Iron Metabolism

Iron (Fe) is a very important element in human metabolism. It is contained mostly in proteins such as hemoglobin, myoglobin, ferritin and hemoxydrin. In a diet you take from 10 to 20 mg of Fe, from red meat, eggs and foods of animal origin, but also vegetable derivatives (here, however, the availability is lower, since it is found bound to substances that prevent assimilation). There are also beverages such as tea and coffee rich in anionic molecules (eg carbonate, sulfate) that can bind iron in the intestinal lumen and hinder absorption.

Iron is not efficiently absorbed: only 10% is absorbed, 1-2 mg per day. Half of the iron is lost in the intestine and half enters the circulation, bound to transferrin, thanks to which it is possible to bring iron to tissues. The tissue that uses the most (about 75%) is the bone marrow, but other tissues are also supplied (such as muscles, where it is needed for myoglobin; in the liver for cytochromes that serve not only for the transport chain but also to detoxify some substances; in the fetus in pregnant women).

Some of the iron is stored in ferritin, an intracellular protein. The amount of iron absorbed also depends on how much iron is taken in: higher intake leads to lower absorption. The problem is that we do not have an efficient system to eliminate iron: the only way is the desquamation of intestinal and skin epithelium in which ferritin with iron is found.

Iron Content

The human body contains 3-4 grams of iron, 2/3 of which is incorporated into the heme of hemoglobin, in erythrocytes. Erythrocytes live about 120 days; every day, some erythrocytes - containing about 20 mg of iron - are destroyed by the reticulo-endothelial system. Fortunately, all this iron is recovered to synthesize new erythrocytes; the only loss for males and females (post-menopause) is the one related to the desquamation mentioned before, which amounts to about 1-2mg per day. In women, there is a loss of iron during the menstrual cycle, and this justifies the lower content of iron in fertile women, so they will have a greater need for iron. Pregnant women also have a greater need for iron.

Biodistribution

In a man of about 60-70 kg, iron is present:

• 60-70% in hemoglobin
• 20-30% in ferritin 7% 
• in myoglobin 
• 0.5% in cytochromes
• 0.1% in circulating transferrin

Ferrous iron and ferric iron

Iron is found in 2 forms: ferrous iron and ferric iron. The former is soluble at neutral pH and is the main form in which it is found in organisms; however, it is oxidized in muscles, rather than in red blood cells. Iron, in the ²⁺ form, binds O₂, a potent oxidant. Hemoglobin and myoglobin frequently convert to beta-hemoglobin and beta-myoglobin (the forms that contain Fe³⁺). Meta-hemoglobin reductase and meta-myoglobin reductase reduce iron instead, turning it into the ²⁺ form.

As a dead organism stops, these enzymes also stop and, therefore, in the corpse iron is all in 3+ form. So there is a continuous alternation between the 2 forms; however ferrous iron is very toxic: if it reacts with oxygen, it can form superoxide anion, which turns into peroxide. In Fenton's reaction there is the reaction of ferrous ion with hydrogen peroxide: iron becomes ferric, giving an electron to hydrogen peroxide and breaking the bond, which forms a normal hydroxide (OH radical). A similar reaction occurs if instead of iron there is a superoxide anion: Haber-Weiss reaction. These are the 2 main reactions by which the production of very toxic radicals is triggered. 

Iron deficiency/excess: effects

Both a deficiency and an excess of iron can be harmful. Iron can accumulate in the pituitary gland, damaging its cells and causing endocrinopathies, in the pancreas causing diabetes and pancreatitis, in the heart, in the skin, in the liver where it causes liver fibrosis leading to cirrhosis, in the testicle where it causes infertility, in the thyroid gland etc.

Even a deficiency of iron causes problems: one of the main is the sideropenic anemia; besides anemia there can be a reduced growth margin, reduced neuronal development, reduced resistance to infections because iron is involved in immune defense.

The iron contained in proteins is detached from them already in the stomach: the acid environment releases iron from organic complexes. The absorption of iron occurs in the duodenum, in the first part of the intestine. Iron arrives either as ferric iron, or as iron contained in the heme. At the level of the intestinal microvilli, ferric iron is reduced to ferrous (the only assimilable form). This reaction is catalyzed by an iron reductase called cytochrome B duodenal, while the electron donor is NADPH. In order for the reaction to take place, vitamin C is also needed, in addition to the acidic pH mentioned before. So anachloridia, rather than a mild scurvy, can cause a sideropenic anemia, because they decrease the amount of iron assimilated.

In the transformation from iron to ferrous, the latter enters the membrane thanks to the bi-valent metal transporter (DMT1). This carrier transports not only ferrous iron, but all bivalent metals; it is a synport, which allows iron to enter together with a proton: this is also a reason why pH is very important for iron metabolism. Thanks to this symport, iron enters the cells and in part is used by the enterocyte, in part by ferritin, the iron storage protein present in all cells. A good part of the iron travels from the apical to the baso-lateral end, since the iron must be poured into the bloodstream.

Along with iron taken in as "free iron", we take in iron contained in heme. There is a heme transporter (HCP1), and within the cell there is a heme oxygenase, which detaches the iron.

Ferrous iron, in order to be released into the bloodstream, must bind to the ferroportin protein. In the blood, however, ferrous iron must be oxidized to ferric iron, since transferrin, the main transporter, recognizes only ferric iron. The iron is then converted to ferric, by means of 2 proteins that contain copper: the one present on the baso-lateral membrane of enterocytes, and the efestin that works as iron oxidase. In some cell types, such as macrophages and hepatocytes, another enzyme is involved: the celluloplasmin, a protein that is bound to the membrane thanks to glycosyl-phosphatidyl inositol. 

Another very important factor is hepcidin, which circulates going to bind to ferroportin and inducing the phosphorylation of tyrosine on ferroportin, accelerating its degradation in proteosomes. In these conditions the cells have less proteins, and therefore less ferroportin able to carry iron out of the cell.

This slide repeats what has been said previously: iron can also be absorbed into the enterocyte by the palaferritin complex, which contains an integrin linked to mobilferrin.

Hepcidin is a small peptide (25 amino acids), identified in 2000, with bactericidal activity. It is produced by the liver and it is an important hormone for the regulation of iron metabolism, as it inactivates ferroportin, blocking the exit of iron from the intestine. The iron in the stool is no longer replaced by spent iron. An increased production of hepcidin by the liver therefore reduces the absorption of iron and this occurs in conditions of excess iron: the absorption of iron is also reduced in macrophages, after digesting the erythrocytes, it limits the release of free iron. Another condition that increases the production of hepcidin is an increase in inflammatory factors: in the presence of processes of this type, especially chronic, there is a reduced absorption of iron in the intestine and a reduced release of iron, when there is a need to decrease iron. The consequence is a reduced supply of iron to the bone marrow, leading to a condition of anemia. If few erythrocytes are produced, the bone marrow goes to reduced levels of epdicidin. If epdicidin is produced in excess, chronic anemia occurs.

Why does an inflammatory process reduce iron absorption? Because it reduces the amount of iron available to bacteria, which use it to infect themselves: therefore anemia is the price you pay to fight a bacterial infection.

Returning to the enterocyte, iron oxidized to ferric is bound to transferrin. Transferrin is produced in the liver and circulates in the blood. In its structure we find 2 domains, each of which can bind ferric iron. Under normal conditions transferrin is present in concentrations greater than the normal need: in fact, its saturation with iron is 40%. The rest of transferrin, called apo-transferrin, is devoid of iron: this is because, in case of need, it is possible to bind larger amounts of iron. If the excess of iron exceeds the amount that can be assembled with transferrin, this surplus, called labile iron, can become ferrous, forming ROS that can cause cell death, as observed in liver cirrhosis due to an excess of iron.

Transferrin is a 78 000 Da protein. It has 2 binding sites, non-cooperative and, in order to keep the iron bound, carbonate is also required; it has a high affinity for iron.

Bacteria, in order to obtain iron, release proteins called siderophores that bind iron and, after that are endocytosed. Iron is used by bacteria both to proliferate and for some defense mechanisms.

In milk there are similar proteins that bind iron, such as lactoferrin, competing with siderophores and thus hindering bacterial proliferation.

Red Blood Cell Demolition

Old red blood cells are eliminated at the level of macrophages in the spleen. Under certain conditions, intra-vascular hemolysis may occur, such as when a very old erythrocyte must pass through a capillary. Because of its inelastic membrane, it is susceptible to oxidative damage, which can break down releasing hemoglobin. This hemoglobin can cause renal failure, so it is essential that there are proteins that can readily bind free iron (haptoglobin and hemisexin). Haptoglobin can bind hemoglobin, preventing it from being eliminated with urine. When hemoglobin is released into the bloodstream, it may lose heme, a very toxic structure (because ferrous iron binds oxygen, giving up the electron, forming a superoxide anion). To prevent this, hemopexin recognizes the heme.

From transferrin to tissues

In order for iron to detach from transferrin, the intervention of receptors is required. The TfR 1 receptor, with high affinity, is normally expressed on proliferating cells, while TfR 2, present mainly in hepatocytes, serves as a sensor, recording the level of iron in the blood and the increase/decrease movement of hepcidin synthesis. The receptor for transferrin is an integral membrane protein, formed by a dimer having 2 subunits capable of binding transferrin (each receptor, can let in 4 iron ions). As for LDL, the receptor for transferrin is associated with a dimple, coated with clathrin: the difference is that, while LDL binds to the receptor and is continuously internalized (the receptor enters, both having and not having LDL bound), here the receptor is internalized only if it binds transferrin. After internalizing transferrin, an endosome is formed; at this point a proton pump, an ATPase, intervenes: thus protons bind to transferrin and iron is detached. Ferric iron is reduced by a reductase, STEAD 3; this happens because the iron must be able to leave the vesicle and be used. Ferrous iron is excreted from the vesicle by the same enterocyte transporter (DMT 1), which can only excrete ferrous iron along with a proton.

The vesicle recycles its contents, the receptor is exposed again on the membrane, and apo-transferrin is expelled. This is repeated about ten times before the endosomes are also digested.

Free iron (which is actually always bound to polyanions, so that it does not form ROS), is handled differently, depending on the tissue. For example, it can enter the mitochondria, where heme is synthesized and here the transporter is the mito-ferrin: inside the mitochondria iron can be used to form iron-sulfur centers and, to do this, the fratassin intervenes.

Unused iron is deposited in ferritin, an intracellular protein found in the cytoplasm of all cell types. Ferritin is a protein complex of 24 subunits, present in all tissues. These subunits are of 2 types: 

H (heavy or heart, since it is the heavy form typical of the heart) or L (light or liver, light form typical of the liver); what changes in tissues is the ratio between these 2 types, but the total number is always 24.

At this point, ferrous iron must be oxidized back to ferric iron. Ferritin has iron oxidase activity, oxidizing it to ferric iron. Ferric iron accumulates in the ferritin core in the form of various oxides. Each molecule of ferritin can contain up to 450 000 iron ions, but usually contains only half that amount. Ferritin is also found in the mitochondrion and the nucleus, where it protects the DNA.

In the female body, ferritin is found in amounts of 150 mg/mL, while in men 300. The elevated amount of ferritin can be a sign of various diseases, such as cardiac or neoplastic diseases.

When intracellular iron exceeds normal ferritin-binding capacity, the excess iron is deposited externally to ferritin, in the form of an amorphous mixture of iron hydroxide, iron phosphate, and iron-binding proteins, becoming hemoxydrin (which is nothing more than ferritin denatured by excess iron); from here the iron no longer comes off. Hemoxydrin can accumulate at scars, making them dark; or in macrophages, spleen and liver, where it can be highlighted by iron dyes, such as Prussian blue.

Regulation of iron absorption

The regulation of intestinal iron absorption is influenced by the concentration of iron already present in the body. An iron deficiency leads to increased intestinal absorption; whereas an overload leads to decreased absorption. Consistent with this, if there is iron deficiency, the synthesis of apo-ferritin is low: the protein binds little iron, which, for its part, is free to circulate in the blood bound to its transport protein. If there is an overload of iron in the body, there is a large synthesis of apo-ferritin, which in the intestinal epithelium can integrate up to 4 500 atoms of iron, although it is usually bound to about 3000 atoms. In this way, ferritin prevents free iron from circulating in the blood, where it might bind to other proteins, deactivating or destroying them. 

IRP1 and IRP2

The two proteins that act as sensors of iron changes in the cytoplasm, in duodenal epithelial cells, are the iron regulatory proteins, IRP1 and IRP2. The regulation of key enzymes involved in iron hemostasis, occurs at the translational level, through Iron Responsiv Element, i.e. regions of m-RNA involved in iron metabolism, and IRP proteins, which bind to IRE regions of m -RNA. Interestingly, IRP1 is an aconitase present in the cytosol; an identical aconitase is found in the mitochondrion, for the Krebs cycle, where it serves to convert citrate to iso-citrate. 

This protein contains a prosthetic group with four iron atoms, 4 sulfur atoms, alternating at the vertices. It was discovered that the enzyme, in the cytoplasm, can lose an iron atom, changing from 4 to 3 iron atoms and becoming able to bind to m-RNA. This structure can give up iron when this is low, while when it is high it works as an aconitase; it can also give up iron when the level of ROS is high.

The enzyme is able to bind to a loop, a hairpin, of the m-RNA: this occurs when iron levels are low. 

IRP2 also belong to the class of aconitases, although they never function as such, even when iron is high. In the latter condition, the protein is at low levels and is oxidized and degraded. Like IRP1, when iron is low it binds to m-RNA, although it does not lose iron from its structure, but is simply present at higher concentrations. 

Whichever IRP it is, it binds to the m-RNA. This has loops, downstream or upstream of the coding region. When IRP binds on the 5'UTR side, it prevents the translation apparatus from working on the m-RNA, consequently protein synthesis will be inhibited. When IRP binds to the 3'UTR, the result is opposite, preventing the destruction of m-RNA by endonucleases: since the interaction side is at the 3', the synthesis will not be disturbed, but the synthesis will increase since the stability of the transcript increases. 

The genes that are inhibited on the 3 UTR side are: the genes for the H and L subunit of ferritin (because in iron deficiency ferritin production is reduced), the enzyme ALA-Synthase that catalyzes the first step of the heme synthesis pathway (because if porphyrins do not bind to iron they are harmful), ferroporitin, the IF factor (sensitive to hypoxia) and the synthesis of mitochondrial aconitase. Thus, iron-using proteins are also inhibited. In general, the sense is to reduce the export of iron, already deficient in itself, in the tissues locally. 

There are other IRPs, which bind to the 3'-UTR of certain m-RNAs, thereby increasing the production of transferrin and thus the mechanism for iron import and also the level of DMT1: the bivalent metal transporter, which is used to move iron out of/into the endosome.

If, on the other hand, iron is increased, the balance is shifted, IRP1 goes back to being an aconitase, and IRP2 is degraded; the IRPs break away from the m-RNA and what was being activated goes back to working less, while what was being inhibited is activated. 

Alongside this rapid translational regulation, there is a transcriptional one that is much slower, but of longer duration. For example, under conditions of hypoxia or increased proliferation, the synthesis of the receptor for transferrin increases, because in apoxia, an attempt is made to increase oxygen uptake by increasing the production of hemoglobin, which requires more iron. Under conditions of increased proliferation, however, there is simply an increased need for protein. Exposure to cytokines increases m-RNA transcription for ferritin, thereby increasing ferritin levels.

Hemochromatosis

This is a genetic disease that leads to iron accumulation. There are inherited diseases in which ferroportin no longer responds to hepcidin: there is a defect in factors for the synthesis of hepcidin. The synthesis of the latter is stimulated by the synthesis of HJV, TFR2 and HFM proteins. The most common form of hemochromatosis is caused by a homozygous mutation in the HFE gene. A mutation in the genes of other proteins can also cause hemochromatosis, but these are very rare cases.

The consequence is that a decreased production of hepcidin leads to an inability to regulate ferroportin: therefore the level of the latter will be high and more iron will enter the enterocytes; or, in some cases, ferroportin binds hepcidin but is not internalized to be degraded. Either way, there is an increase in iron intake.

In addition to genetics there are also dietary habits: if, for example, food is kept in iron containers as it happens in some African tribes, in alcoholics there is hemochromatosis even if the reasons have not yet been fully clarified.

Beta Thalassemia

Storically, Beta Thalassemia has been classified into

• Beta thalassemia, patients are clinically asymptomatic
• Beta thalassemia intermedia, with clinically heterogeneous and genotypic disorders, ranging in severity from mild to severe transfusion-dependent status.
• Beta thalassemia major with severe, transfusion-dependent anemia.

Beta-thalassemia occurs, in which the beta chains of hemoglobin are not produced and there is an accumulation of alpha chains, with formation of tetramers that can precipitate, releasing heme and causing oxidative damage. As a result, red blood cells are weaker and more fragile, living much less, causing hypoxia. Hypoxia causes splenic and hepato-megaly and an alteration of the bone marrow, which increases in size to produce more red blood cells. Increased erythropoiesis causes decreased expression of hepcidin, which in turn results in increased levels of ferroportin and iron availability. Therapy is a periodic administration of transfusions and iron chelators, such as EDTA, which arranges an octahedron, heme-like, around iron. Currently EDTA (too non-specific) is replaced by deferoxamine, a peptide used as a siderophore in bacteria, while in humans it binds iron and, at this point, is eliminated in the urine.