The iron content of the body and its distribution among the various proteins is summarized in Table 9.1. Most of the iron is present in the oxygen-carrying protein of the red blood cell, haemoglobin, the synthesis and breakdown of which dominates iron turnover. Haem of haemoglobin is synthesized in nucleated red cells in the bone marrow by a pathway ending with the incorporation of iron into protoporphyrin IX by ferrochelatase. Haem breakdown from haemoglobin takes place mainly in phagocytic cells, largely those in the spleen, liver and bone marrow. Iron is released from haem by haem oxygenase and is largely reused for haem synthesis. Every day about 30 mg iron is used to make new haemoglobin and most of this is obtained from the breakdown of old red cells.

Table 9.1 Distribution of iron in the body in a 70 kg man ^a

Protein	Location	Iron content (mg)
Haemoglobin	Red blood cells	3000
Myoglobin	Muscle	400
Cytochromes and iron sulphur proteins	All tissues	50
Transferrin	Plasma and extravascular fluid	5
Ferritin and haemosiderin	Liver, spleen and bone marrow	100–1000

a The numbers are different for women.

Relatively little iron is lost from the body (about 1 mg/d in men) and these losses are not influenced by body iron content or the requirement of the body for iron. The body iron content is maintained by variation in the amount of iron absorbed. In women, menstruation and childbirth increase iron losses to about 1.5 mg/d. Iron absorption may not increase sufficiently to compensate for these iron losses and this may eventually lead to the development of iron deficiency anaemia. In most men and postmenopausal women there is some ‘storage’ iron. This is iron in ferritin or its insoluble derivative haemosiderin, which is available for haem synthesis if necessary. Many young women and children have little or no storage iron.

The iron-binding protein transferrin is responsible for extracellular transport. Each molecule of transferrin can bind up to two molecules of ferric iron. Most cells obtain iron from diferric transferrin which binds to transferrin receptors (TfR) on the cell surface. This is followed by internalization into vesicles, release of iron, transport of iron into the cytoplasm and recycling of the apotransferrin (the protein without iron) into the plasma (Fig. 9.1).

Figure 9.1 Iron exchange within the body. Numbers in boxes refer to amount of iron (mg) in the various compartments and the numbers alongside arrows indicate transfer in mg/d. The iron in parenchymal tissues is largely haem in muscle and ferritin/haemosiderin in hepatic parenchymal cells. The dotted line indicates the small daily loss of iron (0.5 mg) into the gut from red cells; the remaining iron loss is mostly in exfoliated gut cells and bile.

Dietary Iron Absorption

Iron absorption ¹ depends on the amount of iron in the diet, its bioavailability and the body’s need for iron. A normal Western diet provides approximately 15 mg iron daily. Of that iron, digestion within the gut lumen releases about half in a soluble form, from which only about 1 mg (5–10% of dietary iron) is transferred to the portal blood in a healthy adult male.

Dietary and Luminal Factors

Most of the dietary iron is non-haem iron derived from cereals (often fortified with additional iron), with a lesser, but well-absorbed, component of haem iron from meat and fish. With iron deficiency, the maximum iron absorption from a mixed Western diet is no more than 3–4 mg daily. This amount is much less in the more vegetarian, cereal-based diets of many populations.

Non-haem iron is released from protein complexes by acid and proteolytic enzymes in the stomach and small intestine. It is maximally absorbed from the duodenum and less well from the jejunum, probably because the increasingly alkaline environment leads to the formation of insoluble ferric hydroxide complexes. Many luminal factors enhance (e.g. meat and vitamin C) or inhibit (e.g. phytates and tannins) non-haem iron absorption. Therapeutic ferrous iron salts are well absorbed (approximately 10–20%) on an empty stomach, but when taken with meals, absorption is reduced by the same dietary interactions that affect non-haem food iron.

Iron Absorption at the Molecular Level

Several membrane transport proteins, regulatory proteins and associated oxidoreductases involved in iron transport through the intestinal cell have been identified. Non-haem iron is released from food as Fe³⁺ (ferric iron) and reduced to Fe²⁺ (ferrous iron) by a membrane-bound, ferrireductase, duodenal cytochrome b (DCYTB). Iron is then transported across the brush-border membrane by the divalent metal transporter, DMT1. Some iron is incorporated into ferritin and lost when the cells are exfoliated. Iron destined for retention by the body is transported across the serosal membrane by ferroportin-1. Before uptake by transferrin, Fe²⁺ is oxidized to Fe³⁺ by hephaestin (a membrane protein homologous to the plasma copper-containing protein caeruloplasmin) or by plasma caeruloplasmin.

Haemoglobin and myoglobin are digested in the stomach and small intestine. Haem is initially bound by haem receptors at the brush border membrane and the iron is released intracellularly by haem oxygenase before entering the labile iron pool and following a common pathway with iron of non-haem origin.

Regulation of Iron Absorption

Iron absorption may be regulated both at the stage of mucosal uptake and at the stage of transfer to the blood. Recent studies show that in mice HIF-2α, a mediator of cellular adaptation to hypoxia, regulates DMT1 transcription and thus mucosal uptake of iron.² As the epithelial cells develop in the crypts of Lieberkuhn, their iron status reflects that of the plasma (transferrin saturation) and this programmes the cells to absorb iron appropriately as they differentiate along the villus. Transfer to the plasma depends on the requirements of the erythron for iron and the level of iron stores. This regulation is mediated directly by hepcidin, a peptide synthesized in the liver in response to iron and inflammation.³

Cellular Iron Uptake and Release

Diferric transferrin binds with high affinity to the transferrin receptor (TfR1) on the membrane of the cell to form a complex which initiates clathrin-dependent endocytosis of the local membrane. The resulting endosome contains the holotransferrin–transferrin receptor complex.⁴ The pH of the endosome is reduced by a proton pump and a conformational change in holotransferrin is induced causing iron release. Iron is then reduced by a membrane-bound ferrireductase (six transmembrane epithelial antigen of the prostate 3, STEAP3 in erythroid cells⁵) and transported into the cytoplasm by DMT1. This iron is then either stored as ferritin or used within the cell. Iron destined for haem and mitochondrial iron sulphur (Fe-S) clusters is transported into mitochondria by mitoferrin for incorporation into protoporphyrin by ferrochelatase⁶ and for Fe-S cluster assembly on the scaffold protein ISCU.⁷ Export by ABCB7 of an as-yet unknown component to the cytoplasm stimulates Fe-S cluster protein assembly there, essential for cellular iron homeostasis.⁷ Apotransferrin and the transferrin receptor of the endosome return rapidly to the cell surface where they dissociate at neutral pH so that the cycle can start again.^7a

The reticuloendothelial macrophages play a major role in recycling iron resulting from the degradation of haemoglobin from senescent erythrocytes. They engulf red blood cells and release the iron within using haem oxygenase. The iron is rapidly released to plasma transferrin or stored as ferritin. The protein transporting iron to plasma is ferroportin or SLC40A1. Ferroportin activity is regulated in a negative manner by the mainly liver-derived peptide, hepcidin that binds to ferroportin externally, inducing endocytosis and degradation.

Hepcidin synthesis in the hepatocyte is controlled at the transcriptional level through the alteration of levels within the nucleus of different transcription factors able to bind to the gene. Interaction of diferric transferrin, bone morphogenetic proteins (BMPs), interleukin (IL)-6 and other inflammatory cytokines with cell surface receptors TfR1, TfR2, hemojuvelin (HJV) and IL-6 receptor lead to upregulation of the hepcidin gene through different and sometimes interacting signalling pathways. TMPRSS6, a plasma membrane serine protease, inhibits the HJV-BMP-SMAD signaling pathway by cleaving HJV from the surface of the cell, thus preventing overproduction of hepcidin and maintaining iron homeostasis.^8,⁹ A soluble form of HJV (sHJV) produced within the cell by the enzyme furin and released into the plasma antagonizes and also inhibits this pathway.¹⁰

The mechanism of action of the HFE protein is less clear. HFE binds to both TfR1 and TfR2, decreasing the affinity of each for transferrin. Stabilization and endocytosis of TfR2 stimulates hepcidin production and there is evidence that diferric transferrin displaces the protein HFE from TfR1, leaving it free to interact with TfR2, thus stimulating hepcidin production in response to plasma iron levels.¹¹^–¹³

Increased erythropoiesis causes decreased hepcidin, increased iron absorption and increased iron availability for haemoglobin production. So far, two plasma antagonists of the BMP-HJV-SMAD pathway, GDF15 and TWSG1, produced by erythroid cells and associated with expanded ineffective erythropoiesis, have been found.^14,¹⁵ Alteration of the function of key regulators leads to body iron overload or iron deficiency (see later). The extent and the manner in which these regulatory pathways vary between different tissues await further investigation.

Iron Storage

All cells require iron for the synthesis of proteins but have the ability also to store excess iron. There are two forms of storage iron: a soluble form, known as ferritin and insoluble haemosiderin.¹⁶ Ferritin consists of a spherical protein (molecular mass 480 000) enclosing a core of ferric-hydroxy-phosphate, which may contain up to 4000 atoms of iron. Haemosiderin is a denatured form of ferritin in which the protein shells have partly degraded, allowing the iron cores to aggregate. Haemosiderin deposits are readily visualized with the aid of the light microscope as areas of Prussian-blue positivity after staining of tissue sections with potassium ferrocyanide in acid (see Chapter 4). Ferritin is found in all cells and in the highest concentration in liver, spleen and bone marrow.

Regulation of Iron Metabolism

The expression of a number of iron proteins involved in both transport and storage is largely controlled posttranscriptionally by iron regulatory proteins (IRP1 and 2).⁴ The conformation of IRP1 required for binding to mRNA iron-responsive elements (IREs) and the turnover of IRP2 are directly affected by the amount of iron within a cell. Depending on where the target IRE elements are found these proteins may inhibit or enhance translation of many proteins involved in iron metabolism.

IREs are regions of mRNA that form hairpin-like stem-loop structures. These consist of a base-paired stem of variable length interrupted by an asymmetrical region including a 5′ unpaired cytosine, followed by an upper stem of five paired bases and a six-membered loop. When the labile iron pool is deficient of iron, IRP1 has an available binding site for IRE. When the labile iron pool is saturated with iron, the iron binds to IRP1 to produce a 4Fe-4S cluster which blocks the IRE binding site and prevents IRP1 binding to the IRE. Fe-saturated IRP1 is then able to function as the enzyme aconitase. In the presence of iron, IRP2 (which is not an Fe-S protein) is degraded. The turnover of IRP1 is also affected by iron levels.

Different iron proteins are regulated by IRP in different ways, depending on where the IRE is located. If the IRE is at the 3′ UTR of the mRNA, IRP binding will stabilize translation by protecting the translation product from endonucleolytic cleavage (e.g. TfR1 and DMT1). If the IRE is at the 5′ UTR of the mRNA, IRP binding will inhibit the translation of mRNA. Both L and H ferritin subunits have 5′ UTR IRE. When iron is abundant, the IRP does not bind to the 5′ IRE so ferritin expression is not inhibited and excess iron can be stored adequately. When iron is scarce, the IRP binds to the IRE and inhibits ferritin synthesis.

Plasma Iron Transport

Almost all the iron in plasma is tightly bound to transferrin, which is usually less than 50% saturated with iron. Transferrin, when incompletely saturated with iron, exists in four forms: apo, monoferric (iron bound at the ‘A’ site), monoferric (B) and diferric. The distribution may be determined by urea-polyacrylamide electrophoresis.¹⁷ Delivery to cells requires specific binding to transferrin receptors. The plasma iron pool (transferrin-bound iron) is about 3 mg, although the daily turnover is more than 10 times his amount. In addition, smaller amounts of iron are carried in the plasma by other proteins.

Haptoglobin is a serum glycoprotein that avidly binds haemoglobin αβ dimers released into the bloodstream by haemolysis. The haemoglobin–haptoglobin complex is rapidly removed from the plasma by a specific receptor, CD163, highly expressed on tissue macrophages.¹⁸

Hemopexin¹⁹ is a plasma glycoprotein of molecular mass approximately 60 kDa that binds haem and transports the haem to cells by a process that involves receptor-mediated endocytosis and recycling of the intact protein.

Low concentrations of ferritin are found in the plasma and ferritin concentrations in healthy subjects reflect body iron stores. Much of this ferritin appears to be glycosylated and has a relatively low iron content.²⁰ Such ferritin has a half life (T_½) of approximately 30 h. Ferritin is also released into the circulation as a result of tissue damage (most strikingly after necrosis of the liver). Tissue ferritin is cleared rapidly from the circulation (T_½ in approximately 10 min) by the liver.

Non-transferrin-bound iron describes a form of iron that is not bound to transferrin, is of low molecular mass and can be bound by specific iron chelators.²¹ Several assays have been described that have demonstrated such a fraction in plasma from patients with iron overload. The chemical form of this iron is unknown, but it is probably rapidly removed from the circulation by the liver. Recent studies implicate the involvement of the zinc transporter ZIP14 in this process.²²

Iron status

Normal iron status implies a level of erythropoiesis that is not limited by the supply of iron and the presence of a small reserve of ‘storage iron’ to cope with normal physiological needs. The ability to survive the acute loss of blood (iron) that may result from injury is also an advantage. The limits of normality are difficult to define and some argue that physiological normality is the presence of only a minimal amount of storage iron ²³ but the extremes (i.e. iron deficiency anaemia and haemochromatosis) are well understood.^23a

Apart from too little or too much iron in the body, there is also the possibility of a maldistribution (Fig. 9.2). An example is anaemia associated with inflammation or infection where there is a partial failure of erythropoiesis and of iron release from the phagocytic cells in liver, spleen and bone marrow, which results in accumulation of iron as ferritin and haemosiderin in these cells. Determination of iron status requires an estimate of the amount of haemoglobin iron (usually by measuring the haemoglobin concentration (Hb) in the blood; see Chapter 3) and the level of storage iron (measuring serum ferritin concentration). Iron deficiency should be suspected in hypochromic, microcytic anaemia, but in the early stages of iron deficiency red cells may be normocytic and normochromic. Another feature of iron deficiency is an increased concentration of protoporphyrin in the red cells; normally, there is a small amount present, but defective haem synthesis caused by lack of iron results in the accumulation of zinc protoporphyrin and occasionally free protoporphyrin may be increased as well.²⁴

Figure 9.2 Body iron content and distribution in different conditions. Additional assays are sometimes required. In genetic haemochromatosis, early iron accumulation is indicated by increased transferrin saturation alone. The serum ferritin concentration increases only later as the level of stored iron increases. In the anaemia of chronic disease, patients often have normal serum ferritin concentrations even when storage iron in the bone marrow is absent (see p. 336). In this situation, the assay of serum transferrin receptor may help detect tissue iron deficiency.

Disorders of iron metabolism

Clinical aspects of iron metabolism have been reviewed ²⁵^–²⁷ and are summarized in Table 9.2. A guideline on the management of genetic haemochromatosis is available.²⁸

Table 9.2 Disorders of iron metabolism

Iron deficiency

Deficient iron intake

Diet of low bioavailability

Increased physiological requirements due to rapid growth in early childhood and in adolescence

Blood loss: physiological (e.g. menstruation) or pathological (e.g. gastrointestinal)

Malabsorption of iron intake

Reduced gastric acid secretion (e.g. after partial gastrectomy)

Reduced duodenal absorptive area (e.g. in coeliac disease)

Rare, inherited iron-refractory iron deficiency anaemia

TMPRSS6 deficiency

Redistribution of iron

Macrophage iron accumulation

Inflammatory, infectious or malignant diseases (‘anaemia of chronic disease’)

Iron overload

Due to increased iron absorption

e.g. Hereditary haemochromatosis – commonly homozygosity for HFE C282Y but sometimes involving other genes (HAMP,HJV,TFR2,SLC40A1)
Substantial ineffective erythropoiesis (e.g. β thalassaemia syndromes of intermediate and major severity, some types of sideroblastic anaemia, congenital dyserythropoietic anaemia)
Sub-Saharan iron overload (‘Bantu siderosis’) – only in combination with increased dietary iron
Other rare inherited disorders (e.g. congenital atransferrinaemia, DMT1 deficiency)
Inappropriate iron therapy (rare)

Due to multiple blood transfusions in refractory anaemias

e.g. Thalassaemia major

Aplastic anaemia

Myelodysplastic syndromes

Methods for assessing iron status

The methods used to assess iron status are summarized in Table 9.3. Some are not generally applicable but have value in the standardization of indirect methods. The determinations of Hb and red cell indices are described in Chapter 3.

Table 9.3 Assessment of body iron status and confounding factors

Serum Ferritin Assay

With the recognition that the small quantity of ferritin in human serum (15–300 μg/l in healthy men) reflects body iron stores, measurement of serum ferritin has been widely adopted as a test for iron deficiency and iron overload. The first reliable method to be introduced was an immunoradiometric assay in which excess radiolabelled antibody was reacted with ferritin and antibody not bound to ferritin was removed with an immunoadsorbent.³¹ This assay was supplanted by the two-site immunoradiometric assay, which is sensitive and convenient. Since then the principle of this assay has been extended to non-radioactive labelling, including enzymes (enzyme-linked immunosorbent assay or ELISA). Most current laboratory immunoassay systems for clinical laboratories include ferritin in the assay repertoire. Factors to be considered when selecting an immunoassay system are discussed later. The method described in the next section is an ELISA. The most sophisticated equipment required is a microtitre plate reader.

Immunoassay for Ferritin

Preparation and Storage of Ferritin

Ferritin may be prepared from iron-loaded human liver or spleen obtained at operation (spleen) or postmortem. The informed consent of the patient or the patient’s relatives should be obtained for use in preparation of ferritin. Tissue should be obtained as soon as possible after death and may be stored at −20°C for 1 year. The usual precautions must be taken to avoid the risk of infection when handling tissues and extracts. Ferritin is purified by methods that exploit its stability at 75°C. Further purification is obtained by precipitation from cadmium sulphate solution and gel-filtration chromatography.³² Purity should be assessed by polyacrylamide gel electrophoresis,³³ and the protein content determined by the method of Lowry and colleagues, as described by Worwood.³³ Buffered solutions of human ferritin may be stored at 4°C, at concentrations of 1–4 mg protein/ml, in the presence of sodium azide as a preservative, for up to 3 years. Such solutions should not be frozen. Ferritin, from human liver or spleen, may be obtained from several suppliers of laboratory reagents. This should be used as an assay standard only after calibration against the international standard (see p. 183).

Antibodies to Human Ferritin

High-affinity antibodies to human liver or spleen ferritin are suitable. Polyclonal antibodies may be raised in rabbits or sheep by conventional methods,³⁴ and the titre checked by precipitation with human ferritin.³³ An immunoglobulin G (IgG)-enriched fraction of antiserum is required for labelling with enzyme in the assay. The simplest method is to precipitate IgG with ammonium sulphate.³⁵ Monoclonal antibodies that are specific for ‘L’ subunit-rich ferritin (liver or spleen ferritin) are also suitable. A mouse monoclonal antibody and a horseradish peroxidase (HRP)-labelled antibody are available from NIBSC (www.nibsc.ac.uk; codes 87/654 and 87/662). Suitable antibodies are available from commercial suppliers.