CHAPTER OUTLINE
High-Yield Terms
Ferrous iron: iron with an oxidation state of +2 (Fe2+ or Fe[II])
Ferric iron: iron with an oxidation state of +3 (Fe3+ of Fe[III])
Cuprous copper: copper with an oxidation state of +1 (Cu1+ or Cu[I])
Cupric copper: copper with an oxidation state of +2 (Cu2+ or Cu[II])
Ferroxidase: any of a class of enzyme that oxidizes ferrous (Fe2+) iron to ferric (Fe3+) iron
Ferrireductase: any of a class of enzyme that reduces ferric (Fe3+) iron to ferrous (Fe2+) iron
Reticuloendothelial cells: comprise phagocytic cells located in different organs of the body, responsible for engulfing, bacteria, viruses, other foreign substances, and abnormal body cells
Hemosiderin: an intracellular complex of iron, ferritin, denatured ferritin and other material, most commonly found in macrophages, accumulates in conditions of hemorrhage and iron excess
Ceruloplasmin: is a ferroxidase that is also a major copper-requiring protein of the blood
Metallothioneins: a family of cysteine-rich proteins that bind a variety of metals and are thought to provide protection against metal toxicities
Role of Iron
Iron serves numerous important functions in the body relating to the metabolism of oxygen, not the least of which is its role in hemoglobin transport of oxygen. Within the body iron exists in 2 oxidation states: ferrous (Fe2+) or ferric (Fe3+). Because iron has an affinity for electronegative atoms such as oxygen, nitrogen, and sulfur, these atoms are found at the heart of the iron-binding centers of macromolecules.
Under conditions of neutral or alkaline pH, iron is found in the Fe3+ state and at acidic pH the Fe2+ state is favored. When in the Fe3+ state, iron will form large complexes with anions, water, and peroxides. These large complexes have poor solubility and upon their aggregation lead to pathological consequences. In addition, iron can bind to and interfere with the structure and function of various macromolecules. For this reason the body must protect itself against the deleterious effects of iron. This is the role served by numerous iron-binding and transport proteins.
Aside from its importance as the prosthetic group of hemoglobin and a number of enzymes (eg, redox cytochromes and the P450 class of detoxifying cytochromes), heme is important because a number of genetic disease states are associated with deficiencies of the enzymes used in its biosynthesis. Some of these disorders are readily diagnosed because they cause δ-aminolevulinic acid (ALA) and other abnormally colored heme intermediates to appear in the circulation, the urine, and in other tissues such as teeth and bones.
Iron Metabolism
Iron is associated with proteins either by incorporation into protoporphyrin IX (forming heme) or by binding to other ligands. There are a number of heme containing proteins involved in the transport of oxygen (hemoglobin), oxygen storage (myoglobin), and enzyme catalysis such as nitric oxide synthase (NOS) and prostaglandin synthase (cyclooxygenase). A number of non-heme iron–containing proteins are also known such as the iron-sulfur proteins of oxidative phosphorylation and the iron transport and storage proteins, transferrin and ferritin, respectively.
Iron, consumed in the diet, is either free iron or heme iron. Free iron in the intestines is reduced from the ferric (Fe3+) to the ferrous (Fe2+) state on the luminal surface of intestinal enterocytes and transported into the cells through the action of the divalent metal transporter 1 (DMT1) (Figure 42-1). Intestinal uptake of heme iron occurs through the interaction of dietary heme with the heme carrier protein (HCP1). The iron in the heme is then released within the enterocytes via the action the heme catabolizing enzyme heme oxygenase (see Figure 33-5). The iron can be stored within intestinal enterocytes bound to ferritin. Iron is transported across the basolateral membrane of intestinal enterocytes into the circulation, through the action of the transport protein ferroportin (also called IREG1 = iron-regulated gene 1). Associated with ferroportin is the enzyme hephaestin (a copper-containing ferroxidase with homology to ceruloplasmin) which oxidizes the ferrous form back to the ferric form. Once in the circulation, ferric iron is bound to transferrin and passes through the portal circulation of the liver. The liver is the major storage site for iron. The major site of iron utilization is the bone marrow where it is used in heme synthesis (see Chapter 33).
FIGURE 42-1: Dietary iron in the form of non-heme iron or heme iron is absorbed in the duodenum. Non-heme iron occurs primarily in the ferric state in the gut and is reduced to the ferrous state through the action of ferrireductases. In the duodenum this reduction is carried out primarily by duodenal cytochrome b (DCYTB). There are additional intestinal brush border ferrireductases since it has been shown in mice that loss of DCYTB does not impair iron absorption. Ferrous iron is then taken up by duodenal enterocytes through the action of divalent metal transporter 1 (DMT1). DMT1 is a member of the solute carrier protein family and is thus, also known as SLC11A2. Heme iron is taken up through the action of heme carrier protein 1 (HCP1). Once in the enterocyte heme is degraded through the action of heme oxygenase releasing the ferrous iron. Ferrous iron can be stored in the enterocyte bound to ferritin or released to the circulation through the action of ferroportin (also called IREG1). Ferroportin is also a member of the solute carrier protein family and has the designation SLC11A3. Iron is transported in the blood bound to transferrin but does so only in the ferric state so during the transport through ferroportin, ferrous iron is oxidized by the ferroxidase called hephaestin. Reproduced with permission of themedicalbiochemistrypage, LLC.
Transferrin, made in the liver, is the serum protein responsible for the transport of iron. Although several metals can bind to transferrin, the highest affinity is for the ferric (Fe3+) form of iron. The ferrous form of iron does not bind to transferrin. Transferrin can bind 2 moles of ferric iron. Cells take up the transported iron through interaction of transferrin with cell-surface receptors (Figure 42-2). Internalization of the iron-transferrin-receptor complexes is initiated following receptor phosphorylation by protein kinase C (PKC). Following internalization, the iron is released due to the acidic nature of the endosomes. The transferrin receptor is then recycled back to the cell surface.
FIGURE 42-2: The main cellular site of iron storage is the liver, specifically in hepatocytes. Iron bound to transferrin is taken up from the blood by hepatocytes due to the binding of transferrin to the transferrin receptor. Free iron (called non-transferrin–bound iron, NTBI) in the plasma can also be absorbed by hepatocytes via the action of DMT1. However, the ferric form predominates in the blood and must first be reduced by ferrireductases prior to DMT1 transport. Upon binding transferrin, the transferrin receptor is internalized via receptor-mediated endocytosis. The acidic environment of the endosome results in the release of ferric iron from transferrin. The ferric iron is reduced in the endosome to the ferrous form via the action of an endosomal ferrireductase, most likely 6-transmembrane epithelial antigen of prostate protein 3 (STEAP3). The ferrous iron is transported out of the endosome via DMT1 action and can then be stored in the hepatocyte bound to ferritin as in intestinal enterocytes. The transferrin-transferrin receptor complexes are recycled back to the surface of the hepatocyte and the transferrin is released to the blood where it can bind more ferric iron in the circulation. Ferrous iron is released from hepatocytes to the circulation through the action of ferroportin. When in the circulation ferrous iron is oxidized to the ferric form by the plasma ferroxidase known as ceruloplasmin. The ferric iron can then be bound by transferrin and delivered to other tissues of the body. Reproduced with permission of themedicalbiochemistrypage, LLC.
Ferritin is the major protein used for intracellular storage of iron. Ferritin without bound iron is referred to as apoferritin. Apoferritin is a large polymer of 24 polypeptide subunits. This multimeric structure of apoferritin is able to bind up to 2000 iron atoms in the form of ferric-phosphate. The majority of intracellularly stored iron is found in the liver, skeletal muscle, and reticuloendothelial cells. If the storage capacity of the ferritin is exceeded, iron will deposit adjacent to the ferritin-iron complexes in the cell. Histologically these amorphous iron deposits are referred to as hemosiderin. Hemosiderin is composed of ferritin, denatured ferritin, and other materials and its molecular structure is poorly defined. The iron present in hemosiderin is not readily available to the cell and thus cannot supply iron to the cell when it is needed. Hemosiderin is found most frequently in macrophages and is most abundant following hemorrhagic events.
In humans approximately 70% of total body iron is found in hemoglobin (Table 42-1). Because of storage and recycling very little (1-2 mg) iron will need to be replaced from the diet on a daily basis. Any excess dietary iron is not absorbed or is stored in intestinal enterocytes. Regulation of iron absorption, recycling, and release from intracellular stores is controlled through the actions of the hepatic iron regulatory protein, hepcidin.
Hepcidin functions by inhibiting the presentation of one or more of the iron transporters (eg, DMT1 and IREG1) in intestinal membranes. With a high iron diet the level of hepcidin mRNA increases and conversely its levels decrease when dietary iron is low. This is occurring simultaneous to reciprocal changes in the levels of the transporters.
The regulation of iron homeostasis in the body is primarily controlled via iron-mediated regulation of mRNA translation (see Chapter 37). Both the transferrin receptor and the ferritin mRNAs contain stem-loop structures termed iron responsive elements, IREs. These IREs are recognized by an iron-binding protein containing an iron-sulfur center similar to that of the tricarboxylic acid (TCA) cycle enzyme aconitase. Other IRE-containing mRNAs include those encoding the erythrocyte protoporphyrin synthesis enzyme, ALA synthase, mitochondrial aconitase, and IREG1 (ferroportin).
Abnormal Iron Metabolism
Iron can bind to and form complexes with numerous macromolecules. Excess intracellular iron results in formation and deposition of hemosiderin which can lead to cellular dysfunction and damage. Thus, the consequences of excess iron intake and storage can have profound consequences, the most significant of which is hemochromatosis (Clinical Box 42-1). However, a reduction in iron intake can also lead to untoward consequences. Most notably, a reduced iron level negatively affects the function of oxygen transport in erythrocytes. Defects in iron metabolism can result from impaired intestinal absorption, excess loss of heme iron due to bleeding, as well as to mutations in the iron response elements of iron-regulated mRNAs.
CLINICAL BOX 42-1: HEMOCHROMATOSIS
Hemochromatosis is defined as a disorder in iron metabolism that is characterized by excess iron absorption, saturation of iron-binding proteins, and deposition of hemosiderin in the tissues. The primary affected tissues are the liver, pancreas, and skin. Iron deposition in the liver leads to cirrhosis and in the pancreas causes diabetes. The excess iron deposition leads to bronze pigmentation of the organs and skin. The bronze skin pigmentation seen in hemochromatosis, coupled with the resultant diabetes lead to the designation of this condition as bronze diabetes. Primary hemochromatosis is referred to as type 1 hemochromatosis. The mutant locus causing type 1 hemochromatosis has been designated the HFE1 locus and it is a major histocompatibility complex (MHC) class 1 gene. Hemochromatosis, that is associated with the HFE1 locus, is one of the most common inherited genetic defects. Manifestation of the symptoms of the disease is modified by several environmental influences. Dietary iron intake and alcohol consumption are especially significant to hemochromatosis. Menstruation and pregnancy can also influence symptoms. Hemochromatosis occurs about 5 to 10 times more frequently in men than in women. Symptoms usually appear between the ages of 40 and 60 in about 70% of individuals. The HFE1 gene encodes an α-chain protein with 3 immunoglobulin-like domains. This α-chain protein associates with β2-microglobulin, typical of MHC class 1 encoded proteins. Normal HFE1 has been shown to form a complex with the transferrin receptor thus, regulating the rate of iron transfer into cells. A mutation in HFE1 will therefore, lead to increased iron uptake and storage. The majority of hereditary hemochromatosis patients have inherited a mutation in HFE1 that results in the substitution of Cys 282 for a Tyr (C282Y). Another mutation found in certain forms of hereditary hemochromatosis also affects the HFE1 locus and causes a change of His 63 to Asp (H63D). This latter mutation is found along with the more common C282Y mutation resulting in a compound heterozygosity. As a result of the C282Y mutation the HFE1 protein remains trapped in the intracellular compartment. Because it cannot associate with the transferrin receptor there is a reduced uptake of iron by intestinal crypt cells. It is thought that this defect in intestinal iron uptake results in an increase in the expression of the DMT1 on the brush border of the intestinal villus cells. Excess DMT1 expression leads to an inappropriate increase in intestinal iron absorption. In hemochromatosis the liver is usually the first organ to be affected. Hepatomegaly will be present in more than 95% of patients manifesting symptoms. Initial symptoms include weakness, abdominal pain, change in skin color, and the onset of symptoms of diabetes mellitus. In advanced cases of hemochromatosis there will likely be cardiac arrhythmias, congestive heart failure, testicular atrophy, jaundice, increased pigmentation, spider angiomas, and splenomegaly. Diagnosis of the disease is usually suggested when there is the presence of hepatomegaly, skin pigmentation, diabetes mellitus, heart disease, arthritis, and hypogonadism. Treatment of hemochromatosis before there is permanent organ damage can restore life expectancy to normal. Treatment involves removal of the excess body iron. This is accomplished by twice-weekly phlebotomy at the beginning of treatment. Alcohol consumption should be curtailed and preferably eliminated in hemochromatosis patients. Iron-chelating agents, such as deferoxamine, can be used to remove around 10 to 20 mg of iron per day. However, phlebotomy is more convenient and safer for most patients.
Non-HFE Hemochromatosis: There are several additional causes of hemochromatosis, although none are as common as classic hemochromatosis. There are at least 4 additional genetic loci, that when defective lead to hemochromatosis. Two of which are juvenile forms identified as type 2A and 2B and 2 additional forms identified as type 3 and type 4.
Juvenile hemochromatosis (JH) type 2A (sometimes called HFE2A) is the result of defects in the gene encoding hemojuvelin (HJV). The function of hemojuvelin is to regulate the expression of the hepcidin gene (HAMP). As suggested by the juvenile nomenclature, this disorder manifests in patients under the age of 30. Cardiomyopathy and hypogonadism are prevalent symptoms with type 2A hemochromatosis. Type 2A hemochromatosis is inherited as an autosomal recessive disorder.
JH type 2B (sometimes called HFE2B) results from defects in the gene encoding hepcidin (HAMP). As with type 2A disease, symptoms of type 2B disease appear in patients under 30 years of age and include cardiomyopathy and hypogonadism. Type 2B hemochromatosis is inherited as an autosomal recessive disorder.
Type 3 hemochromatosis (sometimes called HFE3) is caused by mutations in the transferrin receptor 2 gene (TFR2). TFR2 is a homolog of the classic transferrin receptor (identified as TFR1). Unlike the ubiquitous expression of TFR1, TFR2 expression is almost exclusively found in the liver. The function of TFR2 in the liver is not to act in the uptake of transferrin-bound iron but to sense iron levels and to act as a regulator of hepcidin function. The clinical features of type 3 hemochromatosis are similar to those of classic type 1 disease. Type 3 hemochromatosis is inherited as an autosomal recessive disorder.
Type 4 hemochromatosis (sometimes called HFE4) is also called ferroportin disease because it is caused by mutations in the ferroportin gene. Ferroportin is highly expressed in the liver, duodenum, and reticuloendothelial cells. The major function of ferroportin is to transport dietary iron across the basolateral membranes of intestinal enterocytes into the blood and to recycle iron via the reticuloendothelial system. Symptoms of type 4 hemochromatosis are similar to those of classic type 1 disease but are generally milder. The unique aspect of type 4 hemochromatosis is that it is inherited as an autosomal dominant disease.
Iron-deficiency anemia is characterized by microcytic (small) and hypochromic (low-pigment) erythrocytes. Reduced iron intake and/or excess iron excretion results in decreased globin protein content in erythrocytes as a consequence of the heme control of globin synthesis (see Chapter 37 for details). Table 42-2 shows the most common laboratory test results for determination of iron-deficiency anemia. The most common causes of iron-deficiency anemia are excess menstrual flow or gastrointestinal (GI) bleeding. Causes of GI bleeding can include the use of medications that lead to ulceration or erosion of the gastric mucosa, peptic ulcer disease, gastric tumors, hiatal hernia, or the gastritis associated with chronic alcohol consumption. Treatment of iron-deficiency anemia is to first determine the cause and source of the excess bleeding. Oral administration of ferrous sulfate is commonly used to supplement the iron loss; however, intravenous iron therapy may be called for in some cases. Severe iron-deficiency anemia may necessitate transfusion with packed red blood cells.
Role of Copper
Copper is an essential trace element which plays a pivotal role in cell physiology as it constitutes a core part of copper-containing enzymes termed cuproenzymes. Like iron, copper exists in 2 oxidation states, cuprous (Cu1+ or Cu+) and cupric (Cu2+). Some important copper-containing enzymes include cytochrome oxidase of the electron transport chain and copper-zinc-superoxide dismutase (SOD1) involved in the detoxification of the reactive oxygen species, superoxide. Copper is required for respiration, connective tissue formation, iron metabolism, and many other processes. In human cells, copper is utilized in several cellular compartments, and the intracellular distribution of copper is regulated in response to metabolic demands and changes in cell environment. Although copper is an essential micronutrient, it can be potentially toxic when present in excessive amounts. Copper can also bind with high affinity to histidine, cysteine, and methionine residues of proteins which can result in their inactivation. Copper ions can also readily interact with oxygen leading to the production of the highly damaging hydroxyl radical. The need to provide copper to essential enzymes without causing cellular toxicity has resulted in the evolution of tightly regulated copper homeostatic mechanisms that involve regulated uptake, intracellular transport and efflux, and storage.
Copper Metabolism
Like iron, dietary copper is taken up by intestinal enterocytes via the action of a specific transport protein, copper transporter 1 (CTR1) as well as by the divalent metal transporter 1 (DMT1) which is not specific for any particular divalent metal. CTR1 is responsible for transport of cuprous (Cu+) copper into cells. CTR1 is a member of the solute-carrier family of transporters and as such is also identified as SLC31A1. Uptake by other tissues (primarily the liver and kidneys) also involves CTR1. In the blood 65% to 90% of copper is transported bound to ceruloplasmin, and the rest of copper loosely binds with albumin, transcuprein, and amino acids. In the liver, kidney, placenta, and mammary gland, CTR1 is found mainly in the basolateral membrane where it is responsible for uptake of copper from the blood.
Dietary copper is found primarily in the cupric state (Cu2+) and is reduced to cuprous (Cu+) prior to duodenal uptake. Potential intestinal reductants include ascorbate and the ferrireductase DCYTB (see Figure 42-1). In cells, CTR1 is found at the plasma membrane and in intracellular vesicles. When CTR1 binds copper it is internalized and moves to endocytic vesicles. Copper-transporting P-type ATPases (Cu-ATPases) maintain intracellular copper levels. A low-affinity copper transporter, CTR2, is present intracellularly and functions to release copper from the lysosome or lysosome-like compartments for reutilization. In intestine, CTR1 has an additional role in making dietary copper available for further utilization by facilitating its release from intracellular vesicles. Consistent with this role, a significant fraction of CTR1 in intestinal enterocytes is intracellular and is located in the vicinity of the apical membrane.
Within cells, copper is escorted to specific compartments through the actions of several chaperones referred to as metallochaperones. Copper chaperone for superoxide dismutase (CCS) directly interacts with thereby delivering copper to this enzyme. Cytochrome oxidase assembly protein 17 (COX17) delivers copper to additional chaperones within the mitochondria for synthesis of cytochrome c oxidase. A third chaperone, ATOX1, delivers copper to the secretory pathway by docking with 2 Cu-ATPases, ATP7A and ATP7B. ATP7A is required for cuproenzyme biosynthesis, and in intestinal enterocytes it is required for copper efflux to the portal circulation. ATP7B is mainly expressed in the liver where it is required for copper incorporation into ceruloplasmin and for excretion of copper into the biliary circulation. Mutations in ATP7A result in Menkes disease (Clinical Box 42-2) and mutations in ATP7B are the cause of Wilson disease (Clinical Box 42-3). Other proteins involved in copper homeostasis include the metallothioneins and amyloid precursor protein.
