INTRODUCTION — A tight regulation of iron balance is essential to avoid both iron deficiency and overload. The regulation of iron metabolism involves the interaction of a number of specific proteins as well as the interplay between iron absorption, recycling, and iron loss. This topic review will discuss these factors [1].

Clinical implications are discussed separately:

●Iron deficiency – (See "Iron deficiency in infants and children <12 years: Treatment" and "Iron requirements and iron deficiency in adolescents" and "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults".)

●Iron overload – (See "Approach to the patient with suspected iron overload" and "Clinical manifestations and diagnosis of hereditary hemochromatosis" and "HFE and other hemochromatosis genes".)

ROLE OF SPECIFIC PROTEINS — Our understanding of iron metabolism and the hallmarks of iron deficiency and iron excess is based upon the biology of a number of critical proteins, including but not limited to the following (figure 1):

●Transferrin (Tf), the plasma iron transport protein.

●Transferrin receptor (TfR), the cellular receptor for iron-bound transferrin.

●Ferritin (Ft), the cellular iron storage protein.

●Iron regulatory protein 1 and 2 (IRP1 and IRP2), the cellular iron sensing proteins.

●Divalent metal transporter 1 (DMT1, Nramp2, DCT1, Solute carrier family 11, member 2 [SLC11A2]), the duodenal iron transporter.

●Ferroportin (Ireg1, SLC40A1, Mtp1), the cellular iron exporter. Mutations of ferroportin cause a rare form of hereditary hemochromatosis called ferroportin disease.

●Hephaestin, which likely cooperates with ferroportin for exporting iron from enterocytes to transferrin.

●Ceruloplasmin, a plasma metalloprotein required by ferroportin to export iron from macrophages, hepatocytes, and glial cells.

●Hepcidin, the key negative regulator of intestinal iron absorption as well as macrophage iron release [2]. Mutations of hepcidin cause a rare form of juvenile hemochromatosis [3].

●HFE, mutations of which are responsible for the common form of hereditary hemochromatosis.

●TFR2, mutations of which are responsible for a rare form of hereditary hemochromatosis.

●Hemojuvelin, a hepcidin regulator, mutations of which are responsible for the common form of juvenile hemochromatosis.

●Bone morphogenetic proteins (BMPs; cytokines produced by endothelial cells) such as BMP6 and BMP2, which activate hepcidin binding to BMP receptors and signaling through SMAD proteins [4].

●Matriptase 2/TMPRSS6, the liver hepcidin inhibitor with a major role in iron deficiency.

●Erythroferrone, a hormone produced by erythroblasts stimulated by erythropoietin that downregulates hepcidin in response to erythropoiesis expansion.

Transferrin — The gene for apotransferrin is on the long arm chromosome 3. It codes for a protein (mol wt 80 kDa) that tightly binds one or two ferric (Fe³⁺) iron molecules and is the major transporter for iron trafficking through the plasma. Most of the Tf, which has a half-life of eight days, is made in the liver where its synthesis is considerably increased in states of iron deficiency by unknown mechanisms [1].

ApoTf acquires iron from ferroportin, becoming monoferric or (when iron is abundant) diferric. A 2019 study reported that Tf binds iron at the N-terminal and C-terminal lobes with different affinities; binding to the C-terminal lobe favors the N-terminal lobe binding and the formation of diferric transferrin [5]. Diferric transferrin is the true ligand of both transferrin receptors (TfR1 and TfR2). (See 'Transferrin receptor' below.)

●Tf can be measured in the plasma using an enzyme-linked immunosorbent assay (ELISA) or turbidimetric method to determine the mg of the transferrin protein/dL of plasma.

●The total iron binding capacity of Tf (ie, TIBC) can be measured directly using iron binding methodology (ie, mg of iron binding capacity/dL of plasma), or it can be calculated by multiplying the results of the chemical or immunologic method by a conversion factor calculated by the individual laboratory, as follows:

Complete lack of transferrin is most likely incompatible with life. Hypotransferrinemia is a rare recessive disorder associated with low transferrin levels (<10 mg/dL), severe iron deficiency anemia with hypochromic, microcytic red cells, and iron overloading of the liver and other parenchymal organs [6].

Transferrin receptor — The gene for the TfR (TFRC) is located on the long arm of chromosome 3 (as is the gene for transferrin). TFRC codes for a homodimeric transmembrane protein (mol wt 94 kDa) that is found on most cells, most densely on erythroid precursors and placental cells.

There is a binding site for the transcription factor Stat5 in the first intron of the gene that encodes TfR [7]. Expression of constitutively active Stat5A in an erythroid cell line increased TfR levels, while lethally irradiated mice that received a transplant of Stat5a/b^-/- liver cells developed microcytic, hypochromic anemia [7].

Each TfR molecule can bind two diferric Tf molecules (four Fe³⁺ atoms), which it endocytoses after clustering on clathrin coated pits. The iron is offloaded in acidified vacuoles and the apotransferrin-TfR complex is recycled to the cell surface, where apo-Tf is discharged and released back into the circulation. TfR mRNA has five 3' iron-response elements (IREs) and, as occurs in other iron genes, is post-transcriptionally regulated by iron-response proteins (IRPs), being stabilized in iron deficiency and degraded in iron overload. (See 'Systemic iron homeostasis' below.)

Important roles in several tissues have been illustrated in mouse models:

●Germline inactivation of TfR in mice is embryonically lethal due to severe anemia and abnormal central nervous system development. Haploinsufficiency of TfR causes microcytosis and a reduction of total body iron [8].

●Conditional heart inactivation of TfR in mice causes cardiomegaly, decreased cardiac function, failure of mitochondrial respiration, and early death [9].

●Conditional inactivation of TfR in the epithelial cells of the mouse intestine causes disruption of the epithelial barrier and early death. Since the phenotype was unresponsive to parenteral iron treatment, the authors suggested that TfR is implicated in the maintenance of the intestinal epithelium [10].

●Conditional inactivation of TfR in the liver indicates that TfR is not essential for basal hepatocellular iron supply but is indispensable for the fine-tuning of hepcidin expression in response to hepatocyte iron loading [11].

The only disease associated with TfR mutations causes only mild anemia and leads to a type of combined immunodeficiency. It is due to a homozygous mutation that disrupts the TfR internalization signal, strongly impairing endocytosis and underscoring the essential role of iron in B and T lymphocytes [12].

Serum (soluble) TfR (sTfR) is a product of membrane TfR that is released into the circulation by membrane proteases when TfR is not associated with its ligand, diferric transferrin, as occurs in iron deficiency [13]. Levels of sTfR measured in serum correlate directly with erythropoietic expansion [14]. sTfR levels can be a measure of iron deficiency. (See "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults", section on 'Iron studies (list of available tests)'.)

Ferritin — Ferritin is the cellular storage protein for iron. It is a huge 24 subunit protein (mol wt 440 kDa) consisting of light chains (L ferritin, 20 kd, gene on chromosome 19) and heavy chains (H ferritin, 21 kd, gene on chromosome 11), that can store up to 4500 atoms of iron within its spherical cavity [15]. H Ferritin possesses ferroxidase activity necessary for iron uptake by the ferritin molecule. L ferritin stabilizes the multimeric ferritin shell. An RNA binding protein (poly (rC)-binding protein 1, PCBP1) appears to be required as a cytosolic chaperone to deliver iron to ferritin [16]. PCBP1 gene deletion in mice causes microcytic anemia [17].

Ferritin synthesis is subject to different levels of control, including DNA transcription via its promoter, and mRNA translation via interactions with iron regulatory proteins [18]. (See 'Iron regulatory proteins or iron-responsive element binding protein' below.)

Ferritin is an acute phase reactant, and, along with transferrin and the transferrin receptor, is a member of the protein family that orchestrates cellular defense against oxidative stress and inflammation [18,19]. The gene for H ferritin is activated by oxidative stress via an upstream enhancer antioxidant-responsive element (ARE). (See "Acute phase reactants".)

Mice lacking H ferritin die early in gestation [20]. Mice heterozygous for H ferritin have slightly elevated tissue L ferritin and 7- to 10-fold more serum L ferritin than controls, although they do not have tissue iron overload [21]. These observations suggest that reduced H ferritin expression in humans might be responsible for cases in which high serum ferritin is present in the absence of iron overload [21]. Conditional inactivation of H ferritin in duodenal cells in mice causes iron overload, indicating that ferritin in the gut is important in the control of iron absorption [22].

Much of the iron stored in ferritin is accessible for metabolic needs. Ferritin is degraded by lysosomes through a process called ferritinophagy that requires the cargo protein NCOA4 [23,24]. This process is important for recovering intracellular iron when needed. Inactivation of NCOA4 in mice causes increased ferritin aggregates in spleen and liver macrophages and other organs [23,25]. In vitro downregulation of NCOA4 affects erythroid maturation and hemoglobin synthesis [17]. Ferritin within erythroid precursors may donate iron for heme synthesis, especially at the beginning of hemoglobin accumulation, at a time when the transferrin-transferrin receptor pathway is still insufficient [26]. However, the most important role of NCOA4 is in macrophage ferritinophagy, to recover iron stored in ferritin in iron deficiency [27].

When ferritin accumulates, it aggregates and is proteolyzed by lysosomal enzymes; it is then converted to an iron-rich, poorly characterized hemosiderin which releases its iron slowly and is detected by the Prussian blue reaction, which is used in the common Perls staining for iron in bone marrow aspirates.

Ferritin measured clinically in plasma is usually apoferritin, a non-iron containing molecule. The plasma level generally reflects overall iron storage, with 1 ng of ferritin per mL indicating approximately 10 mg of total iron stores. Thus,

●An adult male with a plasma ferritin level of 50 to 100 ng/mL has iron stores of approximately 500 to 1000 mg [28].

●A serum ferritin less than 15 ng/mL is 99 percent specific for making a diagnosis of iron deficiency. (See "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults", section on 'Iron studies (list of available tests)'.)

●An elevated serum ferritin in the absence of infection or inflammation may suggest the presence of an iron overload state. (See "Approach to the patient with suspected iron overload", section on 'CBC, LFTs, and iron studies'.)

●Ferritin levels may be extremely high in patients with hemophagocytic lymphohistiocytosis or certain rheumatologic disorders. In such cases, the ferritin tends to be less glycosylated than normal. (See "Clinical features and diagnosis of hemophagocytic lymphohistiocytosis", section on 'Serum ferritin levels'.)

A separate ferritin (m-ferritin) is present within mitochondria and is the product of an intronless nuclear gene [15]. Its expression is increased in tissues with high numbers of mitochondria, rather than in tissues involved in iron storage [29]. M-ferritin likely protects mitochondria from oxidative damage [15].

Iron regulatory proteins or iron-responsive element binding protein — The expression of proteins involved in cellular iron uptake and storage is regulated by the iron status of the cell.

Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) are cytosolic RNA-binding proteins that bind to iron-responsive elements (IRE), consisting of a loop configuration of nucleotides, that are located in the 5' or 3' untranslated regions of specific mRNAs encoding for iron and other genes (eg, ferritin, TfR, DMT1, ferroportin, the erythroid specific form of delta-aminolevulinic acid synthase [eALAS], hypoxia inducible factor-2 alpha [HIF-2 alpha; HIF-2α]).

Binding of IRPs to their target sequences occurs when the cell is iron deficient; this has different effects according to whether the IRE position is at the 5' or the 3' UTR:

●When IRPs bind to the 5' IRE of ferritin, ferroportin, or eALAS, the rates of biosynthesis are decreased.

●When IRPs bind to the 3' end of transcripts such as TfR or DMT1, the mRNA half-life is prolonged and rates of biosynthesis are increased (figure 2).

IRP1 and IRP2 sense the state of the iron balance in the cell in different ways. When cellular iron levels increase, assembly with iron sulfur clusters changes IRP1 to aconitase and its binding ability is lost. Under the same conditions of cellular iron increase, IRP2 interacts with the iron stabilized FBXL5 protein, which recruits an E3 ligase [30,31]. This causes IRP2 ubiquitination and proteasomal degradation [32]. FBXL5 has an iron- and oxygen-sensing domain, representing an example of the iron-oxygen connection.

The net effect of these IRPs is that the iron overloaded state is characterized by increased production of ferritin (to permit adequate storage) and ferroportin (to export excess iron) as well as decreased production of TfR (to minimize iron uptake). These changes are reversed in iron deficiency, which is characterized by reduced ferritin and ferroportin and elevated TfR synthesis (figure 2).

Targeted deletion of the gene encoding IRP1 in the mouse leads to no evident hematologic change in adulthood, while targeted deletion of the gene encoding IRP2 causes misregulation of iron metabolism, refractory microcytic anemia, and a neurodegenerative disease due to abnormal neuronal accumulation of iron [33,34].

These observations establish a major role for IRP2 in the regulation of iron uptake in erythroid cells. However, it has been shown that HIF-2 alpha, which has an IRE in the 5' UTR, is specifically controlled by IRP1 [35,36]. This demonstrates another example of the iron-hypoxia connection. Irp1-/- mice, when young or iron deficient, develop polycythemia, pulmonary hypertension, and sudden death from hemorrhages, due to an inability to suppress the synthesis of HIF-2 alpha that stimulates both erythropoietin in the kidney and endothelin 1 in pulmonary endothelial cells [37,38].

There are differences in tissue-specific expression of the two IRPs, and also specific targets. (See "Molecular pathogenesis of congenital polycythemic disorders and polycythemia vera", section on 'Oxygen sensor'.)

HFE — HFE, a product of the HFE gene on the short arm of chromosome 6, is an MHC class I-like protein present ubiquitously at low levels, with high-level expression in hepatocytes. The C282Y variant in the HFE gene is responsible for the vast majority of cases of hereditary hemochromatosis in adults. Iron overload is also noted in mice with HFE gene deletion [39]. Conditional deletion of HFE in the liver produces the phenotype of hemochromatosis [40]. In contrast, conditional deletion of HFE in duodenal cells or macrophages does not alter systemic iron metabolism [41].

The HFE protein is associated in a complex with TfR as a possible iron sensor. Diferric transferrin competes with this binding, releasing HFE from TfR when iron is abundant. Free HFE increases the response of hepcidin to iron, since patients with hemochromatosis due to an HFE mutation have low hepcidin levels and do not respond to oral iron challenge [42,43]. (See 'Hepcidin' below.)

It has been reported that when HFE is free from TfR, it binds to TfR2, encoded by a gene mutated in type 3 hereditary hemochromatosis that is considered to be a sensor of the level of transferrin saturation [44]. (See "HFE and other hemochromatosis genes".)

However, HFE-TFR2 binding has been disputed [45]. Indeed, the diseases caused by pathogenic variants affecting HFE and TfR2 are different [2]. Simultaneous gene deletion of both HFE and TfR2 in mice results in marked dysregulation of hepcidin and more severe iron overload than loss of the single molecules [46]. These observations suggest distinct functions for HFE and TfR2. According to an in vitro study, HFE stabilizes the BMP receptor ALK3 that is degraded when HFE is inactive [47].

Transferrin receptor 2 — Transferrin receptor 2 (TfR2), encoded by a gene on chromosome 7q22, is a member of the TfR family and is homologous to TfR1, but it has no IRE elements and has a lower affinity for diferric transferrin than TfR1 [48]. It displays a restricted expression pattern, being present at high levels in hepatocytes and erythroid cells. TfR2 may bind diferric transferrin and is considered a sensor of Tf saturation [49].

Mutations of TfR2 cause a form of hereditary hemochromatosis. Mice with either germline or liver conditional inactivation of TfR2 develop iron overload [46,50,51]. (See "HFE and other hemochromatosis genes", section on 'Transferrin receptor 2 (TFR2)'.)

TfR2 is expressed in immature erythroid cells, where it interacts with the erythropoietin receptor, stabilizing it on the cell surface [52]. In mice, conditional inactivation of TfR2 in the bone marrow causes erythrocytosis with normal erythropoietin levels. It has been shown that TfR2 modulates the erythropoietin sensitivity of erythroblasts by sensing iron deficiency through decreased diferric transferrin. Through its sensor activity, TfR2 may coordinate erythropoiesis with hepcidin synthesis [53]. TfR2 is also expressed in osteoclasts and osteoblasts, where it controls bone structure by modulating the BMP pathway [54].

Hemojuvelin — Hemojuvelin (HJV) is a glycosylphosphatidylinositol (GPI)-anchored protein that regulates hepcidin production in hepatocytes. It is the product of a gene on chromosome 1q21 and is homologous to RGM (Repulsive Guidance Molecule, also known as RGMc) expressed in the central nervous system. HJV is highly expressed in liver, skeletal muscles and heart. Mutations of the gene encoding HJV (HFE2) produce the common form of juvenile hemochromatosis with extremely low hepcidin levels [55]. (See "HFE and other hemochromatosis genes", section on 'Hemojuvelin (HJV)'.)

HJV is present in a membrane-associated form and also as a soluble form with opposite effects on hepcidin activation [56,57]. Membrane HJV is a coreceptor for bone morphogenetic proteins (BMPs) and is essential in hepcidin activation. In vitro cleavage of HJV by furin in hypoxia and iron deficiency produces soluble HJV components and serves to downregulate hepcidin [58]. Its role in vivo is uncertain. In iron deficiency, membrane HJV is cleaved by the liver serine protease TMPRSS6 to attenuate the BMP signaling and suppress hepcidin [59]. (See 'Matriptase-2/TMPRSS6' below.)

Divalent metal transporter 1 — The duodenal divalent metal transporter 1 (DMT1) is the major route for the uptake of non-heme iron from the intestinal lumen (figure 1). It was identified by positional cloning as gene responsible for a form of microcytic anemia in mice with a missense mutation that had a marked impairment in intestinal iron transport [60].

Parallel functional studies in Xenopus oocytes found a single cDNA that stimulated iron transport. This divalent metal transporter protein (DMT1), was identical to Nramp2 [61]. The transporter also transports other heavy metals such as lead, zinc, and copper coupled with protons. DMT1 is widely expressed, particularly in the proximal duodenum. Expression of the isoform containing an iron responsive element is specifically upregulated in dietary iron deficiency [60] and hypoxia through the action of HIF-2 alpha [62], with greatest expression at the brush border of the apical pole of the enterocytes in the apical two-thirds of the villi, the major site of iron absorption.

Pathogenic variants in the gene that encodes DMT1, SLC11A2, cause life-long microcytic anemia, increased Tf saturation, and liver iron accumulation, but with low or only moderately high serum ferritin levels [63,64].

Local intestinal hypoxia has an important role in increasing the expression of genes involved in iron transport, especially the luminal proteins DMT1 and DCYTB and the iron exporter ferroportin (see 'Ferroportin' below). It has been shown that HIF-2 alpha binds to the DMT1 promoter and regulates DMT1 in duodenal cells; tissue-specific deletion of HIF-2 alpha in mouse enterocytes decreases intestinal iron absorption as well as the expression of DMT1 in enterocytes [62].

Duodenal cytochrome b — Duodenal cytochrome b (DCYTB) is a membrane reductase that facilitates iron absorption as ferrous iron from the lumen (figure 1) [65]. Similar to DMT1, it is regulated by HIF-2 alpha [62].

HIF-2 alpha — Hypoxia-inducible factor 2 alpha (HIF-2α), encoded by the EPAS1 gene, has an IRE in the 5' UTR and is controlled by IRP1 [35,36]. (See 'Iron regulatory proteins or iron-responsive element binding protein' above.)

HIF-2α is an essential mediator of iron absorption that cooperates with low hepcidin to increase absorption in iron deficiency, anemia, and hypoxia [66]. In hypoxia, HIF-2α increases the expression of key genes (DMT1, DCYTB, and ferroportin) that contribute to enhanced iron absorption [67]. (See 'Intestinal iron absorption' below.)

A number of families with gain-of-function mutations in EPAS1 have been described with autosomal dominant erythrocytosis. (See "Molecular pathogenesis of congenital polycythemic disorders and polycythemia vera", section on 'EPAS1 (hypoxia inducible factor 2) gene mutations'.)

HIF-α is regulated by the von Hippel-Lindau (VHL) gene, and its dysregulation may contribute to some of the findings in VHL disease. (See "Molecular biology and pathogenesis of von Hippel-Lindau disease", section on 'Hypoxia-inducible factor 1 and 2'.)

Ferroportin — Ferroportin-1 (Ireg1, SLC40A1, formerly called SLC11A3, Mtp1) is an iron export protein found by positional cloning in the mutant zebrafish with hypochromic anemia [68]. When expressed in Xenopus oocytes, zebrafish ferroportin-1 acts as an iron exporter. Human ferroportin is highly expressed in the basal portion of placental syncytiotrophoblasts, the basolateral surface of duodenal enterocytes, macrophages, hepatocytes, cardiomyocytes, erythrocytes, and other cells [69-72]. The ferroportin gene maps to 2q32.

Ferroportin functions as a major exporter of iron, transporting iron from mother to fetus, transferring absorbed iron from enterocytes into the circulation, and allowing macrophages to recycle iron from damaged and senescent red cells back into the circulation (figure 1).

In animal and human in vitro models, ferroportin is posttranscriptionally regulated by the amount of available iron, due to the presence of an IRE in the 5' UTR [73-76]. An isoform of ferroportin without the 5' IRE (ferroportin B) has been identified in duodenal mucosa. It appears to function to evade iron-mediated post-transcriptional regulation in conditions of iron deficiency [75]. The same isoform is expressed in erythroid cells and may function to "fine-tune" systemic iron usage [77].

The most important control of ferroportin is post-translational, since ferroportin is downregulated through its interaction with hepcidin [70,74,78,79]. When hepcidin levels increase, hepcidin binds to ferroportin, occludes the central cavity that exports iron, and induces ferroportin internalization and lysosomal degradation [80]. This reduces the amount of iron that is released into the circulation from duodenal cells as well as macrophages [78].

Besides post-translational control by IRPs, ferroportin expression is increased by heme independently from iron [81]. Both iron and erythrophagocytosis (through heme increase) stimulate ferroportin transcription [82]. Ferroportin expression is reduced in inflammation via Toll-like receptors [83].

Ferroportin mutations have been found in a number of families with an autosomal dominant form of iron overload called "ferroportin disease" [84-86]. (See "HFE and other hemochromatosis genes", section on 'Ferroportin (SLC40A1; FPN1)'.)

The hepcidin-ferroportin axis may have an important local role (eg, in the heart). Mice with conditional inactivation of ferroportin in cardiomyocytes develop cardiac iron overload and dilated cardiomyopathy and have a decreased survival, strengthening the concept that these cells need to maintain an intact iron export system [82].

Hephaestin and ceruloplasmin — Hephaestin and ceruloplasmin are involved in iron export as multioxidases:

●Hephaestin is the product of an X-linked gene mutated in mice with sex-linked anemia, a disorder in which enterocytes are iron-loaded but efflux of iron through the basolateral membrane and into the plasma is inhibited [87]. Hephaestin cooperates with ferroportin in iron export in enterocytes. It has significant homology to the serum protein ceruloplasmin and has ferroxidase activity as well.

●Ceruloplasmin is a copper-containing protein encoded by the CP gene on chromosome 3q24-25; it is a ferroxidase required for efficient recycling of iron in the liver, reticuloendothelial system, and glial cells. Mutations of ceruloplasmin lead to aceruloplasminemia. (See 'Iron release from macrophages' below.)

FLVCR — A mammalian heme export protein has been described (FLVCR, feline leukemia virus, subgroup C, receptor), which has been postulated to protect developing erythroid cells from the toxicity of unbound cytoplasmic heme. Interference with this protein results in a loss of erythroid progenitors and severe anemia in experimental animals, while its inhibition in human erythroleukemia cells decreases heme export, impairs their erythroid maturation, and leads to apoptosis [88].

The heme exporter FLVCR1 regulates expansion and differentiation of committed erythroid progenitors by controlling intracellular heme accumulation in mice [89] and plays a crucial role in maintaining intestinal heme homeostasis [90].

ZIP14 — ZIP14, a metal transporter of zinc and manganese encoded by the SLC39A1 gene, has been shown to be important for non-transferrin bound iron (NTBI) uptake by hepatocytes, pancreatic acinar cells [91], and beta-cells of the islets, while other NTBI transporters such as L-type calcium channels, DMT1, ZIP8 are active in the heart and anterior pituitary [92].

Hepcidin — Hepcidin (also called liver-expressed antimicrobial peptide [LEAP-1] or hepcidin antimicrobial peptide [HAMP]) is an acute phase reactant with intrinsic antimicrobial activity [93-96]. It is encoded as a propeptide by a gene on 19q13. There are two isoforms of the mature peptide; hepcidin-25 has a central role in iron homeostasis, while the function of hepcidin-20, which lacks the five amino acid sequence crucial for iron regulation, is unknown.

Serum hepcidin levels have correlated directly with serum ferritin in healthy people, are highest in inflammation, and are lowest in iron deficiency anemia. Reference ranges for hepcidin levels in healthy controls were noted to be wide in one study, but when rigorous criteria were required to eliminate individuals with inflammation or renal or hepatic disease, variability was considerably less [97].

Initiatives are ongoing for standardization of hepcidin assays [98]. Two large studies have measured serum hepcidin in the general population. In the first study, hepcidin levels were analyzed using a competitive enzyme-linked immunosorbent assay in 2998 well-characterized participants from the Nijmegen Biomedical Study [99]. In the second report, serum hepcidin levels were measured by mass spectrometry in 1545 individuals from an Italian cohort [100].

●Both studies observed stable values in males across ages but strong variation in females, with low values in younger and higher levels in older females [100].

●There was a trend of increasing hepcidin concentrations during the day, although there was no evidence for a primary or secondary circadian variation in hepcidin levels [99].

●Levels were strongly associated with serum ferritin levels and were less strongly associated with C-reactive protein and total iron binding capacity (TIBC) in men and for TIBC, alanine aminotransferase, and glomerular filtration rate in women [99].

●Because of the lack of a standardized assay, hepcidin testing is not ready for regular clinical use, although a number of test platforms are under active investigation [101]. Serum hepcidin levels may have diagnostic value in certain iron disorders [101].

Hepcidin is produced in many tissues, but the primary site of synthesis is in the liver [102-104]. Other tissues that produce hepcidin include macrophages in inflammation, adipocytes, and retinal cells [105-107]. An essential cell-autonomous role for hepcidin in cardiac iron homeostasis has also been demonstrated [108]. Alpha-2 macroglobulin may be the hepcidin transporter in blood [109].

Hepcidin is rapidly excreted by the kidney and reabsorbed in the proximal tubules; its levels increase in chronic kidney disease. It serves as an important mediator in the pathogenesis of the anemia of chronic disease [103,110,111]. It may decrease in chronic liver disease [101]. Its deficiency or inappropriate production explains the pathogenesis of iron overload in hereditary hemochromatosis [42,103,112-114] (see "HFE and other hemochromatosis genes"). Its excessive inhibition by ineffective erythropoiesis explains iron overload in iron loading anemias [115]. Hepcidin levels are also influenced by hormones, especially inhibited by testosterone [116].

The following examples demonstrate the importance of hepcidin in iron balance and indicate that hepcidin plays a major role as a negative regulator of intestinal iron absorption and iron release from macrophages:

●Hepcidin knockout (KO) mice develop iron overload [94,117]. Liver-specific KO mice fully recapitulate the severe iron overload phenotype observed in the total hepcidin KO mice, demonstrating that the hepatocyte constitutes the predominant reservoir for systemic hepcidin and that the other tissues capable of synthesizing hepcidin are unable to compensate [118].

●In humans, mutation in hepcidin causes a rare form of juvenile hemochromatosis [3]. (See "HFE and other hemochromatosis genes", section on 'Hepcidin (HAMP)'.)

●Injection of hepcidin inhibited intestinal iron absorption in mice independent of their iron status and did not require the HFE gene product. In other experiments, injection of a synthetic hepcidin in mice was associated with a rapid and prolonged reduction in serum iron, along with accumulation of hepcidin in ferroportin-rich organs (eg, liver, spleen, proximal duodenum) [79].

●Constitutive overexpression of hepcidin leads to severe iron deficiency anemia at birth [119] and slows dietary iron absorption and cycling through macrophages, resulting in iron-restricted erythropoiesis and a failure to respond to adequate erythropoietin levels [120].

●Overexpression of hepcidin inhibits the iron accumulation normally observed in HFE-deficient mice [112] and in mouse models of beta thalassemia [121].

●The BMP SMAD signaling pathway activating hepcidin in response to iron, and the IL-6 signaling pathway increasing hepcidin in inflammation are discussed below. (See 'Iron sensing and signaling pathway' below.)

The mechanisms by which hepcidin reduces iron absorption in the intestine and releases iron from macrophages is by interacting with and inactivating the iron export protein ferroportin [78,95]. Hepcidin also appears to regulate levels of non-transferrin-bound iron (NTBI) in a mouse model of bacterial infection; this may be the key mechanism by which hepcidin exerts its antimicrobial properties against "siderophilic" bacterial strains that use NTBI and thrive in an iron-rich environment [96].

Strategies are underway for altering/inhibiting the function of hepcidin and its receptor ferroportin in order to alleviate some of these disorders of iron metabolism [122]. (See "Anemia of chronic disease/anemia of inflammation", section on 'Pathogenesis'.)

BMP and BMP receptors — Bone morphogenetic proteins (BMPs; cytokines produced by endothelial cells) such as BMP6 and BMP2 activate hepcidin binding to BMP receptors and signaling through SMAD proteins [4].

BMP6-null mice have a phenotype resembling hereditary hemochromatosis, with reduced hepcidin expression and tissue iron overload, indicating the essential role of BMP6 in iron regulation in mammals [4,123]. BMP6 is highly expressed in liver sinusoidal endothelial cells (LSEC), and conditional inactivation of BMP6 in LSEC causes iron overload [124]. Also, BMP2, produced by the same cells, regulates hepcidin; its conditional inactivation in LSEC and in endothelial cells in mice causes iron overload, defining its role in signaling for hepcidin, for which the mechanism still needs to be clarified [125].

The phenotype of BMP6 and BMP2 double knock out mice is not more severe than the individual knockouts, suggesting that the two BMPs cooperate in hepcidin activation, possibly through the formation of BMP heterodimers [126].

Matriptase-2/TMPRSS6 — The most powerful inhibitor of hepcidin expression is matriptase-2, which is encoded by TMPRSS6, the liver transmembrane protease, serine 6 gene. Matriptase-2 exerts its hepcidin regulatory effects by cleaving membrane hemojuvelin, a protein that normally signals to promote hepcidin expression [59,127]. (See 'Hemojuvelin' above.)

●In agreement with hemojuvelin being the substrate of matriptase-2, mice that are double knockout for both TMPRSS6 and HJV have the same iron overload phenotype as that of HJV^-/- mice [128].

●The important role of TMPRSS6 in human iron deficiency is indicated by the observation that mutations in TMPRSS6 cause a rare autosomal recessive disorder characterized by iron deficiency anemia unresponsive to treatment with oral iron, but partially responsive to parenteral iron, a condition termed iron-refractory iron deficiency anemia (IRIDA). This condition is discussed in detail separately. (See "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults", section on 'Inherited disorders/IRIDA'.)

●Single nucleotide polymorphisms in the TMPRSS6 gene described in several populations appear to affect serum iron concentrations, hemoglobin levels, erythrocyte characteristics (eg, mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH]), and, indirectly, erythropoiesis [129-133]. These findings suggest a genetic susceptibility to the development of iron deficiency

●Anti-TMPRSS6 compounds increase hepcidin and control iron overload in animal models of iron disorders and are being studied in clinical trials to increase hepcidin to alleviate iron loading.

Erythroferrone — Erythroferrone (ERFE) is encoded by the ERFE gene (also called FAM132B or CTRP15). The protein is a member of the tumor necrosis factor alpha (TNF-alpha) family. Erythroferrone is stimulated by erythropoietin, a circulating hormone essential for the maturation and survival of erythroid progenitor cells to raise the red blood cell count in response to hypoxia or anemia. ERFE downregulates hepcidin to ensure iron supply.

The mechanism is mediated by blocking the binding of a subgroup of BMP proteins (including BMP6 and BMP2) to their receptors [134,135]. ERFE behaves as a ligand trap for BMPs, to attenuate BMP signaling and acquire iron [136]. Antibodies raised against the N-terminal domain of ERFE prevent hepcidin suppression and are proposed as a therapeutic tool for iron loading disorders due to low hepcidin [136]. (See "Regulation of erythropoiesis", section on 'Erythropoietin'.)

SYSTEMIC IRON HOMEOSTASIS — The normal iron content of the body is approximately 3 to 4 grams. It exists in the following forms (figure 3):

●Hemoglobin in circulating red cells and developing erythroblasts – approximately 2.0 to 2.5 g

●Iron-containing proteins (eg, myoglobin, cytochromes, catalase) – 300 to 400 mg

●Plasma transferrin-bound iron – 3 to 4 mg

●The remainder is storage iron in the form of ferritin or hemosiderin

Men have approximately 1 g of storage iron (mostly in liver, spleen, and bone marrow). Women have less storage iron, depending upon the extent of menses, pregnancies, deliveries, lactation, and iron intake. (See "Anemia in pregnancy", section on 'Iron deficiency'.)

Only a small amount of iron (1 to 2 mg) enters and leaves the body on a daily basis. Most iron is recycled from the breakdown of old red blood cells by macrophages of the reticuloendothelial system. Essentially all circulating iron is bound to transferrin. This chelation renders the iron soluble and prevents iron-mediated free radical toxicity. Iron homeostasis is regulated strictly at the level of intestinal absorption and release of iron from macrophages.

Intestinal iron absorption — The gastrointestinal mucosa plays a major role in regulating iron absorption, which varies according to the form of iron in the diet. A Western daily diet contains approximately 15 mg of iron. Some of this is heme iron, of which approximately 30 percent is promptly absorbed, likely via its own transport system. A candidate heme iron transporter, named heme carrier protein 1, has been found in the apical brush border membrane of duodenal enterocytes in the mouse [137]. However, the role in heme transport was subsequently dismissed [138].

The remaining non-heme iron, accounting for almost all of the iron in the diet in non-Western countries, is poorly absorbed, with less than 10 percent being taken into the mucosal cells (figure 3).

Heme dietary sources (fish, poultry, and meat) have a higher bioavailability than do non-heme (vegetable) sources (30 versus <10 percent). In addition, intraluminal factors can affect absorption (table 1).

Ascorbic acid and meat sources enhance the absorption of non-animal sources of iron such as cereal, breads, fruits, and vegetables, whereas tannates (teas), bran foods rich in phosphates, and phytates inhibit iron absorption [139-143]. (See "Treatment of iron deficiency anemia in adults", section on 'Dosing and administration (oral iron)'.)

Molecular mechanisms of intestinal heme absorption are unclear. The proposed heme carrier protein 1, which is highly expressed in the gut and stimulated by hypoxia [137], functions as a folate transporter [138]. A heme exporter, Feline Leukemia Virus Receptor 5 (FLVR5) is expressed in enterocytes, macrophages and erythroblasts with the likely function of exporting heme excess [88]. (See 'FLVCR' above.)

Iron in food is prominently ferric (Fe³⁺), which is poorly soluble above a pH of 3 and is therefore poorly absorbed. In comparison, ferrous iron (Fe²⁺) is more soluble, even at the pH of 7 to 8 seen in the duodenum. As a result, it is more easily absorbed.

Ferrous iron is taken up at the mucosal side by the intestinal transporter, DMT1. In this process, duodenal cytochrome b (DCYTB) reduces ferric iron to ferrous iron. Transcription of both DMT1 and DCYTB is stimulated by hypoxia inducible factor-2 alpha (HIF-2 alpha; HIF-2α) in the hypoxic environment of intestinal mucosa [62,144].

The iron is thought to enter the cell where it binds to cytosolic low molecular weight iron carriers and is transported to the basolateral portion of the cell. To enter the circulation, iron must be transported across the basolateral membrane by the duodenal iron exporter, ferroportin. Upon its release, the ferrous iron is oxidized to the ferric form and loaded onto transferrin. This oxidation process involves hephaestin, a homologue of ceruloplasmin, a known ferroxidase.

Mucosal cells, in addition to the cellular mechanisms noted above, respond to specific physiologic signals (see 'Hepcidin' above). In hypoxia, hepcidin is downregulated to allow increased iron export through ferroportin, while HIF-2α increases the expression of key genes (DMT1, DCYTB, and ferroportin) that contribute to enhanced iron absorption [67]. The rate of iron absorption is appropriately enhanced when iron stores are reduced or absent (figure 1). HIF-2α is an essential mediator of iron absorption that cooperates with low hepcidin to increase absorption in iron deficiency, anemia and hypoxia [66]. (See 'HIF-2 alpha' above.)

The degree of erythropoiesis has an indirect effect, as iron absorption is increased when driven by increased erythropoiesis, a process mediated by hepcidin suppression.

Iron absorption is especially increased in disorders that cause ineffective erythropoiesis, such as beta thalassemia, dyserythropoietic and sideroblastic anemia, or variants of the myelodysplastic syndrome. On the other hand, intestinal cells can hold onto iron in the iron-replete state; this iron is lost when the mucosal cells are sloughed. All of these regulatory processes are mainly mediated by hepcidin through its interaction with ferroportin [1]. This function of hepcidin appears to be especially important when there are competing needs for iron, such as when anemia, iron deficiency, and infection coexist, as shown in African children in the malaria setting [145].

Iron absorption is decreased in conditions of iron excess through hepcidin increase, a process lost in hereditary hemochromatosis. (See "Clinical manifestations and diagnosis of hereditary hemochromatosis", section on 'Pathophysiology'.)

Transferrin saturation — Circulating transferrin normally is approximately one-third saturated with iron (ie, Fe ÷ TIBC = 1/3, when both are expressed as microg of iron per 100 mL of plasma) [146,147]. The formula to change transferrin levels from mg of protein to microg of iron binding capacity (see 'Transferrin' above) can be used if this information has not already been provided by the laboratory.

Conditions in which transferrin saturation (TSAT) is reduced include those in which the supply of iron to the plasma from the macrophage and other storage sites is reduced. These include:

●Iron deficiency anemia

●The anemia of chronic disease (anemia of inflammation)

●Some patients with a ferroportin mutation (see 'Ferroportin' above and "HFE and other hemochromatosis genes", section on 'Ferroportin (SLC40A1; FPN1)')

Conversely, TSAT is increased in those conditions in which the supply of iron is excessive or is greater than the current demand. These include:

●Most cases of hereditary and acquired hemochromatosis

●Aplastic anemia, bone marrow suppression

●Sideroblastic anemias

●Ineffective erythropoiesis

●Heavily transfused patients

●Liver disease with reduced transferrin synthesis

●Monoclonal immunoglobulin with anti-transferrin activity (rare) [148]

The use of TSAT to evaluate disease states is discussed separately:

●Iron deficiency – (See "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults", section on 'Iron studies (list of available tests)'.)

●Iron overload – (See "Approach to the patient with suspected iron overload", section on 'CBC, LFTs, and iron studies' and "Clinical manifestations and diagnosis of hereditary hemochromatosis", section on 'Diagnostic evaluation'.)

Iron loss — There is no normal mechanism of regulated iron loss. Iron is lost in sweat, shed skin cells, and some gastrointestinal loss at a rate of approximately 1 to 2 mg/day (figure 3). A normal adult Western male has 1 to 2 mg of heme iron and 10 to 15 mg of other iron in his diet. If 30 percent of the heme iron and 10 percent of the other iron is absorbed, then the total rate of iron absorption is 1 to 2 mg/day [146]. Thus, a man can easily stay in iron balance and even build up iron stores. On the other hand, a woman with an additional menstrual iron loss of 1 to 2 mg/day generally has lower iron stores than men, and is always delicately poised to become iron deficient (table 2).

There is abundant expression of DMT1 in the proximal tubule and collecting ducts of the kidney [60]; these cells also express TFR1, ZIP14, and ZIP8 [149]. The iron handling by the kidney in terms of uptake and export is not fully elucidated. Iron is filtered in the glomerulus and reabsorbed in the tubules both as transferrin and non-transferrin-bound iron (NTBI). The expression of ferroportin on the apical membrane of proximal tubules suggests a possible contribution of iron excretion in iron overload [150].

Iron release from macrophages — Approximately 20 to 25 mg of iron are released daily from the breakdown of senescent red cells in the macrophages (figure 3). Hemoglobin heme released from phagocytosed red cells is catabolized by microsomal heme oxygenase to biliverdin and carbon monoxide and the resulting iron is released to the circulation through ferroportin or stored in ferritin according to the body needs and to the local concentration of hepcidin [151]. Thus, hepcidin coordinates both duodenal iron absorption as well as macrophage iron release. (See 'Hepcidin' above.)

Upon its release from ferroportin, ferrous iron is oxidized to the ferric form, and loaded onto transferrin. The oxidation process involves ceruloplasmin, a known copper dependent multioxidase. This could explain the iron overload that is seen in aceruloplasminemia, an autosomal recessive disorder of iron metabolism characterized by anemia, diabetes, retinal degeneration, and neurologic symptoms. Affected patients evidence inherited mutations in the ceruloplasmin gene, along with progressive parenchymal iron accumulation in conjunction with strongly decrease of circulating serum ceruloplasmin [152]. (See "Bradykinetic movement disorders in children", section on 'Neurodegeneration with brain iron accumulation'.)

Iron sensing and signaling pathway — Hepcidin is upregulated in response to increased circulating and body iron levels (figure 1), inflammation, infection, endotoxin, and p53; it is downregulated following hypoxia, anemia, iron deficiency, ineffective erythropoiesis, all conditions characterized by increased levels of erythropoietin (figure 4) [153-158]. Hepcidin has also been shown to be regulated by a transferrin-dependent pathway in the zebrafish embryo [159]. In humans hepcidin increases following the absorption of amounts of iron sufficient to acutely increase transferrin saturation [102]. (See 'Transferrin saturation' above.)

Transcription of hepcidin in response to increased plasma or tissue iron is mediated by bone morphogenetic proteins (BMP), requires hemojuvelin (HJV) as a BMP coreceptor, and is SMAD-dependent (figure 4) [57,160,161]. Increased hepcidin production is seen in the acute and chronic inflammation, mediated by lipopolysaccharide, interleukin (IL)-6 and IL-1-beta (figure 4) [110] and is an essential component of anemia of acute inflammation and anemia of chronic diseases [162].

Since most iron is used by maturing erythroid cells, several conditions including iron deficiency anemia, hypoxia, and erythropoietic expansion decrease hepcidin production to favor iron acquisition [115]. Suppression of hepcidin in hypoxia, initially proposed to be mediated by HIF-1 alpha (HIF-1α), occurs indirectly through erythropoietic expansion [163].

Ferrokinetic studies provided earlier information about erythropoiesis. The methods consisted of intravenously injecting a tracer label of Fe-59 bound to plasma transferrin. Three measurements were then made: the disappearance rate of Fe-59 from plasma over a period of minutes provided an index of beginning erythropoiesis; the appearance of Fe-59 by scanning over the spleen, liver, and bone marrow indicated the erythropoietic sites; and the total amount of iron appearing in red blood cells (RBCs) in the circulation 7 to 10 days later provided a measure of effective erythropoiesis. As an example, in severe beta thalassemia, the very rapid disappearance of plasma iron indicated a massive onset of erythropoiesis, and the low incorporation of injected iron in RBCs (20 to 30 percent, versus a normal value of >80 percent) showed that the increased erythropoiesis was severely ineffective. The existence of a regulator of iron absorption produced by erythroblasts was first proposed based on ferrokinetic studies [164]. It was subsequently documented that both effective and ineffective erythropoiesis upregulate iron acquisition by suppressing hepcidin.

Candidates for this hepcidin-suppressing function are cytokines produced by the erythroblasts. Growth differentiation factor 15 (GDF15), which is especially increased in serum of patients with homozygous beta-thalassemia, was first proposed as a major hepcidin inhibitor. However, mice with GDF15 gene deletion treated with phlebotomy are able to suppress hepcidin similarly to wildtype animals [165]. Erythroferrone (ERFE), a member of the tumor necrosis factor alpha (TNF-alpha) family that is strikingly increased in erythroblasts of mice a few hours after phlebotomy or erythropoietin administration, plays a major role in hepcidin inhibition [166]. In vitro, ERFE binds and sequesters different BMPs, including BMP6 and BMP2, attenuating the SMAD signaling [134,135].

However, full hepcidin inhibition by ERFE requires an attenuated BMP pathway, strengthening the complex process of hepcidin inhibitory control [167].

A role for PDGF-BB as a hepcidin inhibitor in hypoxia has been demonstrated in humans [168].

In iron deficiency, the BMP pathway is attenuated. In addition to cleavage of HJV by TMPRSS6, a study has described a role for epigenetic downregulation of the pathway [169].

The iron sensing and signaling pathway involving hepcidin is complex and not fully elucidated [2,95,170]. The proposed model is shown in the figure (figure 4).

●BMP6 in iron overload binds and activates its own receptors (BMPRs) in the presence of the coreceptor HJV. In agreement with this model, BMP6 inactivation in mice causes severe iron overload with low hepcidin [4,123].

●BMP2 participates to hepcidin activation likely setting up the basal hepcidin levels. It does not respond significantly to iron increase and signals prevalently through ALK3.

●Inactivation of BMP2 in mice causes severe iron overload that is not compensated by BMP6.

●BMPs bind to BMPRs. Conditional inactivation of the BMPR Alk2 and Alk3 in mouse liver causes iron overload of different severity [171]. Signal transduction of BMPs occurs through SMAD proteins; conditional inactivation of Smad4 in mouse liver causes liver iron accumulation and the inability to upregulate hepcidin, findings that are similar to those seen in hereditary hemochromatosis [172].

●Whether and how the HFE TFR2 make a complex in the presence of increased diferric transferrin that cooperate with the BMP-HJV-SMAD pathway for hepcidin activation is not fully defined. In HFE^-/- mice, the BMP pathway is indeed less active, and treatment with BMP6 appears to ameliorate iron overload [173].

●Inflammatory cytokines, especially IL-6 (and IL1-beta) activate hepcidin transcription through interaction with the IL-6 receptor and signal transduction through STAT3 [43,57,102,174].

●There is a crosstalk between the two pathways of hepcidin activation (inflammation- and iron-dependent) as shown by improved hepcidin control by compounds that inhibit the BMP-SMAD pathway in inflammation [175].

●In iron deficiency, TMPRSS6 seems to be the major regulator of hepcidin suppression, cleaving the BMP coreceptor HJV. When erythropoiesis is expanded by erythropoietin, ERFE plays a major role in downregulating hepcidin expression, both in effective and ineffective erythropoiesis.

SUMMARY

●The regulation of iron metabolism involves the interaction of a number of specific proteins as well as the interplay between iron absorption from the gastrointestinal tract, recycling of iron from red cells at the end of their life span, release of iron stores from the monocyte-macrophage system, and iron loss from the body (figure 1 and figure 3). (See 'Role of specific proteins' above.)

●Intestinal iron absorption is tightly controlled, since there is no physiologic means of excreting iron from the body once it is absorbed. HIF-2alpha and hepcidin are the most important regulators of apical and basolateral transporters, respectively (See 'Intestinal iron absorption' above.)

●Ferroportin functions as a major exporter of iron, transporting iron from mother to fetus, transferring absorbed iron from enterocytes into the circulation, and allowing macrophages to recycle iron from senescent and damaged red cells back into the circulation. (See 'Ferroportin' above and 'Iron release from macrophages' above.)

●The most important control of iron cycling through ferroportin is post-translational, since ferroportin protein and activity are downregulated through its interaction with hepcidin. (See 'Hepcidin' above.)

●Iron is lost in sweat, shed skin cells, and perhaps some gastrointestinal loss at a rate of approximately 1 mg/day. A woman with an additional menstrual iron loss of 1 to 2 mg/day generally has lower iron stores than men and is always delicately poised to become iron deficient. (See 'Iron loss' above and "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults", section on 'Search for source of blood and iron loss'.)

ACKNOWLEDGMENT — We are saddened by the death of Stanley L Schrier, MD, who passed away in August 2019. The editors at UpToDate gratefully acknowledge Dr. Schrier's role as author on this topic, his tenure as the founding Editor-in-Chief for UpToDate in Hematology, and his dedicated and longstanding involvement with the UpToDate program.