OVERLOAD: CAUSES, CONSEQUENCES, AND CONTROL *Douglas P. Bentley

Department of Pathology, Spire Cardiff Hospital, Cardiff, UK *Correspondence to [email protected]

Disclosure: No potential conflict of interest.

ABSTRACT

The endeavours of physicians taxed with the problem of over the last 100 years have successfully eliminated this as an issue of hematological interest. , although by no means a new problem, has drawn much more attention over the last few decades. The development of reliable indicative tests of iron status since the early 1970s has sparked a revolution in our understanding of the mechanisms of iron absorption, the processes of internal iron metabolism, and the effects of iron in excess of physiological needs. There continues to be regular reviews of specific issues in iron overload but this article sets out our understanding of the principles of iron metabolism in the context of iron overload, and is intended to point the ways by which this potentially fatal disorder, created by nature or by man, might be overcome.

Keywords: Iron overload, hemochromatosis, iron depletion, iron chelation.

THE BASIC PRINCIPLES OF HUMAN IRON iron losses incurred by desquamation and minor METABOLISM bleeding. The major quantity of functional iron is in the form of hemoglobin and iron stores (of At our current state of knowledge, iron is variable magnitude up to 500 mg) and these are essential for all life. It provides the vital functions metabolically inactive. Except in active pathological of electron transport, reversible oxygen binding, conditions, any iron excess above immediate enzyme activation, DNA synthesis, and pro- requirements is retained largely by the RE system oxidant scavenging, but is required only in in the form of and the related compound amounts necessary for these metabolic functions. hemosiderin, but some is also found in the hepatic Small amounts of iron storage, as reflected by parenchymal cells. These stores are exhausted a serum ferritin concentration in the range 20- in uncomplicated iron deficiency prior to the 400 µg/L, merely act as a reserve for when iron development of anaemia. However, iron depletion demands are increased. without anaemia may afford some protection against microbial infection,1 and iron loading may Human iron metabolism is highly conserved: increase susceptibility to infection with some approximately 30 mg of iron are required daily, specific microorganisms.2 Other mammals have a predominantly for hemoglobin synthesis. This iron completely different scale of iron turnover, with is derived mainly from the digested hemoglobin of much of the daily erythropoietic iron requirements senescent red cells recycled by the macrophages of met from the diet and, consequently, there is the reticuloendothelial (RE) system. Iron exchange a greater rate of iron loss and a high level of with the environment is, by comparison, very slow: protection from iron overload and toxicity. Man has plasma iron turns over every 2 hours, whereas total no controlled iron excretory mechanism. body iron turnover time is 10 years. Only 10-20% of the iron is absorbed from the diet (which on average The hepatic antibacterial peptide hepcidin3 is the contains about 10 mg of iron daily) to maintain a central regulator of the iron supply. Its function is total body iron content of about 35-50 mg/kg body to suppress iron release from macrophages and weight, and to compensate for the small insensible hepatocytes, to limit the iron supply to that required

1 Website Article • HEMATOLOGY • December 2014 EMJ EUROPEAN MEDICAL JOURNAL for erythropoiesis and other metabolic functions, saturation is conveyed to the replicated and form enterocytes to maintain an adequate duodenal crypt cell and in low iron states stabilises level of iron stores, but to limit these to non-toxic the iron responsive protein (IRP) binding to the iron levels. Continuous cellular iron release is affected responsive element (IRE) on the 5’-untranslated through the transmembrane (TM) sole iron exporter DMT1 mRNA to signal increased expression. In the ferroportin. Ferroportin expression increases in iron iron replete state mRNA translation is inhibited, deficiency4 but its iron transport role is restrained and in addition, DMT1 can become saturated and by hepcidin binding,5 which leads to internalisation iron absorption becomes limited – the so-called and degradation of both molecules.6 ‘mucosal block’.21 Iron nanocages, largely of non- animal origin,22 are absorbed through a specific Homeostatic hepcidin regulation is determined by receptor, and release from internalised endosomes the degree of plasma transferrin saturation and is controlled by protonation. Once intracellular 7 iron status. Diferric transferrin displaces human iron enters a common intracellular pool it is hemochromatosis protein (high-Fe; HFE), the released into the plasma by ferroportin23 and product of HFE, from the widely expressed cell bound to transferrin through the oxidative action surface membrane transferrin receptor 1 (TfR1). HFE of ceruloplasmin and its homologue hephaestin.24 then forms complexes with hepatic parenchymal 8 cell-derived hemojuvelin (HJV), and in turn, IRON OVERLOAD bonds with TfR2, expressed almost exclusively on the hepatocyte surface and stabilised by Definition holotransferrin. HJV, amongst other functions, is the key regulator of hepcidin expression and, via There is no precise definition of iron overload. The its co-receptor, the uniquely iron-sensitive9 bone presence of excess iron may be determined by a morphogenetic protein 6 (BMP6),10,11 which together gross elevation in serum ferritin concentration or with the essential binding of neogenin,12 initiates a expansion of iron stores demonstrated histologically complex signalling process and by phosphorylation by bone marrow or liver biopsy. Magnetic of some proteins of the intracellular SMAD resonance imaging will detect ferric iron in the liver, family upregulates the nuclear HAMP coding for heart, and the brain which has largely replaced hepcidin.13 The mature protein is a cleavage-induced chemical measurements made on liver biopsy single product of furin action.14 Digestive activity samples.25 Elevated serum ferritin concentrations of the TM serine protease matriptase-2, also may be found in non-iron deficiency anaemias, liver–derived, and whose expression is increased in indicating the displacement of iron unused for iron depletion,15 cleaves HJV into multiple inactive hemoglobin synthesis into stores. Increases in sub-components16 to decrease membrane HJV serum ferritin concentration are seen in acute expression and competitively block intact HJV- liver disease,25 acute leukaemia,26 inflammatory induced hepcidin synthesis. Enterocytes are less disease,27 hyperthyroidism,28 and the rare hereditary sensitive to hepcidin than macrophages,17 which hyperferritinaemia-cataract syndrome,29 and reflects the different order of their respective these do not indicate total body iron excess. volumes of iron exported to the plasma. By lowering Fully saturated transferrin is also a feature of iron the plasma iron concentration, hepcidin may overload. By exception in untreated megaloblastic increase host resistance to infection. anaemia elevated transferrin saturation, non- transferrin bound iron (NTBI), and hyperferritinaemia A well-mixed diet contains three chemically may arise through the severe anaemia and massively different forms of iron. Heme iron, once liberated increased (up to 9-fold30) ineffective erythropoiesis from myoglobin and hemoglobin, is absorbed by and rapid shunting of recycled hemoglobin the heme carrier transport protein HCT1,18 located iron through macrophages to the plasma. NTBI on the apical aspect of the duodenal enterocyte. disappears and transferrin saturation precipitously It also functions as a folate transporter and its falls to normal within the first 2 days,31 before there regulatory role in iron metabolism has not been is any increase in the hemoglobin concentration, elucidated. Inorganic iron is reduced to the Fe2+ after effective replacement therapy. The serum form by duodenal cytochromes prior to absorption ferritin concentration is usually raised due to the by the divalent metal transporter 1 (DMT1), the severe anaemia and with effective treatment returns expression of which on the duodenal enterocyte to normal or occasionally levels indicative of iron is increased in iron deficiency.19,20 The degree of deficiency; iron overload is not a feature.

Website Article • HEMATOLOGY • December 2014 EMJ EUROPEAN MEDICAL JOURNAL 2 Causes of Iron Overload inherited disorders of iron absorption. The less critical H63D allows cell membrane HFE It is evident that iron overload occurs only expression and binding to β-2M on the cell surface. when intake is increased beyond normal daily It therefore produces a milder phenotype as there requirements. The stringent control of iron is a lower suppression of hepcidin expression. In absorption prevents large quantities of dietary compound heterozygotes, clinical disease is often and inappropriate therapeutic iron given by the associated with comorbid factors.44 Many further mouth leading to iron overload, but prolonged of HFE, 18 to date, have been identified iron medication has been identified as a possible but are very rare. Generally the iron accumulation in cause in a small number of patients.32,33 There classical hemochromatosis is insidious, and organ are usually contributing factors such as a genetic damage becomes apparent only in later life, also, mutation causing increased iron absorption in these females are affected less severely than males. recorded instances. Patients with chronic kidney disease are at risk of iron overload if intravenous (JH) is phenotypically iron therapy, given to correct the increased similar, and the autosomal recessive form of HH lost during hemodialysis, is not monitored closely.34 arises as a result of HJV mutation.45 This produces Although it is possible that two-way trafficking of a more severe degree of iron overload with organ iron in enterocytes35 may provide a mechanism damage evident in the first two decades of life. for increased iron loss, there are no data available Over 30 mutations in HJV have been identified, but on iron losses in iron overload patients. Primary G320V is the most common and has been reported or secondary increase in iron absorption and the in several populations worldwide. Hypogonadism administration of iron parenterally, usually as a and cardiac involvement are typical features. blood transfusion, are the responsible causes of Mutation of HAMP, the coding gene for hepcidin iron overload. In inherited states of increased iron causes a much more severe form of JH46 because absorption penetrance is always highly variable. of a total lack of hepcidin. Mutation of TfR247 is an Gender,36 iron-limiting disorders such as coeliac exceedingly rare cause of HH although >30 single disease,37 hepatic dysfunction,38 the effects of nucleotide polymorphisms have been identified. diet, alcohol consumption,39 obesity,40,41 and, of The condition is less severe than classical HH, but course, blood donation, will alter the penetrance of with generally earlier development of iron overload. inherited disorders leading to iron overload. Co-inheritance of C282Y is commonly found in affected patients. Primary Increase in Iron Absorption Ferroportin mutations,48 although rare, are the In hereditary hemochromatosis (HH) a genetic most common causes of dominant HH in East defect in the iron absorption pathway leads to Asia, where the C282Y mutation is rare. The progressive iron accumulation and is a result of unusual phenotype of RE iron loading in the face insensitivity or loss of the ferroportin/hepcidin of low transferrin saturation is found in the typical iron regulatory mechanism. In the classical form, form. The failure of hepcidin to denature mutated mutation of the HFE gene42 prevents expression of ferroportin and consequent loss of iron export the HFE protein by substitution of the cysteine from macrophages and enterocytes explains residue with tyrosine (C282Y), thereby losing these features. In the atypical form, inheritance of a disulphide bond. Defective post-translation ferroportin retains its iron export function but is processing of HFE causes its intracellular insensitive to the effects of hepcidin to produce retention and subsequent degradation and failure a phenotype similar to classical HH, but without of cell surface expression, where bonding with enterocyte iron accumulation.49 Homozygous β-2 microglobulin (β-2M)43 is essential for its ferroportin disease has not been described and is function. Suppression of hepcidin production probably incompatible with life. leads to failure of ferroportin degradation and uncontrolled iron export from enterocytes. Neonatal hemochromatosis is the most common Transferrin saturation thereby rises and iron is cause of rapidly fatal liver disease in the neonate deposited in vulnerable organs. and is caused by intrauterine iron loading. It does not have a known genetic basis.50 However, the The C282Y mutation is very common in the people discovery of maternal anti-liver antibodies, of of Northern and Western Europe, and therefore, unknown antigen, indicates a complement-fixing frequently alters the phenotype of the less common immunoglobulin G alloantibody attack. Hepcidin

3 Website Article • HEMATOLOGY • December 2014 EMJ EUROPEAN MEDICAL JOURNAL expression is lowered secondary to liver dysfunction METABOLIC DISORDERS CAUSING and leads to increased foetal iron uptake from INAPPROPRIATE IRON DEPOSITION the ferroportin-rich placenta. The elevated serum ferritin is typical of inheritance from the mother, Acaerulplasminaemia and a high recurrence rate in siblings suggests a bigenic origin of a mitochondrial disorder. Mutation in the ceruloplasmin gene inhibits transferrin iron uptake because of a lack of Nutritional Iron Overload ferroxidase activity. This is dependent on the trinuclear clusters which stabilise this An iron loading syndrome similar to HH is seen in circulating protein.63 At least 40 mutations have some native Africans. In the past this has been been identified and the majority lead to premature attributed to the effects of an acid beer and alcohol- stops in protein synthesis. The truncated induced hepcidin suppression, before its possible ceruloplasmin is degraded and the oxidation of genetic basis was identified. The Q248H ferroportin Fe2+, essential for binding to transferrin, fails. The mutation is unique to Africans and is associated autosomal recessive syndromes of hepatic iron with increased serum ferritin concentrations in overload, cerebellar ataxia, retinal degeneration, and adults51,52 and children.53 diabetes mellitus result64,65 together with growth retardation. The unusual iron accumulation in the Secondary Increase in Iron Absorption brain is thought to be due to the inability of neuroglial 66 Although iron absorption is controlled mainly by cells to donate iron to plasma transferrin. body iron status, the rate of erythropoiesis also DTM1 mutation plays a role. Conditions in which erythropoiesis is increased, irrespective of its effectiveness, The rare non-functional DTM1 mutations67 will elevate iron absorption non-specifically.inevitably lead to defective duodenal inorganic Congenital hemolytic anaemias, such as hereditary iron absorption and low red cell iron incorporation. spherocytosis and pyruvate kinase deficiency Liver iron overload is a usual feature and leads to iron overload, only if they are co-inherited presumably results from excess iron absorption with an additional mutation such as the C282Y through the alternative pathways. The serum of classical hemochromatosis.54 The massive ferritin concentrations, however, are less markedly increase in ineffective erythropoiesis also occurs in raised68,69 for unknown reasons. Both homozygotes β-thalassaemia, which majorly contributes to the and compound heterozygotes have been identified. iron loading caused by the life-maintaining blood transfusions. Non-syndromic inherited sideroblastic Atransferrinaemia anaemia (SA), usually caused by congenital primary There are many acquired causes of δ-aminolevulinic acid synthetase deficiency, or hypotransferrinaemia but the very rare autosomal GLRX5-dependent ALAS2 deficiency are associated recessive inheritance of atransferrinaemia causes 55 with systemic tissue iron loading. Rarer disorders a microcytic anaemia with absent marrow iron but of effective red cell production leading to iron widespread siderosis. Homozygous mutations and 56 overload include xerocytosis and congenital compound heterozygotes have been described.70 dyserythropoietic anaemias.57 Iron overload is also Excessive iron absorption results from lowered found without blood transfusion in mild SA58 and hepcidin secretion, and iron loading in the liver other myelodysplastic syndromes.59 In erythroid results from the absence of transferrin binding. hyperplasia iron absorption is increased as a result of low hepcidin secretion. There may be additional Transfusional Iron Overload marrow signalling pathways which are not fully Patients with β-thalassaemia constitute the understood. Erythropoietin stabilised duodenal majority of those transfusion-dependent from ferroportin,60 growth derived factor 15 (GDF-15),61 an early age. Young sickle-cell anaemia patients and the twisted gastrulation protein homologue 162 with cerebrovascular occlusion are becoming have been associated with low hepcidin secretion, increasingly common. However, the number of but other candidates continue to be sought. adults requiring maintenance transfusion is increasing with the ageing population in whom refractory anaemias are very common. Each 250 mL red cell transfusion delivers variably up to 250

Website Article • HEMATOLOGY • December 2014 EMJ EUROPEAN MEDICAL JOURNAL 4 mg of iron, which would add as much as 13 g of There is, however, a wide margin of safety in iron annually in a transfusion-dependent adult. pathological iron loading. From the optimal Iron from senescent red cells accumulates in the iron status (a normal hemoglobin concentration RE cells, and although these iron deposits are and adequate iron reserves) iron stores may be relatively non-toxic, the iron load will saturate harmlessly increased by several grams, but transferrin from an early stage while RE iron no threshold of toxicity can be defined for an loading continues. Whenever plasma transferrin individual. RE cells have the largest, but a still- iron binding sites are saturated, variable amounts limited capacity, for iron storage. At some stage of labile (chelatable) iron can be detected in additional iron progressively saturates transferrin developing erythroid cells and this increases and loads parenchymal cells indiscriminately, but β-thalassaemia71 propensity for toxic NTBI to appear the skin, heart, liver, pancreas, and other ductless in the plasma. The greatest morbidity from chronic glands are particularly vulnerable. NTBI appears in transfusions is due to cardiac failure and this is the plasma when all transferrin iron binding sites usually apparent after two decades of transfusion- are occupied and are thought to be the direct cause dependency without iron chelation. of iron toxicity. The associated labile plasma iron (LPI) pool, by definition, may have the greater EFFECTS OF IRON OVERLOAD propensity to cause organ damage by the production of free radicals but also may be more In excess, iron is damaging to biological readily chelated when an iron binding site is macromolecules by the very same properties that available. Indeed, a common observation is that make it essential to life. However, the formation of chelators may eliminate the LPI pool but elevate free radicals through its interaction with oxygen the NTBI.72 It has been suggested that iron forms and water is inevitable in the neutral pH and the LPI pool, which may be internalised by oxygen-rich and humid conditions of mammalian DMT1 expressed on hepatocyte parenchymal cells, existence. Intra-cytoplasmic cell membranes and but the metal transporter ZIP 14 is also a nucleic acids are particularly vulnerable, and iron likely candidate.73 toxicity is manifested by irreversible tissue damage and the risk of malignant disease. Iron in the Fe2+ MANAGEMENT OF IRON OVERLOAD state will induce free radical formation by donating electrons to oxygen to generate superoxide or Venesection hydrogen peroxide to give highly reactive (OH.) oxygen species. However, iron in this reduced In patients with excessive iron absorption the most state is essential for participating in reversible effective means of iron depletion is by venesection, oxygen binding to heme. A complex system of which is highly effective, controlled, and predictable binders, chaperones, and reductases is therefore in its effect. Furthermore, there should be no essential to protect the organism while maintaining adverse systemic effects, although with progressive these metabolic processes. The transport protein iron depletion iron absorption will inevitably 74 transferrin and the storage protein ferritin (i.e. that increase in cases of primary iron hyperabsorption. are not required for metabolic purposes), hold the Most HH patients will require many months of iron in the less catalytic Fe3+ form. weekly or twice-weekly venesection. The volume of iron to be removed is difficult to predict and The location of the increasing iron deposits during lifelong treatment will be required. A target serum positive iron balance is determined through the ferritin of 50 µg/L is recommended but depletion route by which the iron is acquired. Increased iron of NTBI and LPI pools are probably more relevant; absorption initially elevates plasma transferrin liver damage will misleadingly elevate the serum saturation and increases iron uptake in any cells ferritin concentration. Cardiac involvement, liver expressing a transferrin receptor. Iron loading in RE cirrhosis, and arthropathy are the complications cells is derived from senescent red cells acquired most refractory to the effects of iron depletion in by blood transfusion or from ingested parenchymal HH. Devoid of these complications patients should cells damaged by iron toxicity, such as is seen in the have a normal life expectancy.75 Many patients are hepatic Küpffer cells. RE dysfunction, however, is nowadays identified prior to the development of not a notable feature of iron overload, although in iron toxicity by family studies or unexpectedly by HH there is an increased susceptibility to infection routine testing intended to detect iron deficiency. by iron-dependent micro-organisms.2 The exception to response by venesection is typical

5 Website Article • HEMATOLOGY • December 2014 EMJ EUROPEAN MEDICAL JOURNAL ferroportin disease where anaemia will result highly effective50 and rather unexpected for an without iron depletion. Iron depletion, however, alloantibody reaction. Treatment of these new- may not be so critical because of the low transferrin borns, however, with immunoglobulin and iron saturation, which would not lead to excessive NTBI, chelators is now no longer required. and therefore, little risk of tissue damage. Modulation of Iron Absorption Control Iron Chelation As a general rule it is more straightforward to The most powerful iron chelators of necessity are achieve a therapeutic benefit by suppression of those naturally occurring. Although iron is readily a biological process than to promote it, and iron released by metabolic processes, the iron in heme, metabolism is no exception. Inhibition of the transferrin, and ferritin is resistant to currently expression of TMPRSS6 would lower matriptase available therapeutic chelating agents. The size of activity and preserve HJV. It has been shown in the chelatable iron pool limits the amount of iron murine hemochromatosis and β-thalassaemia available at any one time. Pharmacological iron models that targeted antisense oligonucleotides chelators are therefore very inefficient. However lower transferrin saturation and liver iron the amount of chelatable iron increases with accumulation.83 In the anaemic β-thalassaemic progressive iron loading but remains only a fraction mice there was a shift towards more effective of the total iron needed to be removed in order to erythropoiesis. For transfused patients this may achieve a beneficial degree of iron depletion. NBTI, suppress the additional burden of increased iron incorporating the LPI pool and any transitional absorption but would not lead to iron depletion to intracellular iron pool of a low molecular weight the required degree. (e.g. iron-citrate), have been proposed as the source of chelated iron.76 Chelation of this pool might Minihepcidins temporarily remove the toxic iron,77 but equilibrium Hepcidin therapy would be the most obvious with the large iron deposits in storage organs is approach to patients with hepcidin defects and has rapidly re-established. been shown to be effective in mice.84 Hepcidin is a The approaches to iron chelation therapy peptide with a complicated structure, and is difficult have recently been extensively reviewed.78,79 to extract or synthesise. Furthermore, it is unlikely to Desferrioxamine has been in use for >60 years but be effective by mouth. Truncated hepcidin analogue suffers the disadvantages of being ineffective by structures administered parenterally or by mouth mouth, having a short plasma half-life requiring have been effective in lowering the serum iron continuous subcutaneous infusion, and ocular and concentration in normal mice and liver iron content 85 oto-toxicities. In an emergency situation, as in cardiac in hepcidin knockout mice. failure due to siderosis, continuous intravenous Transferrin infusion of Desferal may be considered. Newer orally effective iron chelators such as deferiprone Transferrin therapy has been shown86 to clear the and deferasirox, being more lipophilic, may achieve toxic NTBI, to lower the serum iron concentration, better tissue penetration, giving access to a larger and apparently decrease the iron overload in chelatable iron pool. In order to achieve maximum β-thalassaemic mice. There still remains the need effect, however, trials of combined oral and for total body iron depletion in the highly transfused parental chelators are showing some possibilities affected patients. In inherited atransferrinaemia of greater effectiveness, but negate the value of NTBI accumulation and low hepcidin secretion avoiding the use of desferrioxamine. Erythropoiesis are corrected by apotransferrin infusions87 but can be improved in some SA patients by these do not lower the accumulated iron stores, iron depletion with deferasirox.80 It has been which, however, could be depleted by subsequent postulated81 that chelation removes reactive oxygen venesection or chelation. species and shifts iron from the mitochondria to the cytosol to decrease mitochondrial damage CONCLUSION and limit the ineffective erythropoiesis. Chelators have also been used in the treatment of iron The last two decades have seen a vast overload in acaeruloplasminaemia.82 In neonatal improvement in our understanding of human iron hemochromatosis the antenatal administration metabolism. Iron overload continues to present of high-dose immunoglobulin infusions is great clinical challenges, and new therapeutic

Website Article • HEMATOLOGY • December 2014 EMJ EUROPEAN MEDICAL JOURNAL 6 strategies are required to overcome the obstacles hold the key to improving the lives of the large presented by nature. Modulation of iron absorption numbers of patients yet to benefit from the and development of more powerful iron chelators progress made.

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