HHS Public Access
`Author manuscript
`Free Radic Biol Med. Author manuscript; available in PMC 2016 July 11.
`Published in final edited form as:
`Free Radic Biol Med. 2014 July ; 72: 23–40. doi:10.1016/j.freeradbiomed.2014.03.039.
`
`Physiology and Pathophysiology of Iron in Hemoglobin-
`Associated Diseases
`
`Thomas D Coates, MD [Professor]
`Pediatrics and Pathology, Section Head Hematology, Children’s Center for Cancer and Blood
`Diseases, Children’s Hospital Los Angeles, University of Southern California, Keck School of
`Medicine, 4650 Sunset Blvd, MS 54, Los Angeles, California, 90027, tcoates@chla.usc.edu,
`Voice: 323 361 2352
`
`Abstract
`Iron overload and iron toxicity, whether because of increased absorption or iron loading from
`repeated transfusions, can be major causes of morbidity and mortality in a number of chronic
`anemias. Significant advances have been made in our understanding of iron homeostasis over the
`past decade. At the same time, advances in magnetic resonance imaging have allowed clinicians to
`monitor and quantify iron concentrations non-invasively in specific organs. Furthermore, effective
`iron chelators are now available, including preparations that can be taken orally. This has resulted
`in substantial improvement in mortality and morbidity for patients with severe chronic iron
`overload. This paper reviews the key points of iron homeostasis and attempts to place clinical
`observations in patients with transfusional iron overload in context with the current understanding
`of iron homeostasis in humans.
`
`
`
`Introduction
`Toxicity and increased morbidity due to iron overload are common and well-recognized
`complications associated with various hemoglobin disorders. Chronic iron overload occurs
`primarily from repeated blood transfusions in a number of hematological disorders. In fact,
`the most extensive information regarding severe chronic iron overload comes from decades
`of experience with the management of patients with thalassemia major, a hemoglobinopathy
`where the primary morbidity stems from iron overload and that is fatal, if untreated. The
`toxicity due to transfusional iron overload depends upon a number of factors in addition to
`the degree of tissue iron loading itself. While our experience with thalassemia has been very
`helpful, it is not entirely applicable to all disorders associated with iron loading, as the
`patterns of tissue iron distribution and the severity of tissue damage differ among them.
`
`Many advances in our understanding of the treatment of transfusional overload have
`occurred, particularly in the last 15 years. The ability to noninvasively measure tissue iron in
`humans by magnetic resonance imaging (MRI), major breakthroughs in our understanding
`of the molecular physiology of iron regulation, and the availability of new iron chelating
`
`Conflict of interest: Dr Coates is on the Speaker’s Bureau or consults for Novartis Pharma, Shire Pharma, Apo Pharma, and Celeron.
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`agents have resulted in a dramatic improvement in the survival of patients with severe iron
`overload [1, 2].
`
`The purpose of this review is to summarize our current understanding of iron homeostasis,
`briefly introduce the hematological disorders primarily associated with iron overload, and
`discuss how new knowledge regarding iron homeostasis informs and is validated by
`observations made in course of clinical monitoring and management of humans with
`transfusional iron overload.
`
`
`
`Iron homeostasis
`Biological organisms have evolved to conserve iron and as such, humans have no
`mechanisms for the excretion of iron. Approximately 1 to 2 mg per day, or about 0.05% of
`the total body iron, is lost through desquamation of the gastro-intestinal tract lining and skin,
`and in small amounts, through blood loss [3]. This is balanced through absorption of dietary
`iron, primarily in the duodenum. Iron balance is maintained entirely through the regulation
`of absorption and recycling of iron from red cells. Iron absorption can be increased by as
`much as 20 fold in cases of acute blood loss (reviewed in [4, 5]). Iron absorption can also be
`pathologically increased in certain genetic disorders of iron transport as well as in
`hemoglobin disorders associated with ineffective erythropoiesis. Figure 1 summarizes key
`features of normal and pathologic iron balance.
`
`Patients with hemoglobin disorders have significant differences in iron utilization,
`erythropoietic drive and iron input from transfusion that result in pathological iron
`absorption, iron loading and toxicity. In these patients, the relatively small changes in dietary
`absorption and minimal iron excretion are not sufficient to maintain iron balance.
`
` Regulation of iron proteins
`Iron balance is maintained by controlling the levels and function of iron transport proteins.
`Transferrin is the main plasma iron transporter that binds two molecules of ferric iron
`(Fe3+). Transferrin is usually between 20 and 30% saturated with iron (see below). At the
`systemic level, transferrin saturation is the main iron sensor and plays a role in controlling
`the levels of the iron regulatory peptide, hepcidin. At the cellular level, there are two
`common mechanisms that apply to most of the proteins involved in iron homeostasis. First,
`iron regulatory protein 1 (IRP1) and 2 (IRP2) bind to iron response elements (IRE) in
`untranslated regions (UTR) of mRNA encoding proteins involved in cellular iron uptake,
`storage and export (transferrin receptor-1, TfR; divalent metal transporter-1, DMT1; ferritin-
`H/ferritin-L/ferroportin, FPN). IRP1/2 bind to IRE under conditions of low iron, while they
`dissociate from IRE in high iron states (reviewed in [6]). If the IRE is in the 3’UTR, IRP
`binding stabilizes the mRNA, prevents degradation, and increases protein production. If the
`IRE is in the 5’UTR, mRNA translation is inhibited [6–8]. The second general mechanism
`imparts tissue specific sensitivity to iron balance by modulation of the proportion of iron
`sensitive and iron-insensitive mRNA. At least for DMT1 and FPN, two different splice
`variants of mRNA exist, one with IRE, and the other without. This means that one variant
`responds to iron levels and one does not. The ratio of IRE to non-IRE differs in different
`tissues, resulting in differences in responsiveness to iron and differences in loading [9, 10].
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`In general, the IRP/IRE system protects against iron loss. There are over thirty-five mRNAs
`including hypoxia inducible factor 2(cid:345) that have IRE and are responsive to iron [7, 11].
`
` Dietary uptake
`Under normal circumstances, dietary ferric iron is reduced by cytochrome B (DcytB) to
`ferrous iron (Fe2+) at the apical brush border of duodenal enterocytes, and transported into
`the cell by divalent metal transporter-1 (DMT1). DMT1 expression is highest at the
`duodenum and decreases toward the colon [12]. Dietary heme iron is absorbed into the
`enterocyte via the heme carrier protein-1 (HCP1). Inside the enterocyte, heme is degraded by
`heme oxygenase and iron is released into the cytosol [13–15]. The free iron, referred to as
`labile cellular iron (LCI), is stored in the cells by ferritin or exported to the plasma by FPN.
`As enterocytes recycle about every three days, the iron stored in enterocytes is lost in the
`stool. This and the very small amount of iron excreted in the bile are the only natural
`mechanisms for iron removal in humans and accounts for a 1–2mg loss per day, as
`mentioned above [3].
`
` Macrophage phagocytosis of erythrocytes
`Recycling of iron from heme is a main component of iron homeostasis. Macrophages in the
`reticuloendothelial system recycle iron from senescent red cells via erythrophagocytosis
`[16]. About 90% of senescent endogenous or transfused red cells are eliminated by this
`mechanism. The internalized heme is degraded by heme oxygenase and the iron is either
`stored by ferritin, or released into the plasma through FPN by the macrophages, which are
`the main regulators of plasma iron levels [16–19]. The effects of this regulation are seen
`clinically in the case of acute inflammation. If iron release into plasma by the macrophages
`is blocked, as is the case in response to fever, plasma iron levels drop within hours because
`of the continued requirement for 25 mg/day of iron to make red cells [20, 21]
`
`Free hemoglobin and heme, which may be present in the plasma of patients with
`hemoglobin disorders because of shortened red blood cell survival and intravascular
`hemolysis, bind to haptoglobin and hemopexin, respectively, and are taken up by
`haptoglobin- or hemopexin-mediated binding to the scavenger receptor, CD163 on
`reticuloendothelial macrophages [22–24]. Like with intact red cells, heme oxygenase
`releases iron in the macrophage where it is then stored by ferritin in the cytosol or
`transported back to the plasma via FPN.
`
` DMT1 regulation
`In addition to enterocytes, erythroid precursors, hepatocytes, macrophages and other cells
`also express DMT1, which transports iron released from transferrin in endosomes into the
`cytosol [25]. Its expression is markedly increased by iron deprivation in the intestine, and
`less so in the kidney, liver, brain, and heart [12, 26]. DMT1 mRNA has a splice variant with
`an IRE in the 3’-UTR of the mRNA and one that has no IRE. Hence, when cellular iron
`levels are low, transcription of DMT1 is favored and more iron is transported into the
`enterocytes [26]. The ratio of the two variants differs depending on the tissue. Brain has the
`highest IRE/non-IRE ratio, while spleen, thymus, pancreas have the highest non-IRE/IRE
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`ratio. Thus, for example, iron entry into the pancreas would not be expected to decrease in
`the presence of high iron [5, 10, 12, 26].
`
` Ferritin storage
`In the cytosol, labile cellular iron (LCI) binds to ferritin or is exported in the Fe2+ state to the
`plasma via FPN. Ferritin is a multimeric iron storage protein that is found in animal and
`plant cells as well as in fungi and bacteria, and can bind about 4500 molecules of iron. Iron
`is incorporated into ferritin as Fe2+, but is quickly oxidized to Fe3+ within the ferritin shell
`by H-ferritin ferroxidase. The main function of ferritin within the cells is to protect them
`from iron toxicity. Small amounts of ferritin are released in the plasma by macrophages as
`L-ferritin via a lysosomal secretory pathway [27]. Ferritin mRNA has an IRE, and an
`increase in intracellular free iron leads to translational increase in ferritin production [28].
`Ferritin has been used as an estimate of iron loading, although the correlation between iron
`load and ferritin is only accurate in patient populations[29].
`
` Transferrin transport of iron
`Transferrin (Tf) is the main iron transport protein and binds two molecules of ferric iron.
`Transferrin-bound iron (TBI) is the primary source of iron available to cells under normal
`conditions. Holotransferrin binds to the homologous transferrin receptors, TfR1 and TfR2,
`and is endocytosed. In the acidic lysosomal environment, Fe3+ is released from Tf, and exits
`the lysosomes via DMT1 into the cytosol. In order for the transfer into the cytosol to occur,
`Fe3+ has to be reduced to the ferrous state, Fe2+(reviewed in [5]). Iron can also be
`transported out of the endosomes by the metal iron transporter, ZRT/IRT-like protein 14
`(ZIP14) [30].
`
`Both TfR1 and TfR2 have IREs in the 3’UTR and are post-transcriptionally regulated by
`IRP. TfR1 is expressed in most tissues, but at much higher levels in erythroid precursors and
`liver. TfR1 is also expressed in the heart at about the same level as in the liver, but 7.5 times
`less than in the spleen, and by implication, in splenic macrophages [31]. TfR2 is exclusively
`expressed in the liver and intestine, and at levels 5.8 times higher in the liver than the
`intestine. Levels of TfR2 are much higher than those of TfR1 in human liver [32]. Both
`receptors preferentially bind diferric Tf, but the affinity of TfR1 for iron is 25 times higher
`than that of TfR2. TBI is taken up exclusively by TfR1 in erythroid precursors, but is taken
`up by both TfR1 and TfR2 in the liver [5, 33]. Unlike TfR1, TfR2 does not have IRE and its
`expression does not respond to iron levels [34, 35].
`
` Ferroportin export of cellular iron
`FPN is the only known cellular iron exporter. It is expressed at very low levels in the
`membranes of most cells, but is abundant in macrophages, liver, syncytiotrophoblasts in
`placenta, the basolateral membranes of enterocytes [36, 37], and in erythroid precursors
`[38]. FPN gene expression in the heart is about 3 fold less than in the liver, and does not
`change with iron deficiency [31]. However, FPN mRNA and protein levels do increase in the
`heart about 2 fold with iron loading, which is sufficient to cause a Tf saturation of 70% [39].
`Like DMT1, FPN has two mRNA splice variants, one that contains an IRE and one that does
`not, allowing for tissue iron export variability in response to cellular iron based of the
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` Hepcidin
`
`relative proportion of the two forms of mRNA [38, 40]. FPN exports Fe2+, which must then
`be oxidized to Fe3+ in order to bind Tf. Though the exact mechanism is still unclear, an
`oxidase must be at play in order for iron to be exported. Ceruloplasmin, a multi-copper
`oxidase in plasma, facilitates release of iron and oxidizes Fe2+ into Fe3+ for binding to Tf.
`Low levels of ceruloplasmin in copper deficiency or congenital aceruloplasminemia lead to
`intracellular iron accumulation. The resulting high intracellular iron causes mitochondrial
`damage and can trigger progenitor apoptosis [41–43]. An analogous but membrane-bound
`multi-copper oxidase called hephaestin is present in the basolateral membrane of enterocytes
`and facilitates iron transport from the gut into the plasma [4, 5].
`
`FPN is the target of the iron regulator peptide, hepcidin [44, 45].
`
`Hepcidin is a 25 amino acid, defensin-like peptide that was discovered in the course of
`purifying (cid:346)-defensin 1 from urine [46]. This peptide hormone is made in the liver and
`regulates the flow of iron from enterocytes and macrophages into the plasma by binding to
`FPN, thereby causing its internalization and degradation by the ubiquitin pathway [44, 45].
`It is the primary regulator of the movement of iron into the plasma. Hepcidin is expressed
`almost exclusively in the liver, with 31 and 15 fold lower levels detectable in intestine and
`heart, respectively [31].
`
`Hepcidin levels are very low or absent in iron deficiency, leading to increased transport of
`iron via FPN from enterocytes and macrophages into the plasma. Conversely, hepcidin is
`elevated in iron overload and inflammatory states [4, 20, 47, 48]. This results in decreased
`iron absorption, decreased release of iron into the plasma, and sequestration of iron in tissue
`macrophages.
`
`Iron-mediated regulation of hepcidin levels is through bone morphogenetic protein-6
`(BMP-6) and its receptor on hepatocytes (Figure 2). The regulation is complex, and in
`humans, involves several proteins in addition to the BMP-6 receptor, i.e., the co-receptor
`hemojuvelin (HJV), the hereditary hemochromatosis protein (HFE), TfR1, TfR2, and
`matriptase-2 (coded by TMPRSS6). BMP-6, produced by sinusoidal endothelial cells in the
`liver, binds to the BMP-6 receptor complex on the hepatocyte, which in turn activates
`hepcidin transcription through a SMAD1/5/8 pathway. HJV, which is responsible for
`juvenile hemochromatosis, acts as a co-receptor and increases the sensitivity of the BMP
`receptor to BMP-6. Neogenin, a ubiquitous membrane protein, may also act as part of the
`BMP-6 receptor complex to enhance hepcidin production. High levels of holotransferrin
`stabilize TfR2 and displace HFE from TfR1, allowing it to interact with TfR2. The TfR2-
`HFE complex then associates with the BMP receptor complex, ultimately increasing
`hepcidin production. Thus, TfR2 is acting as an iron sensor that shuts down release of iron
`from enterocytes or reticuloendothelial macrophages into the plasma when iron is high and
`Tf is saturated (reviewed in [49, 50]). Finally, matriptase-2 (TMPRSS6) is a
`metalloproteinase on the hepatocyte membrane that is stabilized by iron deficiency and
`cleaves HJV, leading to decreased activation of the BMP-6/SMAD pathway, and hence
`decreased production of hepcidin. Mutations in TMPRSS6 lead to loss of inhibition of
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`hepcidin production. The ensuing high hepcidin markedly decreases iron absorption and
`results in iron-resistant iron deficiency anemia [51].
`
`Inflammation stimulates hepcidin production. This is mediated through the inflammatory
`cytokine, IL-6 and through activin-B. IL-6 acts through its receptor and the JAK2/STAT3
`pathway to turn on hepcidin production [36], and activin-B activates the BMP-6 receptor
`[47]. This results in sequestration of iron in the macrophages and decreased intestinal
`absorption, leading to the classic picture of chronic inflammatory anemia. High hepcidin
`levels would block release of iron via FPN from any cell.
`
`Hypoxia, anemia, and erythropoiesis reduce hepcidin production, and increase iron
`absorption. Anemia and hypoxia affect hepcidin expression indirectly through their effects
`on erythropoiesis in the bone marrow [38]. Erythropoietin (EPO) activates the JAK2/STAT5
`pathway that turns on erythroid proliferation and inhibits differentiation [52, 53]. Hepcidin
`levels decrease when bone marrow activity increases [54]. It is clear that this effect is from
`the marrow response to anemia and not from the anemia itself [55]. Growth differentiation
`factor15 (GDF-15) is released by erythroid precursors and has been implicated in the
`downregulation of hepcidin [56–58]. GDF-15 is increased in hemoglobinopathies
`(thalassemia, congenital dyserythropoietic anemia type 1), and in refractory anemia with
`ring sideroblasts [59]. GDF-15 is decreased post-transfusion, in parallel with EPO and
`decreased marrow activity, resulting in increase in hepcidin [54]. Twisted gastrulation
`(TWSG1), soluble HJV, and erythroferrone are other factors that increase with increased
`erythroid activity and result in reduced hepcidin production [60, 61].
`
` Non transferrin bound iron (NTBI) and labile plasma iron (LPI)
`About 20–30% of transferrin is normally bound to iron. Non-transferrin bound iron (NTBI)
`refers to a heterogeneous group of potentially toxic iron complexes found in the plasma,
`mainly Fe3+-citrate or albumin complexes. NTBI can be detected in the plasma as soon as
`transferrin saturation reaches 35% [62], and rises significantly when transferrin saturation
`exceeds 70 to 80% [63–65]. Transferrin saturation can be used as a surrogate for NTBI when
`it is above 35%. However, a fraction of NTBI, known as labile plasma iron (LPI), is very
`loosely bound to proteins, is highly redox active and thought to be the main species that
`causes iron mediated oxidative damage [66, 67]. Under normal conditions, NTBI/LPI should
`not be found in the plasma. However, in the presence of iron overload, once Tf becomes
`saturated, NTBI/LPI levels rise significantly, and can easily enter many cell types, resulting
`in increased labile cellular iron (LCI). This is thought to be primarily Fe2+-glutathione [68]
`and is highly reactive, causing organ damage and failure.
`
` Normal iron uptake into organs
`Iron bound to transferrin enters cells by binding to TfR1. TfR1 is expressed in most cells;
`however, the relative expression and activity vary significantly with different cells and is
`higher in tissues with high iron requirements. TfR1-mediated uptake is a major route of
`entry in erythroid precursors [25] and the liver. The TfR1-Fe complex is endocytosed. With
`acidification and in the presence of the ferroreductase Steap3, Fe2+ leaves the endosome
`through DMT1 and is chaperoned in the cytoplasm to ferritin or transported into the
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`mitochondria through mitoferrin [69]. The exact mechanism of this uptake is not known. In
`the mitochondria, iron is passed to ferrochelatase for incorporation into protoporphyrin-IX
`to make heme or is used for production of iron-sulfur clusters [36]. Normally, the pancreas
`and heart do not have high iron requirements. In pathologic states, they take up iron
`primarily by non-transferrin-mediated processes.
`
` Organ uptake of NTBI
`When transferrin becomes saturated, NTBI/LPI levels rise, and NTBI/LPI easily enters the
`liver, pancreas, endocrine glands and cardiomyocytes by non-transferrin dependent
`pathways. Hepatic uptake of NTBI in humans is rapid and efficient [70, 71]. In mice, this
`uptake is thought to involve DMT1 [72] and the zinc transporter, ZIP14 [73], which is
`upregulated in iron-loaded liver and pancreas, while DMT1 is downregulated in iron-loaded
`liver [9]. There is also evidence that ZIP14 may play a role in the uptake of TBI [9, 30], and
`that it may be expressed in the heart [74]. Spleen and pancreas have the highest proportion
`of non-IRE containing DMT1 mRNA. Thus, loading of NTBI/LPI into the spleen and
`pancreas via DMT1 does not decrease in response to high iron [10]. This, in combination
`with pancreatic ZIP14 [9, 30], may explain why rapid pancreas iron loading is observed in
`humans soon after liver iron increases [75].
`
`The liver loads with iron via regulated, transferrin receptor-mediated processes, and via
`uptake of NTBI, possibly through DMT1 on the hepatocyte cell surface [24, 76]. Cells
`normally control the uptake of iron by modulation of the expression of the high affinity
`TfR1. Iron can also enter the liver via the lower affinity TfR2, and that may account for the
`iron uptake increase observed when iron is abundant. In states of iron excess, TfR1 in the
`liver is downregulated whereas NTBI uptake remains the same [71, 77]. The molecular
`mechanisms are not well worked out yet in humans. However, the ability of the liver to load
`both TBI and NTBI may explain the very rapid loading of iron in the liver in humans [75,
`78].
`
` Removal of iron from liver
`Hepatocytes express FPN on the sinusoidal surfaces, with increased expression in periportal
`areas, and thus can export iron [76, 79]. Iron can also be excreted into the bile in humans.
`This may be important in conditions of iron overload [80], but it is not thought to be a major
`pathway under normal conditions [80–82]. In the rat, iron is excreted in the bile and
`reabsorbed in the intestine. This reabsorption is blocked if iron is bound to the chelator,
`deferiprone [83]. The enterohepatic circulation has been suggested to be important in
`humans [71], and significant amounts of iron are excreted in the feces of iron-overloaded
`humans in the presence of iron chelators [84, 85].
`
` Cardiac iron loading
`Transferrin receptors are present in the heart [31] and are downregulated in presence of iron
`overload [39]. However, the rate of NTBI uptake in cultures of heart cells is 300 times that
`of transferrin iron, and is increased significantly by iron loading [86]. Thus, once cardiac
`cells are overloaded with iron, the rate of further loading is increased [86, 87]. NTBI is
`thought to enter cardiac cells through L-type calcium channels [78, 88, 89] where it causes
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`oxidant-mediated cellular injury to cardiac mitochondria [77]. T-type calcium channels may
`also be involved in cardiac iron loading [89–91]. DMT1 is weakly expressed in the heart
`[80, 88], as is ZIP14 [74], but may also serve as portals of iron entry in the heart. FPN is
`expressed in the heart at lower levels than in the liver and increases with iron loading [31,
`39].
`
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`
`Iron regulation during erythropoiesis
`Cellular iron is closely regulated in red cell precursors during erythropoiesis. Iron import
`mechanisms are highly expressed in early committed red cell precursors, allowing high iron
`intake for heme production (reviewed in [38]). As hemoglobin is being made in the later
`stages of erythroblast development, there is an increased need for iron in these cells. Thus,
`high levels of TfR1 are expressed at the cell surface during each nucleated stage of erythroid
`development [92]. When hemoglobin production stops, TfR1 is released from the surface of
`the mature reticulocytes, the last stage of differentiation [93]. High iron levels in these
`precursors would normally decrease FPN through dissociation of IRP1 and IRP2 from the
`IRE in the FPN mRNA. Interestingly, FPN is expressed at all stages of erythroid
`development, even though it would be expected to be low when iron levels are very high.
`The IRE form of FPN mRNA predominates in early erythroid progenitors and late erythroid
`cells, resulting in iron-regulated FPN production while the iron-insensitive form of FPN1
`mRNA is present in pronormoblasts through ortho chromatophilic normoblasts. These
`variant FPN transcripts, which do not contain IRE and thus are insensitive to high iron,
`account for more that half of the total FPN mRNA in erythroid cells. The current hypothesis
`is that the FPN that is not downregulated by high iron levels in the pronormoblast to
`orthochromatic normoblast stages would provide an exit route for iron that might otherwise
`be toxic to the normoblast [38, 40]. Hydrogen peroxide (H2O2) that can diffuse into the
`nucleus, interacts with LCI and produce hydroxyl radical which can directly cause DNA
`damage [94] and can trigger apoptosis of erythroid precursors [52]. Oxidative damage from
`iron during erythropoiesis is thought to be, at least in part, the cause of ineffective
`erythropoiesis.
`
`Ineffective erythropoiesis
`Erythropoiesis is ineffective in some hemoglobin disorders and marrow failure states, and is
`thought to be the result of apoptosis of the erythrocyte precursors. The increased marrow
`activity, driven in part by anemia, leads to low levels of hepcidin and 2 to 3 times the normal
`iron absorption [95]. The increased iron levels should increase hepcidin. However, the effect
`of increased marrow activity on lowering hepcidin levels dominates the effect of iron
`overload on increasing hepcidin. At a minimum, hepcidin does not increase as much as it
`should for the level of iron overload (reviewed in [4]). ROS produced by oxidant interaction
`with iron in hemichromes that are formed from aggregates of heme and (cid:345)-globin chains
`cause hemolysis of the mature red cells and trigger apoptosis of erythroid precursors [52].
`The anemia results in tissue hypoxia and increase in EPO, leading to erythroid hyperplasia,
`usually without a rise in hemoglobin because of the underlying hemoglobinopathy (see
`below).
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`While ineffective erythropoiesis causes iron overload, the converse is also true. Consistent
`with a role for cellular iron toxicity, infusion of apotransferrin (transferrin with no bound
`iron) into iron-loaded thalassemic mice resulted in a decrease in transferrin saturation and
`LPI. This led to an increase in hemoglobin, improvement in red cell survival, correction of
`many of the red cell morphologic abnormalities, decreased deposition of (cid:345)-globin on the red
`cell membrane, decreased spleen size and increased hepcidin production. The improved red
`cell survival is presumably due to the reduction in redox-active iron-containing (cid:345) globin
`chains on the red cell membrane. While infused apotransferrin increased apoptosis of early
`erythroid precursors, apoptosis of mature erythroid precursors was reduced, resulting in
`overall increase in mature precursors and ultimately, an increase in hemoglobin. Hepcidin
`expression was higher in the livers of apotransferrin-treated animals and FPN tended to be
`lower [96]. The increase in hepcidin would be consistent with a decrease in putative
`erythroid-derived suppressors of hepcidin [56, 61, 97] because of reduced ineffective
`erythropoiesis. The increased hepcidin would also decrease iron release from macrophages
`and iron uptake in the gut. Overall, extramedullary erythropoiesis was reduced and there was
`significant decreased in ineffective erythropoiesis [96].
`
`These studies suggest that increasing hepcidin in the presence of iron overload decreases
`ineffective erythropoiesis and appear to significantly improve the anemia, at least in mouse
`models of thalassemia [98]. Other strategies that increase hepcidin also decrease liver iron,
`transferrin saturation, deposition of (cid:345)-globin on red cell membranes, reduce splenomegaly,
`and improve hemoglobin levels in thalassemic mice [99–101], confirming the findings seen
`with apotransferrin infusion [96]. Preliminary data in humans using an Activin IIa receptor
`fusion protein, which also increases hepcidin, demonstrated increased hemoglobin levels in
`patients with thalassemia intermedia (see below) [102]. These data suggest that iron toxicity
`contributes to ineffective erythropoiesis.
`
` Clinical introduction to hematological disorders associated with iron
`overload and toxicity
`The primary classes of disorders associated with clinically important iron overload and
`toxicity are listed in Table 1. The disorders fall into four groups based on their pathology: 1)
`Disorders with ineffective erythropoiesis, i.e., inability to make hemoglobin or red cells.
`They have variable levels of anemia, but all are characterized by hypercellular bone marrow
`with normal to increased erythropoietic activity; 2) Disorders with increased destruction of
`mature RBC and increased effective erythropoiesis with increased RBC precursors in the
`bone marrow; 3) Disorders with marked decrease in erythropoietic activity, which is
`generally ineffective; 4) Genetic disorders of iron absorption or transport. The disorders of
`absorption are not associated with anemia while those associated with transport may
`clinically present like iron deficiency (small RBC with mild anemia), but are actually
`associated with iron loading. The clinical severity and organ distribution of iron overload
`and toxicity as well as the response to treatment with chelators depend in part on the
`underlying marrow activity, the effectiveness of erythropoiesis, and the resulting effects on
`iron regulatory mechanisms as we will discuss below.
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`Free Radic Biol Med. Author manuscript; available in PMC 2016 July 11.
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`Author Manuscript
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`Author Manuscript
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`Apotex Tech.
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` Disorders with anemia and ineffective erythropoiesis
` Thalassemia—The term “thalassemia” generally refers to a family of disorders
`secondary to combinations of over 300 known mutations in the (cid:346)-globin gene ((cid:346)-
`thalassemia) or to a smaller number of mutations in the (cid:345)-globin gene ((cid:345)-thalassemia).
`Humans have one (cid:346)-globin gene on each allele on chromosome 11, and thus may have two
`identical (cid:346)-mutations (homozygotes), two different (cid:346)-mutations (compound heterozygotes),
`or a (cid:346)-gene mutation in only one allele (heterozygous, trait, or carrier state). The
`heterozygous (cid:346)-thalassemia state is also referred to as “thalassemia minor”. There are two (cid:345)-
`globin genes on each chromosome 16, thus humans have four (cid:345)-globin genes. Those missing
`one (cid:345)-gene are called “silent carriers” because there are no hematolo