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 .
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 . 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.
- 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% .
- 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 220.127.116.11) and lipoamide dehydrogenase (EC 18.104.22.168). 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 . Animal tests have shown that, in iron deficiency, the activity of these enzymes is reduced to 30–60% of original activity .
- 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 . Hydroxyl radicals and the super oxide anion oxidize macromolecules and destroy organic structures.
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.
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 .
- 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 .
- 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 (). 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+ .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 22.214.171.124) the porphyrin ring is split, and the iron released to feed the intracellular iron pool .
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 . 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.
- 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 (). 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) . 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.
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 (). 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 /, /.
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 (). 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.
- 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 . 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).
Body iron losses vary with sex and age and are significant when there is blood loss . 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 . 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 .
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.
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 .
- 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 .
The macrophage as a regulatory mechanism
Macrophages of the reticuloendothelial system play an important role in the regulation of iron balance . 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 126.96.36.199). 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 ()
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 .
The hepatocyte can, however, also absorb iron not bound to transferrin (Tf) when the transport capacity of Tf is exhausted . 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 ). This occurs as shown in . 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 (). This regulation ensures that the amount of toxic protoporphyrin IX is aligned with the availability of iron .
Hepcidin as a regulator of iron metabolism
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.
Each somatic cell regulates its own iron balance, and possesses a regulatory system for the coordination of iron uptake, use and storage . 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 ().
- 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 )
- 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.
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– , 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.
- 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).
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 .
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 . 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
The different states associated with iron restriction are shown in . The evaluation of the iron status using ferritin index and hemoglobin content of reticulocytes (CHr, RetHe) as markers, is presented in .
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.
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.
- 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.
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.
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 . 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.
Erythropoiesis is the dominating factor in iron metabolism . 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 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 .
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 .
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 (). 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.
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 a scoring system to provide optimal guidance for the evaluation of iron restriction in non-anemic patients was established (). 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.
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.
- 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 .
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 . 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 . 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 .
Iron deficiency and the neurotransmitter system
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 . 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 .
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 . 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 ().
Acquired forms of iron overload are usually due to transfusional iron. The iron is primarily deposited in 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.
Of the six disorders of the hepcidin-ferroportin axis five have the classic HH phenotype. Laboratory results in these phenotypes are elevated transferrin saturation, elevated ferritin, normal hematocrit and tissue iron overload.
In HH, the 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 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, TfR2-associated, hemojuvelin (HJV)-associated and HAMP-associated HH (). 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 . The results of biomarkers in HH are shown in .
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 C282Y HFE-mutation.
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.
Mutations in the HAMP gene, which encodes the protein hepcidin are responsible for this type. It is a rare form of juvenile hemochromatosis.
This type is caused by mutations in the TfR2 gene and, in terms of clinical symptoms, is an intermediate form between the HFE type and HH type 2.
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.
With the exception of ferroportin disease (type 4), all types of HH are similar in their clinical, diagnostic and histological characteristics, for example:
- Intestinal iron absorption is unregulated and the rapid mobilization of iron from macrophages leads to high TfS:
- The high TfS promotes the uptake of iron by the cells via the TfR and deposition of iron in the cells, in particular 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.
Deposition of iron in the parenchymal organs leads to hepatic fibrosis, hypogonadism, cardiomyopathy, diabetes mellitus, arthropathy and skin pigmentation in HH. The most severe forms of HH are diagnosed in childhood and early adolescence, but usually before the age of 30. Every child with hypo gonadotropic 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.
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.
Liver biopsy is not required to establish the diagnosis of HFE hemochromatosis in C282Y homozygotes . 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 has shown that liver cirrhosis can also be detected by laboratory investigation ().
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 (). 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.
The following laboratory tests are used to diagnose restriction of iron:
- Biomarkers such as ferritin, transferrin saturation (TfS) soluble transferrin receptor (sTfR), zinc protoporphyrin (ZPP), and ferritin index (sTfR/log10 ferritin)
- Hematological tests, such as blood count, erythrocyte indices e.g., proportion of hypochromic red blood cells (% HYPO), and reticulocyte Hb content (Ret-He and CHr)
- Inflammation markers such as C-reactive protein (CRP). Often CRP is elevated, ferritin is normal or elevated. In this condition iron deficiency is often overlooked, because iron deficiency is frequently concomitant with inflammatory disease. The diagnostic value of biomarkers and hematologic indices in the diagnosis of iron restriction is shown in . The results of the markers in patients with disorders in iron metabolism are shown in .
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 .
- 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.
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 .
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 .
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 .
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 . 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 . A CHr value < 29 pg predicts functional iron deficiency in patients receiving ESA therapy . Indicators of iron-restrictive erythropoiesis are the following results %HYPO > 6% or CHr < 29 pg . In isolation transferrin saturation is not recommended as a predictor of responsiveness to intravenous iron therapy .
9. Galy B, Ferring-Appel D, Kaden S, Gröne HJ, Hentze MW. Iron regulatory proteins are essential for intestinal function and control key iron absorption molecules in the duodenum. Cell Metab 2008; 7: 79–85.
15. Preziosi P, Hercberg S, Galan P, Devanlay M, Cherouvrier F, Dupin H. Iron status of a healthy French population: factors determining biochemical markers. Ann Nutr Metab 1994; 38: 192–202.
16. Baart AM, van Noord PAH, Vergouwe Y, Moons KGM, Swinkels D, Wiegerinck ET, et al. High prevalence of subclinical iron deficiency in whole blood donors not deferred for low hemoglobin. Transfusion 2013; 53: 1670–7.
19. Cappellini MD, Comin-Colet J, de Francisco A, Dignass A, Doehner W, Lam CSP, et al. Iron deficiency acoss chronic inflammatory conditions: international expert opinion on definition, diagnosis, and management. Am J Hematol 2017; 92: 1068–78.
23. Steinmetz T, Tsamaloukas A, Schmitz S, Wiegand J, Rohrberg R, Eggert J, et al. A new concept for the differential diagnosis and therapy of anemia in cancer patients. Support Care Cancer 2011; 19: 261–9.
33. Björn-Rasmussen E, Hallberg L, Isaksson B, Arvidson B. Food iron absorption in man. Applications of the two-pool extrinsic tag method to measure haem and non-haem iron absorption from the whole diet. J Clin Invest 1974; 53: 247–55.
38. Crawford DJ, Murphy TL, Ramm LE, Fletcher LM, Clouston AD, Anderson HJ, et al. Serum hyaluronic acid with serum ferritin accurately predicts cirrhosis and reduces the need for liver biopsy in C282 hemochromatosis. Hepatology 2009; 49: 418–25.
41. Gurrin LC, Osborne NJ, Constantine CC, McLaren CE, English DR, Gertig DM, et al. The natural history of serum iron indices for HFE C282Y homozygosity associated with hemochromatosis. Gastroenterology 2008; 135: 1945–52.
43. Brittenham GM, Weiss G, Brissot P, Laine F, Guilygomarc A, Guyader D, Moirand R, Deugnier Y. Clinical consequences of new insights in the pathophysiology of disorders of iron and heme metabolism. Hematology 2000; 65: 39–50.
50. Giannini E, Mastracci L, Botta F, et al. Liver iron accumulation in chronic hepatitis C patients without HFE mutations: relationships with histological damage, viral load and genotype and γ-glutathione S-transferase levels. Eur J Gastroenterol Hepatol 2001; 13: 1355–61.
65. Breymann C, Richter C, Hüttner C, Huch R, Huch A. Effectiveness of recombinant erythropoietin and iron sucrose vs. iron therapy only, in patients with postpartum anaemia and blunted erythropoiesis. Eur J Clin Invest 2000; 30: 154–61.
68. Tessitore N, Solero GP, Lippi G, et al.: The role of iron status markers in predicting response to intravenous iron in haemodialysis patients on maintenance erythropoietin. Nephro Dial Transplant 2001; 16: 1416–23.
- The determination of transferrin saturation
- The iron resorption test
- The detection of acute iron intoxication.
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 . The method of the International Committee for Standardization in Hematology (ICSH) , 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.
Plasma (no EDTA plasma), serum: 1 mL
- 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) .
- 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.
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. .
- 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) .
- 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 , 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.
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 . The WHO recommendations for daily oral iron intake are shown in . 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. .Iron absorption from supplementes is greater with alternate day than with consecutive day dosing in iron-deficient anemic women .
Total iron deficit (mg) = Body weight (kg) × 0.24 × [target Hb (g/L) – actual Hb (g/L)] + 500
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 .
- 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) are diarrhea, vomiting, leukocytosis, hyperglycemia, and positive abdominal X-ray findings.
Method of determination
- 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).
- 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.
Iron in serum or plasma is stable for 3 days if stored at room temperature or for one week if stored at 4 °C.
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.
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 .
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.
- 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.
Immunoassays, such as enzyme-linked immunoassay (ELISA), immunometric assay (IMA), luminescence immunoassay (LIA).
Serum, plasma: 1 mL
Iron is contained in three pools of the organism:
- 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.
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 . 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 ) 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 . 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 .
Serum ferritin is the preferred test for diagnosing iron deficiency (). 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% . 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% . Threshold values of ferritin for the diagnosis of total iron deficiency and storage iron deficiency are shown in .
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 .
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 . 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.
Serum ferritin levels increase in acute-phase response, infection, acute and chronic inflammation, autoimmune disease, chronic renal disease, chronic heart failure, inflammatory bowel disease and malignancy. The 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 hyper ferritinemia 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.
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 ().
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.
Since 1997 the 3rd International Standard for Ferritin (recombinant; NIBSC code 94/572) has been available. It only contains the L subunit of apoferritin. Although commercially available ferritin assays are standardized the results of patients show extraordinary variations. 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 .
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 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 .
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.
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%.
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% .
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.
- 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 ().
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 . 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 . There is a rapid increase in serum ferritin following intestinal iron uptake. Synthesis is regulated by the IRE/IRP system (see also ). 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 .
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 . 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 . A virally-induced cytokine storm is found in a subgroup of patients with SARS-Cov-2 .
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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25. Biesma DH, Kraajenhagen RJ, Poortmann J, Marx JJM, van de Wiel A. The effect of oral iron supplementation on erythropoiesis in autologous blood donors. Transfusion 1992; 32: 162–5. Transfusion 2013; 53: 1637–44.
31. Giannini E, Mastracci L, Botta F, et al. Liver iron accumulation in chronic hepatitis C patients without HFE mutations: relationships with histological damage, viral load and genotype and α-glutathione S-transferase levels. Eur J Gastroenterol Hepatol 2001; 13: 1355–61.
34. Adamkiewicz TV, Abboud MR, Paley C, Olivieri N, Kirby-Allen M, Vichinsky E, et al. Serum ferritin level changes in children with sickle cell disease on chronic transfusion are non-linear, and are associated with iron load and liver injury. Blood 2009; 114: 4632–9.
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41. Kristensen GB, Rustad P, Berg JP, Aakre KM. Analytical bias exceeding desirable quality goal in 4 out of 5 common immunoassays: results of a negative single serum sample external quality assessment program for cobalamin, folate, ferritin, thyroid-stimulating hormone, and free T4 analysis. Clin Chem 2016; 62: 1255–63.
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.
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.
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 . 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 .
Serum, plasma: 1 mL
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.
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 :
- 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).
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 :
- Reduced proliferation through inadequately low EPO production
- Intrinsic, EPO-independent erythropoietic hypo proliferation
- Maturation disorders e.g., ineffective erythropoiesis)
- Reduced erythrocyte lifetime (peripheral hemolysis).
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 ().
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.
An early predictor of response to ESA therapy is the increase of sTfR. In one study , 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.
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 . 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 . 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 .
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 .
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 .
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) . 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 . This does not occur in all cases. shows the sTfR threshold for differentiating iron deficiency anemia from ACD and combined state of ACD with the IRE (ACD/IRE). shows the behavior of sTfR in iron deficient states.
- 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.
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 .
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 /, /. Sex-specific reference ranges have been specified for one manufacturer’s assay .
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 . The cause is reported to be the progressive separation of the TfR from reticulocytes and leukocytes.
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.
- 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 ).
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 .
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 . 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 .
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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 homeostasis
- Suspected functional iron deficiency
- Suspected iron overload
- Evaluation of plasma iron turnover.
- 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 .
Serum, plasma (no EDTA plasma): 1 mL
Blood should be collected in the fasting period.
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 (). 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. In disorders of iron metabolism 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 .
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 .
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 .In anemic patients with chronic kidney disease a probatory iron dose is recommended in cases if TfS is < 30% .
Plasma iron turnover is a measure of the amount of iron transported in the plasma every day (). 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 .
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.
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.
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.
Increased iron stores and inflammation induce hepcidin production, whereas hypoxia, anemia, iron deficiency, increased erythropoiesis and therapy with erythropoiesis stimulating agents (ESA) attenuate hepcidin synthesis. Inflammation decreases the iron availability of the organs, whereas hypoxia or anemia increase iron release and absorption .
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 /, /:
- Increase in 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.
- Diagnosis and differentiation of hemochromatosis.
Serum, fasting (blood collection by 9 a.m.): 1 mL
In most cases bio active hepcidin 25 is determined.
The systemic regulation of iron homeostasis is mediated by hepcidin. More than 80% of anemias result from iron deficiency or functional iron deficiency due to inflammation caused by chronic heart failure, inflammatory bowel disease, chronic kidney disease, immune activation in infections, malignant tumors or autoimmune diseases. Hepcidin can be a useful biomarker 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 . 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% . 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) . Disorders of hepcidin and ferroportin regulation are described in .
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 , the different mass spectrometry assays showed acceptable agreement, but significant disagreement with the immunoassays.
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 .
Hepcidin, the systemic iron regulator
Hepcidin is the principal regulator of plasma iron and ensures a stable concentration of transferrin-bound iron (). 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 ().
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. () . 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 .
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 . 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 .
Hepcidin production is stimulated by the increase of the intracellular labile iron pool or by inflammation induced by IL-6.
Signals that stimulate and inhibit hepcidin production act through receptors on the hepatocyte cell membrane. The receptors responsible for stimulating hepcidin expression respond to an increase in extracellular iron (iron sensing), to inflammatory stimuli and to stress of the endoplasmic reticulum. The receptors responsible for inhibiting hepcidin expression respond to hypoxia, erythropoietin increase, and the release of growth differentiation factors of the erythroblasts when iron is required. Transferrin deficiency also causes reduced hepcidin expression.
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 through bone morphogenetic protein (BMP) receptor is the standard pathway (). 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 . BMP6 activates its receptors BMPR I and BMPR II in the presence of the co receptor hemojuvelin , forming a heterotetrameric complex of BMP6, the receptors and hemojuvelin . 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.
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 .
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 .
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 .
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 . Tf deficiency leads to microcytic hypochromic anemia and insufficient hepcidin expression. Infusion of Tf normalizes plasma hepcidin.
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.
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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.
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.
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Clinical and laboratory findings
Clinical and laboratory findings
R, sTfR assay of Roche; S, sTfR assay of Siemens; ferritin expressed in μg/L
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
Presence of C282Y mutation
Stage 4 fibrosis
Clinical and laboratory findings
Clinical and laboratory findings
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
R, sTfR assay of Roche; S, sTfR assay of Siemens; RetHe CHr, hemoglobin content of the reticulocyte; %Hypo, proportion of hypochromic red cells
Conversion: μg/dL × 0.1791 = μmol/L; pp, post partum.
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%.
Storage iron deficiency
Total iron deficiency
Clinical and laboratory findings
Clinical and laboratory findings
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.
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
Data expressed in %
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. .
Figure 7.1-2 Cellular iron uptake from transferrin (Tf). With kind permission from Ref. . 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.
Figure 7.1-3 Iron distribution within the organism and quantitative exchange, modified according to Ref. . 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.
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.
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).
Figure 7.1-6 Post-transcriptional regulation of cellular iron homeostasis. Adapted from Ref. 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.
Figure 7.1-7 Prevalence of iron deficiency and iron deficiency anemia in men, women and children . 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).
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. . 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.
Figure 7.1-9 Evaluation of iron status .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.
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. .
Figure 7.3-1 Differentiation of non-anemia-related hyperferritinemia based on ferritin, transferrin saturation (TfS) and CRP, Ref. . 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.
Figure 7.4-1 Relationship between serum erythropoietin (EPO) and hematocrit (HCT). Adapted from Ref. 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 .