07

Iron metabolism

07

Iron metabolism

07

Iron metabolism

07

Iron metabolism

7.1 Iron metabolism and disorders

Lothar Thomas

7.1.1 Iron, an essential element

Iron is the second most common metal in the earth’s crust and is present most often as either the insoluble Fe3+ hydroxide or Fe3+ oxide. Iron is a period 4 transition element that can exist in oxidation states ranging from –2 to +6. In biological systems, these oxidation states are limited primarily to the ferrous (+2), ferric (+3), and ferryl (+4) states. The inter conversion of iron oxidation states is not only a mechanism whereby iron participates in electron transfer, but also a mechanism whereby iron can reversibly bind ligands. This is possible by virtue of its vacant δ-orbitals /1/.

Fe2+ and Fe3+ are the commonly used abbreviations for the hydrated ions [Fe(H2O6)]2+ and [Fe(H2O6)]3+. The binding with iron leads to increased acidity of water. This effect increases with the charge of the iron ion: the pKa of water bound to Fe3+ is 2, while that of water bound to Fe2+ is 7. Under physiological conditions, Fe3+ is insoluble due to the formation of polynuclear hydroxo-bridged complexes. Fe2+ is soluble, but can react with H2O2, generating the extremely toxic hydroxyl radical (Fenton reaction):

Fe2+ + H2O2 → Fe3+ + OH· + OH–
Fe3+ + HA– → Fe2+ + A· + H+

Both super oxide anion and hydroxyl radicals are capable of oxidizing biologic macromolecules and, by damaging DNA, may cause heritable defects. The preferred biological ligands for iron are oxygen, nitrogen, and sulfur atoms /1/. Free iron is toxic, Fe3+ more than Fe2+. Therefore, body iron always circulates bound to ligands. In the cells, iron is present in its divalent form, outside the cells in its trivalent form.

Iron is involved in a number of important biological reactions, such as oxygen and electron transport, and is the substrate for oxidation and reduction reactions.

Four major classes of iron-containing proteins carry out biochemical reactions /1/:

  • Hemoproteins such as hemoglobin and myoglobin. The key functions of iron in which oxygen is bound to porphyrin ring and iron-containing molecules, either as part of the prosthetic group of hemoglobin within erythrocytes or as a facilitator of oxygen diffusion in myoglobin in the muscle cell are the transport of oxygen from the environment to the terminal oxidases in the mitochondria. In iron deficiency, the erythrocyte hemoglobin content and the myoglobin content of the muscles can be reduced by 40–60% /2/.
  • Heme-containing enzymes, such as cytochromes. Here too, iron is bound to the porphyrin ring. Cytochromes are present in the mitochondrial electron transport chain coupled with other enzymes. During electron transfer, the iron in the heme group (the active center of the cytochromes) undergoes a valence change (Fe2+ to Fe3+ and vice versa).
  • Iron-sulfur proteins. The iron sulfur enzymes act as electron carriers via the action of iron bound to either 2 or 4 sulfur atoms and cysteine side chains. The enzymes function in association with the coenzymes flavin adenine dinucleotide (FAD), the flavoprotein succinate dehydrogenase (EC 1.3.99.1) and lipoamide dehydrogenase (EC 1.6.4.3). A second group of iron-sulfur proteins are enzymes that contain iron-sulfur complexes as prosthetic group. All of them share the involvement of four cysteine residues of the relevant protein. Enzymes of this group include NADH reductase, succinate dehydrogenase, QH2-cytochrome c reductase, and the iron regulatory protein 1 (IRP-1); refer to Section 7.1.4 – Iron deficiency. Animal tests have shown that, in iron deficiency, the activity of these enzymes is reduced to 30–60% of original activity /2/.
  • Non-enzymatic proteins such as transferrin, ferritin and hemosiderin. They are involved in iron transport as well as iron storage in the cells.

The properties of electron gain and loss that make iron so useful in biochemical reactions also render iron potentially harmful. Iron can react with oxygen to generate the toxic super oxide anion.

Fe2+ + O2 → Fe3+ + O2–

The main source of H2O2 is the respiratory chain, since up to 5% of the oxygen used in mitochondrial respiration is converted to H2O2 /3/. Hydroxyl radicals and the super oxide anion oxidize macromolecules and destroy organic structures.

7.1.2 Critical determinants of iron homeostasis

Critical determinants of serum iron levels are intestinal iron absorption, iron recycling, iron storage, the hepcidin mediated control of ferroportin cellular iron efflux, the cellular iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network, transferrin saturation, and erythropoietic activity.

7.1.2.1 Iron absorption and transport

Increasing the solubility of iron

All organisms that require iron have to overcome the problems of insolubility and toxicity of this metal. In inorganic nature, iron exists as insoluble Fe3+ hydroxide or Fe3+ oxide. The solubility of Fe3+ increases dramatically when the pH is reduced, rising from 10–18 mol/L at pH 7.0 to 10–3 mol/L at pH 2.0 /3/.

Food iron occurs largely as either ferric iron or heme iron. The body has developed the following strategies to promote intestinal uptake of iron /3/:

  • Acidification is the first step making iron biologically available. The gastric lumen can reach a pH of 1. Fe3+ in the stomach binds to organic molecules, generically termed siderophores. Fe3+ chelated as Fe3+-siderophore complex keeps in solution when it enters the less acidic duodenum where iron is absorbed. Siderophores share the common feature of high avidity for Fe3+. and bind Fe2+ at lower affinity... Prolonged achlorhydria can produce iron deficiency because chelation will not occur unless iron is solubilized.
  • The second approach to increase iron bio available is to reduce Fe3+ to the more soluble Fe2+. This is mediated by ferrireductases, which reside on the enterocyte plasma membrane upstream of the iron transport systems. Some proportion of Fe3+ dietary iron is reduced by dietary constituents and intestinal secretions to Fe2+, which is soluble at neutral pH. Ascorbic acid is the best-known reducing agent in the diet /5/.

7.1.2.2 Intestinal iron absorption

Only 1–3 mg of dietary iron is provided daily by the intestinal absorption of dietary iron. Two main forms of iron are taken up by the intestinal mucosa /4, 5, 6/:

  • Non-heme iron exists predominantly in the form of Fe(III) salts and is not bioavailable. Fe3+ is reduced to Fe2+ at the apical cell membrane of the enterocyte by duodenal cytochrome b (DCYTB) reductase, an ascorbic acid dependent enzyme (Fig. 7.1-1 – Iron transport in the small intestine by the enterocytes). Fe2+ is subsequently transported into the cell by divalent metal transporter 1 (DMT 1). Fe2+ transport systems are not specific for iron. DMT1 is a H+ ion divalent cation symporter capable of transporting transition metals such as Fe2+, Zn2+, Cd2+, Mn2+, and Co2+ /7/.The interaction of Fe2+ and Fe3+ with dietary components such as poly phenols (black tea), phytates, oxalic acid, tannins, carbonates and phosphates reduces the bio availability of iron. This applies in particular for the oral ingestion of ferrous sulfate.
  • In diets rich in meat heme accounts for two thirds of the dietary iron supply of the body. Heme is soluble at alkaline pH and precipitates at acidic pH. In the intestinal lumen heme is released from hemoglobin and myoglobin by pancreatic enzymes and enters the intestinal absorptive cell as an intact metalloprotein. Heme is trans located intact across the brush border membrane of the duodenal enterocyte. Within the enterocyte heme is degraded by heme oxygenase (EC 1.14.99.3) the porphyrin ring is split, and the iron released to feed the intracellular iron pool /5/.

In the intracellular iron pool of the enterocyte, iron is bound to proteins and low-molecular substances to eliminate its toxic effects. Part of the iron is exported into the blood stream via the basolaterally expressed efflux chanel ferroportin (FPN1) in conjunction with the ferrooxidase hephaestin. The remainder is stored as ferritin in the cytoplasm and may be lost when the senescent enterocytes are sloughed off and excreted in the feces.

Iron export from cells across the basolateral membrane of the enterocyte is mediated by the iron exporter ferroportin. The exporter is highly tissue specific and expressed only in those tissues clearly involved in exporting iron into plasma /3/. Once Fe2+ transverses the basolateral membrane of the enterocyte it is oxidized by the ferroxidase hephestin to Fe3+ and binds to transferrin (Tf) in the plasma. Tf transports Fe3+ to all cells equipped with surface transferrin receptors.

Intestinal iron absorption can be modulated by recent dietary iron intake, by the size of body iron stores, and by erythropoietic proliferation.

7.1.2.3 Iron uptake of the tissues

The distribution of iron to the tissues occurs through a process known as transferrin cycle /5, 6/:

  • Intestinally absorbed iron which circulates in the blood binds with high affinity to apotransferrin (apo-Tf) in the plasma. Apo-Tf is a specific Fe3+-binding protein that is abundantly available compared to iron. Apo-Tf is responsible for solubilizing Fe3+, neutralizing its reactivity and supplying the tissues, in particular erythropoiesis, with iron. Each apo-Tf molecule can bind two Fe3+ atoms. The apo-Tf content in plasma can bind 12 mg of iron. Normally, only 30% of the iron binding capacity is saturated (i.e., only 4 mg of iron is bound to transferrin). This means that 75% of the iron transport protein is present as apo-Tf, and 25% as holo-Tf.
  • In the tissues, the specific receptor for iron-Tf (holo-Tf), the transferrin receptor (TfR), is expressed on the plasma membrane of the cells, but most highly on those requiring large amounts of iron (Fig. 7.1-2 – Cellular iron uptake from transferrin). The Tf-TfR complex is internalized into the cells by endocytosis. After iron has been released, the TfR-apo-Tf complex is recycled to the cell surface and the TfR is released into the circulation as soluble transferrin receptor (sTfR) /6/. Erythroid precursor cells have the highest number of TfR and two-thirds of the body’s total receptors. The extent of released sTfR is an indicator of cellular iron status and erythroid proliferation. Iron deficiency and erythropoietic hyper proliferation increase the body’s total TfR content and the serum level of sTfR.
  • The Fe3+ released in the cells by acidification of the endosome forms the labile iron pool, from which iron is distributed to the functional sites of the cell or stored as ferritin.

The saturation of transferrin with iron is an important determinant and laboratory diagnostic indicator of systemic iron homeostasis.

7.1.2.4 Plasma iron turnover (iron recycling)

The daily transport of iron in the plasma required to maintain iron balance is referred to as iron turnover. The major source of serum iron comes from the recovery of iron from destruction of senescent red cells (iron recycling) and does not result from intestinal absorption. The total hemoglobin of a person with a weight of 70 kg and a blood volume of 5 L contains 2.5 g of iron. When iron stores are replete, approximate 1 mg of iron per day is absorbed intestinally, thus compensating for the equivalent loss of iron through apoptotic cells of the gut. Approximately 80% of the body’s functional iron is contained in erythrocytes (Fig. 7.1-3 – Iron distribution within the organism). In an individual with a normal iron status 25–35 mg of the total body iron are recycled daily. Most of the iron is needed for the production of hemoglobin. Erythrocytes have a functional life span of 120 days, and every day 0.7% of erythrocytes are removed from the circulation and must be replaced. Every day 20–30 mg (0.8%) of the recycled iron is used for erythrocyte regeneration and another 5 mg are required by non-erythroid body cells /1, 5/.

7.1.2.5 Iron storage

The iron content in the body is 30–40 mg/kg body weight and varies as a function of age, gender and the specific tissues. Approximal 75% of non-storage iron is present in the erythroid mass (Fig. 7.1-3 – Iron distribution within the organism and quantitative exchange). The storage iron content in the body varies from 15 to below 1 mg/kg body weight and 0.5 to 1 g of iron is stored in healthy individuals.

Storage iron is distributed as follows /1/:

  • In macrophages of the reticuloendothelial system (RES) and in hepatocytes. The liver accounts for 60% of the storage capacity and 8% of the plasma iron turnover. Hepatocellular iron is stored in ferritin (80%) and hemosiderin (15%). Iron accumulates mostly in the periportal regions with a decreasing gradient toward the centrilobular areas. Iron stored in ferritin can eventually be mobilized. Approximately 5% of liver iron accumulates in Kupffer cells as hemosiderin. During iron overload, liver damage ensues only when hepatic iron content is about more than tenfold elevated.
  • Approximately 40% of stored iron are found as ferritin in muscle tissues and cells of the RES, which can be overloaded with a maximum of 5 g of iron.

Total body iron content

The total body iron content is maintained by intestinal absorption of dietary iron. Newborn infants have a total body iron content of 250 mg /5/. During growth spurts, iron absorption must exceed the daily iron loss of 1 mg to maintain a serum iron level of about 60 μg/dL (10.7 μmol/L).

7.1.2.6 Physiological iron loss

Body iron losses vary with sex and age and are significant when there is blood loss /1/. Since hemoglobin contains 3.46 mg of iron per gram of hemoglobin, each mL of blood loss (Hb 150 g/L) results in depletion of 0.5 mg of iron /5/. Men lose about 0.9 mg of iron per day through gastrointestinal (0.6 mg) and renal elimination (up to 0.3 mg). In women, iron losses are higher during reproductive years. Menstrual iron loss, estimated from an average blood loss of 33 mL per cycle, equals 1.5 mg/day, but may be as high as 2.1 mg/day. Oral contraceptives reduce this loss while intrauterine devices increase it /1/.

7.1.3 Regulation of iron metabolism

Mechanisms responsible for maintaining iron homeostasis at a systemic and cellular level require adequate iron supply and the prevention of accumulation of toxic iron.

Systemic iron homeostasis depends on the peptide hormone hepcidin, a soluble plasma iron regulator, which is expressed in the liver. Hepcidin expression is decreased in iron deficiency and increased in iron overload and inflammation. Hepcidin regulates the amount of intestinal iron absorption and the liberation of iron from cells of the reticuloendothelial system by degradation of the iron exporter ferroportin.

Cellular iron homeostasis is maintained by the RNA-binding cytoplasmic iron regulatory proteins IRP 1 and IRP 2, which regulate the expression of proteins such as ferritin, mitochondrial aconitase, 5-aminolevulinic acid synthetase and hypoxia-inducible factor 2α (HIF-2α) at the post-transcriptional level.

7.1.3.1 Systemic regulatory mechanisms

To maintain normal iron levels in the plasma, and to supply the tissues with iron recycled from erythrocytes or, in smaller amounts, with iron absorbed by the intestines, systemic regulatory mechanisms are required. Unlike other trace elements for which homeostasis is regulated by elimination, iron homeostasis is regulated by the interaction of intestinal absorption, iron release/uptake by macrophages and hepatocytes, iron uptake by the hematopoiesis, and the hepcidin-mediated control of ferroportin cellular iron efflux.

Intestinal absorption as a regulatory mechanism

The duodenum is an important sensor and regulator of iron absorption, since it also regulates the iron transport proteins. The regulation occurs:

  • Through HIF-2α. This is the main iron-inducible transcription factor of the enterocyte iron transporters. The reduction of intracellular iron, O2 or ascorbic acid decreases the activity of cytoplasmic prolyl hydrolases (PH), which act as sensors. As a result of the reduced PH activity, the DCYTB and DMT1 genes are up regulated, resulting in increased expression of DCTB, DMT1 and ferroportin on the enterocyte membrane, and increased apical iron absorption and basolateral iron transport into the blood. HIF-2α has been reported to have a greater effect on apical uptake than basolateral iron uptake. Hypoxia induces an increase in iron absorption 6–8 h after its onset. This is followed by changes in the increase of plasma iron, activation of erythropoiesis, and an increase in hepcidin /8/.
  • By the enterocyte iron content (labile iron pool). This regulates (i) iron absorption through its effects on the cellular iron-responsive element/iron regulatory protein (IRE/IRP) regulatory network and (ii) the expression of DMT 1 /9/.

The macrophage as a regulatory mechanism

Macrophages of the reticuloendothelial system play an important role in the regulation of iron balance /4/. Senescent and damaged erythrocytes are sequestered by the macrophages of the reticuloendothelial system of the liver and spleen and removed from the circulation. In the macrophages senescent erythrocytes are degraded and the hemoglobin is broken down into heme and the globulin components. The degration is induced by oxidative cleavage of the porphyrin ring, catalyzed by NADP+-dependent heme oxygenase (EC 1.14.99.3). Heme oxygenase is regulated by a number of factors, in particular inflammatory cytokines. Iron is stored in the macrophages in the form of ferritin and hemosiderin. The iron exporter ferroportin mediates the efflux of iron from the macrophages and its transfer to apotransferrin in the circulation (Fig. 7.1-4 – Regulation of iron content of the intracellular pool)

In chronic inflammatory conditions, iron efflux is reduced and the macrophages are overloaded with iron. In HFE hemochromatosis, the macrophages are relatively iron-depleted in relation to total body iron.

The hepatocyte as a regulatory element

Hepatocytes are equipped with a complex array of molecules that affect iron metabolism. It expresses both IRP1 and IRP2, transferrin, hepcidin and two transferrin receptors (TfR1 and TfR2), which can bind diferric transferrin. TfR1 is important for iron uptake of the hepatocyte. TfR2 functions as a sensor of the saturation of TFR1 with iron and stimulates hepcidin expression. Refer to Fig. 7.6-4 – Signals and pathways for the regulation of hepcidin expression.

The hepatocyte can, however, also absorb iron not bound to transferrin (Tf) when the transport capacity of Tf is exhausted /1/. If serum ferritin levels are > 800 μg/L the liver is the most important storage site.

The erythropoiesis as a regulatory mechanism

Erythroid precursor cells are the main consumers of iron. On their cell membrane they express TfR1, which enables the cell to take up Tf-bound iron (see Section 7.4 – Soluble transferrin receptor). This occurs as shown in Fig. 7.1-2 – Cellular iron uptake from transferrin. The iron content of the labile pool determines the influx of iron via the IRE-IRP system which regulates the stability of mRNA for TfR1 and 5-aminolevulinic acid synthase, the first enzyme of heme synthesis (Fig. 7.1-5 – First step of heme synthesis). This regulation ensures that the amount of toxic protoporphyrin IX is aligned with the availability of iron /4/.

Hepcidin as a regulator of iron metabolism

Hepcidin is the body’s systemic iron regulator (see also Section 7.6 – Hepcidin) /10/.

Produced in the liver, hepcidin controls the entry of iron into the plasma mediated by the iron exporter ferroportin:

  • For dietary iron from duodenal enterocytes
  • For recycled iron from senescent erythrocytes and other cells degraded by macrophages
  • For iron stored in hepatocytes
  • For iron released from the placenta into the fetal circulation during pregnancy.

The concentration of hepcidin is feedback-regulated by the transferrin saturation (TfS) and erythropoietic demand for iron. Iron deficiency, increased erythropoietic activity and hypoxia suppress the expression of hepcidin. Low levels of hepcidin facilitate increased intestinal iron absorption and increased synthesis of Hb. Hepcidin is significantly elevated in inflammation and in secondary iron overload. Hereditary hemochromatoses with malfunction of the hepcidin-ferroportin axis are associated with varying degrees of reduction of hepcidin.

TfS reflects the difference between the hepcidin-ferroportin regulated transfer of iron into the plasma and the iron demand for erythropoiesis. Hepcidin levels are positively correlated with transferrin saturation and ferritin levels.

7.1.3.2 Regulation of cellular iron metabolism

Each somatic cell regulates its own iron balance, and possesses a regulatory system for the coordination of iron uptake, use and storage /11/. On a post-transcriptional level cellular iron metabolism is coordinately controlled by the binding of iron regulatory proteins (IRP1 or IRP2) to cis-regulatory mRNA motifs termed iron regulatory elements (IREs). The IRE/IRP interactions regulate the expression of the mRNAs encoding proteins (Fig. 7.1-6 – Post-transcriptional regulation of cellular iron homeostasis).

  • For cellular iron acquisition (transferrin receptor 1; TFR1) and divalent metal transporter 1 (DMT1)
  • For storage of iron (ferritin H and L chains )
  • For utilization of iron (erythroid 5’aminolevulinic synthase, the central enzyme in the synthesis of hemoglobin, see Fig. 7.1-5 – First step of heme synthesis)
  • For mitochondrial aconitase
  • For hypoxia inducible factor.

The IRPs are iron sensor proteins located in the cytoplasma of the cells. Distinct mechanisms control IRP1 and IRP2 activity in response to the cellular labile iron pool. Under iron-replete conditions, a cubic 4Fe-4S cluster assembles in IRP1, preventing IRE binding. This assembly converts IRP1 to a cytosolic aconitase that inter converts citrate to isocitrate.

IRE/IRP complexes formed within the 5’UTR of an mRNA inhibit translation (reduced formation of ferritin and erythroid 5’aminolevulinic synthase; ε-ALAS ), whereas IRP binding to IREs in the 3´UTR of TFR1 mRNA prevents its degradation (TfR1 is active)

In patients with low labile iron pool IRE/IRP complexes

  • Formed within the 5’UTR of ferritin mRNA and ε-ALAS mRNA, inhibits the synthesis of ferritin and ε-ALAS
  • Formed within the 3’UTR of the TfR mRNA prevents degradation by RNAse and TfR1 synthesis is increased.

7.1.3.3 Iron homeostasis and immune system

Activation of the immune system in response to inflammation, infection, autoimmune activity, chronic kidney disease, inflammatory bowel disease, and malignant tumors leads to a change in the body’s systemic iron distribution and intracellular iron metabolism which is caused by inflammatory cytokines and the radicals produced under their influence, such as nitrogen monoxide (NO) and O2– /12/, and by the increased expression of hepcidin. The iron is diverted from the circulation and stored in the hepatocytes and cells of the reticuloendothelial system. This results in functional iron deficiency, characterized by impaired iron release from body stores that is unable to meet the demand for erythropoiesis (also called reticuloendothelial cell iron blockade). The plasma iron level is low, the ferritin concentration normal or usually even high.

The following mechanisms are involved in the defect of iron recycling /13/:

  • Activation of macrophages. In the macrophages, inducible NO synthase (iNOS) is activated and NO production is triggered through stimulation by inflammatory cytokines. NO activates IRP-1 and increases iron absorption and storage in the macrophages by increased TfR1 expression and synthesis of ferritin.
  • Prevention of iron release from hepatocytes and macrophages. Stimulated by interleukin-6, hepcidin inhibits the iron exporter ferroportin.
  • Reduced synthesis of hemoglobin by inhibition of ε-ALAS by NO. At the same time, however, the inflammation-induced increase in hepcidin inhibits iron export from the erythroid precursor cells, resulting in the erythrocytes normal hemoglobin synthesis (normochromic erythropoiesis in anemia of chronic disease).

7.1.4 Iron deficiency

Prevalence of iron deficiency

Iron deficiency, in its various forms, is the most common nutritional disorder in the world. Of a world population of around 6 billion, 2 billion people have iron deficiency and 750 million have iron deficiency anemia. It is estimated that for each case of iron deficiency anemia there are 2.5 cases with reduced iron status /63/.

The following groups are at increased risk for iron deficiency and iron deficiency anemia:

  • Infants, especially in the developing countries, with a prevalence of about 63%
  • Young women of reproductive age. They have increased iron demand during puberty and lose approximately 16 mg of iron with each menstrual cycle
  • Pregnant women, whose iron demand increases from 0.8 mg/day in early pregnancy to 7.5 mg/day in late pregnancy.

In Europe and North America severe forms of iron deficiency are less common than in the developing countries. For industrialized countries, it is estimated that 10% of women of child-bearing age, 10% of children, 1% of men, 30% of elderly individuals and 30% of pregnant women have iron deficiency /15/. Nutritional iron deficiency is the second most common cause of iron deficiency in the world, after blood loss. In the developing countries, one of the main causes of blood loss is parasitosis. In the industrialized nations, the main causes are increased menstrual bleeding in women aged < 50 years, and blood loss due to malignant tumors in men and women aged ≥ 50 years. Nutritional iron deficiency is frequently also caused by an unbalanced diet. Refer also to

7.1.4.1 Iron deficiency states

The different states associated with iron restriction are shown in Tab. 7.1-2 – States of iron restriction. The evaluation of the iron status using ferritin index and hemoglobin content of reticulocytes (CHr, RetHe) as markers, is presented in Fig. 7.1-9 – Evaluation of iron status.

Males have iron reserves of approximately 1000 mg while females have reserves of about 300–500 mg. The gold standard for estimating storage iron is the histological iron determination in the bone marrow aspirate. Stained blue with hexacyanoferrate (III) solution the non-heme iron that is stored in the histiocytes presents as hemosiderin granules. Iron stored in the form of hemosiderin is not readily mobilizable.

7.1.4.1.1 Subclinical iron deficiency

In this condition, also known as mild iron deficiency or iron deficiency without anemia, there is a reduction of storage iron reserves, which manifests in decreased ferritin concentrations. In this state, there usually already is a compensatory borderline increase in sTfR and zinc protoporphyrin concentration and in intestinal iron absorption. Clinically, there are no pathological findings. Groups at risk for this condition include individuals in growth spurts such as children and adolescents, athletes, blood donors, vegetarians, and women of reproductive age.

According to the European Commission Directive, suitable blood donors are men with Hb > 135 g/L and women with Hb > 125 g/L.

In the RISE study /16/, in which the lower threshold was defined as 125 g/L for both sexes, the prevalence rates of subclinical iron deficiency in blood donors were:

  • Approximately 6.9% in male and 9.8% in female donors if a zinc protoporphyrin value > 100 μmol/mol hemoglobin was the threshold
  • Approximately 4.8% in male donors and 9.8% in female donors, if transferrin saturation < 16% was the threshold
  • Approximately 27.4% in male donors and 24.7% in female donors, if a hepcidin value < 0.25 nmol/L was the threshold.
7.1.4.1.2 Iron deficiency anemia

Conditions of total iron deficiency are accompanied by microcytic anemia. The causes are iron-deficient diet, gastrointestinal bleeding, and bleeding associated with chronic inflammatory diseases or malignant tumors. Anemia is the last symptom of iron deficiency. At this stage, all functional sites that require iron attempt to adapt in order to compensate for the lack of iron. For example, the erythropoiesis reduces the red cell Hb content, the muscle cells synthesize less myoglobin and the mitochondria synthesize less of iron-containing enzymes. Thus, a new equilibrium between iron supply and iron demand is established.

7.1.4.1.3 Functional iron deficiency

Functional iron deficiency is characterized by impaired iron release from body stores that is unable to meet the demand for erythropoiesis (also called reticuloendothelial cell iron blockade). See Tab. 7.1-2 – States of iron restriction. These patients have low transferrin saturation and normal or high serum ferritin. A specific form of functional iron deficiency is seen in patients treated with erythropoiesis stimulating agents (ESAs). If erythropoiesis is stimulated by ESA more than 3-fold, the iron required for erythropoiesis can no longer be transported, despite replete iron stores, due to the limited capacity of the transferrin pool. As a result, hypochromic red blood cells are released from the bone marrow.

7.1.4.1.4 Iron deficiency and erythropoiesis

Erythropoiesis is the dominating factor in iron metabolism /17/. Every second, 2–3 million red blood cells are released from the bone marrow, and each day 6 g of Hb is produced. To supply of every red blood cell with 30 pg of hemoglobin, 30–40 mg of iron is delivered to the erythroblasts every day. The amount of iron required for hemoglobin synthesis is 10 times higher than the circulating iron pool, which means that every transferrin molecule is recycled about 10 times per day. Each iron atom liberated from senescent erythrocytes remains in the circulation for 90 min. before it is recycled into the bone marrow. A small percentage of circulating iron is used for supplying non-hematopoietic tissues.

Iron-deficient erythropoiesis is a condition of insufficient supply of iron to the erythroblasts, which results in hypochromic red blood cells. Iron stores are usually depleted (Tab. 7.1-3 – Iron deficiency in healthy individuals and in diseases).

Iron-deficient erythropoiesis with replete iron stores or even elevated iron stores usually indicates the presence of anemia of chronic disease (ACD). A hallmark of ACD is the development of disturbances of iron homeostasis with increased uptake and retention of iron within cells of the reticuloendothelial system. This leads to a diversion of iron from the circulation into storage sites of the reticuloendothelial system, subsequent limitation of iron availability for erythroid progenitor cells, and iron restricted erythropoiesis /18/.

ACD is frequently concomitant with chronic inflammatory conditions (chronic heart failure, chronic kidney disease, and inflammatory bowel disease). In inflammatory conditions, hepcidin production and release is induced by pro inflammatory cytokines, especially IL-6. This results in increased internalization and degradation of ferroportin and subsequent cellular iron retention. This ultimately leads to decreased levels of circulating iron, which may result in insufficient iron availability to meet the body’s needs /19/.

ACD is characterized as mild normocytic, normochromic anemia. This results from increased apoptosis of red cell precursors in the bone marrow, since inflammatory cytokines have an antagonistic effect on erythropoietin. See (Fig. 15.1-2 – Erythropoiesis develops a proliferation pool (upper row) and a maturation pool). Erythropoietin prevents the apopotosis of red cell precursors, while inflammatory cytokines promote it. The more severe the inflammation, the higher the degree of apoptosis.

ACD like conditions in the absence of chronic inflammation are:

  • Erythropoiesis dysfunction which may result from an inadequately low erythropoietin response in relation to the extent of the anemia or by an impaired function of the erythropoietin receptor of the erythroblasts
  • Inadequate intrinsic activity of the erythron in relation to the erythropoietin stimulation. Erythropoiesis may be disturbed by chemotherapy or vitamin B12 and folate deficiency, because of reduced DNA synthesis
  • Inability to produce heme, due to insufficient iron supply, which results from the hepcidin-induced reduced release of iron from macrophages and hepatocytes as well as reduced intestinal absorption of iron.
7.1.4.1.5 Iron restriction in non-anemic patients

Iron restriction may be present in the absence of anemia as shown inpatients with chronic heart failure, in preoperative patients and in whole blood donors. The diagnosis of iron restriction before hemoglobin decreases below the reference limit is an important step in the management of these patients. From the clinical point of view, it would be helpful to detect iron restriction at the earliest stage as a precautionary iron therapy can be initiated. Current standard tests for detection of iron restriction are serum ferritin ≥ 30 μg/L, transferrin saturation < 20%, soluble transferrin receptor, the ferritin index, and hematological indices e.g., proportion of hypochromic red cells (%HYPO), and the reticulocyte hemoglobin content (CHr, RetHe). In a study /14/ a scoring system to provide optimal guidance for the evaluation of iron restriction in non-anemic patients was established (Tab. 7.1-4 – Scoring system for the diagnosis of iron restriction in non-anemic patients). Non-anemic patients with ferritin levels > 300 μg/L had no iron restriction. In patients with ferritin levels in the range of 31–300 μg/L the proportion of iron restriction decreased with increasing ferritin concentration.

7.1.4.2 Clinical manifestation of iron deficiency

Clinical manifestations of iron deficiency include glossitis, angular stomatitis, koilonychia, blue sclerae, esophageal web (Plummer Vinsen syndrome), and restless legs syndrome. Physical manifestations such as fatigue, exhaustion and general weakness are the symptoms of anemia.

The symptoms of iron deficiency result from the following two functional impairments /20/:

  • Anemia limits the capacity for oxygen transport into the tissues
  • Tissue iron deficiency inhibits the oxidative metabolic capacity of the cell and therefore also the energy supply due to reduced activity of iron-containing enzymes.

Besides anemia, the clinical symptoms and manifestations of iron deficiency can be due to impairment of immune function, of mental function, of neurotransmitter function in the central nervous system, and due to impaired muscular function.

Iron deficiency and immune function

It is the aim of the organism during infections to restrain invading microorganisms from iron, without causing an iron deficiency in its own defense cells. Iron deficiency leads to increased susceptibility to infection, since the function of the antigen-nonspecific immune system is impaired during the acute-phase response. The defense function of the polymorphonuclear granulocytes is compromised, due to a reduction of the iron-containing enzyme myeloperoxidase. This enzyme is crucial for the production of reactive oxygen radicals which are responsible for the intracellular killing of microbial pathogens /1/.

Macrophage function is not affected by iron deficiency. Available data on changes in the T-cell response are inconsistent, the humoral immune response appears not to be affected.

Iron deficiency and mental function

The highest cellular iron content in the central nervous system is found in the oligodendrocyte /1/. These cells are responsible for the myelination of the nerve fibers, which requires the production of fatty acids and cholesterol. Both processes involve iron-containing enzymes. Cerebrospinal fluid iron is bound to transferrin. The iron concentration is 15–25 μg/L, which is 30 times lower than the plasma iron concentration. Iron deficiency during the fetal period and in early childhood has been associated with stillbirth, premature birth, and mental retardation /21/. Infants under the age of 2 years with prolonged iron deficiency anemia and hemoglobin levels < 100 g/L have a significantly reduced mental development index /22/.

Iron deficiency and the neurotransmitter system

The dopaminergic system is the only system affected by decreased iron content in the brain /1/.

Iron deficiency and muscle function

In iron deficiency, the muscle content of myoglobin and cytochrome C is decreased proportionally to the amount of hemoglobin in the blood /1/. The extent of reduction in iron is also reflected in the content of iron-sulfur enzymes and mitochondrial enzymes of the skeletal muscle, which can be reduced by 50–90%, resulting in diminished muscle function. Intravenous iron therapy leads to an increase in muscle strength after 4 days. This corresponds to the turnover of iron-containing enzymes. Muscular endurance is dependent on the activity of its iron-containing enzymes and is relatively independent in decline of hemoglobin to concentrations of 100 g/L. However, skeletal muscle metabolism during short, intensive muscular activity is a function of the oxygen supply and therefore of the hemoglobin value /20/.

7.1.5 Iron overload disorders

Iron-overload disorders are classified into primary (hereditary) and secondary (acquired) forms based on their pathophysiology.

Hereditary iron-overload disorders are categorized according to whether the underlying pathopysiological defect is in the hepcidin-ferroportin axis, erythroid maturation, or iron transport /4/. The most common disorders of the hepcidin-ferroportin axis represent a form of primary hemochromatosis. The pathophysiology of these disorders is inadequate or ineffective hepcidin-mediated down-regulation of ferroportin. The iron overload is caused by intestinal iron absorption, is directed to the parenchymal tissue and may cause organ damage (Tab. 7.1-5 – Heritable forms of systemic iron overload).

Acquired forms of iron overload are usually due to transfusional iron. The iron is primarily deposited in hepatocytes and cells of the reticuloendothelial macrophage system and is regarded as relatively harmless.

The excess of body iron in systemic iron overload is stored in the form of ferritin or hemosiderin. This pool, which is normally 0.1–1 g in size depending on age and sex, can be increased 20–30-fold.

7.1.6 Hereditary hemochromatosis

Hereditary hemochromatosis (HH) includes several genetic disorders that cause iron overload. Approximately 95% of the cases of hemochromatosis is a homozygous mutation in the HFE gene. HFE is a hemostatic iron regulator localized on chromosome 6p22.2 exon 4, c.845G-A, rs 1800562, which results in a p.C282Y substitution and is termed hemochromatosis /36, 37/.

7.1.6.1 Inheritance

Hemochromatosis affects about 1 in about 200 individuals of northern European descent. Simple heterozygosity for p.C282Y affects 1 in 7 individuals, and the more minor p.H63D variant in HFE affects 1 in 3 individuals of Northern European descent. Simple or compound heterozygosity for the p.C282Y and p.H63D variants or digenic inheritance of p.C282Y with another mutation in HFE, such as p.S65C, may cause mild elevations in transferrin saturation or ferritin concentrations but not clinically significant iron overload /36, 37/.

Of the six disorders of the hepcidin-ferroportin axis five have the classic HH phenotype. Laboratory results in these phenotypes are elevated transferrin saturation (TfS), elevated ferritin, normal hematocrit and tissue iron overload.

In HH, the hepatocytes and macrophages release more iron than in healthy individuals. The enterocytes also release more iron into the blood, while iron absorption from the lumen is not or only moderately elevated. The abnormal behavior of the enterocytes, hepatocytes, and macrophages is due to unregulated export of iron by ferroportin. This behavior of ferroportin results from deficient synthesis or reduced activity of hepcidin. In general, every genetic defect of the hepcidin-ferroportin axis causes unregulated influx of iron into the bloodstream, followed by iron overload of the organs with potential toxicity and damage.

In humans, hepcidin deficiency is linked to HFE-associated, transferrin receptor 2 (TfR2)-associated, hemojuvelin (HJV)-associated and HAMP-associated HH (Tab. 7.1-5 – Heritable forms of systemic iron overload).

The term HFE refers to the HLA class I-Iike hemochromatosis gene originally named HLA-H. The different forms of HH are numbered (type 1–4) in order of their discovery /4/.

The results of biomarkers in HH are shown in Fig. 7.1-10 – Age of onset of organic diseases and functional disorders and behavior of serum iron and hepcidin as a function of the HH-type.

7.1.6.2 Hereditary hemochromatosis by mutation in HFE gen

In hereditary hemochromatosis (HH) too little functional active hepcidin is synthesized. The most common type of HH results from mutations in the gene HFE.

Type 1

This type is the most common disorder of the hepcidin-ferroportin axis. It is often referred to as HFE hemochromatosis or simply HH. Most individuals carry the p.C282Y HFE-mutation.

Type 2a

This type is also known as juvenile hemochromatosis due to the early age of onset of clinical symptoms. There is a mutation in the HJV gene, which encodes the protein hemojuvelin.

Type 2B

Mutations in the HAMP gene, which encodes the protein hepcidin are responsible for this type. It is a rare form of juvenile hemochromatosis.

Type 3

This type is caused by mutations in the gene of transferrin receptor 2 (TfR2) and, in terms of clinical symptoms, is an intermediate form between the HFE type and HH type 2.

Type 4

This type is a separate disease, which is due to mutations in the SCL40AI gene, which encodes the cellular iron exporter ferroportin. It is also known as ferroportin disease, since it differs from the other forms of HH in its genetic, biochemical and histological characteristics.

Refer also to Tab. 7.1-6 – Clinical and laboratory findings in hereditary hemochromatoses.

7.1.6.3 Clinical significance

Hemochromatosis primarily affects the liver and joints and results from a failure in the regulation of the iron regulatory protein hepcidin. Mutant HFE, hemojuvelin, TFR2, and hepcidin lose the ability to upregulate hepcidin synthesis, causing low concentrations of serum hepcidin.

Liver disease /36, 37/

The most frequent clinical manifestations are advanced liver fibrosis or cirrhosis and primary liver cancer. In men, who are homozygous for p.C282Y have an increased risk of death by the age of 75 years, as compared with those who do not have HFE variants. Among women who are homozygous for p.C282Y the risks of colorectal cancer and breast cancer are doubled. Risk factors are alcohol consumption, ferritin concentrations > 1000 μg/L, diabetes mellitus, low platelet count (< 200 × 109/L), a liver iron content > 200 μmol/gram and elevated activity of aspartate aminotransferase.

Arthritis /36, 37/

The arthropathy affects the metacarpophalangeal joints, followed by the hip ankle, radiocarpal elbow, shoulder and knee joints, as well as the lumbar spine. The complaints are 8 times as common in patients with hemochromatosis as in those without the disorder. Arthritis is strongly associated with advanced liver fibrosis. Risk of arthritis, advanced liver fibrosis, and the subsequent development of primary liver cancer increase with progressive iron loading.

The most severe forms of HH are diagnosed in childhood and early adolescence, but usually before the age of 30. Every child with hypogonadotropic hypogonadism, unexplained cardiomyopathy or liver cirrhosis should be tested for HH.

In adults, the severe forms of HH in patients with diabetes, liver cirrhosis and arthritis do not manifest until the 5th or 6th decade of life in men and even later in women.

In β-thalassemia mutations in both genes encoding globin cause deficiency of hemoglobin and hypoxia. Overall ineffective erythropoiesis results in β-thalassemia because hypoxia stimulates increased levels of erythropoietin that activate generation of red cell precursors in the bone marrow that produce erythroferrone that inhibits hepcidin formation.

7.1.6.4 Criteria of HFE hemochromatosis

The criteria for the diagnosis of HFE hemochromatosis according to the guidelines of the European Association for the Study of Liver Disease (EASL) are shown in Tab. 7.1-7 – Criteria for the diagnosis of HFE hemochromatosis according to EASL guidelines /37/.

7.1.6.5 Rare hereditary hemochromatoses

Types of HH with deficiency of hepcidin which occur less frequently result from mutations of the genes encoding TfR2, hemojuvelin, hepcidin and ferritin. With the exception of ferroportin disease (type 4), all types of HH are similar in their clinical, diagnostic and histological characteristics. That concerns, for example:

  • An unregulated intestinal absorption of iron and the rapid mobilization of iron from hepatocytes and macrophages that cause high TfS
  • The high TfS promotes the uptake of iron by the cells via the TfR and deposition of iron in the cells,especially the hepatocytes
  • The macrophages are relatively depleted in iron and the utilization of iron for hematopoiesis is normal
  • There is inadequate or ineffective hepcidin-mediated down-regulation of ferroportin.

Hemochromatosis is a disorder associated with deposits of excess iron that causes multiple diseases like liver fibrosis, pancreatopathy, cardiac disease, diabetes, arthropathy, and skin pigmentation.

7.1.6.6 Diagnosis of iron overload

Refer to:

7.1.6.7 Molecular basis of iron overload

The key regulator of iron metabolism is hepcidin, because it controls the cellular iron exporter ferroportin. By binding to ferroportin, hepcidin induces the internalization and degradation of ferroportin in cells with high iron metabolism, such as hepatocytes, enterocytes, erythroid precursor cells, and placental cells. Ferroportin exports iron across the cell membrane into the extracellular compartment.

If hepcidin is inactive or ineffective, there is uncontrolled intestinal uptake of iron, release of iron into the circulation, and storage of iron in the parenchymal cells of organs (see also Section 7.6 – Hepcidin).

7.1.6.8 Liver biopsy in hemochromatosis

Liver biopsy is not required to establish the diagnosis of HFE hemochromatosis in C282Y homozygotes /36/. It is, however, frequently performed in patients > 40 years of age to exclude hepatic fibrosis or cirrhosis when hepatomegaly is present, aminotransferases are elevated and ferritin is above 1000 μg/L. One study /38/ has shown that liver cirrhosis can also be detected by laboratory investigation (Tab. 7.1-8 – Findings in C282Y hemochromatosis with and without liver cirrhosis).

In iron loading anemias e.g., thalassemias, intestinal absorption of iron is increased differently, and approximately 5-fold in thalassemia major. The pattern of iron-loading anemias contains hereditary and acquired disorders of erythropoiesis.

7.1.7 Secondary iron overload

Secondary iron overload results from iron loading. Reasons are either excess intestinal absorption of iron, parenteral administration of banked blood units, and hemolysis occurring in hematologic diseases, but not from disorders of the hepcidin-ferroportin axis (Tab. 7.1-9 – Iron overload not due to disorders of the hepcidin-ferroportin axis). Since only about 1 mg of iron is excreted per day, the body is burdened with 200–250 mg of iron with each unit of banked blood.

Increased intestinal absorption of iron occurs in hyper proliferative and ineffective erythropoiesis, hypoxia, and genetic disorders such as HH. In iron-loading anemias, intestinal iron absorption is increased to varying extents. The spectrum of these anemias comprises hereditary and acquired disorders of erythropoiesis. In thalassemia, for example, iron absorption can be increased up to 5-fold compared to normal.

Clinically, liver function is normal in secondary iron overload. Like in HH, the consequences can be reduced glucose tolerance, diabetes mellitus, and cardiomyopathy. Patients with thalassemia can develop liver cirrhosis decades after onset of the disease.

7.1.8 Diagnosis of iron restriction

The following laboratory tests are used to diagnose restriction of iron:

A scoring system /14/ to provide optimal guidance for the evaluation of iron restriction in anemic patients in the presence and absence of inflammatory conditions is shown in Tab. 7.1-12 – Scoring system for the diagnosis of irion restriction in anemic patients.

7.1.8.1 Monitoring of iron therapy

The treatment of patients with iron deficiency depends on the type of iron restriction /19/. See Tab. 7.1-2 – States of iron restriction.

7.1.8.1.1 Monitoring of oral iron therapy

Therapeutic efficacy of oral iron therapy is available under the following conditions:

  • Increase of reticulocyte count by at least 20% after 10 days
  • Elevation of reticulocyte hemoglobin content (CHr, RetHe) within 5 days. Normalization of the reticulocyte hemoglobin content during therapy means a normal iron supply of erythropoiesis, however no repletion of iron stores.
  • Decrease in soluble transferrin receptor level by more than 20% after 10 days
  • Increase in transferrin saturation by more than 20% after 10 days. The increase to > 20% indicates repletion of iron stores /58/.
  • Daily increase in hemoglobin level of 1–2 g/l approximately 2–3 weeks after starting therapy
  • Normalization of serum ferritin after 2–3 months.
7.1.8.1.2 Monitoring of intravenous iron therapy

Intravenous iron treatment can deliver a larger iron supply than oral iron and effectively replenish iron stores more rapidly. However, in inflammatory conditions much of the intravenous iron is transported into the reticuloendothelial system, where it cannot be mobilized. In iron deficiency anemia, 50% of the iron administered is incorporated into hemoglobin within 3–4 weeks. In patients with chronic inflammation, renal anemia and tumor anemia this does not occur to this extent, but they also show a mild increase in hemoglobin levels /23/.

Intravenous iron treatment preferred in conditions of high hepcidin levels (chronic inflammatory conditions), which prevent effective oral iron therapy e.g., chronic heart failure, chronic kidney disease, and inflammatory bowel disease. High bolus dosage of intravenous iron have a limited effect in patients with hemodialysis because of sequestration of a large proportion in the liver and the reticuloendothelial system. Sequestration of iron causes further increase in existing hepcidin levels /64/.

Intravenous iron in patients with postpartum anemia

Therapy of hemoglobin levels of below 100 g/L in women with postpartum anemia using each 200 mg iron sucrose on 7 days caused changes in hematologic markers: increase in hemoglobin (15 g/l), reticulocyte count, transferrin saturation (50%), and ferritin (5 fold), respectively /65/.

Chronic kidney disease (CKD)

The iron requirement in patients after kidney transplantation is about 500 mg and in patients with peritoneal dialysis approximately 3000 mg every year /66/. A serum ferritin concentration < 100 μg/l in non dialyzed CKD patients or < 200 μg/l in chronic hemodialysis patients is associated with high likelihood of iron deficiency and a potentially good response to intravenous iron therapy. Serum ferritin values > 1,200 μg/l should be used to ascertain whether investigation of potential iron overload should be undertaken /67/. A CHr value < 29 pg predicts functional iron deficiency in patients receiving ESA therapy /67/. Indicators of iron-restrictive erythropoiesis are the following results %HYPO > 6% /68/ or CHr < 29 pg /69/. In isolation transferrin saturation is not recommended as a predictor of responsiveness to intravenous iron therapy /67/.

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7.2 Iron (Fe)

Lothar Thomas

7.2.1 Indication

Parameter for:

  • The determination of transferrin saturation
  • The iron resorption test
  • The detection of acute iron intoxication.

7.2.2 Method of determination

Spectrophotometric method

Principle: to measure serum iron, Fe3+ is released from transferrin at acidic pH and the proteins are precipitated. The Fe3+ is reduced to Fe2+ and the free iron forms a color complex with a chromogen. The concentration of the iron-chromogen complex is measured spectrophotometrically.

A variety of methods have been described /1/. The method of the International Committee for Standardization in Hematology (ICSH) /2/, revised in 1990, uses HCl to release iron, trichloracetic acid for protein precipitation, thioglycolic acid to reduce Fe3+, and ferrozine as the chromogen.

The calibration is performed using the standard reference material (SRM) 937 provided by the National Institute of Standards and Technology (NIST). The standard contains iron, 5 mmol/L dissolved in HCl.

7.2.3 Specimen

Plasma (no EDTA plasma), serum: 1 mL

7.2.4 Reference interval

See Tab. 7.2-1 – Reference intervals for iron.

7.2.5 Clinical significance

The concentration of iron in plasma is regulated by the hepcidin-ferroportin system. In conditions of empty iron stores production of hepcidin is inhibited and ferroportin mediated iron export of intestinal mucus cells to transferrin is possible.

The adult without anemia has about 4.0 g of total body iron. The functional form of iron is about 80% of the total body iron and includes iron in hemoglobin (2.3 g), myoglobin (0.35 g), and enzymes (catalases and cytochromes). Of the remaining iron 15 to 20% is stored (ferritin or hemosiderin). The liver contains about 0.2 g, the macrophages about 0.5 g and the bone marrow 0.15 g of iron. Conditions increasing iron absorption include Fe(II), vitamin C, hydrochloric acid, low gastric pH, sugars and amino acids present in the food, pregnancy, and low concentration of hepcidin in serum. Factors that culminate in decreased iron absorption are Fe(III), phytates, calcium present in the food, systemic inflammation, and high levels of serum hepcidin /17/.

Serum iron is unsuitable for assessing body iron status for the following reasons /7/:

  • High diurnal intraindividual variation in serum iron levels both diurnally and from day to day. For example, measurements made in healthy males in the morning at 9 a.m. revealed values of 155 ± 16 μg/dL (27.8 ± 2.9 μmol/L) and 12 hours later of 65 ± 5 μg/dL (11.6 ± 0.9 μmol/L) /8/.
  • High inter individual variation. The range of variation of the aforementioned population group is 100–300 μg/dL (17.9–54 μmol/L) at 9 a.m. and 20–100 μg/dL (3.6–17.9 μmol/L) 12 hours later. Due to the high inter individual variation, the reference interval is extremely broad.

7.2.5.1 Diagnosis of iron deficiency

In addition to the aforementioned reasons, the following factors also make the serum iron concentration unsuitable for diagnosing iron deficiency:

  • The serum iron concentration depends on the acute-phase response, since Fe3+ is bound to transferrin, which is a negative acute-phase protein. Low serum iron levels can therefore be due to depleted iron stores, but can also occur in response to inflammation with normal or even elevated storage iron.
  • Dependency of the serum iron level on dietary intake; levels may rise within 10 min. /9/.
  • Hemolysis may cause normal values when iron is determined in serum in suspected cases of iron deficiency. Serum obtained by routine procedure of blood sampling contains 10–30 mg/L of free hemoglobin. Since each mg of hemoglobin adds 3.5 μg of hemoglobin-bound iron to the serum, the serum iron level rises by 3.5–10.5 μg/dL (0.6–2 μmol/L) /10/.
  • The serum iron level is a late indicator of iron deficiency. Results below the reference interval are measured only once storage iron reserves are depleted and functional iron levels are reduced.
  • The serum iron concentration does not generally reliably indicates iron deficiency. In one study /6/, in which iron deficiency anemia of a patient population had been confirmed by bone marrow iron staining, iron deficiency was correctly diagnosed by serum iron in only 41% of cases while ferritin yielded a correct diagnosis in 90% of cases. This diagnosis was accepted on the basis of an iron level less than 60 μg/dL (11 μmol/L) and a ferritin concentration less than 13 μg/L for females and less than 25 μg/L for males.

7.2.5.2 Daily iron requirement and treatment of iron deficiency

The daily iron requirement is about 1 mg for adults above the age of 50, and 1.6 mg for females of reproductive age. The recommended dietary iron intake in industrialized countries is about 10 mg, 10% of which is absorbed in the absence of iron deficiency. Patients receiving total parenteral nutrition should be administered 1 mg of iron intravenously per day /11/. The WHO recommendations for daily oral iron intake are shown in Tab. 7.2-2 – WHO recommended intakes of iron. The parenteral iron dose that should be administered to patients with iron deficiency is calculated using the formula below. The treatment of iron deficiency without anemia in otherwise healthy persons is shown in Ref. /12/.Iron absorption from supplementes is greater with alternate day than with consecutive day dosing in iron-deficient anemic women /13/.

Total iron deficit (mg) = Body weight (kg) × 0.24 × [target Hb (g/L) – actual Hb (g/L)] + 500

7.2.5.3 Diagnosis of iron overload

Elevated iron levels can occur temporarily in healthy individuals, but often they are due to diseases such as:

  • Ineffective erythropoiesis with increased destruction of red blood cells in the bone marrow
  • Liver damage caused by alcohol or hepatitis C
  • Hereditary hemochromatosis
  • Transfusion-induced iron overload in chronic anemias. For each unit of banked blood, 200–250 mg of iron are stored. Clinical symptoms and complaints of iron overload do not occur only after transfusion of 100–150 units /14/.
  • Iron overdosage due to intoxication with iron-containing substances. This is mainly a problem in children. Serum iron levels above 300 μg/dL (54 μmol/L) are associated with clinical symptoms and require treatment. The most important findings in addition to serum iron values averaging 500 μg/dL (90 μmol/L) /15/ are diarrhea, vomiting, leukocytosis, hyperglycemia, and positive abdominal X-ray findings.

See also:

7.2.6 Comments and problems

Method of determination

The precision of routine assays for the determination of iron is acceptable, however its accuracy is suspect. Compared with the revised ICSH method /2/, the routine methods exhibit /1/:

  • A negative bias across the whole measuring range
  • A significant negative intercept
  • Poor correlation with the ICSH method in the concentration range below 75 μg/dL (13.4 μmol/L).

Critical factors influencing accuracy include /16/:

  • pH of the reaction mixture which is influenced by the plasma proteins. At pH ≤ 1.65, iron is easily released from transferrin, but does not readily complex with ferrozine. At pH 4–5, the rate of iron release from transferrin is poor.
  • Loss of iron during protein precipitation
  • High ferritin concentrations. When ferritin levels are above 1200 μg/L, iron is mobilized from ferritin and also included in the measurement, even in the ICSH method.
  • Hyperlipidemia and hyperbilirubinemia interfere with spectrophotometry.

Hemolysis, contamination, and chelating agents

Hemolytic serum gives falsely high levels of iron, especially when using methods without deproteinization. Therefore the serum should be separated within 2 h after blood collection.

No glassware should be used in the determination. Using plastic materials contamination is usually not a problem. EDTA plasma can only be used if the iron is measured by atomic absorption spectroscopy.

Stability

Iron in serum or plasma is stable for 3 days if stored at room temperature or for one week if stored at 4 °C.

References

1. Tietz NW, Rinker AD, Morrison SR. When is a serum iron really a serum iron? The status of serum iron measurements. Clin Chem 1994; 40: 546–51.

2. Iron Panel of the International Committee for Standardization in Hematology. Revised recommendations for the measurements of serum iron in human blood. Br J Haematol 1990; 75: 615–6.

3. Saarinen UM, Siimes MA. Developmental changes in serum iron, total iron-binding capacity, and transferrin saturation in infancy. J Pediatr 1977; 91: 875–9.

4. Koerper MA, Dallman PR. Serum iron concentration and transferrin saturation in the diagnosis of iron deficiency in children: normal developmental changes. J Pediatr 1977; 91: 870–4.

5. Quigley G, Lokitch G, Jacobson B, Wittmann B, Ross P, Lucas B. The effect of normal gestation on indices of iron status. Clin Chem 1990; 36: 972.

6. Burns ER, Goldberg N, Lawrence C, Wenz B. Clinical utility of serum tests for iron deficiency in hospitalized patients. Ann J Clin Pathol 1990; 93: 240–5.

7. Heimpel H, Riedel M, Wennauer R, Thomas L. Die Plasmaeisenbestimmung – nützlich, unnötig oder irreführend? Med Klinik 2003; 98: 104–7.

8. Hamilton LD, Gubler CH, Cartwright GE, et al. Diurnal variation in the plasma iron level of man. Proc Soc Exp Biol (NY) 1950; 75: 65–8.

9. Cavill I, Jacobs A, Worwood M. Diagnostic methods for iron status. Ann Clin Biochem 1986; 23: 168–71.

10. Eckfeldt JH, Witte DL. Serum iron: would analytical improvement enhance patient outcome? Clin Chem 1994; 40: 505–7.

11. Forbes A. Iron and parenteral nutrition. Gastroenterology 2009; 137: S47–S54.

12. Clenin GE. The treatment of iron deficiency without anemia (in otherwise healthy persons). Swiss Medical Weekly 2017; 147: w 14434.

13. Stoffel NU, Zeder C, Brittenham GM, Moretti D, Zimmermann MB. Iron absorption from supplements is greater with alternate day than with consecutive day dosing in iron-deficient anemic women.Haematologica 2020; 105, 5: 1232–9.

14. Marcus RE, Huehns ER. Transfusional iron overload. Clin Lab Haematol 1985; 7: 195–212.

15. Lacouture PG, Wason S, Temple AR, Wallace DK, Lovejoy FH. Emergency assessment of severity in iron overdose by clinical and laboratory methods. J Pediatr 1981; 99: 89–91.

16. Tietz W, Rinker AD, Morrison SR. When is a serum iron really a serum iron? A follow-up study on the status of iron measurements in serum. Clin Chem 1996; 42: 109–11.

17. Saboor M, Zehra A, Hamali AA, Mobarki AA. Revisiting iron metabolism, iron homeostasis and iron deficiency anemia Clin lab 2021; 67: 660–6.

7.3 Ferritin

Lothar Thomas

Ferritin plays an important role in iron homeostasis since it binds, sequesters and stores intracellular iron, thus serving the dual functions of maintaining iron in a biologically available form and protecting the organism against the toxic effects of free iron. Ferritin is present in the cytoplasm and mitochondria of virtually all cells, although most of the body’s ferritin is found in hepatocytes and in the iron-storing cells of the reticuloendothelial system, such as macrophages and Kupffer cells /1/.

The serum hepcidin level regulates the iron load of the storage cells and the release of iron into the circulation. Serum ferritin reflects the content of stored iron and is therefore widely used in diagnosing iron-related disorders and in population surveys of iron status.

7.3.1 Indication

  • Suspected iron deficiency without anemia
  • Microcytic, hypochromic anemia
  • Monitoring of risk groups for iron deficiency (e.g., pregnant women, blood donors, children, hemodialysis patients)
  • Monitoring of oral iron therapy
  • Assessment of iron stores prior to treatment with erythropoiesis stimulating agents (ESA)
  • Suspected hereditary hemochromatosis or secondary iron overload
  • Monitoring of iron mobilization therapy in iron overload
  • Patients with metabolic syndrome
  • In combination with soluble transferrin receptor (sTfR) to calculate the ferritin index (sTfR/log ferritin) a marker of iron supply for erythropoiesis.

7.3.2 Method of determination

Immunoassays, such as enzyme-linked immunoassay (ELISA), immunometric assay (IMA), luminescence immunoassay (LIA).

7.3.3 Specimen

Serum, plasma: 1 mL

7.3.4 Reference interval

Refer to Tab. 7.3-1 – Reference intervals for ferritin.

7.3.5 Clinical significance

The iron content of the body is distributed into three compartments:

  • The red blood cells which contain the bulk of the body’s iron. An indirect measure of the iron content of this pool is the hemoglobin concentration.
  • The functional iron pool, also known as transit pool or transferrin pool. It contains only small amounts of iron and its size can be assessed by measuring transferrin saturation (TfS).
  • The storage iron pool. Non-heme iron is contained within the iron-storage protein, ferritin. This protein is present in most tissues as a cytosolic component and plays an important role in the storage of intracellular iron.

In total body iron deficiency all three iron pools are depleted and the ferritin level in serum is lower than only in storage iron deficiency (Tab. 7.3-2 – Ferritin levels in iron deficiency).

For iron supply of the tissues, ferritin-bound iron is rapidly mobilized from the storage iron pool. Ferritin is constantly broken down within lysosomes or released into the blood. The plasma ferritin concentration closely parallels storage iron reserves. Quantities of 1 μg/L of serum ferritin represent 8–10 mg of stored iron or are equivalent to approximately 140 μg of stored iron per kg of body weight /7/. This relationship provides a useful correlation up to a serum ferritin level of 200 μg/L.

A comparison between serum ferritin levels with microscopically determined iron in bone marrow aspirates reveals an acceptable relationship between these two indicators of storage iron reserve. However, this is only the case if there is equilibrium, because the iron in bone marrow is not readily mobilized since it is stored as hemosiderin.

Changes in intracellular iron homeostasis (see Section 7.1.4 – Iron deficiency) are rapidly reflected by a change in serum ferritin. For example, 59Fe, administered intravenously in the form of denatured erythrocytes, is taken up in the macrophages of the reticuloendothelial system and appears in the circulation as 59Fe-labeled ferritin 20–40 min. later /8/. Depending on dosage, 59Fe-labeled ferritin is removed from circulation with a half-life of 4–40 min. The hepatocyte can take up 160,000 iron atoms per minute /1/.

7.3.5.1 Ferritin for diagnosing iron deficiency

Serum ferritin is the preferred test for diagnosing iron deficiency (Tab. 7.3-3 – Diseases and conditions associated with decreased serum ferritin levels). The ferritin level allows the differentiation between latent iron deficiency and total iron deficiency. If in adults a ferritin level ≤ 12 μg/L is accepted as an indicator of total body iron deficiency, the diagnostic sensitivity for iron deficiency is only 25% /9/. However, the diagnostic sensitivity and specificity of ferritin for storage iron deficiency is 92% and 98%, respectively, by using a threshold of ≤ 30 μg/L, resulting in a positive predictive value of 92% /9/. Threshold values of ferritin for the diagnosis of total iron deficiency and storage iron deficiency are shown in Tab. 7.3-1 – Reference intervals for ferritin.

Total iron deficiency is associated with highly ineffective erythropoiesis and microcytic, hypochromic anemia. Reduced ferritin levels usually precede iron deficiency anemia. However, in adolescents, iron deficiency is more than twice as prevalent as iron deficiency anemia /10/.

The situation may be different for other populations. A study that used WHO cutoffs of hemoglobin < 110 g/L for diagnosing anemia and ferritin < 10 μg/L for diagnosing total iron deficiency in Pakistani, Bangladeshi and Indian children living in England, reported a prevalence of 20–29% for anemia, but a prevalence of only 8–13% for hypo ferritinemia /11/. The normal ferritin levels are thought to be due to the higher prevalence of infections in this group compared to children of the native population.

7.3.5.2 Ferritin in conditions of inflammation

Serum ferritin is an acute-phase protein and the diagnostic significance is limited in patients with inflammation. Therefore acute and chronic inflammation, autoimmune disease, infections, chronic renal disease, chronic heart failure, inflammatory bowel disease and malignancy are associated with elevated values. The ferritin concentrations are in the range of > 100 to above 1,000 μg/L. This is also the case in liver diseases when ferritin is released from hepatocytes, or in Still’s disease where hyperferritinemia is an indicator of disease activity.

If these conditions are not evident, the determination of the following biomarkers are useful for evaluation of the iron status:

  • C-reactive protein; if inflammation is present, CRP is > 5 (10) mg/L
  • Transferrin saturation (TfS); if inflammation is present, TfS is decreased (less than 20%).

In inflammation and cytolysis, ferritin levels are falsely elevated in relation to iron stores, because:

  • Due to IL-6 activated hepcidin synthesis, there is reduced release of iron from macrophages and increased release of ferritin
  • Ferritin release is increased from damaged hepatocytes due to cytolysis
  • There is increased release of ferritin from leukocytes in leukemias and lymphomas.

The sTfR/log10 ferritin index is a better indicator of iron deficiency than the ferritin concentration in patients with inflammation. See also Section 7.4 – Soluble transferrin receptor.

In adult patients admitted to a hospital about 30% of the anemic group with iron restriction had ferritin levels > 300 g/l, but no patient of the non-anemic group /45/.

7.3.5.3 Ferritin and iron overload

Iron overload results in an increase in total body iron and only affects the iron stores. It is primarily the parenchymal cells, in particular hepatocytes, that are overloaded with iron in hereditary hemochromatosis, while the reticuloendothelial system macrophages are preferentially overloaded in secondary iron overload.

The serum ferritin is a useful marker in diagnosis and estimation of iron overload. An additional important test is the transferrin saturation (TfS), especially for differentiating iron overload from hyper ferritinemia in anemia of chronic disease (Fig. 7.3-1 – Differentiation of non anemia-related hyper ferritinemia based on ferritin, transferrin saturation and CRP).

In cases of iron overload, with the exception of type 4 hereditary hemochromatosis (ferroportin disease) and aceruloplasminemia, hyper ferritinemia is accompanied by an increase in TfS. However, the ferritin value can be elevated in chronic inflammation without the TfS being raised. As transferrin is a negative acute-phase protein, hyper ferritinemia during inflammation is accompanied by a normal or decreased TfS.

Often, elevated ferritin levels are diagnosed during routine examinations, especially in elderly individuals. A significant number of cases remain clinically unclear.

Refer to Tab. 7.3-4 – Diseases and conditions associated with elevated serum ferritin concentrations.

7.3.6 Comments and problems

Standardization

The WHO International Standard (IS) 94/572 for ferritin (recombinant; NIBSC code 94/572) is available. It only contains the L subunit of apoferritin. The harmonization of ferritin measuring systems is far from optimal with the implementation of traceability to WHO IS being a a factor of confusion /52/.

Refer to Tab. 7.3-5 – Development of reference material for ferritin immonassays.

A serum concentration of 62.3 μg/L was estimated in 4 of 5 test kits between 48.5 and 73.4 μg/L and the lower reference value (15 μg/L) was measured between 7.7 and 18.7 μg/L /41/. A new study showed /52/ that current ferritin measuring systems (MS) are still not well harmonized. Intercomparison study of four measuring systems (MS) showed that IS 94/572 was commutable for use with only one MS recovering its assigned value.

Method of determination

The results obtained with assays from different manufacturers are only moderately compatible, although most manufacturers calibrate their assays against the same reference preparation. This is due to the immunologic heterogeneity of the isoferritines in serum, different antibody specificities, different handling of the reference preparation during calibration of the assays, and the different principle of the immunoassay. Most assays provide better detection results for alkaline isoferritins than for liver ferritin, which has more L than H subunits. The ferritin measured in serum is mostly iron-free apoferritin. The iron content is important for antibody binding.

Reference interval

Reference ranges in the laboratories vary and depend on the gold standard used for diagnosing iron deficiency. In men, the upper reference interval value increases slightly but continuously from about 350 μg/L to about 400 μg/L between the ages of 30 and 55. In women, the upper reference interval value increases from an average 150 μg/L to about 300 μg/l between the ages of 50 and 70 /28/. Lower reference limits for serum ferritin collected from data sources for children < 19 years were 18.8 μg/L in female and 24.4 μg/L in male individuals /49/.

Stability

Stable for 6 days at 4–8 °C and at 20 °C when stored in a sealed container, or for at least 12 months when stored at –20 °C.

Hemolysis

Low-degree intravasal or in vitro hemolysis has no effect on the result, but intense red coloring of the serum due to the release of intra erythrocytic ferritin can cause an increase in ferritin levels by up to 60%.

7.3.7 Pathophysiology

Ferritin is a ubiquitous iron-binding protein that is evolutionary highly conserved and has the sole task of sequestering and storing atomic iron. It consists of a protein shell, apoferritin, which can store up to 4500 iron atoms in its internal cavity. The protein shell has a molecular weight of 430–460 kDa, is approximately 25 Angstrom thick, and is made up of 24 symmetrically configurated subunits of two types, a light subunit (L-subunit) of about 19 kDa and a heavy subunit (H-subunit) of about 21 kDa. The amino acid sequences of both subunits differ by about 50% /1/.

The heterogeneity of ferritin observed in different tissues is explained by the fact that isoferritins are composed of variable proportions of H-type and L- type subunits.

Depending on the tissue type and physiological status of the cell, the ratio of H to L subunits in ferritin can vary /42/:

  • From the isolated H-type in HeLa cells (H24L0) through isoferritins mainly composed of H-type, such as in muscles, thymus, red blood cells, brain and heart
  • Through the intermediate type in lymphocytes
  • And the dominant H-type in liver and spleen
  • To the type H0L24, that occurs in serum.

The ratio of H- to L-type is not fixed but relatively flexible and can change in response to stimuli such as inflammation, cell differentiation, or xenobiotics.

The ferritin molecule has an internal diameter of 70–80 Angstrom and an external diameter of 120–130 Angstrom. Iron is stored as ferric-oxyhydroxide phosphate of the composition (FeOOH)8 (FeOOPO3H2). Iron-containing apoferritin, or holo ferritin as it is also known, can incorporate approximately 4500 iron atoms and thus double its molecular weight to 900 kDa (Fig. 7.3-2 – Ferritin molecular structure showing the H and L subunits and a crystalline iron core).

In an empty ferritin molecule:

  • The inner surface of the H subunits comprise a shell that has ferrooxidase activity for the conversion of Fe (II) to Fe (III), since iron atoms can enter into the apoferritin cavity only in the form of Fe (II), but is stored as Fe (III).
  • The L subunit has a nucleation site that is involved in the formation of the iron core.
  • The cell regulates iron metabolism through changes in the ratio of H to L subunits /43/. This is important as intracellular ferritin plays a key role in regulation of the labile iron pool through uptake and release of iron into and from the pool. Expression of apoferritin with increased numbers of H subunits therefore permits enhanced iron uptake into the molecule. Homozygous murine H-subunit knockouts are lethal.

Serum ferritin is poor in iron, is immunologically similar to L type apoferritin and can contain a glycolyzed side chain. It is assumed that the same gene product produces both serum ferritin and L type ferritin /44/. There is a rapid increase in serum ferritin following intestinal iron uptake. Synthesis is regulated by the IRE/IRP system (see also Fig. 7.1-6 – Post-transcriptional regulation of cellular iron homeostasis). Ferritin synthesis in macrophages is of particular importance. These cells play a central role in iron homeostasis as they take up the iron from senescent red blood cells and then release it as functional iron. Serum ferritin concentration basically reflects reticuloendothelial iron, and changes in storage iron content can be measured within 40 min. based on changes in ferritin concentrations.

The pro inflammatory cytokines TNF-α and IL-1β that are produced in greater quantities during the acute-phase reaction have a regulatory effect on iron homeostasis at the level of apoferritin subunit production. For example, stimulation of mRNA synthesis of the H subunit in mesenchymal cells and macrophages leads to the production of apoferritin rich in H subunits and thereby prioritizes iron storage /43/.

Ferritin cannot transfer stored iron directly to apotransferrin. The removal of Fe (II) is supported by salt-forming low molecular substances like citrate. Fe(III) is then released to apotransferrin from the salts formed in this process.

Following infection, inflammation, or injury, an Acute-phase response occurs involving the synthesis and release from the liver of acute phase reactants. Activated macrophages invade damaged tissues and release IL-1β into the bloodstream. IL-1β induces ferritin synthesis in hepatocytes and the translational efficiency of the L-subunit mRNA increases /46/. The synthesis of IL-1β and the L-subunit of ferritin are strongly increased in cytokine storm. The cytokine storm is an umbrella term encompassing disorders of immune dysregulation characterized by constitutional symptoms, systemic inflammation, and multiorgan dysfunction that can lead to multiorgan failure /47/. A virally-induced cytokine storm is found in a subgroup of patients with SARS-Cov-2 /48/.

Apoferritin is produced in excess in the cells, and ferritin is subject to continual degradation in the lysosomes. The iron that has been released is incorporated into newly produced apoferritin. When storage iron reserves are high, little iron is taken up into the macrophages and the excess apoferritin is released into the circulation. In cases of iron deficiency, the hepatocytes and the reticuloendothelial system release little apoferritin into the circulation, so that serum ferritin concentrations are low.

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51. Girelli D, Corrocher R, Bisceglia L, Olivieri O, De Franceschi L, Zelante L, Gasparini P. Molecular basis for recently described hereditary hyperferritinemia-cataract syndrome: a mutation of the iron-responsive element of ferritin L-subunit gene (the Verona mutation). Blood 1995; 86 (11): 4050–53.

52. Braga F, Pasqualetti S, Frusciante E, Borrillo F, Chibireva M, Panthegini M. Harmonization status of serum ferritin measurements and implications for use as marker of iron-related disorders. Clin Chem 2022; 68 (9): 1202–10.

7.4 Soluble transferrin receptor

Lothar Thomas

The transferrin receptor (TfR) is a transmembrane protein that mediates iron delivery from the functional pool into cells, mainly erythroblasts, by receptor-mediated endocytosis. Nearly all cells have TfR on their cytoplasma membrane, but TfR is mostly located in the erythroid precursors. Like many receptor types, TfR is regularly released by shedding into the circulation where it is present as soluble form (sTfR) in a truncated form. The sTfr for the most part circulates attached to transferrin. The serum sTfR concentration is proportional to cellular expression of the membrane-associated TfR and reflects the degree of erythropoiesis, being increased in states of hyper proliferative and decreased with hypo proliferative erythropoiesis. The serum sTfR concentration provides a quantitative measure of functional iron status, in that the level is increased in the presence of iron-restricted erythropoieses, functional iron deficiency, and iron-deficient anemia. Serum sTfR is not influenced by infections or chronic inflammation and may distinguish iron-deficient anemia from anemia of chronic disease.

7.4.1 Indication

Assessment of iron status

  • Iron deficiency, in particular in individuals with a high prevalence of subclinical iron deficiency e.g., women with menstrual blood loss, healthy adolescents, athletes in endurance training, multiple blood donors
  • Confirmation of iron deficiency in individuals with borderline ferritin levels
  • Differentiation of iron deficiency anemia from that caused by inflammation and detection the presence of functional iron deficiency when the two coexist
  • Calculation of the ferritin index (sTfR/log10 ferritin) for the assessment of iron status
  • Evaluation of iron status in anemic patients in combination with ferritin determination prior to treatment with erythropoiesis stimulating agents (ESA)
  • Disorders associated with an expanded erythroid marrow.

7.4.2 Method of determination

Enzyme immunoassay, latex-enhanced immuno-nephelometric and -turbidimetric assay. A standard preparation, which contains recombinant TfR is available. Currently, the assays are still calibrated with preparations of intact sTfR, sTfR-transferrin complex, or mixtures /1/. This leads to non-comparable results between assays. The WHO reference reagent 07/202, which contains 21.7 mg/L (303 nmol/L) of sTfR when reconstituted with 0.5 mL of water, will be in use in near future /2/.

7.4.3 Specimen

Serum, plasma: 1 mL

7.4.4 Reference interval

Refer to Tab. 7.4-1 – Reference intervals for sTfR.

7.4.5 Clinical significance

The sTfR level in serum is dependent on the proteolytic cleavage of TfR expressed on the cell surface. The cells in individual tissues express TfR at different intensity levels. The highest loading is found in organs with high iron requirements such as bone marrow and the placenta. In the healthy adult, 80% of serum sTfR level originates in the bone marrow, where it is released by the erythropoietic precursor cells. The erythrocytes have no TfR.

The clinical significance of the sTfR can only be assessed by taking the clinical question into account, as this biomarker reflects erythropoietic activity as well as the iron status.

7.4.5.1 sTfR in the assessment of erythropoietic activity

The sTfR concentration varies with erythropoietic activity and therefore with the quantity of the erythropoietic precursor cells. It is a marker for the size of the erythroblast compartment. However, this is only the case when the storage iron reserves is adequate and available. Erythropoietic activity is /7/:

  • Elevated during hemolysis such as in autoimmune hemolytic anemia, hereditary spherocytosis, sickle cell anemia, secondary polycythemia or in stimulated ineffective erythropoiesis such as thalassemia, megaloblastic anemia or myelodysplastic syndrome
  • Reduced during hypo regenerative erythropoiesis such as in chronic kidney disease, intensive chemotherapy, aplastic anemia and after transfusion of banked blood.

Estimation of erythropoietic activity and functional classification of erythropoietic disorders

  • In combination with the reticulocyte count and the erythropoietin (EPO) concentration
  • In relation to the severity of the anemia (e.g., hemoglobin level or hematocrit).

Hypoproliferative erythropoiesis

The productive capacity of erythropoiesis for the maintenance of a normal Hct is reflected in the reticulocyte count and the sTfR level. An anemia associated with a low reticulocyte count indicates a hypo proliferative erythropoiesis. In terms of its functional classification and for therapy, it is important to know whether the anemia results from /8/:

  • Reduced proliferation through inadequately low EPO production
  • Intrinsic, EPO-independent erythropoietic hypo proliferation
  • Maturation disorders e.g., ineffective erythropoiesis)
  • Reduced erythrocyte lifetime (peripheral hemolysis).

The first step in differential diagnosis is to assess whether the EPO concentration is inadequately low (Fig. 7.4-1 – Relationship between serum erythropoietin and Hct). This is the case in chronic kidney disease, for example.

If the EPO concentration is adequate or even elevated, relative EPO deficiency or ineffective erythropoiesis (EPO > 100 U/L) can be excluded, and the anemia results from intrinsic hypo proliferative erythropoiesis. In this case, sTfR is assayed and evaluated in relation to the Hct (Fig. 7.4-2 – Relationship between serum sTfR concentration and HCT).

An inadequately low sTfR level in relation to the Hct indicates intrinsic hypo proliferative erythropoiesis. The causes are:

  • Deficiency in iron, vitamin B12, folic acid
  • Inflammatory conditions
  • Aplastic anemia or pure red blood cell aplasia. The sTfR level is lower than one third of the mean value of the reference range.

Hyper proliferative erythropoiesis

In hyper proliferative erythropoiesis, the sTfR level is increased due to expansion of the erythroblast compartment. Erythropoiesis can be:

  • Effective, which is the case in hemolytic anemias; the reticulocyte count is elevated
  • Ineffective, as is the case in vitamin B12 deficiency and folic acid deficiency anemia, iron deficiency anemia, and myelodysplastic syndrome. The reticulocyte count is not elevated.
  • In response to ESA therapy, sTfR concentration rises over the first 2 weeks following administration of EPO and are due to an increase in the erythroblast compartment.

7.4.5.2 sTfR in response to ESA therapy

An early predictor of response to ESA therapy is the increase of sTfR. In one study /9/, the response to ESA was effective when the sTfR concentration was normal at baseline and increased by more than 20% within 2 weeks of starting ESA treatment. The increase in sTfR is, however, not a recommended indicator of effective erythropoiesis.

7.4.5.3 sTfR in the assessment of iron status

The hemoglobin level has low diagnostic sensitivity for the detection of iron deficiency because:

  • In nutritional iron deficiency, the anemia is relatively mild, and in the early phase there is an overlap in the hemoglobin levels between iron-sufficient and iron-deficient patients
  • In developing countries, iron deficiency is associated with malnutrition and infections, resulting in a high prevalence of ACD. Serum ferritin is suitable for assessing the adequacy of iron stores in ACD only if it is reduced.

The plasma sTfR level is always elevated in iron deficiency anemia and reflects the iron demand of the erythroblast compartment /10/. Quantitative phlebotomy studies in healthy individuals have shown that the decrease in tissue iron following the depletion of iron stores is always accompanied by an increase in the sTfR concentration, which occurs before changes in the other biochemical markers of iron deficiency (e.g., transferrin saturation and zinc protoporphyrin) and also clearly before MCV and MCH levels start to decline /11/. In a study investigating the diagnostic efficacy of sTfR for the detection of iron deficient anemia in young women, the sTfR exhibited a diagnostic sensitivity of 79% and a specificity of 63% when hemoglobin was < 120 g/L, ferritin < 20 μg/L and zinc protoporphyrin > 1.4 μg/g hemoglobin /12/.

7.4.5.3.1 sTfR in conditions with reduced functional iron

Functional iron is the transferrin-bound extracellular iron content, which is approximately 4 mg. An increased sTfR concentration is an indicator of reduced circulating iron (i.e., the imbalance between tissue iron demand and iron supply). The supply depends on iron stores and the degree of their mobilization. The sTfR level in iron deficiency is a direct measure of plasma iron turnover (i.e., the amount of iron that is transported from the plasma to the bone marrow and tissues every day). When the sTfR concentration is elevated, little iron is bound to transferrin and transported (TfS < 16%).

When iron stores decline, serum ferritin levels drop until iron stores are depleted, at which time the ferritin concentration falls below the lower limit of the reference interval. With further iron loss, and as iron-deficient erythropoiesis begins, sTfR begins to increase and continues to do so as the severity of iron-deficient erythropoiesis increases, reflecting the increasing number of receptors on the erythroid cells /11/.

The sTfR (mg/L)/log10 ferritin (μg/L) ratio, or ferritin index, is inversely related to iron status. It is an indicator of iron supply for erythropoiesis and shows better correlation with iron deficiency than the isolated determination of ferritin and sTfR /13/.

7.4.5.3.2 sTfR in anemia of chronic disease

One problem in the differential diagnosis of microcytic/borderline normocytic erythropoiesis in patients with inflammatory conditions or malignant tumors (anemia of chronic disease; ACD) is the detection of iron-restricted erythropoiesis.

The ACD is normally normocytic and normochromic, but in about 10% of cases it is hypochromic. To establish whether the hypochromia is due to ACD combined with iron deficiency, it can be useful to determine the sTfR level, or better, the ferritin index, because, in contrast to ferritin and TfS, the sTfR level does not change in inflammatory conditions. An increase in sTfR or in the ferritin index in ACD indicates ACD/IRE, a combined state of ACD and iron-restricted erythropoiesis (IRE) /14/. However, not all cases of IRE are detected, because sTfR can be low normal in ACD and there must be marked iron deficiency for sTfR to exceed the upper reference interval value /14/. This does not occur in all cases. Tab. 7.4-2 – Differentiation of iron deficiency anemia from ACD and the combined state of ACD/IRE shows the sTfR threshold for differentiating iron deficiency anemia from ACD and combined state of ACD with the IRE (ACD/IRE). Tab. 7.4-3 – sTfR in diseases and various clinical settings shows the behavior of sTfR in iron deficient states.

In ACD, the hypo proliferative erythropoiesis is usually due to a combined disorder /8/:

  • EPO production is not increased adequately in relation to the decrease in the Hct
  • In addition there is intrinsic hypo proliferative erythropoiesis due to inhibition by inflammatory cytokines and increase in hepcidin.

7.4.6 Comments and problems

Method of determination

Immunoassays with monoclonal and polyclonal antibodies are available commercially. Due to the lack of use of the standard preparation, there is variation in the results obtained with assays from different diagnostics manufacturers.

One problem in the preparation of a standard is that the iron status influences the structure of the sTfR-Tf complex in the circulation. The immunoreactivity of sTfR is considerably reduced when sTfR is not complexed with transferrin. A stable complex is only formed when Tf is saturated with iron /26/.

Reference interval

Some assays are reported to be age- and sex-dependent, others aren’t. Children have higher levels than adults. In children, levels decrease with increasing age /4, 27/. Sex-specific reference ranges have been specified for one manufacturer’s assay /6/.

Stability

Stable for at least 1 week at room temperature (20 °C) and at 4–8 °C. In whole blood, levels rise progressively with storage time. This is also reported to be the case with EDTA whole blood and EDTA plasma /28/. The cause is reported to be the progressive separation of the TfR from reticulocytes and leukocytes.

7.4.7 Pathophysiology

The TfR is a glycoprotein that is located on the cell membrane of nucleated cells. Its function is to transport Tf-bound Fe(III) into the cytoplasm.

TfR is a heterodimer composed of two identical transmembrane subunits of 85 kDa each in size. Each of the two units is composed of the following domains (Fig. 7.4-3 – Transferrin receptor (TfR)) /29/:

  • The C-terminal unit composed of 671 amino acids
  • The transmembrane unit composed of 28 amino acids
  • The N-terminal intracellular unit composed of 61 amino acids.

The extracellular domain contains two N-glycosylated side chains on asparagine residues and one O-glycosylated side chain on threonine. The carbohydrate side chains are functionally important. Mutations with the loss of the glycosyl chains exhibit a lower affinity for Tf. The receptor undergoes rapid proteolytic cleavage if the glycosyl chain on the threonine is lost. The extracellular domain possesses a trypsin-sensitive region at which it can be proteolytically cleaved from the cell membrane. This results in a 70 kDa fragment that retains its capacity for binding Tf extracellularly, but not intracellularly.

Each TFR has binding sites for two Tf molecules loaded with iron. Signal transmission into the cytoplasm via phosphorylated serine residues on the N-terminal intracellular unit follows the binding of Tf to TfR. This triggers endocytosis, leading to internalization of the iron-loaded Tf-TfR complex into the cytoplasm. See Fig. 7.1-2 – Cellular iron uptake from transferrin).

TfR exhibits a binding constant for iron-saturated (diferric) Tf that is 30- to 500-fold higher than for apo-Tf and mono ferric Tf. Even when the proportion of iron-saturated Tf makes up only 10% of total plasma Tf, this is sufficient to saturate TfR with its ligand /29/.

TfR expression occurs post-transcriptionally via iron regulatory elements (IREs) and iron regulatory proteins (IRPs). See also Fig. 7.1-6 – Post-transcriptional regulation of cellular iron homeostasis.

Highest receptor densities in any organ are found in the erythroblast compartment and the placenta. The polychromatic normoblast has a receptor density of 800,000/cell, the orthochromatic normoblast and reticulocytes exhibit densities of 500,000 to 100,000. The presence of the receptor on the cell membrane can be determined using flow cytometry with the monoclonal antibody CD 71. The cells shed their receptors during maturation, in particular during maturation of orthochromatic erythroblast to erythrocyte, and with the loss of hemoglobin synthesis.

Approximately 70–80% of sTfR that are detected in plasma stems from erythropoiesis. Multi vesicular corpuscles appear in the plasma following transport to the cell surface of the TfR-Apo-Tf complexes that have been relieved of their iron. The complexes have the extracellular TfR domain on their surfaces. In the plasma, this domain is then proteolytically cleaved from the surface by proteases (e.g., by leukocytes) and is then a soluble fragment of approximately 85 kDa, the sTfR.

Depending on the antibody used, the sTfR concentration measured in the immunoassay can include:

  • The 85 kDa fragment
  • A Tf complex and one, possibly even two receptor fragments (MW of 250 kDa)
  • The TfR-Tf complex that is still bound to vesiculas.

There is a constant relationship between tissue TfR content and serum sTfR level. This depends on:

  • Cell turnover in the erythroblast compartment (i.e., erythropoietic proliferation)
  • TfR expression on the cell membranes of erythroid precursor cells. There is a direct relationship between the number of TfR on the cell surface and the serum sTfR concentration in iron-restricted erythropoiesis.
  • sTfR is supposed as a possible iron-requirement regulator. This is supported by the fact that patients suffering from thalassemia exhibit increased iron absorption and elevated sTfR values despite full iron stores.
  • TfR expression on the cell membrane and the cellularity of the erythroblast compartment is subnormal to normal in combinations of ACD with iron-restricted erythropoiesis. Even so, elevated sTfR levels are measured in some of these patients /14/. One possible explanation is increased TfR shedding by maturing erythroblasts.

sTfR concentrations are normal, and in some cases elevated in myelodysplastic syndrome (MDS). However, receptor expression on the cell membrane is reduced. Massive hyper cellularity in the erythroblast compartment due to ineffective erythropoiesis is the cause of sTfR concentrations that are normal or even elevated in MDS /30/.

References

1. Cook JD, Skikne BS, Baynes RD. Serum transferrin receptor. Annu Rev Med 1993; 44: 63–7.

2. Thorpe SJ, Heath A, Sharp G, Cook J, Ellis R, Worwood M. A WHO reference reagent for the serum transferrin receptor (sTfR): international collaborative study to evaluate a recombinant soluble transferrin receptor preparation. Clin Chem Lab Med 2010; 48: 815–20.

3. Vernet M, Doyen C. Assessment of iron status with a new fully automated assay for transferrin receptor in human serum. Clin Chem Lab Med 2000; 38: 437–42.

4. Ooi CL, Lepage N, Nieuwenhuys E, Sharma AJ, Filler G. Pediatric reference intervals for soluble transferrin and transferrin receptor-ferritin index. World J Pediatr 2009; 5: 122–6.

5. Suominen P, Punnonen K, Rajamäki A, Irjala K. Evaluation of a new immunoenzymometric assay for measuring soluble transferrin receptor to detect iron deficiency in anemic patients. Clin Chem 1997; 43: 1641–6.

6. Kolbe-Busch S, Lotz J, Hafner G, Blanckaert NJC, Claeys G, Togni G, Carlsen R, Röddiger R, Thomas L. Multicenter evaluation of a fully mechanized soluble transferrin receptor assay on the Hitachi and Cobas Integra analyzers. The determination of reference ranges. Clin Chem Lab Med 2002; 40: 529–36.

7. Beguin Y, Fillet G. Monitoring of erythropoiesis by the serum transferrin receptor and erythropoietin. Acta Clin Belgica 2001; 56: 146–54.

8. Beguin Y, Clemons GK, Pootrakul P, Fillet G. Quantitative assessment of erythropoiesis and functional classification of anemia based on measurements of serum transferrin receptor and erythropoietin. Blood 1993; 81: 1067–76.

9. Beguin Y, Loo M, R’Zik S, et al. Early prediction of response to recombinant human erythropoietin in patients with anemia of renal failure by serum transferrin receptor and fibrinogen. Blood 1993; 82: 2010–6.

10. Baynes RD, Skikne BS, Cook JD. Circulating transferrin receptors and assessment of iron status. J Nutr Biochem 1994; 5: 322–30.

11. Skikne BS. Circulating transferrin receptor assay – coming of age. Clin Chem 1998; 44: 7–9.

12. Lin XM, Zhang J, Zou ZY, Long Z, Tian W. Evaluation of serum transferrin receptor for iron deficiency in women of childbearing age. Br J Nutr 2008; 100: 1104–8.

13. Punnonen K, Irjala K, Rajarnäki A. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood 1997; 89: 1052–7.

14. Thomas C, Thomas L. Biochemical markers and hematologic indices in the diagnosis of functional iron deficiency. Clin Chem 2002; 48: 1066–76.

15. Huebers HA, Beguin Y, Pootrakul P, Einspahr D, Finch CA. Intact transferrin receptors in human plasma and their relation to erythropoiesis. Blood 1990; 75: 102–7.

16. Kogho Y, Niitsu Y, Kondo H, et al. Serum transferrin receptor as a new index of erythropoiesis. Blood 1987; 70: 1955–8.

17. Carmel R, Skikne BS. Serum transferrin receptor in megaloblastic anemia of cobalamin deficiency. Eur J Haematol 1992; 49: 246–50.

18. Beguin Y, Lipscei G, Thoumsin H, Fillet G. Inappropriate erythropoietin production is responsible for defective erythropoiesis in early pregnancy. Blood 1991; 78: 89–93.

19. Choi JW, Woon WI, Soo HP. Serum transferrin receptor during normal pregnancy. Clin Chem 2000; 46: 725–7.

20. Sweet DG, Savage GA, Tubman TRJ, Lappin TR, Halliday HL. Study on maternal influences on fetal iron status at term using cord blood transferrin receptors. Arch Dis Child Fetal Neonatal Ed 2001; 84: F40–F43.

21. Kuiper-Kramer EPA, Baerts W, Bakker R, van Eyck J, van Raan J, van Eijk HG. Evaluation of the iron status of the newborn by soluble transferrin receptor. Clin Chem Lab Med 1998; 36: 17–21.

22. Sweet DG, Savage GA, Tubman R, Lappin TR, Halliday HL. Cord blood transferrin receptors to assess fetal iron status. Arch Dis Child Fetal Neonatal Ed 2001; 85: F46–F48.

23. Kivivuora SM, Anttila R, Vinikka L, Pesonen K, Siimes MA. Serum transferrin receptor for assessment of iron status in healthy prepubertal and early pubertal boys. Pediatr Res 1993; 34: 297–9.

24. Mehta AB, McIntire N. Haematological disorders in liver disease. Forum 1998; 8: 8–25.

25. Nagral S, Mehta AB, Gomes ATB, Ellis G, Jackson BFA, Sabin CA, McIntire N. Serum soluble transferrin receptor in the diagnosis of iron deficiency in chronic liver disease. Clin Lab Haem 1999; 21: 93–7.

26. Kato J, Kubone M, Kohgo Y, Fujikawa K, Takimoto R, Torimoto Y, et al. Ratio of transferrin (Tf) to Tf-receptor complex in circulation differs depending on Tf iron saturation. Clin Chem 2002; 48: 181–3.

27. Choi JW, Soo HP, Woon WI, Soon KK. Change in transferrin receptor concentrations with age. Clin Chem 1999; 45: 1562–3.

28. de Jongh, Vranken J, Vundelinckx G, Bosmans E, Maes M, Heylen R. The effects of anticoagulation and processing on assays of IL-6, sIL-6R, sIL-2R and soluble transferrin receptor. Cytokine 1997; 9: 696–701.

29. Wang S, He X, Wu Q, Chen JL, Yu Y, Zhang P, Huang X, et al Transferrin receptor 1-mediated iron uptake plays an essential role in hematopoiesis. Haematologica 2020; 105. doi: 10.3324/haematol.2019.224899.

30. Kuiper-Kramer EPA, Coenen JLLM, Huisman CMS, Abbes A, Van Raan J, van Eijk HG. Relationship between soluble transferrin receptors in serum and membrane-bound transferrin receptors. Acta Haematol 1998; 99: 8–11.

31. Aisen P. Transferrin, transferrin receptor, and the uptake of iron by cells. In: Sigel H (ed.) Metal ions in biological systems, vol 35. New York; Marcel Dekker 1998: 585–631.

32. Garbowski MW, Evans P, Vlachodimitropoulou. Hider R, Porter JB. Residual erythropoiesis protects against myocardiak´l hemosiderosis in transfusion dependent thalassemia by lowering labile plasma iron via transient generation of apotransferrin. Heamatologica 2017; 102: 1640–9

7.5 Transferrin saturation (TfS)

Lothar Thomas

Iron is transported in plasma bound to the protein transferrin (Tf). Each transferrin molecule can carry a maximum of two Fe(III) atoms, which corresponds to 1.4 mg of iron per gram of transferrin. Diferric transferrin circulates in the blood and provides iron to most cells of the body. TfS is the ratio of iron/transferrin concentration in serum or plasma and is expressed in %. The saturation of transferrin with iron is a major indicator and determinant of systemic iron availability.

7.5.1 Indication

  • Suspected lack of iron availability
  • Suspected iron overload
  • Evaluation of plasma iron turnover.

7.5.2 Method of determination

Iron and Tf concentrations are determined from the same serum sample after blood sampling in the morning. TfS (%) is calculated using the equations shown in Tab. 7.5-1 – Calculation of transferrin saturation and is based on the following:

  • The molecular weight of Tf is 79,570 Da
  • Each molecule has two binding sites for iron. Therefore, 1 g of Tf binds 1.4 mg (25.1 μmol) of iron /1/.

7.5.3 Specimen

Serum, plasma (no EDTA plasma): 1 mL

Blood should be collected in the fasting period.

7.5.4 Reference interval

Refer to Tab. 7.5-2 – Reference intervals for transferrin saturation.

7.5.5 Clinical significance

Tf is responsible for the body’s iron turnover and supplies the tissues, in particular erythropoiesis, with iron. Tf-bound iron is available as functional iron for the synthesis of hemoglobin and iron-containing enzymes. The iron is derived predominantly from hemoglobin released in the breakdown of senescent erythrocytes (Fig. 7.5-1 – Iron turnover between the different compartments). The iron content of the hepatocyte regulates the Tf concentration in plasma. If it is low, Tf synthesis is increased; if it is high, Tf synthesis is down regulated.

Does the doctor want to know if iron deficiency is the reason for anemia TfS is ordered.

TfS may show the following pattern:

  • TfS is decreased if there is insufficient intestinal iron absorption with unchanged iron demand. This is the case in nutritional iron deficiency.
  • TfS is below 20% if in inflammatory conditions, Tf synthesis is down regulated and iron turnover is reduced because iron is captured in macrophages and hepatocytes. This is the case in anemia of chronic disease (ACD), for example.

TfS is elevated if there is increased intestinal iron absorption with unchanged iron demand, as is the case in hereditary hemochromatosis. For diagnosis and differentiation of iron overload using TfS and ferritin refer to Fig. 7.3-1 – Differentiation of non-anemia-related hyper ferritinemia based on ferritin, transferrin saturation and CRP.

A TfS < 16% indicates inadequate iron supply for erythropoiesis (iron-restricted erythropoiesis), TfS < 10% indicates total body iron deficiency, and TfS < 20% is common in inflammatory conditions because of iron sequestration in the reticulo endothelial system /5/.

According to the Network for Advancement of Transfusion Alternatives (NATA), orthopedic surgery patients should have normal hemoglobin levels (women ≥ 120 g/L, men ≥ 130 g/L). If hemoglobin is decreased, pre- and intraoperative iron therapy is recommended if TfS is < 20% and/or ferritin < 30 μg/L /6/.In anemic patients with chronic kidney disease a probatory iron dose is recommended in cases if TfS is < 30% /15/.

The behavior of TfS in iron deficiency is shown in Tab. 7.5-3 – Behavior of transferrin saturation in conditions of iron deficiency.

The behavior of TfS in iron overload is shown in Tab. 7.5-4 – Behavior of transferrin saturation in conditions of iron overload.

The limitations of TfS are presented in Tab. 7.5-5 – Limitations of TfS.

Plasma iron turnover is a measure of the amount of iron transported in the plasma every day (Fig. 7.5-1 – Iron turnover between the different compartments). TfS is an indicator of the turnover. If TfS is low (iron deficiency), turnover is reduced, if it is high (iron overload), turnover is high. This correlation applies only to a limited extent in ACD, since the relationship between iron turnover and serum transferrin is impaired due to down regulation of transferrin synthesis.

Used in isolation, TSAT has poor sensitivity and specificity in detecting those who respond to intravenous iron. Under oral iron therapy with a dose of up to 5 mg/day, approximately 60% is absorbed intestinally, with a dose of 100 mg only 10%. Part of the remainder is then passively transported into the blood via the enterocytes. If TfS is greater than 60%, the iron in blood no longer binds only to Tf, but also to other plasma proteins such as albumin.

Patients with iron overload begin to have non-transferrin bound iron when the transferrin saturation exceeds 70%. The species that form this iron pool are dominated by iron citrate and iron-albumin complexes. Non-transferrin bound iron is taken up inappropriately by high vascular organs such as the liver, heart and pancreas leading to elevated levels of intracellular iron, with non-transferrin bound iron gaining intracellular access through iron permeases. Ideally the administration of therapeutic iron chelators should remove non-transferrin-bound iron. Deferiprone rapidly scavenges iron from non-transferrin-bound iron /14/.

7.5.6 Comments and problems

Sampling

Blood should always be sampled in the morning. When screening for hereditary hemochromatosis or secondary iron overload, two results from samples collected on different days should be evaluated. Samples should be collected in a fasting period, because food intake causes iron levels to rise, leading to a falsely elevated result for TfS.

Calculation of transferrin saturation

TfS must only be calculated from results obtained from the same sample. There must be no acute-phase response (CRP normal), otherwise TfS will be falsely low.

References

1. Vernet M. Immunochemical assay of transferrin and iron saturation in serum. Clin Chem 1993; 39: 2352–3.

2. Lentjes EGWM, Lindeman JHN, van de Bent W. Measured versus calculated iron binding capacity inplasma of newborns. Ann Clin Biochem 1995; 32: 478–81.

3. Higgins V, Cham MK, Adeli K. Pediatric reference intervals for transferrin saturation in the CALIPER cohort of healthy children and adolescents. eJIFCC 2017; 28: 77–84.

4. Koerper MA, Dallman PR. Serum iron concentration and transferrin saturation in the diagnosis of iron deficiency in children: normal developmental changes. J Pediatr 1977; 91: 870–4.

5. Bothwell TH, Charlton RW, Cook JD, Finch CA. Iron metabolism in man. Oxford, England 1979: Blackwell Scientific

6. Goodnough LT, Maniatis A, Earnshaw P, Benoni G, Beis P, Bisbe E, et al. Detection, evaluation, and management of preoperative anaemia in the elective orthopaedic surgical patient: NATA guidelines. Br J Anaesthesia 2011; 106: 13–22.

7. Baynes RD. Assessment of iron status. Clin Biochem 1996; 29: 209–15.

8. Yip R, Johnson C, Dallman PR. Age-related changes in laboratory values used in the diagnosis of anemia and iron deficiency. Am J Clin Nutr 1984; 39: 427–36.

9. Ullrich C, Wu A, Armsby C, Rieber S, Wingerter S, Brugnara C, et al. Screening healthy infants for iron deficiency using reticulocyte hemoglobin content. JAMA 2005; 294: 924–30.

10. Means RT, Krantz SB. Progress in understanding the anemia of chronic disease. Blood 1992; 80: 1639–47.

11. European Best Practice Guidelines for the Management of Anemia in Patients with Chronic Renal Failure. Nephro Dial Transpl 1999; 14, Suppl 5: 14–5.

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14. Hider RC, Hoffbrand AV. The role of deferiprone in iron chelation. N Engl J Med 2018; 379 (22): 2140–50.

15. KDIGO 2012 Clinical Practice Guideline for the evaluation and management of chronic kidney disease. Kidney Int 2013 (suppl) 2013; 3 (1).

7.6 Hepcidin

Lothar Thomas

7.6.1 Hepcidin regulates iron metabolism

Human hepcidin is produced as an 84-amino acid pre pro hepcidin. Subsequent post translational processing results in the biologically active 25 amino acid form (hepcidin-25) that is secreted in the plasma. Although this peptide was first isolated as antimicrobial peptide from urine hepcidin is predominately expressed and produced by hepatocytes and in smaller amounts in the heart and brain. Hepcidin is the key regulator of systemic iron homeostasis. After entering the circulation hepcidin negatively regulates the export of iron in reticuloendothelial macrophages and enterocytes. In addition to these effects on body iron distribution, hepcidin directly inhibits erythroid-progenitor proliferation and survival.

The effects of hepcidin in the tissues are as follows:

  • Increasing concentrations negatively regulate the export of iron from enterocytes, hepatocytes, and reticuloendothelial macrophages. Increased plasma concentrations of hepcidin are indicators of increased storage iron and/or an inflammatory disorder. In addition to these effects on body iron distribution, hepcidin directly inhibits erythroid-progenitor proliferation and survival.
  • Diminished concentrations increase intestinal iron absorption and the release of iron from hepatocytes and macrophages. The following disorders are combined with low concentrations of hepcidin: iron overload, stimulation of erythropoiesis e.g., in hypoxia or with stimulating agents (ESA). Required iron is provided by increase in intestinal iron absorption /1/.

7.6.1.1 Regulation of iron transport in reticuloendothelial macrophages and duodenal enterocytes

Hepcidin blocks the intestinal absorption of iron and the release of iron from stores by inducing the internalization and degradation of the cellular iron exporter ferroportin. Iron retention in the macrophages reduces the release into plasma and the availability of iron for erythropoiesis. The iron retention in enterocytes decreases dietary iron absorption. Thus, an increase in hepcidin leads to anemia because of /2, 3/:

  • Increase in hepatocyte and macrophage iron
  • Decrease in dietary iron absorption
  • Decrease in circulating iron.

Hepcidin synthesis is strongly influenced by inflammation. The physiologic regulation of hepcidin formation is superseded by the up regulatory effects of inflammatory cytokines. Binding of interleukin-6 (IL-6) to its receptor results in phosphorylation of the intracellular signal transducer and activator of transcription 3 (STAT3) which acts in the nucleus with an IL-6 responsive elements in the hepcidin promoter. The synthesis of hepcidin is enhanced and increased concentrations of hepcidin restrict the iron supply for erythropoiesis and can lead to anemia of chronic disease. The increased synthesis of hepcidin during infection, inflammation and in cancer patients couples iron metabolism to host defense and decreases iron availability to invading pathogens.

Genetic disorders of the hepcidin-ferroportin axis cause diseases with iron overload or iron-restricted erythropoiesis. Characteristics and functions of hepcidin are summarized in Tab. 7.6-1 – Characteristics and functions of hepcidin.

7.6.2 Indication

  • Diagnosis and differentiation of hemochromatosis.

7.6.3 Method of determination

Isotope dilution micro HPLC tandem mass spectrometry /4/ and immunoassays /5/.

7.6.4 Specimen

Serum, fasting (blood collection by 9 a.m.): 1 mL

In most cases bio active hepcidin 25 is determined.

7.6.5 Reference interval

Depends on the method used. Isotope dilution micro HPLC tandem mass spectrometry for hepcidin-25 in serum: 0.5–23 nmol/L /6/.

7.6.6 Clinical significance

The systemic regulation of iron homeostasis is mediated by hepcidin. More than 80 % of anemias result from storage iron deficiency or functional iron deficiency. In functional iron deficiency iron stores are repleted, however due to inflammation iron is not released by hepatocytes and cells of the reticuloendothelial system causing functional iron deficiency. Patients with chronic heart failure, inflammatory bowel disease, chronic kidney disease, immune activation in infection, malignant tumor, and autoimmune disease often suffer from functional iron deficiency. Hepcidin can be a useful marker in the diagnosis and differentiation of impaired iron regulation and anemia.

There is a hyperbolic relationship between ferritin and hepcidin-25. Hepcidin-25 rises disproportionately with increasing ferritin levels. At a hepcidin concentration ≤ 0.2 nmol/L (limit of detection), serum ferritin is 9 μg/L /7/. A significant relationship was observed between hepcidin-25 and CRP, transferrin saturation (TSAT), sTfR, and the ferritin index, respectively. At a hepcidin level ≤ 0.2 nmol/L, TSAT is ≤ 14,3% /7/. There is no relationship between hepcidin and the hematological markers of iron metabolism, such as hemoglobin level, MCH,% HYPO, and the reticulocyte Hb content (CHr, RetHe) /8/. Disorders of hepcidin and ferroportin regulation are described in Tab. 7.6-2 – Disorders of hepcidin and ferroportin regulation.

Combining hepcidin and CHr in a diagnostic plot allows the differentiation between IDA, ACD, ACD/IDA and ACD/IRE (Fig. 7.6-1 – Differentiation of iron deficiency anemia using a diagnostic plot).

7.6.7 Comments and problems

Method of determination

Measuring ranges are between 0.2 and 200 nmol/L. Synthetic hepcidin-25 is used as a standard. 1 nmol corresponds to 2.789 μg. The detection limit of mass spectrometry assays is 0.2 nmol/L. Immunoassays are more or less sensitive. In an international round robin /14/, the different mass spectrometry assays showed acceptable agreement, but significant disagreement with the immunoassays. For predicting iron deficiency by ferritin < 15 ug/L yielded diagnostic sensitivity of 93.1% and specificity of 85.5%, whereas the same hepcidin cutoff for ferritin < 30 ug/L yielded sensitivity of 67.7% and specificity of 91.7% /29/.

Blood sampling

Blood should be sampled in the morning, since hepcidin levels rise during the day /4/.

Influence factors

Iron therapy and blood transfusions cause the hepcidin level to rise throughout the day. Findings suggest that these daily variations are mediated by an innate diurnal rhythm rather than dietary iron /15/.

7.6.8 Pathophysiology

Hepcidin, the systemic iron regulator

Hepcidin is the principal regulator of plasma iron and ensures a stable concentration of transferrin-bound iron (Tab. 7.6-1 – Characteristics and functions of hepcidin). Hepcidin is synthesized in the liver as an 84-amino acid precursor protein, including a 24 amino acid leader peptide. The human circulating active form consists of the C-terminal 25-amino acids of the protein (Fig. 7.6-2 – Structure of hepcidin-25).

Systemic iron release from the tissues is mediated by the cellular iron exporter ferroportin, which is expressed by all cells that are important in iron metabolism, such as macrophages, hepatocytes, and syncytiotrophoblasts. Hepcidin regulates the release of iron upon binding to ferroportin. After binding ferroportin is internalized into the cell and degraded in the lysosomes. (Fig. 7.6-3 – Binding of hepcidin to ferroportin in the enterocyte and macrophage cell membrane) /16/. The functionally relevant signal for the endocytosis of ferroportin is its ubiquitination which is triggered by the binding of hepcidin to ferroportin. Substitution of lysine in ferroportin region 229–269 inhibits ubiquitination /16/.

When iron stores are adequate or high, the liver produces hepcidin which circulates to the small intestine and causes ferroportin to be internalized, blocking the absorption of iron. When iron stores are low, hepcidin production is suppressed and ferroportin molecules are expressed on basolateral membranes of enterocytes transporting iron from the enterocyte to plasma transferrin. Similarly, the hepcidin ferroportin interaction also explains how macrophage recycling of iron is regulated /17/. The export of iron from enterocytes, hepatocytes and macrophages requires besides the hepcidin-ferroportin axis a ferrioxidase (hephaestin in enterocytes and ceruloplasmin in macrophages) to transform Fe2+ to Fe3+ for binding iron to transferrin. Refer to Fig. 7.1-4 – Regulation of iron content of the intracellular iron pool of the enterocyte.

Hepcidin production is stimulated by the increase of the intracellular labile iron pool or by inflammation induced by IL-6.

Mutations in the hepcidin gene lead to an ineffective hepcidin molecule. Intestinal iron absorption is therefore not regulated, and as a result iron overload develops /17/.

7.6.8.1 Regulation of hepcidin synthesis

Synthesis of hepcidin is influenced by two important regulators:

  • Stores regulator: the regulator influences the content of body iron stores. Replenning iron stores induce non-parenchymal cells to produce bone morphogenetic protein 6 (BMP6) The protein binds to its correspondend receptor on the surface of hepatocytes and activates SMAD signalling pathway for production of hepcidin. Increasing concentrations of hepcidin decrease the release of iron into the circulation and stimulate the storage of iron as ferritin or hemosiderin in the intestinal villi.
  • Erythroferrone: the regulator is produced in the bone marrow by erythroid precursor cells. In hemolytic disorders ineffective erythropoiesis needs more iron and lower concentrations of hepcidin. In ineffective erythropoiesis the proportion of erythroid precursor cells in the bone marrow is higher than in effective erythropoesis. Erythroid precursor cells produce erythroferrone that inhibits production of hepcidin and provides erythropoiesis with more iron.

7.6.8.2 Iron sensing

The sensing of iron is an important step for the stimulation and regulation of hepcidin /15, 17/:

The main iron-sensing tissue for systemic iron regulation are the hepatocytes. Iron released by macrophages or taken up by enterocytes is delivered to plasma transferrin and sensed by the hepatocytes.

Iron sensing by the hepatocytes

Iron sensing by the hepatocyte is mediated via bone morphogenetic proteins and the TfR-1 /18, 20/.

Iron sensing through bone morphogenetic protein (BMP) receptor is the standard pathway (Fig 7.6-4 – Signals and pathways for the regulation of hepcidin expression). The synthesis of BMP depends on the iron content of the hepatocyte. Excess iron in the labile iron pool of the hepatocyte causes the latter to release BMP, in particular BMP6, into the plasma. BMP6 is the key endogenous regulator of hepcidin expression. It is produced only in the hepatocyte, not in the enterocyte /18/. BMP6 activates its receptors BMPR I and BMPR II in the presence of the co receptor hemojuvelin /19/, forming a heterotetrameric complex of BMP6, the receptors and hemojuvelin /21/. The latter, after phosphorylation, complex with SMAD4 which trans locates to the nucleus to activate hepcidin (HAMP) transcription. In the presence of diferric transferrin the HFE-TfR-2 complex functions as an iron sensor on the hepatocyte surface and activates hepcidin to a still undefined pathway. Binding of hepcidin to ferroportin causes internalization and degradation of the iron exporter in duodenal enterocyte and blocking of iron release from macrophages.

Transmembrane serine protease 6 (TMPRSS6) inhibits hepcidin response by cleaving hemojuvelin from the heterotetrameric complex /22/. Neogenin, on the other hand, stabilizes the complex.

If the labile iron pool in the hepatocyte decreases, hemojuvelin (HJV) is released into the plasma. HJV competes with and can displace its membrane-bound form, and since it is likely functionally inactive, the function of the heterotetrameric BMP complex and thus the expression of hepcidin are inhibited /23/.

7.6.8.3 Hepcidin elevation in inflammation

Inflammatory conditions induce hepcidin expression, with the main regulator being IL-6. The IL-6 receptor activates the STAT-3 signaling pathway (STAT, signal transducers and activators of transcription). Inflammatory cytokines and lipopolysaccharides can also activate the endoplasmic reticulum. This induces hepcidin synthesis by forming c-AMP-responsive-element-binding protein H (CREBH). Hepcidin increase during infection causes depletion of extracellular iron, which is thought to be a general defense mechanism against many infections by withholding iron from invading pathogens. Conversely, by iron sequestration in macrophages, hepcidin action may be detrimental to cellular defense against certain intracellular pathogens /24/.

7.6.8.4 Suppression of hepcidin in hypoxia, anemia and iron deficiency

Hypoxia, anemia and iron deficiency inhibit hepcidin expression via hypoxic inducible factor, erythropoietin and the erythroid regulating factor erythroferrone (ERFE).

ERFE mediates hepcidin suppression during increased erythropoietic activity stimulated by endogenous erythropoietin. ERFE is produced by erythroid precursors in the marrow and the spleen and acts directly on the liver to decrease hepcidin production thereby increase iron availability for new blood cell synthesis /25/.

Hepcidin expression is also inhibited by iron-deficiency-induced elevated concentrations of soluble hemojuvelin, which competes with its membrane-bound form for binding to the heterotetrameric signal complex.

The plasma Tf concentration is a key determinant of hepcidin expression /26/. Tf deficiency leads to microcytic hypochromic anemia and insufficient hepcidin expression. Infusion of Tf normalizes plasma hepcidin.

Hepcidin concentration in serum is lower in patients with hepatitis C and alcoholic liver disease than in controls /30/.

References

1. Swinkels DW, Wetzels JFM. Hepcidin: a new tool in the management of anemia in patients with chronic kidney disease? Nephrol Dial Transplant 2008. doi: 10.1093/ndt/gfn267.

2. Ukarma L, Johannes H, Beyer U, Zaug M, Osterwalder B, Scherhag A. Hepcidin as a predictor of response to epoetin therapy in anemic cancer patients. Clin Chem 2009; 55: 1354–60.

3. Ganz T. Hepcidin and iron regulation, 10 years later. Blood 2011; 11/. 4425–33.

4. Kobold U, Dülffer T, Dangl M, Escherich A, Kubbies M, Röddiger R, Wright JA. Quantification of hepcidin-25 in human serum by isotope dilution mico-HPLC-tandem mass spectrometry. Clin Chem 2008; 54: 1584–1586.

5. Stoffel NU, Zeder C, Fort E, Swinkels DW, Zimmermann MB, Moretti D. Prediction of human iron bioavailability using rapid c-ELISAs for human plasma hepcidin. Clin Chem Lab Med 2017; 1186–92.

6. Galesloot TE, Vermeulen SH, Geurts-Moespot AJ, Klaver SM, Kroot JJ, van Tienoven D, et al. Serum hepcidin: reference ranges and biochemical correlates in the general population. Blood 2011; 117: e218–e225.

7. Thomas C, Kobold U, Thomas L. Serum hepcidin-25 in comparison to biochemical markers and haematological indices for the differentiation of iron-restricted erythropoiesis. Clin Chem Lab Med 2011; 49: 207–13.

8. Thomas C, Kobold U, Balan S, Roeddiger R, Thomas L. Serum hepcidin-25 may replace the ferritin index in the Thomas plot in assessing iron status in anemic patients. Internat J Hematology 2011; 33: 187–93

9. Price EA, Schrier SL. Unexplained aspects of anemia of inflammation. Advances in Hematology 2010. doi: 10.1155%2F2010%2F508739.

10. Agarwal N, Prchal JT. Anemia of chronic disease. Acta Haematol 2009; 122: 103–8.

11. Nemeth E. Hepcidin in β-thalassemia. Ann NY Acad Sci 2010; 1202: 31–5.

12. Van der Weerd NC, Grooteman MPC, Bots ML, van den Dorpel MA, den Hoedt CH, Mazairac AHA, et al. Hepcidin-25 in chronic hemodialysis patients is related to residual kidney function and not to treatment with erythropoiesis stimulating agents. Plos One 2012; 7: e39783s.

13. Peters HPE, Laarakkers CMM, Picckers P, Maserreeuv OC, Eek A, Cornelissen EAM, et al. Tubular reabsorption and local production of urine hepcidin-25. BMC Nephrology 2013; 14: 70.

14. Kroot JJC, Kemna EHJM, Bansal SS, Busbridge M, Campostrini N, Girelli D, et al. Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization. Haematologica 2009; 94: 1748–52.

15. Schaap CCM, Hendricks JCM, Kortman GAM, Klaver SM, Kroot JJC, Laarakkers CMM, et al. Diurnal rhythm rather than dietary iron mediates daily hepcidin variations. Clin Chem 2013; 59: 527–35.

16. Pietrangelo A. Hepcidin in human iron disorders: Therapeutic implications. J Hepatol 2010. doi: 10.1016/j.jhep.2010.08.004.

17. Ganz T. Hepcidin – a regulator of intestinal iron absorption and iron recycling by macrophages. Best Practice&research Clin Haematol 2005; 18: 171–82.

18. Camaschella C, Nai A, Silvestri L. Iron metabolism and iron disorders revisited in the hepcidin era. Haematologica 2020; 105 (2): 260–72.

19. Kautz L, Besson-Fournier C, Meynard D, Latour C, Roth MP, Copin H. Iron overload induces Bmpg expression in the liver but not in the duodenum. Haematologica 2010. doi: 10.3324%2Fhaematol.2010.031963.

20. Truska J, Peng H, Lee P, Beutler E. Bone morphogenetic proteins 2,4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6. PNAS 2006; 103: 10289–93.

21. Lin L, Valore EV, Nemeth E, Goodnough JB, Gabayan V, Ganz T. Iron transferrin regulates hepcidin synthesis in primary hepatocyte culture through hemojuvelin and BMP 2/4. Blood 2007; 110: 2182–9.

22. Ramsay AJ, Hooper JD, Folgueras AR, Velasco G, Lopez-Otin C. Matriptase-2 (TMPRSS6): a proteolytic regulator of iron homeostasis. Haematologica 2009; 94: 840–9.

23. Schmidt PJ, Toran PT, Giannetti PJ, Andrews NC. The transferrin receptor modulates HFE-dependent regulation of hepcidin expression. Cell Metabolism 2008; 7: 205–14.

24. Michels K, Nemeth E, Ganz T, Mehrad B. Hepcidin and host defense against infectious diseases. PloS Pathog 2015; 11 (8): e1004998. doi: 10.1371/journal.ppat.1004998.

25. Mirciov CSG, Wilkins SJ, Hung GCC, Helman SL, Anderson GJ, Frazer DM. Circulating iron levels influence the regulation of hepcidin following stimulated erythropoiesis.Haematologica 2018; 103 (10): 1616–26.

26. Bartnikas TB, Andrews NC Fleming MD. Transferrin is a major determinant of hepcidin expression in hypotransferric mice. Blood 2010. doi: 10.1182/blood-2010-05-287359.

27. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary hemochromatosis. Nature Genetics 1996; 13: 399–408.

28. Millot S, Delaby C, Moulouel B, Lefebvre T, Pilard N, Ducrot N, et al. Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model. Haematologica 2017; 102: 260–70.

29. Gutschow P, Han H, Olbina G, Westerman K, Nemeth E, Ganz T, et al. Clinical immunoassay for human hepcidin predicts iron deficiency in first-time blood donors. JALM 2020. doi: 10.1093/jalm/jfaa038.

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Table 7.1-1 Prevalence of iron deficiency (ID) and iron deficiency anemia (IDA)

Group

Age

ID

(%)

IDA

(%)

Women /63/

20–49 yrs

11

5

50–69 yrs

5

2

> 70 yrs

7

–

Men /63/

20–49 yrs

< 1

< 1

50–69 yrs

2

–

> 70 yrs

4

–

Pregnant women /63/

1. trimester

9

–

2. trimester

14

–

3. trimester

37

–

Children  /58, 59/

9–12 months

11

3

1–2 yrs

9

3

> 2–20 yrs

9–11

2–5

Global anemia prevalence /60/

Children
(%)

Women
(%)

Men
(%)

Africa

60

41

28

Latin America

46

23

11

Eastern Mediterranean

63

44

17

Southeast Asia (Indonesia, Sri Lanka, Thailand)

49

49

32

Southeast Asia (Bangladesh, India, Myanmar, Nepal)

66

60

36

North America

7

8

5

Table 7.1-2 States of iron restriction

Clinical and laboratory findings

Storage iron deficiency (ID)

A negative iron balance primarily reduces storage iron (mobilizable iron). When iron stores are depleted, but functional iron (circulating iron) is unchanged, then the body iron content is reduced. This is the earliest stage of iron deficiency.

Laboratory findings: ferritin < 30 μg/L, transferrin saturation (TfS) is normal (≥ 16–20%).

Subclinical (ID)

Condition is identical with storage iron deficiency.

Absolute (total) ID

All compartments and iron-dependent functions of the body are affected by iron deficiency.

Iron-deficiency anemia (IDA)

If functional iron levels are markedly reduced and iron stores are depleted, the iron supply for erythropoiesis required for normal hemoglobin synthesis can no longer be maintained, anemia results.

Laboratory findings: microcytic hypochromic anemia, ferritin ≤ 30 μg/L, TfS < 16–20%, soluble transferrin receptor (sTfR) level elevated, ferritin index (sTfR/log10 ferritin) increased, zinc protoporphyrin ≥ 100 μmol/mol heme, reticulocyte hemoglobin content (CHr, RetHe)< 28 pg, and the proportion of hypochromic red cells (%HYPO) > 3,8%.

Iron restriction /61/

Iron restriction is the common mechanism in several clinical settings via four conditions:

  • Absolute iron deficiency
  • Iron sequestration (impaired iron trafficking). The common mechanism of iron sequestration reflects reticuloendothelial blockade of iron and occurs in chronic inflammatory conditions. In this setting, increased hepcidin stops reticuloendothelial iron export. However, the high capacity of transferrin uptake preserves hemoglobin synthesis of the erythroblasts despite systemic iron restriction. Red cells are normocytic normochromic, however the red cell count is decreased (hepcidin inhibits proliferation of erythropoiesis).
  • Functional iron deficiency (imbalance between the surging iron requirements of the stimulated erythroid marrow and iron availability)
  • Hereditary conditions with impaired iron transport and utilization.

Functional ID

Functional iron deficiency is characterized by impaired iron release from body stores that is unable to meet the demand for erythropoiesis. Iron sequestration syndrome is the most important reasons for functional iron deficiency. Conditions related to functional ID are chronic heart failure, chronic kidney disease, and inflammatory bowel disease.

Laboratory findings: ferritin ≥ 100 μg/L, transferrin saturation < 20%, normocytic normochromic anemia in most cases.

Thrombocyte Apheresis Donors /72/

Repeat apheresis donation has a noticeable effect on the iron store of the blood donors leading to a high percentage of iron-deficient donors, especially in females. Thrombocyte donors have lower ferritin values and show higher rates of iron deficiency than people who do not donate blood.

Table 7.1-3 Iron deficiency in healthy individuals and diseases

Clinical and laboratory findings

Children

During the first years of life the iron status changes significantly due to the child growing and the consecutive increase in the red blood cell mass. Neonatal iron stores are usually relatively large compared to those at the end of the first year of life (see also Section 7.3 – Ferritin). In the USA 65% of newborns to diabetic mothers, 50% of newborns with growth retardation and 5% of newborns without complications have iron deficiency /24/. Children born to mothers with iron deficiency anemia are 6.6 times more likely to develop the condition themselves by the end of the first year of life than children born to mothers who had normal iron status during pregnancy /25/. The cause of iron deficiency in children born to diabetic mothers and children with intrauterine growth retardation is thought to be impaired feto-maternal transfer of iron or increased erythropoiesis due to intrauterine hypoxemia. One of the main causes of iron deficiency in the first year of life is the feeding of cow’s milk, which contains only 0.6 mg of iron per liter. Approximately 26% of infants who are started on cow’s milk before the age of 6 months have iron deficiency at 2 years, while those fed breast milk or formula diet have normal iron status /25/. The prevalence of iron deficiency in children under the age of 5 is 4–20% in industrialized countries and up to 80% in developing countries. During puberty, erythropoiesis is increased 2–3-fold. Boys require large amounts of iron for muscle development and increasing erythrocyte mass over 2 years. During this period, the amount of storage iron increases by about 50%, and the amount of iron absorbed by the intestine is at least 4 times higher than that of available storage iron /26/. The iron deficiency not only causes anemia, but also has negative effects on cognitive function, growth, motor functions, and immune function in protecting against infectious pathogens.

Blood donor

Blood donors lose 200–250 mg of iron when donating one banked blood unit, corresponding to 0.5 mg of iron per mL of blood with an Hb level of 150 g/L. Approximately 6% of male multiple blood donors exhibit a higher prevalence of iron deficiency if 4 units are donated per year /27/. The prevalence of latent iron deficiency in blood donors is approximately the same (6% in men, 11% in women) /28/. Studies of elderly blood donors showed that donating 5 units of blood over one year results in depletion of iron stores within 3 years despite iron substitution, but does not lead to anemia. It is believed that, once ferritin levels have declined to 20 μg/L, intestinal iron absorption increases sufficiently to meet the body’s iron requirement /29/. Preoperative autologous blood donation results in a significant decrease in total body iron within a short period of time.

Pregnancy

During the 280 days of gestation, pregnant women lose about 3 mg of iron/day (840 mg) through iron transfer to the fetus and through delivery. Up to 2 years of normal dietary iron intake is required to replace this loss. To avoid iron deficiency during pregnancy, 500 mg of stored iron is theoretically required. These amounts are present in only 20% of women of childbearing age, 40% have 100–500 mg, and 40% have no stored iron. Daily iron requirement increases from 0.8 mg/day in early pregnancy to 7.5 mg/day in late pregnancy. Despite increased iron absorption, about 20% of pregnant women have anemia due to insufficient or lack of iron supplementation. Multiple successive pregnancies and breast feeding add to the iron deficit, with lactation resulting in an additional loss of 0.5–1 mg of iron per day /30/. Iron deficiency anemia during pregnancy is associated with a more than 2-fold increased risk of premature birth. The same applies to anemia during the first half of pregnancy. Early iron therapy reduces this risk.

Hemorrhage

Gastrointestinal bleeding is the most common cause of iron deficiency. In one study /31/, 62 of 100 consecutive inpatients with iron deficiency anemia suffered bleeding in the upper or lower gastrointestinal tract. Approximately 58% of patients exhibited a bleeding source in the upper gastrointestinal tract, most commonly a peptic ulcer, while 40% exhibited bleeding in the colon, most commonly due to polyps or a carcinoma.

Nutritional iron deficiency

Heme and elementary iron are the sources of daily dietary iron. Heme iron is contained in meat, fish and poultry and has a high bio availability since it is absorbed bound to the porphyrin ring. The gastrointestinal tract absorbs only 2–10 mg of elementary iron per day, even if doses of 50–100 mg are administered. If an iron dose of 5 mg is administered, only 60% of it is absorbed, if 100 mg is given, only about 10% is absorbed. Fe3+ is not absorbed, since it is not soluble at a pH above 3. The absorption of elementary iron depends on variables such as food preparation, digestion, galenics of the iron preparation, and the content of natural ligands which may inhibit iron absorption. Such ligands include /32/:

  • Phytates, which make up 1–2% of cereals, nuts, and legumes. Adding 10 mg of phytates to a phytate-free bread-based meal reduces iron absorption to 41%.
  • Poly phenols, which can be found in vegetables, tea and legumes, have an inhibitory effect, which is mainly due to the iron-binding by galactans
  • Doses of 165 mg of calcium in the form of milk, cheese or calcium chloride reduce elementary iron absorption by 50%.

The high proportion of cereals and vegetables and the low proportion of meat is one of the main causes of dietary iron deficiency in developing countries. In industrialized countries, the iron content is about 6 mg per 1,000 kcal. One study /15/ reported a daily iron intake of 12–15 mg for men and 7.5–11 mg for women, with heme iron accounting for 10–13%. Since heme iron is about 4 times more easily absorbed than elementary iron, it is the dietary composition rather than the absolute iron content in food that is important /33/. Since meat also increases elementary iron absorption, it can be assumed that in industrialized countries, which have a higher meat consumption than developing countries, the direct and indirect effects of meat provide more than 50% of the daily iron supply /32/.

Worm infection

Infection with hookworm, Ancylostoma duodenale and Necator americanus can cause a daily blood loss of 5 mL and thus a 2.5 mg loss of iron. Over the years this leads to iron deficiency. Infection with Trichuris trichiuria only rarely causes iron deficiency /34/.

HookWorm infestation

Hookworm infestation is associated with iron deficiency anemia. Nector americanus and Ancylostoma duodenale are the most soil transmitted helminth that cause anemia. It is frequently in Sub Saharan Africa and South Asia. Adult worms attach the mucosa and sub-mucosa of the small intestine, damage the capillaries and arterioles, release anticoagulant and ingest 0.03 to 0.15 mL of extravascular blood every day /34/.

Malabsorption

Ferrokinetic studies have shown that patients with chronic blood loss or nutritional iron deficiency absorb ≥ 50% of the 59Fe administered. Patients with incomplete (partial) malabsorption, idiopathic or secondary after gastrectomy, absorb 10–49%, and those with serious gastrointestinal diseases such as celiac disease or Crohn’s disease of the upper gastrointestinal tract less than 10% /35/. The lack of iron absorption is thought to be due to increased loss of iron-containing enterocytes.

Table 7.1-4 Scoring system for the diagnosis of iron-restriction in non-anemic patients with ferritin levels > 30 μg/L. A number of 3 points indicates iron restriction /18/.

Test

Score

sTfR > 1.17 mg/L (S), > 3.0 mg/L (R)

1

Ferritin index (sTfR/log10 ferritin) > 0,81 (S), > 2,28 (R)

2

R, sTfR assay of Roche; S, sTfR assay of Siemens; ferritin expressed in μg/L

Table 7.1-5 Heritable forms of systemic iron overload /4/

Type

Presentation

Gene

Protein

Inheritance

Pathophysiology

Impaired hepcidin-ferroportin axis

Type 1 HH

A

HFE

HFE

AR

Inactive protein due to mutation in the HFE gene; signaling for hepcidin synthesis is therefore impaired

Type 2A HH

J

HJV

HJV

AR

Mutation in HJV, synthesis of hemojuvelin is impaired

Type 2B HH

J

HAMP

Hepcidin

AR

Mutation in HAMP, synthesis of hepcidin is impaired

Type 3 HH

A

TFR2

TfR2

AR

Mutation in TFR2, synthesis of TFR2 is impaired

Type 4 A HH

A

FP (LOF)

FP

AD

Loss-of-function mutation in FP, synthesis of ferroportin is impaired

Type 4 B HH

A

FP (GOF)

FP

ADn

Gain-of-function mutation in FP, synthesis of ferroportin is impaired

Impaired iron transport

Acerulo­plasminemia

A

Cp

Cp

AR

Inadequate uptake of iron by the erythron

DMT1 mutation

J

DVT

DVT

AR

Inadequate absorption of iron by the erythron and enterocyte

Ineffective erythropoiesis

Thalassemia

J

Globin

Globin

AR

Ineffective erythropoiesis, down-regulation of hepcidin, therefore increased intestinal absorption of iron

Congenital sideroblastic anemia

V

ALAS2

XL

Usually mutation in genes that produce heme precursors. Iron which should be incorporated into protoporphyrin IX accumulates in the mitochondria.

SLC25A38

AR

GLRX5

AR

ABCB7

XL

Congenital dyserythro­poietic anemia

J

DAN1
(Type 1)

Defective production of red cell proteins and erythrocytes of different origin. Mild hemolysis, characteristic erythroblast morphology.

SEC23B
(Type 2)

Unknown
(Type 3)

A, adult; j, juvenile; V. aaribleAD, autosomal dominant; AR, autosomal recessive; HH, hereditary hemochromatosis; XL, X-linked; HFE, high Fe; HJV, hemojuvelin; TfR, transferrin receptor; Fp, ferroportin; Cp, ceruloplasmin; DMT, DVT divalenter Metallionentransporter

Table 7.1-6 Clinical and laboratory findings in hereditary hemochromatoses (HH)

Clinical and laboratory findings

HFE hemochromatosis – Generally

The HFE gene encodes the HFE protein. Mutations in this HLA-associated gene lead to inactivity of the HFE protein, which can result in HH. A single homozygous nucleotide replacement (845G>A/845G>A) results in the substitution of tyrosine by cysteine (Cys282Tyr) at amino acid position 282 in the HFE protein, also known as C282Y/C282Y. The mutation is found in 80% of HH patients in Northern Europe. Another common found heterozygous type 845G>A/387C>G, which encodes a Cys282Tyr and His63Asp mutation, is known as C282Y/H63D. With the homozygous C282Y mutation, no disulfide bond, which is required for binding β2-microglobulin, is formed in the HFE protein. The interaction of the two proteins is necessary for the transport of HFE to the endosomal membrane and into the cell membrane, where HFE interacts with TfR1 to trigger the signaling for hepcidin synthesis. The hepcidin deficiency leads to uncontrolled release of iron from enterocytes and macrophages by ferroportin. The average C282Y allele frequency in the Central and Northern European population is about 6%, and the prevalence of C282 homozygosity in the white population is 1 : 200 to 1 : 300. The H63D polymorphism in HFE has a higher prevalence with an allele frequency of about 14%, but is associated with a lower risk for HH. The HFE S65C polymorphism is also associated with HH in rare cases if it is inherited together with the C282Y allele.

– Genotype C282Y/C282Y /4, 36/

Penetrance: the penetrance of the homozygous mutation is not very high. Although one in 200 Northern Europeans are affected, only one in four have symptoms of HH.

Clinical findings: in terms of health, there is generally no significant difference between C282Y homozygous patients and healthy controls. If there are clinical symptoms (usually not until the 5th decade of life), they do not correlate with the diagnostic markers of iron overload. Globally, there is no statistically significant difference in the prevalence of diabetes mellitus, joint trouble and chronic fatigue syndrome compared to healthy controls. The prevalence of skin pigmentation, however, is higher. Due to the increased iron storage in hepatocytes (prevalence 73–96%), the prevalences of hepatic fibroses (8–27%) and liver cirrhoses (3–9%) are likely higher compared to the normal population. Hepatic steatosis, male gender and excessive alcohol consumption are the main risk factors in the progression of liver cirrhosis.

First symptoms usually appear at age 40–60, in women typically not until menopause. Men are affected 5–10 times more often than women. The iron deposition in the tissues can lead to restricted function of the liver, endocrine organs, heart, and joints. The initial symptoms of weakness, fatigue, weight loss, skin discoloration, abdominal pain and loss of libido can progress to liver cirrhosis, cardiomyopathy, or diabetes mellitus.

Laboratory findings: due to the low penetrance, laboratory diagnostic population screening is of little value. A better screening strategy is cascade screening in individuals with a familial predisposition or liver problems. The basic biomarker is TfS. TfS levels ≥ 45% in women and children and ≥ 50% in men are indicative of the condition. Levels increase with increasing age. In the Copenhagen Heart Study, which investigated individuals aged 25–85 years over a 25-year period, TfS increased from 70% to 80% in men and from 50% to 70% in women /39/. If ferritin is assayed additionally, levels greater than 200 μg/L in women and greater than 300 μg/L in men are indicative. In one study /40/, 227 of the 99,711 participants of different races were homozygous for the C282Y mutation. Of these, 84% of the men had TfS levels above 50% and 73% of the women had levels above 45%. 88% of the men had ferritin levels above 300 μg/L and 57% of the women had levels above 200 μg/L. A ferritin concentration ≥ 1000 μg/L, which can be indicative of liver disease, was found in 13% of the homozygous C282Y carriers. Hepcidin is mildly to moderately reduced.

According to the Melbourne Health Iron Study /41/, the likelihood of C282Y homozygotes with ferritin levels in the range of 300–1000 μg/L progressing to a ferritin concentration above 1000 μg/L within the following 12 years is 25%. If TfS and ferritin are elevated, the HFE genotype is identified using molecular genetic methods.

The gold standard for assessing the degree of liver fibrosis/cirrhosis is liver biopsy. Biomarkers assayed for this purpose include AST, which is elevated in 50% of C282Y carriers with fibrosis, thrombocyte count, and ferritin. Elevated AST, a thrombocyte count below 200 × 109/L and ferritin above 1000 μg/L are reported to predict cirrhosis in 90% of cases /42/, but according to other studies only in less than 30% of cases /43/. Serum hyaluronic acid has been shown to be a better marker. In one study /43/, no C282Y carrier with a ferritin concentration below 1000 μg/L had liver cirrhosis. Those with higher levels had a hyaluronic acid concentration of 42.0 ± 9.8 μg/L compared to controls with 19.3 ± 1,8 μg/L. A hyaluronic acid concentration above 46.5 μg/L showed 100% sensitivity and specificity for identifying patients with liver cirrhosis (Tab. 7.1-7 – Criteria for the diagnosis of HFE hemochromatosis according to EASL guidelines) /43/.

Treatment approach: during the induction phase of venesection, removal of 400–500 mL of blood per week until ferritin has decreased to about 50 μg/L (recommended: 50–100 μg/L). During maintenance therapy venesection every 3–4 months. Iron deficiency anemia should be prevented as it is contra productive, since iron deficiency induces negative feedback with deficient expression of hepcidin and increased intestinal absorption of iron /36/.

– Compound heterozygotes (genotype C282/H63D)

Compound heterozygotes have genotype C282/H63D and account for 5–10% of patients with HH. The incidence and penetrance of this disease is very low.

Laboratory findings: TfS and ferritin are elevated, but lower than in C282Y homo- zygotes, hepcidin is mildly to moderately reduced. The mean TfS and ferritin levels are 39.2% and 185 μg/L, respectively, in men and 32.2% and 71 μg/L, respectively, in women /4/. The mean levels for genotype C282Y/C282Y determined in the same study were 65.3% and 420 μg/L, respectively, in men and 47.1% and 161 μg/L, respectively, in women. In another study /40/, 32% of men had ferritin levels above 300 μg/L, and 20% of women had concentrations above 200 μg/L.

– Genotype C282Y/wt (C282Y heterozygotes)

Individuals with a normal gene (wt, wild type) and a mutated C282Y gene do not have HH. Due to its high prevalence, this genotype is rather associated with longer patient survival. TfS and ferritin levels in the wild type were 26.7% and 111 μg/L, respectively, for men and 22.8% and 53 μg/L, respectively, for women and only slightly higher than those in C282Y heterozygotes (30.6% and 118 μg/L, respectively, for men, 26.9% and 57 μg/L, respectively, for women) /4/. Hepcidin is mildly to moderately reduced.

– H63D/H63D and H63/wt

Essentially the same statements and TfS and ferritin levels as for C282Y heterozygotes apply for patients with these genotypes. Hepcidin is mildly to moderately reduced.

– S65C

Mild, rare form of HH. There is a missense mutation from adenine to thymine in the HFE gene, which results in an amino acid replacement of serine by cysteine at position 65 of the protein.

– E168X

There is a nonsense mutation with a guanine to thymine base substitution at position 502 of the HFE gene, which leads to the formation of a stop codon. This results in the formation of an incomplete peptide which lacks the cytoplasmic, transmembrane and alpha 3 domains. The HH is rare and clinically severe.

Type 2A and type 2B hemochromatosis /4, 44/

Both are juvenile forms of HH. Type 2A is caused by mutations in the HJV gene, which encodes the hemojuvelin protein, type 2B results from mutations in the HAMP gene, which encodes hepcidin. Both types are autosomal recessive diseases with full penetrance that occur in both genders. Iron overload begins early in childhood and is more severe than in HFE hemochromatosis.

Clinical findings: the average age at presentation of type 2A is 23.5 ± 5.9 years, that of the less common type 2B is below 30 years. Like HFE hemochromatosis, juvenile hemochromatosis causes hypogonadism, cardiomyopathy, liver cirrhosis, diabetes mellitus, and skin pigmentation, but is associated with earlier and more severe manifestation of these conditions than HFE hemochromatosis. Clinically, hypogonadism and cardiomyopathy are more frequent manifestations than liver disease.

Laboratory findings: 37 patients aged around 24 years had serum ferritin levels of 3200 μg/L and a TfS of 91%. 6 children with an average age of 7.5 years had ferritin levels of 409 μg/L and a TfS of 87.5% /44/. Hepcidin is significantly reduced or completely lacking.

Therapy assessment: if iron overload is clearly documented, phlebotomy should be started early (5–7 mL/kg per week). Assessment criteria see genotype C282Y/C282Y.

Type 3 hemochromatosis /37, 45/

This rare form of HH is linked to mutations in the gene encoding TfR2. Known mutations include nonsense mutations (Y250X) in TfR2 on 7q22 and TfR2 deactivating mutations.

Clinical findings: type 3 hemochromatosis is essentially similar to type 1, but shows high variability ranging from asymptomatic cases to severe iron overload. It also resembles type 1 in its low penetrance in premenopausal women. When iron overload is present, type 3 must be considered as a possible diagnosis even in children.

Laboratory findings: TfS levels are similar to those in the C282Y/C282Y genotype, ferritin is normal in some patients. Hepcidin is moderately reduced.

Type 4 hemochromatosis /45/

Type 4 hemochromatosis is a rare form of iron overload with an autosomal dominant pattern of inheritance. It is due to heterozygous mutations in the SCL40AI gene located on chromosome 2q32. The protein ferroportin, which is responsible for exporting iron from cells, is mutated.

The disease is associated with the following characteristics:

  • Unlike the other forms of hemochromatosis, type 4 has an autosomal dominant pattern of inheritance
  • Iron is preferentially stored in the hepatic Kupffer cells of the reticuloendothelial system and not in the hepatocytes, which is an unusual pathophysiology in hemochromatosis. However, as the disease progresses, iron is also deposited in the hepatocytes, resulting in a mixed storage pattern.
  • Phlebotomy therapy is not well tolerated with iron storage in the reticuloendothelial system, since some patients develop microcytic hypochromic anemia despite elevated ferritin levels. This form of treatment is successful only in patients with parenchymal iron overload /46/.

Laboratory findings: patients have low TfS with high ferritin levels in the late stages of the disease. Hepcidin is high normal or elevated.

Table 7.1-7 Criteria for the diagnosis of HFE hemochromatosis according to EASL guidelines /37/

Criteria

Presence of C282Y mutation

Prevalence

80.6% of patients with HFE hemochromatosis and 0.6% of Northern and Central Europeans are homozygous for C282Y. The prevalence of compound heterozygosity for C282Y/H63D is 5.3% in hemochromatosis patients and 1.3% in the general population in Central and Northern Europe.

Penetrance

Measured using liver iron content (above 25 μmol/g): 75% in men, 52% in women.

Biomarkers

Ferritin: men> 300 μg/L (prevalence 32%), women > 200 μg/L (prevalence 26%).

TfS elevated in 4.3–21.7% and AST elevated in 24–32% of patients with HFE hemochromatosis.

Morbidity

10–33% in general, 32–35% if detected in hemochromatosis families.

Liver disease

Patients with liver disease are 10 times more likely to be homozygous for C282Y than healthy individuals, and the prevalence of liver cell carcinoma is 5.5–10%.

Diabetes mellitus

Type 1 diabetics and individuals with the more complicated type 2 diabetes are more likely to carry the C282Y allele.

Porphyria

The prevalence of C282Y homozygosity in porphyria cutanea tarda is 9–17%.

Table 7.1-8 Findings in C282Y hemochromatosis with and without liver cirrhosis /36/

Assay

No cirrhosis

Stage 4 fibrosis
(cirrhosis)

Ferritin > 1,000 μg/L

19 of 44 patients

10/10 patients

AST (U/L)

34.2 ± 2.8

61.6 ± 7.0

Hyaluronic acid (μg/L)

18.6 ± 1.5

137.7 ± 34.4

Sensitivity/Specificity

100%

100%

Table 7.1-9 Iron overload not due to disorders of the hepcidin-ferroportin axis

Clinical and laboratory findings

Transfusional iron overload

At a hematocrit of 0.45, blood contains 0.5 g iron per liter, and 1 mL of erythrocytes contains 1 mg of iron. Transfusional iron is deposited in the macrophages of the reticuloendothelial system (RES) and is regarded as relatively harmless. However, when the iron storage capacity of the RES is exceeded, the iron accumulates in the hepatocytes of the liver, in the pancreas and other endocrine organs.

This is the case with hyper transfusions such as in /47/:

  • Transfusion-dependent patients with β-thalassemia major with an annual iron load of 116–232 mg/kg body weight, corresponding to 7–14 g of iron for a body weight of 60 kg
  • Patients with myelodysplastic syndrome who may require transfusion of up to 8 units of banked blood per month, which corresponds to an annual iron load of 19 g.

Hyper transfusion does not lead to liver injury, measured as elevated aminotransferases, if the liver iron content does not exceed 350 μmol/g of dry weight /48/.

Clinical findings: iron overload becomes symptomatic only when the total body iron load reaches 0.4–0.5 g of iron/kg of body weight, corresponding to a dosage of 100–150 units of banked blood /49/. The accumulation of iron in the liver and other parenchymatous organs leads to symptoms comparable to those in type 1 hemochromatosis. This usually does not occur until at least a decade after transfusion. Treatment with chelating agents should be started as soon as possible.

Laboratory findings: TfS levels above 50% are measured after transfusion of 20–30 units of banked blood. The TfS level is not an indicator of the extent of iron overload. Ferritin levels are generally above 500 μg/L, but the ferritin concentration does not significantly correlate with the number of units of banked blood /48/. In transfusion-dependent patients with β-thalassemia major, ferritin levels are in the range of 1500–3000 μg/L. Iron chelation therapy should be initiated if ferritin levels are > 1000 μg/L. The treatment goal here is a ferritin value of below 500 μg/L.

Liver disease

Hepatitis C: histologically, mild iron overload of the liver is found in approximately 35% of patients. Ferritin concentrations are 179 ± 139 μg/L in men and 71 ± 100 μg/L in women (x ± s) /50/. It is assumed that the iron status of the liver in hepatitis C may influence histological activity and the degree of fibrosis.

Alcoholic liver injury: chronic alcoholics without hemochromatosis usually have moderate iron overload /51/. The stored iron accumulates in the RES and the Kupffer cells and is thought to be released from damaged hepatocytes. It is believed that alcoholics have increased intestinal iron absorption, since Fe3+ is readily available due to the high secretion of hydrochloric acid. This is no longer the case if gastritis is present. If liver cirrhosis exists, the uptake of Tf-bound iron in the liver is reported to be increased /50/. According to one study, 10% of severe alcoholics who present for rehabilitation therapy had a TfS above 60% and serum ferritin above 1000 μg/L /50/. There is a positive correlation between the aminotransferases and the ferritin concentration in alcoholics /52/.

Hemolytic anemia

Hereditary hemolytic anemias can lead to iron overload. The combination of iron overload and hereditary spherocytosis occurs frequently in heterozygous mutation carriers of HH /53/. Iron overload also develops after splenectomy-induced remission in hereditary spherocytosis. Iron overload can also occur as a result of hereditary pyruvate kinase deficiency /54/. Refer also to Tab. 7.6-2 – Disorders of hepcidin and ferroportin regulation.

Thalassemia syndrome

Thalassemias are the most common forms of anemia worldwide that are associated with ineffective erythropoiesis. The iron overload is not only transfusional, but due to increased intestinal iron absorption even before transfusion treatment is initiated.

Patients with thalassemia minor, regardless of whether they are heterozygous for a β-globin defect or one of the four β-globin genes, have a mild form of anemia, which is usually not associated with iron overload, since erythropoiesis is only minimally ineffective /55/.

Patients with thalassemia major (homozygous β-thalassemia), thalassemia intermedia and the combined state of β-thalassemia and HbE have severe ineffective erythropoiesis and significant iron accumulation in the parenchymatous organs and the reticuloendothelial system. In hemoglobin H disease, three of four β-globin genes are defective. Iron overload does not develop until advanced age.

Laboratory findings: in heterozygous β-thalassemia, TfS is > 35%, serum ferritin is above the reference range. In thalassemia major, TfS is > 50% and ferritin 1500–3000 μg/L.

Treatment: chelate mediated urinary iron excretion is recommended in young patients with a liver iron content of 125 μmol/g of dry weight and higher /48/.

Hereditary hyperferritinemia/cataract syndrome (HHCS)

HHCS arises from various point mutations or deletions within a protein-binding sequence in the 5’ UTR of the L-ferritin mRNA (Fig. 7.1-5 – First step of heme synthesis) that results in increased efficiency of L-ferritin translation. HHCS is a rare disease with a prevalence of about 1 in 200,000. Apart from the presence of a cataract, these patients have no pathological findings. The cataract consists of slowly progressive spots, vacuoles and distinct crystalline deposits mostly in the cortex, but also in the nucleus of the lens. The deposits are homo polymers of L-ferritin, which deposit not only in the lens, but also in other tissues /46/.

Laboratory findings: high ferritin, normal serum iron and normal TfS. Normal iron content in the liver biopsy sample. Development of hypochromic anemia under phlebotomy without decrease in ferritin.

Sideroblastic anemia

Sideroblasic anemias are a heterogenous group of congenital or acquired disorders of the bone marrow caused by pathological deposition of iron in the mitochondria of the erythroblast. The best-characterized congenital forms are caused by mutations in genes required for the production of heme precursors.

Hereditary sideroblastic anemias: these are classified into X-linked sideroblastic anemia (XLSA), XLSA with ataxia, erythropoietic protoporphyria (EPP), thiamin-responsive megaloblastic anemia (TRMA), and Pearson marrow-pancreas syndrome (PMPS).

Only XLSA is associated with systemic iron overload. In XLSA there is a deficiency of erythroid aminolevulinic acid synthase (ALAS) (Fig. 7.1-5 – Post-transcriptional regulation of cellular iron homeostasis). With one exception, all of the more than 20 known mutations are alterations of a base in the encoding region of the gene /55/.

Clinical findings: the disease can occur as early as in utero or as late as at age 90. Due to the X-linked pattern of inheritance, it mainly affects men. In some cases the illness can be alleviated by administering pyridoxine.

Laboratory findings: hypochromic microcytic anemia, but two populations of erythrocytes (dimorphism); one population is microcytic, the other normocytic. There are iron-positive inclusions in the erythrocytes (Pappenheim bodies). Increased erythropoiesis with ring sideroblasts in the bone marrow. Iron deposits are seen especially in the late maturation stages of the erythroblasts. TfS > 35%, serum ferritin elevated /56/.

Acquired idiopathic sideroblastic anemia: this is a clonal disorder which usually presents as mild to moderate refractory anemia /55/. If it occurs in combination with dysplastic mutations of other hematopoietic cell lines, it can develop either into myelodysplastic syndrome or leukemia. In combination with ineffective erythropoiesis there is increased intestinal absorption of iron. The anemia is often erroneously treated by administration of iron until the correct diagnosis is established. Administration of banked blood units also leads to increased accumulation of iron in the parenchymatous organs and in the reticuloendothelial system. TfS is > 35%, serum ferritin elevated.

Congenital dyserythropoietic anemias (CDA)

CDAs are rare diseases. There are three types of CDA, of which type 2 is the most common one. It is caused by a mutation of the gene encoding α-mannosidase. The consequence is deficient glycosylation of surface proteins of the erythroid cells, which leads to destruction of the cytoskeleton, resulting in ineffective erythropoiesis and iron overload /43/.

Laboratory findings: mild to moderate normocytic or macrocytic anemia, mild hemolysis, TfS above 50%, serum ferritin elevated.

African iron overload

In countries like Zimbabwe and South Africa, the prevalence of iron overload is about 10–15%. Patients are typically middle-aged or elderly adults with hepatomegaly and a history of consuming over 1000 liters of home-brewed beer in their lifetime.

Clinical findings: micronodulary liver cirrhosis, diabetes mellitus, and osteoporosis. In contrast to HH, iron is also present in large amounts in the reticuloendothelial system, so the spleen and bone marrow are also affected in addition to the liver. Portal hypertension, esophageal varices and liver failure are common complications /57/.

Laboratory findings: TfS above 55%, serum ferritin above 700 μg/L, sometimes above 4000 μg/L /57/.

Porphyria cutanea tarda

In this disease there is a deficiency of uroporphyrinogen decarboxylase, which leads to increased excretion of uroporphyrines and photosensitive bullous dermatosis. Clinical symptoms become manifest at middle or old age, some patients have a history of heavy drinking. Besides alcohol, the disease can also be triggered by hormone therapy or viral hepatitis. Due to the ineffective erythropoiesis in combination with increased intestinal iron absorption, there is increased iron storage, which improves with adequate iron mobilization therapy /55/.

Atransferrinemia

This is a very rare disorder. Despite the deficiency in transferrin (Tf), intestinal iron absorption and iron turnover are increased. The iron exists in plasma in a non-Tf-bound form and cannot be used for erythropoiesis. The result is hypochromic microcytic anemia and increased storage of iron in the liver, pancreas, myocardium, thyroid gland, and kidneys /4/. See also Section 7.5 – Transferrin saturation (TfS). The disease usually becomes clinically evident in the first months of life, less frequently at school age.

Aceruloplasminemia

Hereditary aceruloplasminemia is caused by mutations in the Ceruloplasmin (Cp) gene on chromosome 3q23-q24 or a pseudo gene on chromosome 8 /4, 56/. Aceruloplasminemia is a disorder of iron homeostasis. In terms of pathophysiology it is comparable to the ferroportin 1 mutation (type 4 hemochromatosis), which is also characterized by impaired release of iron from macrophages and iron-absorbing enterocytes. This results in iron overload, including in the basal ganglia, where the iron content is increased 10-fold.

Clinical findings: basal ganglia symptoms such as dementia, dystonia, dysarthria and diabetes mellitus, which manifest in the 4th–5th decade of life. Iron accumulation in the hepatocytes and reticuloendothelial system, but no hepatic fibrosis.

Laboratory findings: mild microcytic anemia, TfS decreased, serum ferritin in the range of 1000–2000 μg/L, ALT normal. In CSF total protein and iron elevated, absence of pleocytosis, no reduction in glucose concentration.

Table 7.1-10 Significance of biomarkers and hematologic indices in the diagnosis of iron restriction

Clinical and laboratory findings

Iron

None with regard to iron deficiency or iron overload unless chronic inflammation, acute infection or a malignant tumor have been excluded. In addition, iron levels are subject to diurnal variations. Further information can be found in Section 7.2 – Iron.

Transferrin saturation (TfS)

Evaluation of plasma iron turnover. TfS is low with low iron turnover (functional iron deficiency) and high with iron overload. TfS can be assessed only, if chronic inflammation, acute infection or liver disease have been excluded. Screening for hemochromatosis, which can be diagnosed if levels are above 45 (50)%.

Ferritin

Evaluation of iron stores. Low ferritin levels always indicate body iron deficiency. Elevated levels indicate iron overload.

Soluble transferrin receptor (sTfR)

Indicator of functional iron deficiency (iron-restricted erythropoiesis) in association with low, normal or elevated ferritin levels. Evaluation of the erythron mass (hypo-, normo- or hyper regenerative erythropoiesis).

Ferritin index

The sTfR/log10 ferritin index is an indicator of iron supply for erythropoiesis. It covers the whole bandwidth of iron availability from iron deficiency to iron overload.

% HYPO

The proportion of hypochromic erythrocytes is an indicator of the iron requirement for erythropoiesis. A value above 5% indicates that the patient has been in a state of iron-restricted erythropoiesis for several weeks.

Ret-Hb (CHr, RetHe)

The reticulocyte hemoglobin content is an early indicator of the iron requirement for erythropoiesis. A Ret-Hb < 28 pg indicates that there has been an acute need for iron since about 5 days, and erythrocytes with a reduced hemoglobin content are produced.

Zinc protoporphyrin

The zinc protoporphyrin content of the red blood cells is a marker of iron supply for erythropoiesis. However, changes in the iron supply are shown only with a delay as they depend on the erythrocyte life span.

Tabelle 7.1-11 Disorders of iron metabolism: results of biochemical markers and hematologic indices

Disorder

Ferritin

(μg/l)

Iron
(μmol/L)

TfS
(%)

sTfR

FI

Ret

HYPO
(%)

Ret-Hb
(pg)

Latent ID

< 30

≥ 7

> 16

N

N

N

< 3.8

≥ 28

Total ID

< 15

≤ 7

< 16

E

E

D

≥ 3.8

< 28

ACD

≥ 100

≤ 7

≤ 16

N

D

D

≥ 3.8

< 28

Functional ID

≥ 100

≤ 7

≤ 16

N

D

D

E

≥ 28

Acute hemolytic disorder

≥ 30

≥ 7

> 16

N

N

E

N

≥ 28

Bone marrow insufficiency

≥ 30

≥ 7

> 16

N, D

D

D

N

≥ 28

Iron overload

≥ 300

≥ 7

> 45

N

N

N

N

≥ 28

ID, iron deficiency; Fer, Ferritin; FI, Ferritinindex; E, elevated; D, decreased; Ret, reticulocyte count; N, normal; TfS, Transferrin saturation; sTfR, soluble transferrin rezeptor; sTfR/log10 Ferritin; HYPO %, Proportion of hypochromic red cells Ret-Hb, reticulocyte hemoglobin content

Table 7.1-12 Scoring system for the diagnosis of iron-restriction in anemic patients with ferritin levels > 30 μg/L. A number of 3 points indicates iron restriction /18/.

Test

Score

Transferrin saturation ≤ 20.6%

1

sTfR > 1.88 mg/L (S), > 5.27 mg/L (R)

1

%Hypo > 3.8% or CHr (RetHe) ≤ 27.9 pg

1

R, sTfR assay of Roche; S, sTfR assay of Siemens; RetHe CHr, hemoglobin content of the reticulocyte; %Hypo, proportion of hypochromic red cells

Table 7.2-1 Reference intervals for iron

Group

Age

μg/dL (μmol/L)

Children

2 weeks
/3/

63–201
(11–36)

6 months
/3/

28–135
(5–24)

12 months
/3/

35–155
(6–28)

2–12 yrs
/4/

22–135
(4–24)

Females /5/(not pregnant)

25 yrs

37–165
(6.6–29.5)

40 yrs

23–134
(4.1–24.0)

60 yrs

39–149
(7.0–26.7)

Pregnant women /6/

12th week

42–177
(7.6–31.6)

at delivery

25–137
(4.5–24.5)

6 weeks
post partum

16–150
(2.9–26.9)

Men /5/

25 yrs

40–155
(7.2–27.7)

40 yrs

35–168
(6.3–30.1)

60 yrs

40–120
(7.2–21.5)

Conversion: μg/dL × 0.1791 = μmol/L; pp, post partum.

Table 7.2-2 WHO recommended intakes of iron

Group

Intake/day

Infants 4–12 months

8.5 mg

Children 1–3 yrs

5 mg

Children ≤ 6 yrs

5.5 mg

Children ≤ 10 yrs

9.5 mg

Puberty

Males 15 mg

Females 16 mg

Adults

Males 9 mg

Females 12.5 mg

Women > 50 years 9.5 mg

Table 7.3-1 Reference intervals for ferritin

Population

Age

μg/L

Cord blooda) /2/

> 34. gestational week

> 70

Infantsb)
/3/

0.5 months

90–628

1 month

144–399

2 months

87–430

4 months

37–223

6 months

19–142

9 months

14–103

12 months

1–99

Childrenb)
/4/

2–15 yrs

9–59

16–18 yrs

♀

10–63

♂

12–78

Adultsc)
/5/

20–60 yrs

♀

9–140

♂

18–360

Adultsd)
/6/

19–95 yrs

♀

≥ 13

28–96 yrs

♂

≥ 21

a) The 5th. percentile is expressed; calibration to 2nd International Ferritin Standard (Code 80/578).

b) Values are the 2.5th and 97.5th percentiles; calibration to 2nd International Ferritin Standard (Code 80/578).

c) Values are the 2.5th and 97.5th percentiles; recombinant Ferritin Standard (NIBSC Code 94/572); the average levels obtained with immunoassays from three different manufacturers which are readily comparable are shown. The gold standard was a normal hemoglobin level.

d) Values are the 2.5th and 97.5th percentiles. The lower reference interval values were determined on hospitalized patients without acute-phase response with normal erythrocyte and reticulocyte hemoglobinization. Recombinant Ferritin Standard (NIBSC Code 94/572). The gold standard was a reticulocyte hemoglobin content ≥ 28 pg and a proportion of hypochromic erythrocytes (%HYPO) < 5%.

Table 7.3-2 Ferritin levels (μg/L) in iron deficiency

Storage iron deficiency

Total iron deficiency

♀ 9–19

♀ < 9

♂ 18–29

♂ < 18

Iron deficiency in adults according to Ref. /4, 5, 8/.

Table 7.3-3 Diseases and conditions associated with decreased serum ferritin levels

Clinical and laboratory findings

Iron depletion

Iron depletion begins with the loss of storage iron. Once these stores are depleted, functional iron levels begin to fall. With ferritin concentrations below the reference interval, storage iron can no longer be detected histologically in the bone marrow. However, if serum ferritin is within a range of 20–200 μg/L, the concentration can be used to assess iron stores, since 1 μg/L of serum ferritin is representative for 8–10 mg of storage iron. This is only the case in the absence acute-phase response (i.e., if C-reactive protein is normal). If ferritin is below 20 (30) μg/L, it is no longer useful for assessing iron stores. This stage is then referred to as storage iron deficiency. The reduction of iron stores is often measured in adolescents with growth spurts, women of menstruating age, and blood donors. The body tries to compensate and replenish iron stores by increased intestinal absorption.

Reduction of functional iron /13/

Iron deficiency in tissues such as the erythron only occurs once the storage iron reserves have been depleted and the serum ferritin level is low. In iron metabolism, this marks the point of transition from storage iron deficiency to iron depletion. The reduction of functional iron initially causes a decrease in TfS and subsequently microcytic hypochromic anemia, which can be measured in the complete blood count about 8 weeks after storage iron deficiency has developed.

The situation is different if there is a chronic inflammation with borderline storage iron values. The inflammation leads to a reduction of functional iron in the first weeks of inflammation, since IL-6 mediated hepcidin increase inhibits the release of stored iron into the circulation and increases the iron content of the reticuloendothelial system macrophages. As a result, iron distribution is disturbed and the previously borderline ferritin reaches a level within the reference interval. Deficiency of functional iron in the presence of inflammation cannot be detected by ferritin measurements, but only by biomarkers that are representative of functional iron, such as the reticuloctye Hb content (CHr, RetHe), the proportion of hypochromic erythrocytes (% HYPO), and soluble transferrin receptor (sTfR).

Iron deficiency anemia

In iron deficiency anemia there is a total deficiency of body iron (iron depletion). Ferritin levels consistent with storage iron deficiency are 2–5 times more common than microcytic hypochromic anemia /10/. Since about 20% of patients with folate or vitamin B12 deficiency anemia also have reduced ferritin levels, the anemia is not microcytic in these cases (mixture of normocytic and hypochromic erythrocytes).

Pregnancy

Epidemiological studies in industrialized nations reveal that 10–30% of women of childbearing age have reduced ferritin levels and 1.5–14% exhibit an iron deficiency anemia /14/. It is estimated that, in the industrialized nations, about 20% of women enter into pregnancy with empty iron stores. Depending on the study, ferritin levels less than 12–15 μg/L are regarded as storage iron deficiency in pregnant women /15/. A storage iron deficiency is ruled out if the ferritin level is higher provided there is no acute-phase response (i.e., CRP is not above 5 mg/L).

For women who enter into pregnancy with normal ferritin values, lowered levels can be expected starting from the end of the 2nd trimester. At this point, daily iron requirements average 5.6 mg (range 3.5 to 8.8 mg) /16/ and 50–80% of maternal iron turnover is unidirectionally transferred to the fetus from the mother via the placenta /17/. There is a significant relationship between premature uterus contractions and serum ferritin. For example, only 11% of pregnant women exhibit premature contractions when ferritin is > 20 μg/L, but 48% do so when the concentration is < 10 μg/L /18/.

Childhood

The ferritin serum level changes markedly during the first year of life. While the 5th percentile is still as high as 70 μg/L 48 hours post partum, it then falls rapidly as iron is mobilized for hemoglobin synthesis. In the 9th month of life, the lower reference interval value for ferritin is 10 μg/L /19/. Ferritin then gradually increases up to 25–35 μg/L /4, 20/ before it decreases again at the beginning of puberty. Prepubertal boys with a mean age of 11.7 years have average levels of 35 μg/L, which then decrease during puberty to reach approximately 23 μg/L at the mean age of 13.6 years /21/. Neonates born with low ferritin levels of 44 ± 20 μg/L (x ± s) compared to normal controls (175 ± 55 μg/L) still have lower levels (30 ± 17 μg/L) than the control group (57 ± 33 μg/L) even at the age of 6–12 months /19/.

Endurance athletes

Adolescent endurance athletes easily become iron deficient after short periods of intense training. For example, 12 of 20 female runners had serum ferritin levels below 20 μg/L after 5 or 10 weeks of endurance training, and 8 out of 12 even had levels below 12 μg/L. In men, only 1 of 30 had a ferritin level below 12 μg/L /22/.

Blood donors

In first-time blood donors, donating one banked blood reduces the ferritin level in men by 50% from an average 127 μg/L to 66 μg/L. In women, it is decreased from an average of 46 μg/L to 33 μg/L /23/. Multiple blood donors generally have a lower ferritin concentration than non-donors. In one study /24/, average levels were 80 μg/L in non-donating men and 38 μg/l in non-donating women, respectively, compared to 37 μg/L and 17 μg/L, respectively, in multiple donors. When four units of autologous blood were collected over a period of 4 weeks, ferritin levels changed as follows /25/: prior to donation 105 μg/L, after first donation 81 μg/L, after second donation 63 μg/L, after third donation 53 μg/L, after fourth donation 40 μg/L, and pre-operation 32 μg/L. Pica is a persistent craving to eat non-nutritional substances (e.g., ice). One study reported pica in 11% of donors with iron depletion or deficiency compared with 4% of iron-replete donors /26/.

Iron therapy (see also Section 7.1.7.1 – Monitoring of iron therapy)

Intravenous application: parenteral iron therapy induces enhanced ferritin synthesis. This simulates inadequately high concentration of serum ferritin for storage iron reserves. The ferritin can not be used as a measure of storage iron until 2–4 weeks after administration /27/.

Oral application: monthly measurement of ferritin over the course of therapy provides a quantitative impression of the status of the corresponding iron storage reserves. In patients with chronic-hemorrhagic anemia and a daily blood loss of 22 ± 21 mL, a daily oral dose of 105 mg of Fe (II) leads to an increase in ferritin from 8 ± 7 μg/L to 25 ± 18 μg/L in the 2nd month with no further increase in the following two months. Posthemorrhagic anemias without further blood loss and with baseline ferritin values of 7 ± 5 μg/L result in an increase to 40 ± 28 μg/L within the first month of therapy, with a further increase up to 55 ± 10 μg/L in the 4th month /28/.

Table 7.3-4 Diseases and conditions associated with elevated serum ferritin concentrations

Clinical and laboratory findings

Anemia of chronic disease (ACD)

ACD is normocytic and normochromic due to inadequately low erythropoietin levels in relation to the extent of the anemia. There is systemic hepcidin-induced hypo regenerative erythropoiesis and iron absorption via the erythroblast transferrin receptors is reduced. However, since hepcidin inhibits the release of iron by causing the degradation of ferroportin, the erythroblasts are still sufficiently supplied with iron so that reticulocytes released from the bone marrow are normocytic normochromic. Due to the increased iron content of the storage cells, ferritin is elevated. Approximately 10% of ACD patients have iron-restricted erythropoiesis with microcytic anemia due to chronic blood loss /29/. Ferritin levels are usually over 100 μg/L. While not generally performed in ACD, iron substitution is necessary if ACD occurs in combination with hypochromic reticulocytes.

Hereditary hemochromatosis (HH)

HH may be suspected if TfS is above 50% in men and above 45% in women and children. A test for HH should be performed if, in addition to elevated TfS, ferritin levels are > 300 μg/L in men and > 200 μg/L in women.

The main clinical manifestation of HH is liver cirrhosis. However, cirrhosis occurs only rarely in hemochromatosis patients with serum ferritin levels < 1000 μg/L. A study of 30,000 white individuals showed that only 59 had ferritin levels of greater than 1.000 μg/L, of which 24 had homozygous mutant or compound heterozygous mutant HFE genotypes /1/.

Liver disease

Some patients with non-alcoholic steatohepatitis (NASH) exhibit a hyper ferritinemia with levels > 1000 μg/L. TfS is normal. In acute liver injury, ALT correlates positively with serum ferritin. This means that a substantial proportion of patients with ALT activity > 1000 U/L also have ferritin levels > 1000 μg/L. Ferritin levels > 45,000 μg/L have been measured /30/. Patients with chronic hepatitis C had ferritin levels in the range of 266 ± 145 μg/L (x ± s) with histologically proven liver iron /31/. Cases of alcoholic liver disease such as alcohol-induced toxic hepatitis and cirrhosis are primarily characterized by increased intestinal iron absorption that leads to moderate siderosis of the liver. Of 159 chronic alcoholics studied, 23 had TfS above 50% and 8 had ferritin levels > 1000 μg/L /32/. A common pathological process in acute and chronic liver disease is cytolysis, an event that releases ferritin from the hepatocytes.

Critically ill COVID-19 patients

Patients infected with the Coronavirus 2 may develop severe pneumonia and damage of liver, heart and kidneys (SARS-COV-2). The inflammatory cytokine storm, which is defined by the excessive and uncontrolled release of pro-inflammatory cytokines as has been reported in other infections with sepsis has been recognized as the primary cause of death in SARS-COV-2. The ferritin concentration in serum is used to predict COVID-19 progression which is defined by fever, cytopenia affecting at least two hematopoietic lineages, hypertriglyceridemia and/or hypo fibrinogenemia, ferritin > 500 μg/L, hemophagocytosis, elevated IL-1β and soluble IL-2 receptor (CD25), low natural killer cell activity, and splenomegaly. Macrophages, which produce cytokines and account for the majority of the immune cells in the lung parechyma, might be responsible for the secretion of ferritin /48/.

Hemodialysis patients

Hemodialysis patients develop a normocytic, normochromic anemia. Its main cause is inadequate synthesis of erythropoietin in the renal peritububular cells in relation to the reduced hematocrit. Because of the reduced erythrocyte life span due to inflammation, some patients have serum ferritin levels > 600 μg/L. To raise Hb levels, ESA therapy is initiated, and 80–90% of patients are additionally given intravenous iron supplementation. The European Best Practice Guidelines /33/ define the optimal target range for iron balance in these patients as follows: ferritin ≥ 500 μg/L, TfS ≥ 30%,%HYPO< 2,5%. The definition of a ferritin level ≥ 500 μg/L for ESA therapy was based on the fact that the success rate of ESA therapy for ferritin levels < 100 μg/L is below 50%. Over the long term, ferritin concentrations should not exceed 800–1000 μg/L in patients who are on parenteral iron therapy, because this results in increased iron storage outside the reticuloendothelial system (e.g., in hepatocytes). The ferritin concentration is considered an indicator of morbidity and mortality in hemodialysis patients.

Transfusion-associated iron overload /34/

This form of iron overload occurs as a result of chronic blood transfusions in myelodysplastic syndrome, thalassemia, and hemoglobinopathy. Here, the ferritin level predicts organ involvement in iron overload. Levels < 1500 μg/L indicate acceptable iron overload, levels > 3000 μg/L significant overload with liver injury. With chronic transfusion, ferritin initially rises rapidly linearly with the number of banked blood units transfused, but then may slow down after reaching 1500–2500 μg/L, despite increasing iron load.

Post transplantation

Patients with iron overload, indicated by ferritin levels > 1500 μg/L, who undergo autologous stem cell transplantation are at increased risk for infections, such as aspergillosis /35/. Patients who have undergone a kidney transplant and have ferritin levels > 1100 μg/L due to pre transplant administration of 40 units of banked blood have a 3-fold increased 10-year mortality risk /36/.

Systemic hematological disease

Ferritin synthesis is normal in polymorphonuclear granulocytes, lymphocytes and monocytes at normal plasma iron concentration. However, it is increased in leukemia cells. In both acute and chronic myeloid leukemia, ferritin levels can be in the range of several thousand μg/L /37/. This is also the case in acute myelomonocytic leukemia. Hodgkin’s and non-Hodgkin lymphomas with involvement of the liver also cause hyper ferritinemia. During remission, ferritin levels decline.

Solid tumor

Some of these patients have elevated ferritin levels of unknown origin.

AIDS

AIDS patients with infections, such as disseminated histoplasmosis, tuberculosis, or Pneumocystis carinii pneumonia, have hyper ferritinemia, sometimes with concentrations > 10,000 μg/L. Due to the HI virus-induced deregulation of monocytes and macrophages, these cells produce excess amounts of ferritin /38/.

Hyperferritinemic syndromes

High levels of ferritin occur in hyperferritinemic syndromes including hemophagocytic lymphohistiocytosis, macrophage activation syndrome, adult onset Still’s disease, catastrophic antiphospholipid syndrome, and septic shock. A typical hallmark of the hyperferritinemic syndromes seems to be a proinflammatory condition with markedly high levels of IL-1B, IL-1RA and TNF-alpha in the early phase and higher levels of IL-2, IL-10 and TNF-alpha in intensive care unit patients. Such cytokine storm has been already described in SARS-CoV-2. Three mechanisms provoke ferritin concentration higher than 500 ug/L: overactivation of T-lymphocytes, over-activity of Interferon-gamma, and the direct role of the H-chain of ferritin in activating macrophages to increase the secretion of inflammatory cytokines /46/.

Metabolic syndrome (MetS)

Overweight patients with MetS have higher ferritin levels than those of normal weight. MetS patients had average ferritin levels of 133.9 μg/L (median 70 μg/L), while individuals without MetS had average levels of 66.8 μg/L (median 40.1 μg/L). The elevated ferritin in MetS patients, who generally also have slightly elevated CRP levels, are considered to be a sign of subclinical inflammation /39/.

Missense mutation in L-ferritin

A single mutation in the coding sequence (p.Thr30He) of L-ferritin is associated with transferrin saturation < 45% and serum ferritin > 200 μg/L in women and > 300 μg/L in males. The serum iron level is < 140 μg/dl (25 μmol/L) and no excessive tissue iron is determined by liver biopsy /40/.

Porphyria Cutanea tarda

Hyperferritinemia is the indicator of iron overload and a trigger of porphyria attack.

Hyperferritinemia-Cataract Syndrome (HHCS)

Hereditary hyperferritinemia cataract syndrome (OMIM 600866) is an autosomal dominant disease characterized by elevated serum ferritin concentration in the absence of total body iron overload. Patients consult the ophthalmologist due to an early onset of a reduction in visual acuity. The disease is characterized by bilateral cataract that can arise at different age. Light diffracting crystalline deposits are present in cataractous lenses with typical turbidity pattern. Analysis shows L-ferritin in the crystalline deposits. Ferritin represents a soluble protein in the lens even in physiologic conditions however, the amount is 15-fold increased in HHCS patient lens /50/. The disease presents high phenotypic in visual impairment, age of onset, severity of cataract and serum ferritin concentration. The pathogenetic events responsible of HHCS in the Iron Responsive Element (IRE) are mutations localized in the 5’UTR of the light chain ferritin messenger RNA. The mutations alter the iron dependent post transcriptional regulation in which the IRE is involved. Mutations in the IRE of L-ferritin RNA prevent or alter the binding of Iron regulatory Proteins (IRPs) leading to a constitutive and iron independent translation of L-ferritin /50/. Mutations may be classified as major mutations, that prevent the IRE-IRP binding, and minor mutations that cause a decline of the IRP binding affinity. Refer to Fig. 7.1-6 – Post-transcriptional regulation of cellular iron homeostasis.

Laboratory findings: Increase in serum ferritin, normal serum iron and normal transferrin saturation, mutations of ferritin iron responsive element. A correlation between the serum ferritin concentrations and the severity of cataract has been published. In affected patients with HHCS ferritin concentrations ranged from 950 to 2.259 ug/L /51/.

Table 7.3-5 Development of reference material for ferritin immunoassays

Year

Preparation

Comment

1985

WHO international standard (IS) code 80/602

Human liver ferritin, stock depleted, value-assigned based on ferritin protein content

1993

WHO second international standard (IS) code 80/578

Human spleen ferritin, value-assigned based on ferritin protein content

1997

ISO 17511:2020, code 94/572. Third IS.

Ferritin (recombinant L-chain preparation) Traceability to an International Conventional Calibrator

2022

Forth IS (code 19/118)

Values are calibrated against the third IS. According to a preliminary experiment a bias of 5 to 10% among commercial immunoassays exists.

Manufacturers claim calibration alignment to different preparations

Table 7.4-1 Reference intervals for soluble transferrin receptor (sTfR)

Adults

♀ + ♂a) /3/

0.8–1.8

♀ + ♂b) /4/

0.8–2.3

♀ + ♂b)/5/

1.3–3.3*

♀ + ♂c)/5/

3.0–8.5

♀ + ♂d) /5/

0.9–3.1

♀e) /6/

1.9–4.4**

♂e) /6/

2.2–5.0

Children /4/

4–6 monthsa)

1.3–3.2

6–12 monthsa)

1.1–2.9

12–18 monthsa)

1.4–2.5

18 months – yrsa)

1.4–2.9

2–3 yrsa)

1.0–2.9

3–4 yrsa)

1.1–2.6

4–6 yrsa)

1.1–2.7

6–9 yrsa)

0.9–2.6

9–12 yrsa)

0.8–2.7

12–18 yrsa)

0.9–1.9

Data expressed in mg/L; values are 2.5th and 97.5th percentiles; the reference ranges of the following assay kit manufacturers are specified for assays using mechanical analysis systems: a) Siemens; b) Orion; c) Ramco; d) R+D Systems; e) Roche Diagnostics; * manual test; ** premenopausal.

Table 7.4-2 Differentiation of iron deficiency anemia from ACD and the combined state of ACD/IRE

Disorder

Ferritin
(μg/L)

TfS
(%)

sTfR

Iron deficiency anemia (IDA)

< 30

≤ 16

↑

Anemia of chronic disease
(ACD)

≥ 100

≤ 16

n

3. ACD and IRE

≥ 30

≤ 16

↑

For ferritin the lower reference interval value for storage iron for women was used. The ferritin index behaves the same as the sTfR. ↑, elevated; n, normal. IRE, iron-restricted erythropoiesis. IRE, iron restricted erythropoiesis

Table 7.4-3 sTfR in diseases and various clinical settings

Clinical and laboratory findings

Iron depletion

The level of sTfR exceeds the upper reference interval value only when iron stores are depleted and the serum ferritin level has declined below the low reference interval value /11/. If there is no decrease in the transferrin saturation, the sTfR concentration remains normal.

Reduction of functional iron

Elevated sTfR levels are an indicator of iron deficiency in the tissues, provided that erythropoiesis is normo regenerative /11/. Hyper proliferative erythropoiesis is indicated by reticulocytosis.

Iron deficiency anemia

In iron deficiency anemia, iron stores are depleted, functional iron levels are reduced, and the concentration of sTfR is increased. If sTfR is normal in microcytic anemia, a differential diagnosis must consider ACD, β-thalassemia or recently started or intermittent oral iron substitution.

Anemia of chronic disease (ACD)

ACD is normocytic in about 80–90% of cases. In combination with iron-restricted erythropoiesis (IRE), which can also occur with normal or even elevated ferritin levels, the sTfR concentration may be increased. According to one study /14/, the diagnostic sensitivity of sTfR in detecting ACD/IRE is 68% in women and men with a specificity of 66% and 76%, respectively, using the reticulocyte hemoglobin content (CHr, RetHe) < 28 pg as the gold standard.

β-thalassemia

In patients with β-thalassemia and HbE disease, the sTfR concentration can be elevated up to 8-fold. The cause is enhanced cell proliferation in the erythroblast compartment due to ineffective erythropoiesis /15/. These patients frequently also have elevated ferritin levels due to increased intestinal iron absorption.

Hemolytic anemia

In autoimmune hemolytic anemia and in hereditary spherocytosis the sTfR concentration is elevated 3–5-fold /15, 16/. The cause is increased cell proliferation in the erythroblast compartment.

Megaloblastic anemia

In one study /17/, 22 of 33 patients with confirmed vitamin B12 deficiency were anemic. 12 of the anemic patients had elevated sTfR and LD levels. LD activity was positively correlated with the sTfR concentration and negatively with the hemoglobin level. There was no correlation between sTfR and the hemolytic component. After starting treatment with vitamin B12, the sTfR concentration increased within 1–3 days, peaked after 2 weeks and normalized again around the 5th week. In the patients with vitamin B12 deficiency with elevated sTfR, the increase was mild (2-fold on average), with another 2-fold increase occurring during treatment. The elevation in sTfR is due to ineffective erythropoiesis, the decrease during treatment due to increasing effectiveness of erythropoiesis.

Pregnancy

During pregnancy, the plasma volume and mass of the erythroblast compartment increase. Compared to the conditions before pregnancy the plasma volume increases by a factor of 1.5 and the red blood cell mass by a factor of 1.25 by the end of pregnancy. In early pregnancy, the mass of the erythroblast compartment, the erythropoietin (EPO) concentration and the sTfR concentration decrease due to inadequately low EPO secretion before beginning to continuously increase again from pregnancy week 30. During the last weeks of pregnancy, EPO and sTfR levels can be higher than before the pregnancy and above the upper reference interval value. In pregnancy week 16, approximately 50% of pregnant women have a sTfR concentration below the lower reference interval value /18/. Elevated sTfR levels in the 1st and 2nd trimester therefore indicate iron deficiency, while elevated levels in the last trimester reflect increased erythropoietic activity /19/.

Fetus, neonate

Maternal iron deficiency does not cause an increased sTfR level in the fetal cord blood, but a decrease in hemoglobin and serum ferritin levels /20/. In neonates there also is no relationship between the sTfR concentration, gestational age and iron status /21, 22/.

Childhood

In a group of healthy boys aged 11–13 with hemoglobin values of 116–144 g/L and ferritin levels of 12–87 μg/L, none of the boys had an elevated sTfR concentration. In contrast to ferritin, the determination of sTfR levels is not considered to be useful in assessing the iron status in this age group /23/.

Chronic liver disease

Up to 70% of patients with chronic liver disease have iron deficiency, often caused by latent or manifest blood loss, in particular in liver cirrhosis /24/. In chronic liver disease, ferritin and TfS do not reflect the iron status, and MCV can be normal despite the presence of iron deficiency anemia. Using the absence of stainable bone marrow iron as the gold standard test for the diagnosis of iron deficiency, the elevated sTfR concentration is a good indicator of depleted iron stores in patients with chronic liver disease /25/. This presupposes that acute blood loss and acute hemolysis have been excluded.

Cardiosiderosis

Cardiosiderosis is a leading cause of mortality in transfusion-dependent thalassemias. sTfR levels are significantly lower in patients with cardiosiderosis (odds ratio 21). The risk increases when tranfusion-iron loading rates exceed the erythroid transferrin uptake rate by > 0.21 mg/kg/day /32/.

Table 7.5-1 Calculation of transferrin saturation

TfS (%) = Serum iron (μmol/L) × 398 Serum transferrin (mg/dL) TfS (%) = Serum iron (μg/dL) × 70.9 Serum transferrin (mg/dL)

Table 7.5-2 Reference intervals for transferrin saturation

Premature infants /2/

27.8 ± 16.4*

Term infants /2/

37.7 ± 8.3*

Children /3/

♂

♀
  • 0 – < 1 year

4.1–59

4.1–59
  • 1 – < 14 years

6.5–39

6.5–39
  • 14–19 years

9.6–58

5.2–44

Adults /4/ ♀ + ♂

16–45

Data expressed in %

Table 7.5-3 Transferrin saturation in iron-deficient states

Clinical and laboratory findings

Iron deficiency

Decreased levels of TfS are usually associated with iron deficiency. Iron stores are depleted and there is already a deficiency of functional iron. Even though TfS reflects the behavior of the functional iron and may in individual cases decrease once iron stores are depleted, in general it is an unreliable marker for indicating the iron supply for hematopoiesis in critical populations such as pregnant women, children with growth spurts, alcoholics, and athletes (see Tab. 7.1-2 – Iron deficiency states) /7/.

The Second Health and Nutrition Examination Survey and the American Academy of Pediatrics recommend the following thresholds for the diagnosis of iron deficiency in children: serum iron in children aged 1–5 years less than 30 μg/dL (5.4 μmol/L), TfS in children aged 1–2 years < 8% and in children aged 3–5 years < 9% /8/. In a study /9/ of children with hemoglobin levels < 110 g/L, the diagnostic sensitivity of a TfS < 10% was comparable to a reticulocyte Hb content (CHr, Ret-He) ≤ 27.5 pg for the diagnosis of iron deficiency.

Anemia of chronic disease

In infections, chronic inflammation and renal insufficiency, iron cannot be mobilized from the stores. The ferritin concentration is normal or elevated, anemia is present, TfS is usually reduced to levels below 16% or, less frequently, normal /10/.

ESA therapy

In order to correct the anemia in patients with chronic kidney disease, erythropoiesis is stimulated by administering erythropoiesis-stimulating agents (ESA). However, hemoglobin levels will only rise if sufficient functional iron is available. This is confirmed if (e.g., in hemodialysis patients) TfS is ≥ 20% /11/.

Table 7.5-4 Transferrin saturation in iron over load

Clinical and laboratory findings

Hereditary hemochromatosis (HH)

HH affects approximately 0.5% of Northern Europeans. If women with ferritin levels > 200 μg/L and men with ferritin levels > 300 μg/L in addition have elevated TfS, then HH is suspected (see Tab. 7.1-6 – Clinical and laboratory findings in hereditary hemochromatoses). Levels above 45% in women and children and above 50% in men are suspicious and require molecular-biologically testing /12/.

Secondary iron overload

Secondary iron overload can be caused by:

Non-Transferrin-bound iron

Binding capacity of transferrin (Tf) for binding of iron is exceeded in patients with TSAT > 70% and non transferrin bound iron (NTBI) is active. NTBI is also known as the labile plasma iron (LPI). LPI penetrates the cells, functions redox active (refer to Section 19.2 – Oxidative stress) and damages cells of the parenchyma.

Table 7.5-5 Limitations of transferrin saturation (TfS)

  • The synthesis of Tf is suppressed during the acute-phase response
  • In the presence of liver parenchymal damage, Tf is released into the plasma by hepatocytes
  • During pregnancy, Tf synthesis is greater than the decrease in total body iron
  • An increase in Tf and decrease in TfS occurs only if hemoglobin declines by at least 20 g/L in the presence of empty iron stores /5/.

Table 7.6-1 Characteristics and functions of hepcidin

Criterion

Function and characteristics

Structure

The human gene encodes a 84-amino acid precursor protein including a 24 amino acid leader peptide, a 35-amino-acid pro region and the C-terminal, 25-amino acid bio active iron-regulating peptide (hepcidin-25)

Metabolism

Hepcidin is synthesized in hepatocytes, serum concentration of hepcidin-25 can be below 0.5 nmol/L and above 100 nmol/L. Hepcidin is excreted via the kidneys.

Regulation: hepcidin formation is stimulated by elevated iron levels in plasma and hepatocytes, by inflammatory cytokines, and by bone morphogenetic proteins. The formation is inhibited by low iron levels in plasma and hepatocytes, by anemia and hypoxia, bone marrow factors (GDF15, TWSG1), and erythropoietin.

Activity

Hepcidin-25 interacts with the iron exporter ferroportin from macrophages, enterocytes, hepatocytes and placental syncytiotrophoblasts. It causes the internalization and degradation of ferroportin, leading to reduced iron level in plasma.

Effect

Low plasma hepcidin levels lead to increased intestinal iron absorption, enhanced release of iron from the stores and increased supply of iron for erythropoiesis. Elevated hepcidin levels are associated with inflammation, chronic kidney disease, and iron-refractory anemia.

Table 7.6-2 Disorders of hepcidin and ferroportin regulation

Clinical and laboratory findings

Genetic disease – Generally

These are disorders which result from the mutation of genes that encode hepcidin, ferroportin or their physiological regulators /3/.

– Hereditary hemochromatosis (HH)

HH is characterized by excessive deposition of iron in the liver and other organs as a result of unregulated intestinal absorption of iron. Genetic hepcidin deficiency may be associated with homozygous and compound heterozygous mutations in the HFE gene and autosomal recessive mutations in TFR2, in HJV, and in HAMP (see Tab. 7.1-6 – Clinical and laboratory findings in hereditary hemochromatoses (HH).

Enterocytes and macrophages are depleted in iron due to the increased expression of ferroportin, while hepatocytes are overloaded with iron. The overloading is due to the capability of the hepatocytes to take up and store iron that is not bound to transferrin /3/.

Hepcidin: serum concentration is low or inadequately normal.

– Ferroportin disease

This is a rare form of HH which can occur as early as in adolescence and is due to an autosomal dominant mutation in the Ferroportin gene. The most well-known mutation is C326S. In HH, ferroportin is resistant to hepcidin.

Hepcidin: serum concentration is high or high normal.

– Iron-refractory iron deficiency anemia (IRIDA)

IRIDA is due to a mutation in the TMPRSS6 gene, which encodes membrane-bound serine protease matriptase 2. There is increased BMP signaling, which results in stimulated secretion of hepcidin since the mutated serine protease can no longer cleave hemojuvelin and thereby inhibit hepcidin activation (Fig. 7.6-4 – Signals und pathways for the regulation of hepcidin expression by stimulating factors). There is severe iron deficiency.

Hepcidin: serum concentration is high or high normal.

– Transferrin deficiency

Transferrin saturation plays an important role in the regulation of hepcidin. Deficiency of transferrin causes severe hypochromic anemia. This results in hepcidin deficiency, because either the hepatocyte iron sensors fail to be activated, or the bone marrow erythropoieitic factor, which stimulates hepcidin synthesis, is suppressed by blood transfusions.

Hepcidin: serum concentration is low.

Secondary disorders – Generally

These are disorders that affect the hepcidin-ferroportin system.

– Iron deficiency anemia

In iron deficiency anemia and iron deficiency without anemia, hepcidin-25 levels are below 1 nmol/L and in the combined state of anemia of chronic disease with iron demand of erythropoiesis, below 4 nmol/L /6/.

– Anemia of chronic disease (ACD)

In ACD, erythropoiesis is compromised in response to inflammation. Hepcidin is an important biomarker for the diagnosis of this form of anemia /9/. After the stimulation of hepcidin synthesis by IL-6, there is insufficient release of iron from macrophages and hepatocytes via the ferroportin channels, and iron absorption in the duodenum is blocked. This results in deficiency of functional iron. Consequentially, there is iron-restricted erythropoiesis and reduced uptake of iron by the erythroblast transferrin receptors. However, since hepcidin inhibits the release of iron by causing the degradation of ferroportin, the erythroblasts are still sufficiently supplied with iron via sTfR-2 so that erythropoiesis remains normocytic and normochromic. ACD is characterized by moderate anemia due to a reduced erythrocyte count, hypo regenerative erythropoiesis due to a reduced bone marrow response to erythropoietin, and decreased iron turnover /10/. ACD may be present in inflammation, infection, malignant hematologic diseases, solid tumors, and autoimmune diseases.

The serum level of hepcidin-25 is generally ≥ 15 nmol/L and can increase to levels ≥ 100 nmol/L depending on the presence and extent of inflammation. In patients with inflammation, hepcidin-25 can differentiate ACD from IDA. Patients with ACD have a hepcidin concentration ≥ 4 nmol/L, in IDA patients hepcidin levels are ≤ 1 nmol/L. Hepcidin can, however, not differentiate patients with ACD from those with ACD/IDA. Combination of hepcidin-25 as a marker of iron availability with the reticulocyte hemoglobin content (CHr) as an indicator of iron demand for erythropoiesis allows the differentiation of IDA, ACD and ACD/IDA and ACD/IRE (Fig. 7.6-1 – Differentiation of iron deficiency anemia (IDA) using a diagnostic plot). Patients in the ACD/IRE quadrant already have a reduced iron supply, but have not yet developed hypochromia of the reticulocytes.

– β-thalassemia /11/

The high erythropoietic activity in β-thalassemia suppresses hepcidin synthesis and leads to intestinal hyper absorption of iron and elevated iron stores.

Hepcidin: serum concentration is low.

– Chronic hemodialysis (CHD) patients /12/

In CHD patients, hepcidin correlates with storage iron and the extent of inflammation. In patients with chronic renal insufficiency but without hemodialysis, the hepcidin concentration increases as renal function decreases. In these patients, the fractional excretion of hepcidin in urine correlates with that of β2-microglobulin /13/.

– Hemolytic anemia

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. The iron overload is the result of entrance of free hemoglobin in the circulation. Massive hemolysis is confirmed by a complete decrease of haptoglobin and hemopexin, increased lactate dehydrogenase, an increase in red cell distribution width, a reduced half-life of red blood cells, an increase in ferroportin, and a decrease in hepcidin. Tissue iron overload derived from heme or hemoglobin is primarily localized in the liver and spleen macrophages rather than hepatocytes. Because of depressed hepcidin formation of the hepatocytes enteral iron absorption is increased. Serum ferritin is increased but transferrin saturation remains in the normal range. The absence of hepatocyte iron overload is a consequence of both the huge increase in erythroblast production and urinary iron losses /28/.

Table 7.6-3 Important iron-sensing proteins /19, 23/

Iron-sensing proteins

Holotransferrin (holo-Tf)

Holo-Tf is one of three forms of iron-laden transferrin (see Section 7.5 – Transferrin saturation (TfS)). It plays an important role in sensing blood iron levels, since it is the only form that increases continuously with rising iron levels. Holo-Tf is important in the regulation of hepcidin-mRNA via the hemojuvelin/BMP 2/4-dependent pathway.

HFE protein

The HFE protein forms a complex with TfR-2 on the hepatocyte cell membrane (Fig. 7.6-5 – Model of the HLA-H protein (HFE protein) based on its homology with MHC class I molecules). However, the HFE protein competes with holotransferrin for the same binding site on TfR-2. If there is excess iron in the blood, holotransferrin binds to TfR-2, displacing the HFE protein. HFE then binds to holotransferrin-laden TfR-2 and triggers a signal. The signal is transferred to the nucleus via the SMAD signaling pathway (SMAD, soluble mothers against decapentaplegic...) and causes hepcidin expression to be activated. A mutation in the HFE gene (HAMP gene), as in C282Y homozygosity for example, leads to loss of function of the HFE protein and therefore impaired BMP/SMAD signaling to the nucleus and reduced hepcidin synthesis. This results in unregulated release of iron from enterocytes into the circulation (see Fig. 7.1-6 – Post-transcriptional regulation of cellular iron homeostasis). The structure of the HFE protein is shown in Fig. 7.6-5 – Model of the HLA-H protein based on its homology with MHC class I molecules.

Hemojuvelin (HJV)

HJV is an iron transporter which resides on all cells, in particular hepatocytes and muscle cells. It is bound to the cell membrane via neogenin. HJV has the following functions:

  • HJV is a co-receptor of the BMP. Interaction with the HFE-TfR-2 complex causes the HJV-BMP complex to stimulate hepcidin synthesis via the SMAD signaling pathway.
  • HJV is an iron-transporting protein. HJV binds holo-Tf, whereby the HJV-neogenin complex is destroyed and HJV-bound holo-Tf is internalized into the cell by endocytosis. HJV is subsequently reversely trans located into the extracellular space and the circulation.

Bone morphogenetic proteins (BMPs)

BMPs are cytokines and a subgroup of the transforming growth factor β (TGF-β) super-family. They play a role in the regulation of cellular proliferation, differentiation and apoptosis. When the BMP receptors on the hepatocyte membrane are activated by binding of BMPs, the TfR2-HFE complex can interact with the BMPs and activate hepcidin expression via the SMAD signaling pathway.

SMAD proteins constitute a signaling pathway to hepcidin synthesis from the BMP receptors on the hepatocyte membrane through the cytoplasm to the nucleus, where the HAMP gene, which encodes hepcidin, is activated. The BMC receptor-associated SMAD1 molecule is activated by receptor kinase-mediated phosphorylation, which in turn activates the subsequent SMAD protein by phosphorylation. The activated SMAD complex translocates into the nucleus of the cell and regulates the transcription of the HAMP gene.

Figure 7.1-1 Iron transport in the small intestine by the enterocytes. The transformation of Fe2+ to Fe3+ on the basolateral cell membrane is mediated by hephestin or ceruloplasmin. Adapted from Ref. /6/.

Apical surface Basolateral surface DMT 1 Ferritin Ferrireductase Ferroportin Hephestin Fe 3+ Fe 3+ Fe 2+ Fe 2+ Intestinal lumen Blood

Figure 7.1-2 Cellular iron uptake from transferrin (Tf). With kind permission from Ref. /7/. Iron-laden transferrin (Fe2-Tf) binds to transferrin receptors (TfR) expressed at the cell surface. The TfR-iron (Fe2-Tf)2 complexes are localized in clathrin-coated pits which invaginate by forming a special endosome. By acidification of the endosome, iron is released into the cytoplasm via the divalent metal transporter 1 (DMT 1). In the cytoplasm, iron forms the labile iron pool from where it is distributed to the functional sites or stored as ferritin. The apo-Tf-TfR complex is recycled to the cell surface, TfR is enzymatically cleaved from the wall of the endosome and released into the plasma as circulating (soluble) transferrin receptor. The concentration of soluble TfR depends on the mass of erythroid precursor cells in the bone marrow (normo-, hypo- or hyperregenerative erythropoiesis) and their iron requirements.

Extracellular space (pH 7.4) Cytoplasm Fe 2 -Tf Fe 2+ -transporter (DMT 1) Apo-Tf TfR DMT 1 Clathrin-coated pit Endosome Proton pump Acidified endosome (pH 5.5) Mitochondria Non erythroidcells Released Fe Ferritin Hemosiderin H + H + Cell membrane

Figure 7.1-3 Iron distribution within the organism and quantitative exchange, modified according to Ref. /5/. The daily iron requirement of the functional sites is about 25 mg: 20 mg is required for erythropoiesis and 5 mg is utilized for the synthesis of myoglobin and iron-containing enzymes. Iron not required for the functional sites is stored as ferritin, mainly in the hepatocytes and cells of the reticuloendothelial system. Iron turnover is ensured by the plasma transferrin which generally carries 3–4 mg of iron, corresponding to a transferrin saturation of 25–30%. The iron mobilized by the degradation of senescent erythrocytes is immediately resupplied to the functional sites by transferrin for the new synthesis of iron-containing proteins and hemoglobin. In iron-restricted erythropoiesis there is an imbalance between iron requirements and iron supply of the functional sites.

Erythrocytes2500 mg Reticuloendothelialsystem Red blood celldestruction Red blood cellproduction Bone marrow 20 mg daily 20 mg daily ~ 5 mg daily Loss1–2 mg daily Absorption1–2 mg daily Iron store1000 mg Myoglobin& enzymes of therespiratory chain300 mg Plasma4 mg

Figure 7.1-4 Regulation of iron content of the intracellular pool of the enterocyte by the hepcidin-ferroportin-axis. If plasma TfS is above 20%, Fe2+, which is transported from the blood plasma of the contra luminal site into the labile iron pool via the TfR1-HFE complex, signals sufficient iron supply of the tissues. As a result intestinal iron absorption from the luminal site of the enterocyte to the labile iron pool and the release of iron from the labile iron pool via ferroportin on the contra luminal site is inhibited. β2M, β2-microglobulin; DMT1, divalent metal transporter; HFE, HFE protein; TfR1, transferrin receptor 1.

Heme iron Fe 2+ Fe 2+ Fe 2+ Fe 2+ Fe 3+ Fe 3+ Fe 2+ Ferritin Labile iron pool Intestinal lumen Enterocyte Blood plasma Apotransferrin + Fe-Transferrin Apotransferrin Bone marrow Hepcidin β 2 M TfR1 HFE Transferrin Ferro- portin Hephe- stin

Figure 7.1-5 First step of heme synthesis. Catalyzed by aminolevulinic acid synthase (ALAS), succhinyl-CoA and glycine are converted to aminolevulinic acid (ALA). In the next step, aminolevulinic acid dehydrase (ALAD) catalyzes the condensation of two molecules of ALA to porphobilinogen (PBG).

Mitochondrium Cytosol COOH CH 2 CH 2 COOH + + CH 2 – NH 2 COOH Succinate Glycine COOH δ ALA CH 2 CH 2 C = O CH 2 NH 2 COOH δ ALA CH 2 CH 2 C = O CH 2 NH 2 ALAS H 2 O CO 2 2H 2 O ALAD COOH CH 2 NH PBG CH 2 COOH CH 2 CH 2 CH 2 NH 2 C = O δ ALA COOH CH 2 CH 2

Figure 7.1-6 Post-transcriptional regulation of cellular iron homeostasis. Adapted from Ref. /62/ with kind permission. The levels of iron and nitric oxide (NO) affect the structure of iron regulatory protein (IRP), and the regulatory consequences of this steric change result in cellular responses. Under conditions of a high intracellular iron pool or reduced NO formation, IRP is a four-domain protein with a 4Fe-4S cluster. This form of IRP serves as the cytoplasmic aconitase, and does not bind to IRE stem-loop structures in the untranslated mRNA regions of iron proteins. The result is increased iron uptake, and increased iron storage and heme biosynthesis. By contrast, under conditions of a low labile iron pool or increased NO formation, IRE-binding activity is induced in IRP by an allosteric switch. The result is increased iron uptake, and decreased iron storage and heme biosynthesis.

Iron uptakeIron storageHeme synthesis Iron uptakeIron storageHeme synthesis IRE ε-ALAS 5' AAAA 3' 5' AAAA Blocked High 3' ε-ALAS IRP 5' AAAA 3' Transferrin receptor IRE 5' AAAA Blocked 3' Transferrin receptor IRP Ferritin IRE High 5' AAAA 3' 5' AAAA Blocked 3' Ferritin IRP IRP = cytoplasmic aconitase 1–3 4 IRP = IRE-binding protein 1–3 4 7 mG AUG A G U G N C C Iron high, NO low Iron low, NO high IRP Consequence Response

Figure 7.1-7 Prevalence of iron deficiency and iron deficiency anemia in men, women and children /15/. Iron deficiency is defined as abnormal values for at least two of the following four independent iron status indicators: serum ferritin, erythrocyte zinc protoporphyrin, transferrin saturation, and mean corpuscular volume (MCV).

%35302520151050 0,5–2 2–6 6–10 10–14 14–18 Age (years) Both genderWomenMen 18 –30 30 –40 40 –50 50 –65 >65

Figure 7.1-8 Percentage of pathological results for the iron status indicators hemoglobin, mean corpuscular volume (MCV), transferrin saturation, erythrocyte zinc protoporphyrin and serum ferritin. With kind permission from Ref. /15/. Results for these biomarkers are considered pathologic if they are below the following thresholds: ferritin < 12 μg/L; transferrin saturation < 16%; erythrocyte zinc protoporphyrin < 3 μg/g Hb; MCV < 70 fl, < 73 fl, < 75 fl, < 80 fl in children aged < 2 yrs, 2–6 yrs, 6–14 yrs and adults, respectively; hemoglobin (Hb) < 110 g/L, < 120 g/L, < 130 g/L in children aged < 6 yrs, ≥ 6 yrs and adults, respectively.

50403020100 Women (%) Hemoglobin, MCV, Transferrin saturation, Erythrocyte protoporphyrin Serum ferritin Age (years) 0,6–2 2–6 6–10 10–14 14–18 18–30 30–40 40–50 50–65 >65 6050403020100 Men (%) Age (years) 0,6–2 2–6 6–10 10–14 14–18 18–30 30–40 40–50 50–65 >65

Figure 7.1-9 Evaluation of iron status /70/.The combination of the reticulocyte hemoglobin content (CHr, RetHe) with the ferritin index allows the division of iron restriction into the following 4 states of iron supply of erythropoiesis:

Q1) normal iron supply, Q2) latent iron deficiency, Q3) total (absolute) iron deficiency, (Q4) functional iron deficiency (imbalance between the surging iron requirements of the erythroid marrow and iron availability) in chronic inflammatory conditions. Der cutoff of the ferritin index using the sTfR assay of Siemens is 0,8 and 2,0 using the assay of Roche.

Normal iron supply Iron stores reduced, functional iron normal = latent iron deficiency Iron stores depleted, functional iron reduced = total iron deficiency Iron stores normal/ increased = functional iron deficiency (iron sequestration) sTFR/log ferritin (Ferritin Index) 0 0.8 (2.0) CHr (pg) 362820 Q1 Q2 Q4 Q3

Figure 7.1-10 Age of onset of organic diseases and functional disorders and behavior of serum iron and hepcidin concentrations as a function of the type of hereditary hemochromatosis. Modified from Ref. /36/.

0 10 20 30 40 50 Years Gen HAMP, HJV TfR2 FPN HFE FibrosisCirrhosisCarcinoma CardiomyopathyCongestive heart failure Glucose intolerancegonadaldysfunction Serum iron Hepcidin Years Organs

Figure 7.3-1 Differentiation of non-anemia-related hyperferritinemia based on ferritin, transferrin saturation (TfS) and CRP, Ref. /12/. CRP, C-reactive protein; HHCS, hereditary hyperferritinemia/cataract syndrome; HAMP, gene encoding hepcidin; HFE, gene encoding HFE protein, HJV, gene encoding hemojuvelin; SCL40A1, gene encoding ferroportin; TfR2, gene encoding transferrin receptor 2.

Transferrin saturation Ferritin ♀ > 200 μg/L ♂ > 300 μg/L ♀ ≤ 45% ♂ ≤ 50% ♀ > 45% ♂ > 50% Hereditary hemochromatosis Inflammation (CRP ↑) Hemo- chromatosis Typ 4 HHCS Benigne hyperferri- tinemia Adult HFE Typ 1 TfR2 Typ 3 SCL40A1 Typ 4b Juvenile HJV Typ 2 HAMP Typ 5

Figure 7.3-2 Ferritin molecular structure showing the H and L subunits and a crystalline iron core.

L H L L H H H L L H

Figure 7.4-1 Relationship between serum erythropoietin (EPO) and hematocrit (HCT). Adapted from Ref. /7/ with kind permission. Effective erythropoiesis results in an inverse logarithmic relationship between EPO concentration and Hct if it falls below 38%. This results in an adequate rise in EPO with erythropoietic activity raised by a factor of 3–5, and in reticulocytosis (hyper regenerative erythropoiesis). Inadequately low EPO concentrations indicate the presence of a hypo proliferative anemia through deficient erythropoietic stimulation. This is the case in renal anemia, for example. Some laboratories use the O/P ratio (O, observed = measured EPO concentration; P, predicted = expected EPO concentration based on the Hct). An O/P ratio below 0.8 in the presence of anemia indicates inadequately low EPO synthesis /7/.

1000100101 20 25 30 35 40 45% HCT Inappropriate erythro-poietin response to anemia Appropriate response EPO (U/L)

Figure 7.4-2 Relationship between serum sTfR concentration and hematocrit (HCT). Inadequately low sTfR levels indicate the presence of intrinsic marrow hypoproliferation in the case of normal EPO production. Adapted from Ref. /7/ with kind permission.

100101 20 25 30 35 40 45% HCT Defective marrowresponse to anemia Appropriate response sTfR (mg/L)

Figure 7.4-3 Transferrin receptor (TfR). The TfR is a heterodimer composed of two transmembrane subunits. Each subunit possesses one binding site for a transferrin molecule saturated with two iron atoms. The extracellular domains are cleaved off by the proteolytic action of trypsin and can be measured in plasma as soluble transferrin receptor. Adapted from Ref. /31/ with kind permission.

Iron-saturatedtransferrin Transferrin-binding site COOH 70 20 5 M r × 10 –3 COOH Asn 727 N-ligated glycans O-ligatedglycans Site of trypsincleavageArg 100 Plasma membrane NH 2 Cys-89 S S PO 4 -Ser 24 Asn 317 Asn 251 Ser 24-PO 4 H 2 N Covalentlyboundfatty acids S S Cys-98

Figure 7.5-1 Iron turnover between the different compartments. Modified according to Ref. /13/.

20 mg 2500 mg 20 mg 1000 mg Intestine Intestinalmucosa Functional iron Iron stores Myoglobin, enzymes Diet Transferrin Tissue Blood Absorption1 mg/day 10–15 mg/day 1 mg/day Ironloss Epithelialcells,blood loss 500 mg 5 mg Red blood cells Ferritin

Figure 7.6-1 Differentiation of iron deficiency anemia (IDA), anemia of chronic disease (ACD), the combined state of ACD with the IDA (ACD/IDA), and iron-restricted erythropoiesis in ACD (ACD/IRE) using a diagnostic plot. Serum hepcidin-25 and reticulocyte hemoglobin content (CHr) were determined /8/.

< 0.2 2 4 6 8 10 > 20 15 Hepcidin-25 (nmol/L) CHr (pg) IDA ACD/IRE ACD/IDA ACD 20 25 30 35 40 28

Figure 7.6-2 Structure of hepcidin-25. The three-dimensional structure is stabilized by four disulfide bonds.

C19 C11 C10 C7 C22 C13 C14 C23

Figure 7.6-3 Binding of hepcidin to ferroportin in enterocyte and macrophage cell membrane.

Ferritin Enterocyte Ferroportin Ferroportin Hepcidin Fe(II) Fe(II) Macrophage Ferritin

Figure 7.6-4 Signals und pathways for the regulation of hepcidin expression by stimulating factors (iron overload, inflammation) and inhibiting factors (iron deficiency, hypoxia, increase in erythropoietin). Modified according to Ref. /14, 16/. Abbreviations refer to Tab. 7.5-3 – Behavior of transferrin saturation in conditions of iron deficiency.

Macrophage Fe Fe Fe Fe Fe TF Erythrocytes TF FPN FPN FPN FPN TF BMP6 SMAD1/5/8 1/5/8 Stat3 Nucleus Cytoplasm BMP6 Fe HJV HJV Hepcidin Hepcidin Hepatocyte TFR2 BMPRI-II HFE BMP6 HJV TFR2 Enterocyte DMT1 P IL-6R IL-6 HAMP SMAD1/5/8 SMAD4 P Inhibition:– Iron deficiency– Hypoxia– Erythropoietin

Figure 7.6-5 Model of the HLA-H protein (HFE protein) based on its homology with MHC class I molecules. The protein is a single polypeptide with three extracellular domains which are analogous to the α1, α2, and α3 domains of the MHC class I proteins. β2-microglobulin is a separate protein and interacts with the HLA-H gene product in a non-covalent manner in the α3 homologous region. In addition, the HLA-H protein contains a membrane spanning region and a short cytoplasmatic tail. The appropriate locations of Cys282Tyr and His63Asp are indicated. With kind permission of Ref. /27/.

S H63D mutation COOH COOH Intracellular Plasma membrane Extracellular β2-microglobulin α 2 α 1 α 3 NH2 S S S S S NH2 α-heavy chain C282Y mutation
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