intrinsic factor complex, and megalin, an associated
membrane transport protein (78). After endocytosis in the ileal epithelium, the
vitamin B
12
is freed from the intrinsic factor and binds to transcobalamin II (apo-
transcobalamin) that is produced by the microvascular endothelium of ileal villi
(95). The vitamin B
12
–transcobalamin II complex, termed holotranscobalamin II,
enters the blood where it is the functional carrier of vitamin B
12
to the other cells of
the body. Holotranscobalamin II binds to specific homodimerized receptor proteins
that are displayed on the surface of many different types of cells (107). Although
this receptor has not been examined directly in hematopoetic tissue, its mecha-
nism of transport in the other cells is via endocytosis with subsequent intracellular
release of vitamin B
12
from its complex with transcobalamin II. The incidence
of vitamin B
12
deficiency increases significantly with age such that up to 15% of
older individuals are deficient in developed countries (8, 110). Most often this age-
related deficiency appears to be due to atrophic gastritis and the resultant inability
to dissociate vitamin B
12
from the proteins to which it is bound in food (19). Other

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NUTRITION AND ERYTHROPOIESIS 111
causes include the autoimmune gastropathy termed pernicious anemia, in which
both intrinsic factor and gastric acid are not produced; intestinal malabsorption
involving the terminal ileum; previous surgery that removed the stomach or ileum;
medications that interfere with gastric acid secretion such as H
2
-histamine receptor
blockers or proton pump inhibitors; and a strict vegan diet.
ROLES OF FOLATE AND VITAMIN B
12
IN ERYTHROPOIESIS
The importance of adequate folate and vitamin B
12
in erythropoiesis is demon-
strated by megaloblastic anemia, the clinical disease that can occur with deficiency
of either vitamin. Megaloblastic anemia affects all hematopoietic lineages, but it is
most prominent in the erythroid lineage. Megaloblastic anemia is characterized by
pancytopenia with macrocytic erythrocytes, hypersegmented neutrophilic granu-
locytes, and reticulocytopenia. The bone marrow has increased numbers of large
immature-appearing erythroblasts and myeloblasts (i.e., megaloblasts) that are un-
dergoing increased rates of premature death as shown by elevated serum bilirubin,
lactate dehydrogenase, myeloperoxidase, and by increased iron turnover. This in-
creased death of hematopoietic cells prior to their maturation is termed ineffective
hematopoiesis. Studies in patients with anemia due to folate or vitamin B
12
defi-
ciency have shown that impaired DNA synthesis and its sequelae are key elements
in the increased hematopoietic cell death that characterizes these anemias. The pe-
riod of high proliferation rates during erythropoiesis (Figure 1) makes the erythroid
progenitor cells more susceptible than other types of cells to the impaired DNA
synthesis in folate or vitamin B
12
deficiency. Erythroblasts from patients with fo-
late or vitamin B
12
deficiency anemia had no active incorporation of
3
H-thymidine
into DNA despite total DNA content between 2N and 4N that characterizes cells in
DNA synthesis (i.e., in S-phase of the cell cycle) (75, 115, 121). Flow cytometry
of bone marrow cells from patients with folate or vitamin deficiency had increased
percentages of cells in S-phase compared to controls (50). When rates of DNA syn-
thesis were examined directly in mitogen-stimulated blood lymphocytes of patients
with folate- or vitamin B
12
–deficiency anemia, they were decreased (117), but a
similar study using bone marrow cells did not show a decreased rate (13). Impaired
DNA synthesis would be expected to result in chromosomal breakage and pos-
sibly nuclear damage. Previous studies have shown that chromosomal breakage
is markedly increased in the bone marrow cells of patients with folate or vita-
min B
12
deficiency anemia (44, 75). Also, erythrocyte micronuclei (Howell-Jolly
bodies), a marker of genetic damage when they are increased in splenectomized
patients, are most increased in those patients who have folate or vitamin B
12
deficiency (70).
One-carbon units are required in three biochemical pathways involved in the
synthesis of DNA. These pathways are shown in Figure 2. They are (a) two steps
in the de novo synthesis of purines in which 10-formyl-THF provides two carbons

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of the purine ring structure; (b) the reaction catalyzed by thymidylate synthase in
which 5,10-methylene-THF provides the methylene group and reducing equiva-
lents for the methylation of deoxyuridylate to form thymidylate; and (c) the re-
action catalyzed by DNA methyltransferase in which 5-methyl-THF provides the
methyl group (indirectly through remethylation of homocysteine to form methio-
nine and subsequently S-adenosylmethionine) for the methylation of cytosines
in DNA. As mentioned above, the methylcobalamin form of vitamin B
12
is the
coenzyme involved in the transfer of the methyl group from 5-methyl-THF to ho-
mocysteine, thereby regenerating methionine and THF (Figure 2c). With vitamin
B
12
deficiency, not only does inhibited methionine regeneration lead to decreased
S-adenosylmethionine and increased homocysteine and S-adenosylhomocysteine,
but 5-methyl-THF accumulates intracellularly, while other forms of THF, specif-
ically the 10-formyl-THF required for purine synthesis and the 5,10-methylene-
THF required for thymidylate synthesis, decrease (108). This predicted “trapping”
of intracellular folate in the 5-methyl-THF form (46) resulting in intracellular de-
ficiencies of other forms of folate including those required for de novo synthesis of
deoxynucleotides was demonstrated in the bone marrow cells of rats made func-
tionally vitamin B
12
deficient by nitrous oxide exposure (49). In folate or vitamin
B
12
deficiency, the de novo synthesis of deoxynucleotides is decreased, resulting in
impaired synthesis and repair of DNA, and ultimately, in cell death. Erythropoiesis
under these deficiency conditions is termed ineffective because the erythroid cells
are present in the hematopoietic tissue, but most of them cannot mature to the
late stages of differentiation before undergoing apoptosis. The decreased numbers
of erythroid cells surviving until the postmitotic, terminal stages in ineffective
erythropoiesis leads to anemia.
THE RELATIONSHIP BETWEEN IMPAIRED DNA
SYNTHESIS AND ERYTHROID CELL APOPTOSIS
An in vivo murine model (10) and its in vitro extension (57) of folate-deficient
erythropoiesis have provided some new insights into the cellular events that lead to
erythroid cell apoptosis in folate deficiency. In this model, mice are fed an amino
acid–based, folate-free diet that induces a pancytopenia with all of the charac-
teristics of the human hematopoietic disease that results from folate or vitamin
B
12
deficiency (10). To study the cellular events of folate-deficient erythropoiesis
in a purified population of developmentally synchronized cells, mice are fed the
folate-free diet before and during the acute erythroblastosis phase of Friend virus
disease. This virus induces a proliferation of erythroid cells that accumulate at
the proerythroblast stage of differentiation, and when combined with the folate-
free diet yield a population of folate-deficient proerythroblasts. When cultured
with EPO under folate-sufficient conditions almost all of these proerythroblasts
differentiate into reticulocytes, but when cultured with EPO under folate-deficient
conditions, most of these proerythroblasts undergo apoptosis before differentiating

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into reticulocytes (57). Proerythroblasts freshly isolated from folate-deficient mice
have decreases in all forms of folate (58). During their differentiation in vitro, the
erythroblasts cultured under folate-deficient conditions accumulate in S-phase of
the cell cycle. The majority of these cells undergo apoptosis in S-phase (59). The
folate-deficient erythroid cells can be saved from their apoptotic fate if they are
supplied in vitrowith sufficient amounts of both thymidine and a purine that can
be salvaged to provide the necessary deoxynucleotides that permit DNA synthesis
(59). Hypoxanthine, inosine, adenosine, and deoxyadenosine are effective for this
purine salvage, but guanosine and deoxyguanosine are not. The medium supple-
mentation required in vitro for the survival and completion of erythroid differenti-
ation is 60 µmol/L for the purine and 20 µmol/L for thymidine, indicating that the
defects in DNA replication and repair that lead to apoptosis in folate-deficient ery-
throid cells are due to impaired de novo synthesis of primarily purines (Figure 2a)
and secondarily thymidylate (Figure 2b). The methylation of cytosines in the DNA
of folate-deficient murine erythroblasts is the same as in control erythroblasts (DJ
Park and MJ Koury, unpublished data). Similarly, bone marrow cells of patients
with vitamin B
12
deficiency anemia and bone marrow cells of controls had similar
percentages of methylated cytosines in their respective DNAs (97). These results
in mice and humans suggest that inhibition of DNA methylation by folate or vita-
min B
12
deficiency (Figure 2c) does not play a role in the anemias resulting from
deficiency of these vitamins.
The mechanism by which the DNA damage in folate or vitamin B
12
deficiency
leads to apoptosis in hematopoietic cells has not been established. The inhibited
conversion of deoxyuridylate to thymidylate has been associated with increased
uracil misincorporation into DNA due to the utilization by DNA polymerase of de-
oxyuridine triphosphate in lieu of thymidine triphosphate (11, 12, 116). However,
one study in vitamin B
12
–deficient patients (97), and another in folate-deficient
patients (99), did not find this increased incorporation of uracil in DNA of blood
cells. Uracils misincorporated close to one another on opposite DNA strands have
been proposed as a source of double-stranded DNA breakage in eukaryotic cells
(38) and lead to double-stranded DNA cleavage in an experimental prokaryotic
system (28). The DNA of the folate-deficient erythroblasts in the in vitro murine
model have only a two- to threefold increased proportion of misincorporated uracil
compared to controls (58), suggesting that uracil misincorporation may not be a
significant source of DNA strand breakage leading to apoptosis. These two- to
threefold increases in uracil misincorporation in folate-deficient erythroblasts are
similar to the changes seen in lymphocytes of folate-deficient rats that have ev-
idence of DNA damage (31), but less than found in patients with megaloblastic
anemia (116). The rescue of folate-deficient erythroblasts by exogenous purines
and thymidine suggests that insufficient deoxynucleotide triphosphates may be
the cause of DNA damage and apoptosis. Murine granulocyte progenitors treated
with the antifolate methotrexate are similarly rescued by exogenous purines and
thymidine (84). Although one study of bone marrow from patients with mega-
loblastic anemia found increases in all deoxynucleotides (51), others have shown

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specific deoxynucleotide depletions in splenic cells of folate-deficient rats (53),
human lymphocytes cultured under folate-deficient conditions (111), and cell lines
treated with antifolates (9, 122). Murine erythroblasts accumulate increased p53
protein when they are cultured under folate-deficient conditions (58), suggesting
that p53 expression is an indicator of DNA damage in folate-deficient erythrob-
lasts. However, when erythroblasts from p53-null mice are cultured under folate-
deficient conditions, they have similar rates of apoptosis as do their p53 wild-type
littermates, which indicates that p53 is not necessary for the apoptosis that results
from folate deficiency-induced DNA damage (59).
The murine in vivo and in vitro systems have also provided insights into the
morphological changes of ineffective erythropoiesis that occur in folate or vitamin
B
12
deficiency. When mice are made folate-deficient by being fed the folate-free
diet, they develop a macrocytic anemia with decreased reticulocytes (10, 58). As the
folate-deficiency anemia progresses, the bone marrow hematopoietic cells of the
mice, including the erythroid cells, have decreased numbers of total nucleated cells,
increased size of the individual cells, and increased numbers of cells undergoing
apoptosis. While the absolute numbers of reticulocytes are decreased in folate-
deficient mice, the absolute numbers of CFU-Es are increased in their bone marrow
and spleen compared to controls (10). This result indicates that the folate-deficient
mice, like their human counterparts, have increased EPO levels in response to the
anemia, with a resultant increased survival of erythroid cells in the CFU-E and
other early stages of the EPO-dependent period. However, most of these increased
CFU-Es do not survive during the subsequent stages of erythropoiesis, but rather
they succumb to apoptosis, most often while in S-phase of the cell cycle. Erythroid
cells that are in the CFU-E stage or in S-phase during the post-CFU-E stages of
differentiation are larger and more immature appearing than the normal erythroid
cells which accumulate in the G
0
/G
1
phase during the terminal stages of erythroid
differentiation. Together, the shift to earlier stages of erythroid differentiation and
the accumulation of cells in S-phase contribute to the increased size and immature
appearance of erythroid cells in the bone marrow that characterize megaloblastic
anemias (58).
IRON METABOLISM AND THE IRON-DEFICIENCY STATE
Iron is an essential element that is a component of heme-containing proteins
(i.e., hemoglobin, myoglobin, and cytochromes) and innumerable nonheme iron-
containing proteins with vital functions in many metabolic processes of all cells.
However, at pH 7.4 and physiological oxygen tension, the relatively soluble fer-
rous ion is readily oxidized to the ferric ion, which forms virtually insoluble ferric
hydroxides. Moreover, unless bound to specific ligands, iron plays a key role in
the formation of harmful oxygen radicals, which ultimately cause peroxidative
damage to vital cell structures. Because of this virtual insolubility and potential
toxicity, specialized mechanisms and molecules for the acquisition, transport, and

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NUTRITION AND ERYTHROPOIESIS 115
storage of iron in a soluble, nontoxic form have evolved to meet cellular and or-
ganismal iron requirements. Moreover, organisms are equipped with sophisticated
mechanisms that prevent the expansion of a catalytically active intracellular iron
pool, while maintaining sufficient concentrations for metabolic use (2, 90, 100,
104).
Cellular iron acquisition and its proper intracellular targeting into functional
iron proteins depend on an array of other proteins. “Traditional” proteins involved
in iron metabolism include transferrin, transferrin receptor, and ferritin, but re-
cent research has identified a number of novel genes whose products emerge as
important players in iron metabolism (Table 1).
Iron represents 55 and 45 mg per kilogram of body weight in adult men and
women, respectively. Normally, about 60% to 70% of total body iron is present in
hemoglobin in circulating erythrocytes. In vertebrates, iron is transported within
the body between sites of absorption, storage, and utilization by the plasma glyco-
protein, transferrin, which binds ferric iron very tightly but reversibly. The daily
turnover of transferrin iron is roughly 30 mg and, normally, about 80% of this
iron is transported to the bone marrow for hemoglobin synthesis in developing
erythroid cells. Senescent erythrocytes are phagocytosed by macrophages of the
reticuloendothelial system where the heme moiety is split from hemoglobin and
catabolized enzymatically via heme oxygenase-1 (HO-1) (71). Iron, which is lib-
erated from its confinement within the protoporphyrin ring inside macrophages, is
returned almost quantitatively to the circulation. The remaining 5 mg of the daily
plasma iron turnover is exchanged with nonerythroid tissues, namely, the liver.
About 1 mg of dietary iron is absorbed daily, and the total organismal iron balance
is maintained by a daily loss of 1 mg via nonspecific mechanisms (mostly cell
desquamation) (100).
Several important features of organismal iron metabolism must be mentioned.
First, iron turnover is virtually an internal event in the body, and most of the iron
turning over is used for the synthesis of hemoglobin in erythroid cells. Second,
at least some nonerythroid cells can acquire nontransferrin-bound iron (NTBI),
and this process likely operates in vivo only in severely iron-overloaded patients
who have NTBI in their plasma. However, hemoglobin synthesis is stringently
dependent on transferrin as the source of iron for erythroid cells. Third, although
iron absorption is required for efficient erythrocyte formation on a long-term ba-
sis, quantitatively the most important source of iron for day-to-day erythropoiesis
is macrophages that recycle hemoglobin iron. Fourth, erythrocytes contain about
45,000-fold more heme iron (20 mmol/L) than nonheme iron (440 nmol/L) (100).
The fact that all iron for hemoglobin synthesis comes from transferrin and that
this delivery system operates so efficiently, leaving mature erythrocytes with neg-
ligible amounts of nonheme iron, suggests that the iron transport machinery in
erythroid cells is an integral part of the heme biosynthesis pathway. It seems rea-
sonable to propose that the evolutionary forces that led to the development of highly
hemoglobinized erythrocytes also dramatically affected numerous aspects of iron
metabolism in developing erythroid cells, making them unique in this regard.

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TABLE 1 Some proteins involved in iron metabolism
Protein Function Result of deficiency Reference
Transferrin (Tf) Fe
3+
-carrier in plasma Severe Fe deficiency anemia; (88, 90)
generalized Fe overload
Tf receptor Membrane receptor for diferric-Tf Embryonic lethality (67, 91)
Ferritin (H and L) Cellular Fe storage H: embryonic lethality (7, 32, 90)
IRP (1 and 2) Fe “sensors”; bind to IREs IRP2: brain Fe overload (2, 15, 65, 76, 104)
DMT1/DCT1/Nramp2 Membrane transporter for Fe
2+
Hypochromic microcytic anemia (16,17, 34, 41)
Duodenal cytochrome b Ferric reductase (provides Fe
2+
Unknown (73)
(Dcytb) for DMT1 in duodenum)
Ferroportin 1/Ireg1/MTP1 Fe export from cells Hemochromatosis type 4 (1, 29, 74, 79, 88)
Ceruloplasmin (Cp) Regulation of Fe export from cells Hypochromic microcytic anemia (45)
Hephaestin Regulation of Fe export from enterocytes Hypochromic microcytic anemia (3, 112)
(membrane-bound Cp homolog)
ALA-S2/eALA-S First enzyme of heme synthesis; X-linked sideroblastic anemia (33, 85)
erythroid-specific
5-aminolevulinic-acid synthase
Ferrochelatase Last enzyme of heme synthesis; Fe
2+
Erythropoietic protoporphyria (24, 85)
insertion into protoporphyrin IX
Mitochondrial ferritin Mitochondrial Fe storage (?) Unknown; high expression in (20, 30)
“ring” sideroblasts
Heme oxygenase-1(HO-1) Recycling of hemoglobin Fe Severe anemia and inflammation (71, 88, 93, 94, 120)
Hepcidin Plasma peptide which appears to Fe overload; overexpression of hepcidin (35, 80–82)
inhibit Fe absorption causes severe Fe deficiency anemia
Abbreviations: ALA-S2/eALA-S, erythroid-specific 5-aminolevulinic-acid synthase; DCT, divalent cation transporter; DMT, divalent metal transporter; IRE, iron-responsive element;
IRP, iron regulatory protein.

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NUTRITION AND ERYTHROPOIESIS 117
Iron deficiency is the most prevalent cause of anemia, affecting more than half a
billion people worldwide. The anemia of iron deficiency is caused by a decreased
supply of iron for heme synthesis and, consequently, hemoglobin formation in
developing erythroid cells. Decreased hemoglobinization leads to the production
of erythrocytes that are smaller than normal (microcytic) and contain reduced
amounts of hemoglobin (hypochromic). Blood loss is the most common cause of
iron deficiency. One milliliter of blood contains about 0.5 mg of iron and, hence,
a steady blood loss of as little as 3 to 4 mL per day (1.5 to 2 mg of iron) can
result in a negative iron balance. In men and postmenopausal women, unexplained
iron deficiency is nearly always due to occult bleeding from the gastrointestinal
(GI) tract. Sources of GI bleeding include hemorrhoids, hiatus hernia, peptic ul-
ceration, diverticulosis, tumors of the stomach and colon, adenomatous polyps,
colitis, esophageal varices, and ingestion of salicylates, steroids, and nonsteroidal
anti-inflammatory agents. Worldwide, the leading cause of GI blood loss is hook-
worm infection (87). In premenopausal women, menstrual blood loss is the most
common cause of iron deficiency. The average menstrual blood loss in normal
healthy women is about 40 mL, and women who lose 80 mL or more become iron-
deficient. Increased iron requirements during periods of rapid growth, diminished
iron absorption, or both may also cause iron deficiency.
In the anemia of chronic disease, iron-deficient erythropoiesis results from a
defect in the recycling of hemoglobin iron in the reticuloendothelial system (109).
In patients with anemia of chronic inflammation, there appears to be a defect in
the release of iron from macrophages that is probably caused by cytokine-induced
ferritin synthesis. As a result, iron is plentiful in macrophages, but this iron is not
available to erythroid precursors.
IRON EXPORT FROM CELLS TO TRANSFERRIN: A
NECESSARY PREREQUISITE FOR ERYTHROPOIESIS
There are specialized mammalian cells that must export iron. Absorption of di-
etary iron for transfer to transferrin in plasma requires iron efflux across the ba-
solateral surface of the intestinal epithelia. A second major site of iron release
is from macrophages where senescent or damaged red cells are degraded to ex-
port the metal from hemoglobin and provide it for binding to transferrin. Iron
release from these “donor cells” to plasma transferrin is poorly understood, but
a number of recent studies have provided new clues in this important area of
iron metabolism. A likely candidate for iron export from cells is ferroportin 1
(29), also known as Ireg1 (74) or MTP1 (1), with the ferroxidase activity of hep-
haestin (112) and ceruloplasmin (45) facilitating the movement of iron across
the membranes of enterocytes and macrophages, respectively. Ceruloplasmin and
hephaestin exhibit a high degree of homology; both proteins contain several cop-
per atoms that are necessary for their ferroxidase (i.e., oxidation of Fe
2+
to Fe
3+
)
activity.

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Figure 3 Scheme of possible iron (Fe) pathways in reticuloendothelial macrophages
involved in the recycling of hemoglobin iron. Following phagocytosis of senescent red
blood cells (RBCs), the erythrocyte membrane is lysed and heme is transported to the
endoplasmic reticulum (E.R.) to be degraded by heme oxygenase-1 (HO-1). Most of
the iron derived from hemoglobin catabolism is promptly returned to the circulation,
likely being transported across the plasma membrane by ferroportin 1. In Kupffer cells,
ferroportin 1 (MTP1, 1reg1) is present not only at the plasma membrane but is also
present in the cytoplasm (1). (Reprinted from Reference 89 and used with permission.)
At the end of an erythrocyte’s life, it is phagocytosed by cells of the reticuloen-
dothelial system and iron is liberated from its confinement within the protopor-
phyrin ring by HO-1. These cells have an enormous capacity to purge themselves
of iron, which is likely exported via ferroportin 1 (Figure 3). However, the mecha-
nism involved in the regulation of macrophage iron output is unknown (47). It has
recently been proposed (35, 37) that the plasma peptide hepcidin may be involved
in the regulation of iron release from macrophages, but direct evidence to support
this hypothesis is missing.
Normally, the body iron content in humans is maintained within narrow limits
by the regulation of intestinal iron absorption (77). Both heme and elemental
iron are absorbed through the brush border of the upper small intestine. Heme
iron is more readily available for absorption but usually constitutes only a small
fraction of dietary iron. Heme (derived from hemoglobin or myoglobin) is taken
up intact, probably via specific high-affinity heme-binding sites in the mucosal
brush border (39, 118) (Figure 4). After entering the intestinal epithelial cells, iron
is enzymatically released from heme by HO-1.

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NUTRITION AND ERYTHROPOIESIS 119
Figure 4 Iron transport across the intestinal epithelium. Iron (Fe) must cross two
membranes to be transferred across the absorptive epithelium. The apical transporter
has been identified as Nramp2/divalent metal transporter 1 (DMT1). It acts in concert
with duodenal cytochrome b (Dcytb) that reduces ferric iron. The basolateral trans-
porter, ferroportin 1, requires ferroxidase activity of hephaestin (ceruloplasmin-like
molecule) for the transfer of iron to the plasma. Hephaestin is depicted here at the
basolateral surface of the cell, but it is not known whether it functions at this location.
Heme iron is taken up by a separate process that is not well characterized. Excess iron
within enterocytes is stored as ferritin. (Reprinted from Reference 89 and used with
permission.)
Elemental Fe
3+
is virtually insoluble at neutral pH and, therefore, the availability
of dietary iron for intestinal absorption depends on the composition of intestinal
secretions as well as ligands and reducing agents present in the diet. Ascorbic acid is
the most powerful promoter of nonheme iron absorption, which is also enhanced by
the organic acids (e.g., citric acid and amino acids). On the other hand, compounds
that form insoluble complexes with iron (e.g., phosphates, phytates, and tannin)
prevent absorption. Similarly, conditions in which there is a failure of gastric acid
secretion (e.g., atrophic gastritis) may significantly reduce the availability of iron
for absorption.

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The process of inorganic iron absorption is not fully understood, but a com-
pelling candidate for an iron transporter has recently been identified. Nramp2/
divalent metal transporter 1 (DMT1), which is involved in iron transport across
the endosomal membrane (see below), is also a principal transporter of iron in
the intestine (41, 101). Nramp2/DMT1 transports only the ferrous (reduced) form
of iron, and this explains why reducing agents enhance iron absorption. More-
over, the duodenal brush border contains a ferric reductase, duodenal cytochrome
b (Dcytb) (73), which plays a role in the formation of Fe
2+
prior to its transport
into the enterocyte. The chemical nature of iron in the labile intermediate pool
in enterocytes is unknown, but recently a novel protein necessary for iron egress
from enterocytes was identified. This protein, ferroportin 1 (1, 29, 74), is identical
to the Fe
2+
exporter involved in iron egress from macrophages (Figure 3). The
ferroxidase activity of hephaestin (3, 112), a membrane-bound ceruloplasmin (45)
homologue, also plays an important role in iron export from intestinal epithelial
cells to the circulation. Hephaestin is not an iron transporter itself but likely inter-
acts with the ferroportin 1 to facilitate the movement of iron across the membrane
(Figure 4). Hephaestin is mutated in sex-linked anemia (sla/sla) mice that take up
iron from the intestinal lumen into the epithelial cells normally, but the subsequent
exit of iron into the circulation is diminished (112). It is of interest that during the
process of absorption, iron undergoes at least two changes in its oxidation status:
reduction at the brush border and oxidation at the basolateral membrane.
Physiologically, the major factors affecting iron absorption are the amount of
body iron stores and the rate of erythropoiesis (77). The uptake of iron by mucosal
cells is inversely proportional to total body iron content but seems to be independent
of changes in plasma iron or transferrin concentration. The 3

UTR of mRNA for
Nramp2/DMT1 expressed in intestinal cells contains the iron-responsive element
(IRE) (16, 101); hence, based on the IRE/iron regulatory protein (IRP) paradigm
(see below), diminished Fe levels would be expected to increase Nramp2/DMT1
expression and vice versa. It is unclear how increased erythropoietic activity
(increased plasma iron turnover?) enhances iron absorption. Hypoxia can di-
rectly stimulate iron absorption, independent of changes in erythroid activity.
Interestingly, the gene for Nramp2/DMT1 seems to contain regulatory elements
that can be responsible for its increased transcription under hypoxic conditions
(66).
Recent research, based on genetic studies, revealed that hepcidin probably plays,
either directly or indirectly, an important role in iron metabolism. In its presumed
active form, hepcidin is a 22- or 25-amino acid peptide that has intrinsic antimicro-
bial activity (82). Mice that are unable to express hepcidin develop iron overload
associated with decreased iron levels in macrophages (80), whereas animals that
overexpress hepcidin develop lethal iron deficiency anemia (81). Hence, it has been
suggested (35, 37) that hepcidin may be a putative signaling molecule mediating
communication between the sites of iron storage (hepatocytes and macrophages)
and iron release from duodenal enterocytes or macrophages. However, thus far no
study has demonstrated that circulating hepcidin itself plays a direct role in iron
metabolism.

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NUTRITION AND ERYTHROPOIESIS 121
IRON ACQUISITION FROM TRANSFERRIN
BY DEVELOPING ERYTHROID CELLS
With some notable exceptions (e.g., enterocytes), physiologically, virtually all the
cells in the organism, including erythroid precursors, take up iron from transferrin.
Delivery of iron to cells occurs following the binding of transferrin to transferrin
receptors on the cell membrane (91, 100). The transferrin receptor complexes are
then internalized by endocytosis, and iron is released from transferrin by a process
involving endosomal acidification. Identifying the mechanism of iron transport
across the endosomal membrane was elusive, but a compelling candidate for an
endosomal iron transporter has been identified (34, 41). The transporter, Nramp2
(also known as DMT1 or DCT1, divalent cation transporter 1), is encoded by
a gene that belongs to the Nramp (“natural resistance-associated macrophage
protein”) family of genes (21). Interestingly, Nramp2 generates two alternatively
spliced mRNAs that differ at their 3

untranslated regions (UTRs) by the presence
or absence of the IRE and that encode two proteins with distinct carboxy termini
(16, 17). Isoform II (derived from non-IRE-containing mRNA; for the definition
of IRE see below) has been identified as the major Nramp2 protein isoform that is
expressed in the developing erythroid cells (17). Also, Nramp2 was not found to be
a limiting factor in erythroid cell iron acquisition via the physiological, transferrin-
dependent, pathway. Because the substrate for Nramp2/DMT1 is Fe
2+
, reduction
of Fe
3+
must occur in endosomes, but little is known about this process. A cDNA
encoding a plasma membrane di-heme protein present in mouse duodenal cells
was found to exhibit ferric reductase activity (73). This protein (Dcytb) belongs to
the cytochrome b561 family of plasma membrane reductases, and it would seem
important to examine whether this or a similar b-type cytochrome is involved in
Fe
3+
reduction within endosomes. Following its escape from endosomes, iron is
transported to intracellular sites of use and/or storage in ferritin, but this aspect
of iron metabolism, including the nature of the elusive intermediary pool of iron
and its cellular trafficking, remains enigmatic. Only in erythroid cells does some
evidence exist for specific targeting of iron toward mitochondria, the sites of heme
production by ferrochelatase, the enzyme that inserts Fe
2+
into protoporphyrin
IX. This targeting is demonstrated in hemoglobin-synthesizing cells, where iron
acquired from transferrin continues to flow into mitochondria, even when the
synthesis of protoporphyrin IX is markedly suppressed (85). Moreover, inhibition
of endosome motility decreases the rate of
59
Fe incorporation into heme from
59
Fe-
labeled endosomes, suggesting that in erythroid cells a transient mitochondrion-
endosome interaction may be involved in iron translocation to ferrochelatase (92).
DISTINCT CONTROL OF IRON METABOLISM IN
ERYTHROID CELLS
In general, cells are equipped with a remarkable regulatory system that tightly con-
trols iron levels in the labile iron pool (LIP), that is, iron-in-transit among various
intracellular compartments. Sensitive control mechanisms exist that monitor iron

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levels in the LIP and prevent its expansion, while still making the metal available
for iron-dependent proteins and enzymes. In general, enlargement of the LIP leads
to a stimulation of ferritin synthesis and to a decrease in the expression of trans-
ferrin receptors; the opposite scenario develops when this pool is depleted of iron.
Pivotal players in this regulation are IRP1 and IRP2, which “sense” iron levels in
the LIP.
Iron-dependent regulation of both ferritin and transferrin receptors occurs post-
transcriptionally and is mediated by virtually identical IREs. IREs present in the
5

UTRs of mRNAs, as in ferritin and erythroid-specific 5-aminolevulinic-acid
synthase (ALA-S2, the first enzyme of heme biosynthesis), mediate inhibition
of translation of the respective mRNAs in iron-deprived cells. Similar IREs are
also present in the 3

UTR of the transferrin receptor mRNA. These IREs confer
differential stability to transferrin receptor mRNAs as a function of cellular iron
levels. The IREs are nucleotide sequences that are recognized by specific cytosolic
RNA-binding proteins known as IRP1 and IRP2. An IRE-binding form of each
IRP accumulates in iron-depleted cells, but the mechanism of accumulation differs.
When cellular iron is low, IRP1 is in a form that can bind to IREs, and IRP2 (which
has constitutive RNA-binding activity) is stable. Binding of IRPs to IREs found in
the 5

end of mRNA (ferritin, erythroid-specific ALA-S2) inhibits translation of
these transcripts, whereas binding to IREs in the 3

UTR of the transferrin receptor
mRNA (and probably also in the intestinal form of mRNA for Nramp2/DMT1)
stabilizes the transcripts. Hence, iron deficiency promotes cellular iron acquisi-
tion and possibly intestinal iron absorption while it decreases levels of the cellular
iron-storing protein, ferritin. On the other hand, the expansion of the LIP inacti-
vates IRP1 and leads to a degradation of IRP2, resulting in efficient translation of
ferritin mRNA (and ALA-S2 mRNA in erythroid cells) and rapid degradation of
transferrin receptor mRNA (2, 15, 76, 91, 100, 104).
Some cells and tissues with specific requirements for iron evolved mechanisms
that can override the IRE/IRP-dependent control of transferrin receptor formation.
Erythroid cells, which are the most avid consumers of iron in organisms, use
primarily a transcriptional mechanism to maintain very high transferrin receptor
levels (68, 85). Moreover, erythroid cells are equipped with an important regulatory
mechanism that coordinates protoporphyrin IX formation with iron supply (85).
Because the 5

UTR of mRNA for erythroid-specific ALA-S2 contains the IRE,
the formation of ALA-S2 (the rate-limiting enzyme of porphyrin biosynthesis)
and, consequently, protoporphyrin depends on the availability of iron.
THE AVAILABILITY OF IRON CONTROLS
HEMOGLOBIN SYNTHESIS
Although three different and totally distinct pathways are involved in hemoglobin
synthesis, virtually no intermediates, i.e., globin chains, porphyrin intermedi-
ates, or iron, accumulate in the developing erythroblasts and reticulocytes. This

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NUTRITION AND ERYTHROPOIESIS 123
regulation is achieved, at least in part, by a series of negative and positive feedback
mechanisms in which both iron and heme play important roles. First and foremost,
the supply of iron via the transferrin-receptor pathway limits, and thus controls,
heme synthesis rate in erythroid precursors. Moreover, in erythroid cells “uncom-
mitted” heme inhibits cellular iron acquisition and, consequently, heme synthesis.
Furthermore, availability of heme is essential for the synthesis of globin at both the
transcriptional and, more importantly, the translational levels (85, 86). Numerous
reports indicate that heme stimulates globin gene transcription and is probably in-
volved in promoting some other aspects of erythroid differentiation (105). Hemin
treatment of erythroid precursors leads to rapid accumulation of globin mRNA,
whereas heme deficiency leads to a decrease in globin mRNA levels (27, 36, 102,
103, 105). These effects can probably be explained by heme-mediated upregulation
of the erythroid transcription factor NF-E2 binding activity (105).
It has long been known that the translation of globin in intact reticulocytes and
their lysates is dependent on the availability of heme (14, 23, 69, 124). Heme de-
ficiency inhibits protein synthesis through activation of heme-regulated inhibitor
(HRI). HRI is a cyclic adenosine monophosphate (AMP)-independent protein ki-
nase that specifically phosphorylates the α-subunit of eukaryotic initiation factor
2 (eIF-2). Recent research has revealed that autophosphorylation of threonine 485
is essential for the phosphorylation and activation of HRI and is required for the
acquisition of the eIF-2α kinase activity (96). During translation initiation, eIF-
2-GTP, associated with Met-tRNA
Met
, binds to 40 S subunit and participates in
the recognition of the initiation codon. After translation initiation, eIF-2-GTP is
hydrolyzed to eIF-2-GDP. Because eIF-2 has a much greater affinity for GDP, a
guanine nucleotide exchange factor, eIF-2B, is required to recycle eIF-2 to the
GTP-bound form. Phosphorylation of eIF-α at serine 51 blocks the activity of
eIF-2B, reducing the level of eIF-2-GTP. Heme binding to HRI inhibits the phos-
phorylation of eIF-2α by HRI, resulting in an efficient translation of globin and
probably other proteins in erythroid cells (23, 113). Association of heme with
HRI inhibits the enzyme by promoting the formation of disulfide bonds, perhaps
between two HRI subunits (23). Disulfide bond formation reverses the inhibition
of protein synthesis seen during heme deficiency. Interestingly, HRI contains two
sequences that are similar to the heme regulatory motif found in numerous other
proteins whose functions are regulated by heme. Importantly, the HRI homodimer
has two distinct types of heme-binding sites (22). Binding of heme to the first site
is stable (i.e., HRI is a hemoprotein), whereas binding of heme to the second site
is responsible for the rapid down-regulation of HRI activity (22). The mRNA for
HRI is present in uninduced murine erythroleukemia cells and is increased after
the induction of erythroid differentiation. This accumulation of HRI mRNA in dif-
ferentiating murine erythroleukemia cells is dependent upon the presence of heme
because an inhibitor of heme synthesis markedly reduces HRI mRNA accumula-
tion (26); hence, HRI plays an important physiological role in the translation of
globin and probably other proteins synthesized in erythroid cells. This conclusion
is further supported by the finding that expression of dominant-negative mutants

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of HRI in murine erythroleukemia cells increased hemoglobin production in these
cells upon DMSO induction of erythroid differentiation (25).
Mice rendered null for the HRI gene (HRI
−/−
) appear to be normal, fertile,
and without gross abnormalities of hematologic parameters (43). However, in
erythroid cells from iron-deficient HRI
−/−
mice, a marked increase in both α-
and β-globin synthesis led to accumulated globins that were devoid of heme and
aggregated within the erythrocytes and their precursors. This resulted in a hy-
perchromic normocytic anemia with decreased erythrocyte counts, compensatory
erythroid hyperplasia, and accelerated apoptosis in bone marrow and spleen (i.e.,
ineffective erythropoiesis). These important results established the physiological
role of HRI in balancing the synthesis of α- and β-globins with the availability of
heme in developing erythroid cells. Moreover, these results have demonstrated that
the translational regulation of HRI in iron deficiency is essential for the survival
of erythroid precursors (43).
In conclusion, in erythroid cells iron is not only the substrate for the synthesis
of hemoglobin but also participates in its regulation. Moreover, the iron proto-
porphyrin complex appears to enhance globin gene transcription, is essential for
globin translation, and supplies the prosthetic group for hemoglobin assembly.
CONCLUSION: ERYTHROPOIESIS UNDER NORMAL,
FOLATE-DEFICIENT, AND IRON-DEFICIENT
CONDITIONS
Erythropoiesis during normal conditions, folate deficiency, and iron deficiency is
shown in Figure 5 (see color insert). Erythropoiesis during vitamin B
12
deficiency
is similar to that shown for folate deficiency. In normal erythropoiesis, a minor-
ity of the EPO-dependent cells survives the EPO-dependent period, giving rise
to basophilic erythroblasts that divide and mature into orthochromatic erythrob-
lasts and reticulocytes. Because the periods of EPO dependence and hemoglobin
synthesis do not overlap, apoptosis of progenitors during normal erythropoiesis
does not increase serum bilirubin. During folate-deficient erythropoiesis, increased
apoptosis due to DNA damage extends into the post-EPO-dependent stages, where
hemoglobin synthesis has begun but has not yet reached high levels. This apopto-
sis of cells that have begun hemoglobin synthesis causes slightly increased serum
bilirubin. Those folate-deficient erythroblasts surviving to the late stages produce
fewer but larger reticulocytes, leading to macrocytic anemia. The anemia induces
EPO production, which in turn increases the survival of cells in the EPO-dependent
stages compared to normal erythropoiesis. However, the expansion of these EPO-
dependent populations is relatively incomplete due to increased apoptosis from
the folate deficiency. In iron deficiency, decreased synthesis of heme results in
the decreased protein translation, especially of globins, through the enhanced
action of HRI. This decreased protein translation in the iron-deficient erythroid
cells results in retarded reticulocyte production and smaller, less hemoglobinized

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NUTRITION AND ERYTHROPOIESIS 125
reticulocytes, leading to microcytic anemia. The resultant anemia induces EPO
production, which decreases the apoptosis in the EPO-dependent cells relative
to normal erythropoiesis. This increased survival in the EPO-dependent stages,
however, does not result in increased reticulocyte production due to the inhibitory
effect of HRI during the subsequent hemoglobin synthesis stages.
ACKNOWLEDGMENTS
MJK is supported by a Merit Review Award from the Department of Veterans
Affairs. PP thanks the Canadian Institutes of Health Research for support. The
authors thank Alex Sheftel, Conrad Wagner, and Maurice Bondurant for their
helpful discussions and suggestions, and Sandy Fraiberg and Michael Forbes for
excellent editorial assistance.
The Annual Review of Nutrition is online at http://nutr.annualreviews.org
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NUTRITION AND ERYTHROPOIESIS C-1
Figure 5 Models of erythropoiesis. Period of erythroid differentiation shown extends
from the actively dividing CFU-E through the postmitotic Ortho-EB and Retics, which
are shown with their enucleated nuclei. Irregular nuclear fragments in the Poly-EB and
earlier stages represent apoptotic cells. The arrows between the stages represent rela-
tive rates of progression between stages. Colors are as stained with 3,3’-dimethoxy-
benzidine and hematoxylin. Orange represents accumulated hemoglobin.
Abbreviations: Baso-EB, basophilic erythroblast; CFU-E, colony-forming unit-ery-
throid; Ortho-EB, Orthochromatic erythroblast; Poly-EB, polychromatophilic ery-
throblast; Pro-EB, proerythroblast; Retic, reticulocyte. Reprinted with modifications
from Koury MJ, Horne DW, Brown ZA, Pietenpol JA, Blount BC, Ames BN, Hard R,
and Koury ST. 1997. Apoptosis of late-stage erythroblasts in megaloblastic anemia:
association with DNA damage and macrocyte production. Blood 89:4617–23.
Copyright American Society of Hematology, used with permission.
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