screen-shot-2016-11-02-at-10-03-03-pmGlobal Iron Homeostasis

Under normal conditions, dietary iron is usually 15-25 mg daily, of which 5%-10% (1-2 mg) is absorbed through the gastrointestinal (GI) tract and the same amount lost by desquanmation of GI epithelial cells, epidermal cells of the skin, and, in menstruating women, red bood cells. The average total body content of iron in men is 35-45 mg/kg; and lower in menstruating women. Most iron (about 1800 mg) is present in hemoglobin. Men and women, respectively, have approximately 2 or 1.5 g of erythrocyte iron. Iron is stored in cells, predominantly macrophages of the spleen, bone marrow, and liver, but also in hepatocytes, as ferritin or hemosiderin (partially denatured ferritin). At steady state, the serum ferritin level is a reasonably good reflection of total body iron stores. Total storage iron is approximately 1 g in men and 600 mg in women. Additional iron is found as myoglobin in muscle and in cytochromes and other enzymes.

PS: The loss of iron

Iron is eliminated only through the loss of epithelial cells from the gastrointestinal tract, epidermal cells of the skin, and, in menstruating women, red blood cells. On the basis of long-term studies of body iron turnover, the total average daily loss of iron has been estimated at ~1 to 2 mg in normal adult men and nonmenstruating women. Although iron is a physiologic component of sweat, only a tiny amount of iron (22.5 ug/L) is lost by this route. Urinary iron excretion amounts to <0.05 mg/day and is largely accounted for by sloughed cells. Menstruating women lose an additional, highly variable amount over each menstrual cycle, from 0.006 (average) to more than 0.025 mg/kg/day.

The release of iron into the circulation is regulated by ferroportin, expressed on the basolateral GI epithelial cell surface (and on cells of the reticuloendothelial system [RES] and hepatocytes). Ferroportin is downregulated by hepcidin, and when iron is low, hepcidin is low, allowing GI iron absorption to increase and stores to be mobilized from the RES. When iron is plentiful, hepcidin levels increase and result in decreased iron absorption and RES export. Hepcidin, in turn, is downregulated by the recently described hormone erythroferrone (ERFE), produced by erythroblasts during stress erythropoiesis. Absorbed iron is transported by transferrin and taken up into cells via the transferrin receptor. Each molecule of transferrin can bind two molecules of ferric (Fe3+) iron. Transferrin-bound iron turns over as iron is used, particularly by developing red blood cells in the bone marrow. The distribution of iron is influenced by multiple factors, and under normal conditions cells maintain a pool of labile iron by controlling uptake via expression of transferrin receptors and storage via ferritin. Most cells have no mechanism for iron efflux.

Iron balance is regulated such that the amount of iron absorbed equals the amount lost. There is, however, no physiologically regulated pathway for excretion of excess iron in iron overload.

Intestinal Iron Absorption

Iron is found in food as inorganic iron and heme (iron complexed to protoporphyrin IX). The typical diet consists of 90% inorganic and 10% heme iron, though diets in the industrial world can contain up to 50% heme iron from iron-rich meats. The bioavailability of inorganic but not heme iron is influenced by multiple factors such as other dietary constituents, for example, ascorbic acid (enhanced) and phytates and polyphenols in cereals and plants (inhibited). Iron absorption is strongly inhibited by tea, and less so by coffee.

PS: Intestinal Absorption of Iron

Iron is absorbed in the duodenum, and humans and other omnivorous mammals have at least two distinct pathways for iron absorption: one for uptake of heme iron and another for ferrous (Fe2+) iron. Heme iron is derived from hemoglobin, myoglobin, and other heme proteins in foods of animal origin, representing approximately 10% to 15% iron content in the typical Western diet, although heme-derived iron accounts for 2/3 of absorbed iron in meat-eating humans. Exposure to acid and proteases present in gastric juices frees the heme from its apoprotein. Heme is taken up by mucosal cells, but the specific receptor is still unkonwn. Once heme iron has entered the cell, the porphyrin ring is enzymatically cleaved by heme oxygenase. The liberated iron then probably follows the same pathways as those used by nonheme iron. A small proportion of the heme iron may pass into the plasma intact via heme exporter protein FLVCR (feline leukemia virus, subgroup C receptor), which transfers heme onto a heme-binding protein, hemopexin. Absorption of heme iron is relatively unafected by the overall composition of the diet.

Dietary nonheme iron is largely in the form of ferric hydroxide or loosely bound to organic molecules such as phytates, oxalate, sugars, citrate, lactate, and amino acids. Low gastric pH is thought be important for the solubility of inorganic iron. Dietary constituents may also have profound effects on the absorption of nonheme iron, making the bioavailability of food iron highly variable. Ascorbate, animal tissues, keto sugars, organic acids, and amino acids enhance inorganic iron absorption, whereas phytates, polyphenols, and calcium inhibit it. Depending on various combinations of enhancing and inhibitory factors, dietary iron assimilation can vary as much as tenfold.

The rate of iron absorption is influenced by several factors, including body iron stores, the degree of erythropoietic activity, blood hemoglobin and oxygen content, and the presence of inflammation. Iron absorption increases when stores are low or when increased erythropoietic activity is required, such as during anemia or hypoxemia. Conversely, the physiologically appropriate response to iron overload is downregulation of intestinal iron absorption; this downregulation fails in patients with hereditary hemochromatosis or chronic iron-loading anemias.

Iron is absorbed in the intestine via two pathways: one for inorganic iron and the other for heme-bound iron. Little is known about heme iron absorption. Nonheme iron in the diet is largely in the form of ferric-oxyhydroxides (Fe3+, ie, rust), but the intestinal epithelial cell apical iron importer, divalent metal transporter 1 (DMT1 or SLC11A2), transports only ferrous iron (Fe2+). DMT1 is a protein with 12 predicted transmembrane segments, which is expressed on the apical surface of absorptive enterocytes. Iron must therefore be reduced to be absorbed, and this is facilitated by duodenal cytochrome B (Dcytb), a heme-dependent ferrireductase. However, since knockout mice appear to have normal metabolism, Dcytb may not be the only ferrireductase enzyme involved in absorption of nonheme ironOnce transported across the apical border of the enterocyte, iron may be stored within the cell. For this purpose, iron is oxidized to Fe3+ by the H-subunit of ferritin and stored in this form. Eventually, the cell senesces and sloughs off into the feces, and stored iron is lost to the system. Alternatively, iron may be transported across the basolateral membrane into the portal circulation via ferroportin. Ferroportin 1 (FPN1) is the only known iron exporter in mammals and, like DMT1, transports only ferrous iron. FPN1 is a multi-transmembrane segment protein expressed on the basolateral surface of enterocytes. Aslo they are expressed in other tissues involved in handling large iron fluxes including macrophages, hepatocytes, and placental trophoblast. Similar to apical iron uptake, basolateral iron efflux is aided by an enzyme that changes the oxidation state of iron. Once reduced, ferrous iron is transported across the basolateral membrane by ferroportin, then oxidized to ferric iron by hephaestin. Intestinal iron absorption is regulated by hepcidin, which binds to ferroportin, inducing its internalization and degradation.

Cellular Iron Uptake, Storage, and Recycling

Each molecule of transferrin binds two ferric (Fe3+) iron atoms. Diferric transferrin (holotransferrin) binds to the transferrin receptor (TfR1) on target cells and enters by receptor-mediated endocytosis; it is then released from the TfR1 by acidification and transported into the cytoplasm by DMT1. The TfR is recycled to the cell surface.

Most iron in erythroid cells binds protoporphyrin to form heme, which complexes with globin proteins, forming hemoglobin. Erythrocytes survive in the circulation for approximately 120 days, after which aging red blood cells are phagocytized by macrophages of the RES. Hemoglobin is catabolized and iron released to transferrin via ferroportin, or stored within the RES as ferritin or hemosiderin.

PS: Reticuloendothelial System/RES

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The main form of cellular iron storage is ferritin, a complex of subunits that binds iron and renders it insoluble and redox inactive. Circulating ferritin is present in a different subunit form than cellular storage ferritin. The function of circulating ferritin is incompletely understood.

Regulation of Iron Physiology

Because the total body iron content is largely determined by the efficiency of absorption of iron, the regulation of absorption has been of great interest for many years.

Hepcidin is a 25-amino-acid peptide produced in the liver and is the major regulator of iron absorption and storage. Hepcidin regulates cellular iron egress by binding to ferroportin, leading to its internalization and degradation. In this way, elevated levels of hepcidin inhibit iron absorption from the GI tract and promote storage (inhibit release) of iron within hepatocytes and macrophages. Hepcidin production is induced by interleukin (IL)-6 and IL-1 via the JAK/STAT pathway, and can be increased more than 100-fold in inflammatory states. The dysregulation of iron balance seen in the anemia of inflammation (anemia of chronic disease) can be attributed to an inappropriate increase in hepcidin levels, which leads to decreased circulating iron. Hepcidin levels are downregulated by anemia and iron deficiency. Hepcidin agonists and antagonists are under clinical development for the treatment of disorders of inappropriately low or high hepcidin levels, respectively.

Hepcidin, in turn, is negatively regulated by the recently described hormone ERFE. ERFE is produced by erythroblasts during stress erythropoiesis, feeding back on hepcidin and reducing its levels, thus allowing iron absorption and export via ferroportin to proceed.

The Molecular Mechanism for Hepcidin Regualtion

Systematic Mechanism

The two transferrin receptors TfR1 and TfR2, and HFE, an MHC class I-like membrane protein, may serve as holotransferrin sensors. HFE can interact with both transferrin receptors, but this interaction is modulated by holotransferrin concentrations. Because HFE and holotransferrin binding sites on TfR1 overlap, increasing concentrations of holotransferrin result in displacement of HFE from TfR1, and free HFE then interacts with TfR2. TfR2 protein is further stabilized by binding of holotransferrin. The holotransferrin/HFE/TfR2 complex then stimulates hepcidin expression through an incompletely understood pathway, possibly by potentiating BMP pathway signaling. HFE, however, may also regulate hepcidin expression without complexing with TfR2. The role of HFE or TfR2 in hepcidin regulation by iron is supported by genetic evidence: HFE and TfR2 mutations in humans or mice cause hepcidin deficiency and an adult form of hemochromatosis.

screen-shot-2016-11-03-at-3-19-33-pmThe BMP pathway with its canonical signaling via Smad proteins has a central role in the regulation of hepcidin transcription. BMP receptors are tetramers of serine/threonine kinase receptors, with two type I and two type II subunits. Recent data indicate that type I subunits Alk2 and Alk3 and type II subunit ActRIIA and BMPRII are specific BMP receptors involved in iron regulation. In the liver, BMP pathway signaling to hepcidin is modulated by a coreceptor hemojuvelin and, at least in mice, by the ligand BMP6. Loss of hemojuvelin or BMP6 in mice decrease hepcidin expression and impairs hepcidin response to acute or chronic iron loading. In humans, hemojuvelin mutations result in severe hepcidin deficiency and cause juvenile hemochromatosis. It remains to be clarified how BMP receptors and hemojuvelin interact with iron-sensing molecules that regulate hepcidin expression.

Local Mechanism (intestine)

In addtition to the regulaltion by systemic signals, iron absorption is subject to local regualtion by intracellular mechanisms in duodenal enterocytes. At least two mechanisms have been described: one related to the enterocyte iron levels and the other to the hypoxia pathway.

Regulation of the synthesis of multiple proteins involved in iron physiology, including TfR1, DMT1, FPN1, and ferritin, is controlled at a posttranscriptional level by influencing mRNA stability and translation. The mRNA of these proteins contain iron response elements (IREs), conserved nucleotide sequences with a stem-loop structure that binds iron regulatory proteins (IRPs)-1 and -2. The mRNAs for ferritin, DMT1, and FPN1 have IREs in the 5' untranslated region (UTR), and the mRNA for the TfR has multiple IREs in the 3' UTR. In low-iron states, IRP-1 is in a conformation that allows it to bind to IREs; for example, it binds the 3' IRE of the TfR mRNA, stabilizing it and allowing transcription of more TfR protein, and to the 5' UTR of ferritin mRNA, decreasing translation of ferritin for iron storage. Intracellular iron induces ubiquitination and degradation of IRP-2. Iron deficiency by this mechanism upregulates IRP activity via increased IRE binding, resulting in increased cellular iron uptake and decreased iron storage.

Dcytb mRNA does not contain an IRE but is strongly upregulated in iron-deficient duodenum, indicating additional regulation of transcription of iron-related genes. Hypoxia-inducible factor (HIF) transcriptin factors may serve as important local regulators of intestinal iron absorption. Iron deficiency induced HIF signaling in duodenum of mice, and caused increased Dcytb and DMT1 expression, and an increase in iron uptake. Accordingly, targeted deletion of Hif-2alpha in the intestine resulted in dramatic decrease in the expressions of DMT1-IRE and Dcytb mRNA. In vitro studies further demonstrated that HIF-2alpha directly binds to the promoters of DMT1 and Dcytb, activating their transcription.

Summary: Factors That Affecting Hepcidin Levels

Systemic mechanisms: 1) by transferrin levels, including holotransferrin/HFE/TfR2 complex + hemojuvelin (coreceptor) + BMP6 (ligand), which increases hepcidin gene expression; 2) by erythropoiesis, however, the mechanisms by which erythropoiesis regulates hepcidin production are not well understood, but it is thought that erythroid precursors in the bone marrow secrete a factor which exerts its effect on hepatocytes and causes hepcidin supression (so the ERFE previously mentioned?); and 3) by cytokines in inflammation state, via STAT-3 pathway (already described above).

Local mechanisms: 1) posttranscriptional level by influencing mRNA stability and tanslation, via IRPs; 2) HIF's impact on cis-acting regulatory elements of DMT1 and Dcytb genes.


Iron Cycle

screen-shot-2016-11-03-at-5-12-27-pmMost functional iron in the body is not derived from daily intestinal absorption (1 to 2 mg/day) but rather from recycling of iron (20 to 25 mg/day) from senescent erythrocytes and other cells. The most important source and destination of recycled iron is the erythron. At the end of a 4-month lifespan, effete erythrocytes are engulfed by reticuloendothelial macrophages, which lyse the cells and degrade hemoglobin to liberate their iron. This process is poorly understood, but it appears to involve the action of heme oxygenase for enzymatic degradation of heme. Some of this iron may remain stored in macrophages as ferritin or hemosiderin, but most is delivered to the plasma, and the rate of iron export is determined by the hepcidin-ferroportin interaction. In plasma, iron becomes bound to transferrin, completing the cycle. A small amount of iron, probably <2 mg, leaves the plasma each day to enter hepatic parenchymal cells and other tissue. Here, the iron is stored or used for synthesis of cellular heme proteins, such as myoglobin and the cytochromes.

Macrophage Iron Recycling

Although there are many types of tissue macrophages, those that participate in the catabolism of red blood cells can be subdivided into two categories. One type, exemplified by pulmonary alveolar macrophages, is able to phagocytose erythrocytes or other cells and convert the iron they contain into storage forms, but lacks the ability to return the iron to the circulation. This type of macrophage appears to retain the iron throughout its life span. The second type of macrophage, comprising the reticuloendothelial system, acquires iron in a similar fashion but is able to return it to the plasma. The latter macrophages, found espeically in the sinuses of the spleen and the liver, play a primary role in the normal reutilization of iron from destroyed red cells, allowing completion of the iron cycle shown in Figure 23.4.


Plasma Transport

The plasma iron-binding protein, transferrin, is a glycoprotein with a molecular weight of approximately 80 kDa. Transferrin is synthesized chiefly in the liver and actively secreted by hepatocytes, but lesser amounts are made in other tissues, including the central nervous system, the ovary, the testis, and helper T lymphocytes (CD4+ subset). The rate of synthesis shows an inverse relationship to iron in stores; when iron stores are depleted, more transferrin is synthesized, and when iron stores are overfilled, the level of transferrin decreases. Transferrin keeps iron nonreactive in the circulation and extravascular fluid, delivering it to cells bearing transferrin receptors.

Transferrin can be measured directly using immunologic techniques; and normal concentration in the plasma is approximately 2 to 3 g/L. Alternatively, transferrin is quantified in terms of the amount of iron it will bind, a measure called the total iron-binding capacity (TIBC: normal values for plasma iron and TIBC are given in Appendix A). In the average subject, the plasma iron concentration is 100 ug/dL, and the TIBC is 300 ug/dL. Thus, only about one third of the available transferrin binding sites are occupied, leaving a large capacity to deal with excess iron. Plasma iron concentration varies over the course of the day, with the highest values in the morning and the lowest in the evening. Levels of serum transferrin are more constant, and there is no apparent diurnal variation in TIBC. General practice has been to evaluate transferrin saturation using a first morning, fasting sample to standardize the results, but this may not be helpful.

Transferrin has two homologous iron-binding domains, each of which binds an atom of trivalent (ferric) iron. The iron atoms are incorporated one at a time and appear to bind randomly at either or both of the two sites. When binding is complete, the iron lies in a pocket formed by two polypeptide loops. One mole of anion, usually carbonate or bicarbonate, is taken up, and 3 moles of hydrogen ion are released from each mole of iron bouond. There are functional differences between teh two iron-binding sites, but it is not clear that these have physiologic importance.

Under physiologic circumstances, ferric iron binds to transferrin with very high affinity, with an affinity constant of ~1-6 x 1022 M-1. The affinity of iron-transferrin interaction is pH-dependent, decreasing as pH is lowered. Other transition metals, such as copper, chromium, manganese, gallium, aluminum, indium, and cobalt, can be bound by transferrin but with less affinity than iron.

Iron Delivery to Erythroid Precursors

The biologic importance of transferrin in erythropoiesis is illustrated by abnormalities observed in patients and mice with congenital atransferrinemia. When transferrin is severely deficient, red cells display the morphologic stigmata of iron deficiency. This occurs despite the fact that intestinal iron absorption is markedly increased in response to a perceived need for iron for erythropoiesis. Nonhematopoietic tissues avidly assimilate the non-transferrin-bound metal. Similarly, mutant mice lacking tissue receptors for transferrin die during embryonic development from severe anemia, apparently resulting from ineffective iron delivery to erythroid precursor cells.

Transferrin delivers its iron to developing normoblasts and other cells by binding to specific cell-surface receptors. The transferrin receptor (TfR1) is a disulfide-linked homodimer of a glycoprotein with a single membrane-spanning segment and a short cytoplasmic segment. It is a type II membrane protein, with its N terminus located within the cell. The native molecular weight of TfR1 is ~180 kDa. Each TfR1 homodimer can bind two tranferrin molecules. Diferric transferrin is bound with higher affinity than monoferric transferrin. As a result, diferric transferrin has a competitive advantage in delivering iron to the erythroid precursors. Apotransferrin has little affinity for the receptor at physiologic pH but considerable affinity at lower pH, an important factor in intracellular iron release.

TfR1 numbers are modulated during erythroid cell maturation, reaching their peak in intermediate normoblasts. Very few TfR1 molecules are found on burst-forming-unit erythroid cells, and only slightly greater numbers are found on colony-forming-unit erythroid cells. However, by the early normoblast stage, approximately 300,000 receptors are found on each cell, increasing to 800,000 at the intermediate stages. The rate of iron uptake is directly related to the number of receptors. The number decreases as reticulocytes mature, and late in maturation, erythroid cells shed all remaining receptors by exocytosis and by proteolytic cleavage. The shed receptors, referred to as soluble transferrin receptors (sTfR) is a sensitive indicator of erythroid mass and tissue iron deficiency.

After ligand and receptor interact, iron-loaded transferrin undergoes receptor-mediated endocytosis. Specialized endocytic vesicles form, which are acidified to a pH of 5 to 6 by the influx of protons. The low pH facilitates release of iron from transferrin and strengthens the apotransferrin-receptor interaction. Released iron is reduced by an endosomal ferrireductase, STEAP3, and transferred to the cytosol by DMT1. Because DMT1 must cotransport protons with iron atoms, vesicle acidification is also important for the function of this transporter. After the iron enters the cytosol, the protein components of the endosome return to the membrane surface, where neutral pH promotes the release of apotransferrin to the plasma.

After Entering into Erythroid Precursor Cells

In the normal subject, ~80% to 90% of the iron that enters erythoid precursor cells is ultimately taken up by mitochondria and incorporated into heme. Most of the remainder is stored in ferritin. Granules of ferritin may sometimes be detected using the Prussian blue reaction. Normoblasts with Prussian blue-positive (siderotic) granules are called sideroblasts, and, if the granules persist after enucleation, the mature cells are called siderocytes. In normal individuals, approximately half of the normoblasts are sideroblasts, each containing less than five small granules.

It is intriguing that even though erythroid precursors require large amounts of iron and heme for hemoglobin synthesis, they have been found to express the heme exporter FLVCR and the iron exporter ferroportin. Studies suggest that FLVCR is necessary for survival of erythroid precursors. Because heme is toxic to cells at high concentrations, it is thought that FLVCR functions as a safety valve to prevent accumulation of excess heme early during erythroid differentiation.

Erythorid precursors also express ferroportin but its role in erythroid maturation is yet unclear. It is interesting that erythoid cells preferentially express non-IRE ferroportin transcript during the stages of rapid iron uptake, thus avoiding any iron-induced increase in ferroportin translation dependent on the IRE/IRP system.