Iron Absorption and Metabolism

Iron is an essential element for almost all forms of life, but most important as an oxygen transporter. When iron is in its ferrous state, oxygen binds to it within the hemoglobin molecule allowing erythrocytes to circulate and deliver oxygen to all the human bodies cells and tissues. The human body also requires iron in order to obtain ATP from cellular respiration (Oxidative phosphorylation). Although iron is an essential element to the body, like anything in nature, too much of it can be toxic. Its ability to donate and accept electrons readily means that it can spontaneously catalyze the conversion of hydrogen peroxide into free radicals. Free radicals cause a wide array of damage to cellular structures and tissues. To minimize the chances of toxicity, almost every iron atom is bound to protein structures, an example being hemoglobin. The iron is bound to the globin protein. To learn more about the structure of hemoglobin, review the previous article written. There is tight regulation of iron metabolism that allows the body to remain in homeostasis. Understanding iron metabolism is important for understanding multiple diseases of iron overload, and iron deficiency.

Iron Absorption

Most of the bodies iron comes from dietary uptake. There is continuous iron recycling occurring within the body from the sequela of hemoglobin metabolism by the spleen. The macrophages of the reticuloendothelial system store iron from the process of breaking down engulfed red blood cells. Its stored as hemosiderin. Hemosiderin is just a defined deposit of protein and iron that occurs as a result of iron overload, either systemically or locally. The metabolic functions of iron depend on the ability to change its valence state from reduced ferrous state (Fe2+) to the oxidized ferric state (Fe3+). Ferrous iron in the lumen of the duodenum is transported across the luminal side of the enterocyte by a protein called divalent metal transporter-1 (DMT1). Once iron has been absorbed across the cell membrane of the enterocyte, it can either be stored by binding to apoferritin or the cell can release the iron through the help of another transporter called ferroportin. Ferroportin is the only know protein that exports iron across cell membranes. One of the ways that the human body manages iron homeostasis is by the production of hepcidin. When iron stores are adequate, the liver will produce hepcidin, which competitively binds to ferroportin and inactivates it. When iron stores begin to drop, suppression of synthesis of hepcidin allows ferroportin to transport iron again. Before iron is taken by ferroportin across the membrane, it must be converted to its ferric form. Hephaestin, another protein on the enterocyte cell membrane oxidizes iron as it exits to its ferric form (Fe3+). Once oxidized and in its ferric state, the iron binds to apotransferrin (ApoTf). This iron:apotransferrin complex is known as transferrin (Tf). Its important to note that two molecules of ferric iron can bind to one molecule of apotransferrin.

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Iron Uptake into Cells

Individual cells regulate the amount of iron they absorb to avoid adverse toxicity. Cells possess a receptor for transferrin (Tf), called transferrin receptor-1 (TfR1). The physiological pH of the plasma and extracellular fluid allow for a strong affinity to transferrin for TfR1. Through receptor mediated endocytosis transferrin saturates the TfR1 and once a critical mass has accumulated, endocytosis begins. The iron is passed into the cell into an endosome vesicle. Hydrogen ions are then pumped into the endosome and as a result the pH drops causing dissociation of the iron from the transferrin. Almost simultaneously the affinity for TfR1 to apotransferrin increases so it remains bound to the receptor while the iron remains free. The iron is then exported from the endosome vesicle into the cytoplasm by divalent metal transporter 1 (DMT1). The molecules of iron are then either stored, or transported into the mitochondria where they are incorporated into cytochromes or heme for the production of hemoglobin. While the iron is transported in the cytoplasm, the endosome fuses again with the cell membrane and in the extracellular space pH, TfR1 has a low affinity for apotransferrin so it dissociates and begins circulating again in the plasma for free transferrin. Again transferrin being a diiron:apotransferrin complex. Cells are able to store iron so they have a reserve if needed. Ferric iron (Fe3+) is stored in a protein called apoferritin. When iron binds to it it known as ferritin. Ferritin can be used at anytime during iron depletion by lysosomal degradation of the protein.

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Regulation

Just like hepcidin, there are other ways that the body maintains iron homeostasis. Transcription of TfR1 on the surface of the cells can either decrease or increase depending on iron stores within the cell. When iron stores are sufficient, production of TFR1 decreases, and vice versa. This is also useful in diagnosis of iron deficiency. Turns out there is a truncated form of TfR1 that circulates in serum as soluble transferrin receptors (sTfR). These sTfRs reflect the amount of tFR1 in the body. So in iron depletion there will be more circulating sTfRs indicating more production of TfR1 on the cells surface. A useful tool in the diagnosis of iron deficiency anemia.

Iron Recycling

When cells die, they are sequestered by the spleen and liver in which mechanisms salvage iron. These mechanisms are often referred to as the haptoglobin-hemopexin-methemalbumin system. Free hemoglobin in the plasma is quickly complexed with haptoglobin. By binding haptoglobin, the hemoglobin, and consequently, the iron avoid filtration by the glomerulus in the kidneys. This complex is taken up by macrophages, primarily those in the liver, spleen, bone marrow and even in the lungs. These macrophages express CD163, which is the haptoglobin scavenger receptor. The entire complex is internalized into the macrophage within a lysosome. Inside this lysosome, the iron is salvaged, the globin is catabolized as any protein would be, and the protoporphyrin is converted to unconjugated bilirubin. To learn more about the process of bilirubin metabolism, review the previous article. The haptoglobin is also degraded by the lysosome. The iron in free hemoglobin becomes oxidized to its ferric state (Fe3+), and as a result, forms methemoglobin. The heme (metheme) molecule of the free hemoglobin binds to hemopexin, preventing oxidative injury to the cells and tissues, as well as prevents loss of iron through glomerulus filtration. Albumin acts as a carrier for many proteins, including metheme. So albumin acts as a carrier for metheme to find hemopexin, which has a much higher affinity for the metheme itself. This allows for more rapid degradation of the toxic metheme.

There was a lot to learn in this article. Read carefully and go back and refer. I will try to highlight certain areas that I think are more important to the bigger picture. The next step is what happens in certain physiological disease states that leads to either iron overload or iron deficiency.

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DARA-T Workup

Daratumumab (Darzalex) is an IgG1k monoclonal antibody directed against CD38, which is over expressed on the plasma cells in patients with multiple myeloma. Daratumumab binds to CD38 and causes apoptosis through antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. In 2015 the FDA approved daratumumab for the treatment of refractory multiple myeloma. Refractory meaning that patients have received at least three previous treatment protocols that failed to show sustained efficacy or any efficacy at all. Recently in May of 2018, the FDA approved daratumumab for first line therapy in combination with bortezomid, melphalan, and prednisone. The names of the drugs aren’t important, what is important is that this monoclonal antibody approach has become more common and now has moved into first line therapy meaning that more patients are going to receive this treatment. Its no secret that patients with multiple myeloma when undergoing treatment and throughout the course of the disease progression need blood component transfusions.

Typing and screening patients that are receiving daratumumab is extremely difficult and time consuming. The daratumumab not only binds to the CD38 on the malignant lymphoma cells, but it also binds to the red cells who express CD38. This causes interference in transfusion testing. Part of normal pre-transfusion testing is an antibody screen. An antibody screen is important as it tells the transfusion team if there are any alloantibodies. Alloantibodies are antibodie directed towards red cell antigens on the donor cells. If a patient has an alloantibody, it makes selecting red cells for transfusion difficult. Additional testing must be done to select antigen negative donor cells for the antibody that the recipient or the patient has. Daratumumab causes the antibody screen and corresponding antibody panel panreactive, including a positive autocontrol. This may mask any additional clinically significant alloantibody that the patient may have.

The blood bank team must perform testing prior to the patient receiving this daratumumab. The clinical team must be in communication with the blood bank. Before the patient receives the medication, the team must get a baseline type and screen. Normally they are negative, but in the off chance that they have an alloantibody, the blood bank can identify the antibody before daratumumab interferes with testing. Other testing must include a complete phenotype of the patients cell. A complete phenotype will identify all the antigens that are present on the patients cells. This tells the blood bank and clinician vital information. If the patient does NOT have the antigen present on their red cells, there is a chance that they can produce an antibody towards that antigen on donor cells making it hard to find correct donors for transfusion. For example, if the patient is negative for the E antigen, they may or may not develop an antibody towards the E antigen, so in the event that the donor red cells have the E antigen present, the patients antibody will attack those cells and cause a transfusion reaction. For the characteristics of different transfusion reactions, reference transfusion reactions.

Once the daratumumab has been given there are techniques that must be followed to obtain a sample that is suitable for testing. An enzyme called dithiothreitol (DTT) is used to negate the binding of DARA-T to CD38 on the red cell surface. This will allow for an antibody screen to be run. Unfortunately, DTT destroys the Kell antigen on the red cell surface. Kell is a clinically significant antibody in transfusions so its important to know whether or not if the patient has the antigen or not. Patients treated with DTT, MUST have Kell negative donor units, because of the risk of developing an anti-K antibody and not being able to identify it.

 

Thalassemias

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The major hemoglobin that is present in adults is hemoglobin A (HbA). This is a heterotetramer that consists of one pair of alpha-globin chains and one pair of beta-globin chains. Alpha-globin chains are encoded by two copies of the alpha gene present on chromosome 16. Beta-globin chains are driven by one gene on chromosome 11. Normally there is tight regulation of the production of alpha and beta-globin chains and the ratio of production, but sometimes that regulation can be interrupted. This offsets the balance of globin chains being produced. These types of hematological disorders are coined thalassemias. They are a quantitative defect characterized by reduced or absent production of one and rarely two of the globin chains.

Alpha thalassemia is largely due to the inadequate production of alpha globin chains, which leads to an excessive production of either gamma-globin chains or beta-globin chains. In the fetus alpha thalassemia leads to excess gamma chains and in adults it largely leads to excess beta chains. In neonates the absence of alpha-globin chains is incompatible with life, leading to hydrops fetalis or hemoglobin Barts and absolute death after delivery. Hb Barts cannot deliver the oxygen to the tissues because its affinity to oxygen is too high. The hydronic state is reflected in the fetus by heart failure and massive total body edema. Excess beta-globin chains are capable of forming homotetramers and precipitate that leads to a variety of clinical manifestations.

Beta thalassemia is an inherited hemoglobinopathy in which production of beta-globin chains is impaired. There are different classifications corresponding to the degree of reduction in the beta chains. Beta thalassemia major is due to mutations that completely stop all production of beta-globin chains. These are individuals who are homozygous for the disease. They lose the ability to make HbA and because of this will experience severe manifestations and are transfusion-dependent for the rest of life. Symptoms typically begin during late infancy (6-12 months), but some newborns are asymptomatic because the major hemoglobin in newborns is HbF (4A:4G) which is constructed by gamma-globin chains and not beta-globin chains. Beta-thalassemia major presents with pallor, jaundice, and bilirubin in the urine which indicates hemolysis. Hepatosplenomegaly is present as well as heart failure. Failure to thrive and recurrent infections are also other signs. There is so much hemolysis because of the faulty hemoglobin present in the red cells that the bone marrow can’t keep up with production so extra medullary hematopoiesis occurs that results in skeletal abnormalities in the face and long bones. Iron overload is often a symptom of late untreated disease which can affect almost every organ in the body. Mortality is upwards of 85% by age five if untreated. If treated the survival rate is only 60 years of age if lucky.

Beta thalassemia major is also called transfusion-dependent beta thalassemia. There is also a subtype called non-transfusion-dependent beta thalassemia otherwise known as beta thalassemia intermedia. These individuals present with a less severe phenotype of the disease. There is significant variability with the clinical findings in individuals with beta thalassemia intermedia; from osteoporosis to thrombosis to diabetes mellitus. Some individuals will develop hepatosplenomegaly and extramedullary hematopoiesis and some won’t. Also some individuals will have to become transfusion-dependent, but that is typically in the late decades of life.

Anemia is a severe clinical manifestation of both alpha and beta thalassemia. The pathophysiology of beta thalassemia causes excess alpha-globin chains to precipitate in the developing erythrocytes in the bone marrow. This causes inclusion bodies. The inclusion bodies create oxidative stress and damages the cellular membranes. Apoptosis gets activated downstream and the red cell precursors are subsequently phagocytized and destroyed in the bone marrow by activated macrophages. This is also called ineffective erythropoiesis. The bone marrow in an effort to compensate releases these red cell precursors into the peripheral blood riddled with these inclusion bodies. These cells are subsequently sequestered by extravascular hemolysis by the RES which  further contributes to the anemia. The red cells that survive are microcytic and hypochromic and have a significantly shortened life span. Severe tissue hypoxia is seen due to the increased HbF as a compensatory mechanism. HbF has an increased affinity for oxygen and causes a shift to the left on the oxygen dissociation curve.

Typical laboratory findings for an individual with beta thalassemia is a slightly decreased red cell count and a marked decrease in hemoglobin of usually about 2-3 g/dL (12.5-16.5 g/dL). There will be marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, slight increase in reticulocytes and nucleated red cells.

The pathophysiology for anemia associated with alpha thalassemia is associated with precipitation of HbH. Remember HbH is formed when there is decreased production of the alpha-globin chains so there is an excess of beta-globin chains. The precipitation of HbH creates inclusion bodies, typically called Heinz Bodies. These inclusions are recognized by the RES and remove the red cells via extravascular hemolysis.

Laboratory findings for an individual with alpha thalassemia is very similar to that of an individual with beta thalassemia. Decreased hemoglobin, marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, and reticulocytes and NRBCs. The HbH inclusions can be see seen using a cresyl blue stain.

Thalassemias are a quantitative hemoglobinopathy meaning that there is a deficiency or an excess of production of globin chains leading to clinical manifestations. They are inherited and some subtypes can significantly elevate mortality. It is important to diagnose early and to treat early.