Iron, heme, and hemoglobin

Iron uptake in the duodenum

The human body contains about 3 to 4 g of iron , of which 70% to 80% is complexed to protoporphyrin IX to form heme , the oxygen-binding prosthetic group of hemoglobin and myoglobin . Although iron plays a role in many enzymatic reactions, most nonheme iron is stored in the marrow (80%) and liver (20%) as ferritin and hemosiderin . A small amount (1/1000th) of total body iron circulates in blood plasma, complexed to the iron transport protein transferrin . Free iron (esp. ferrous iron) is toxic, with formation of reactive oxygen species (ROS). To minimize damage, a small amount of intracellular ferrous iron necessary for immediate needs is bound to iron chaperone protein (ICP) , forming the labile iron pool (LIP) . Surplus iron, on the other hand, is stored in ferric form as ferritin and hemosiderin. Iron is stored primarily in hepatocytes and macrophages in the spleen and marrow. There is no physiologic mechanism for controlled release of excess iron. A small amount of iron is lost through daily shedding of intestinal epithelial cells and keratinocytes as well as intermittent menstrual blood loss. Chronic iron loss leading to iron-deficiency anemia may be seen in gastrointestinal (GI) bleeding, menorrhagia, and hemolytic anemia.

Iron in food is present as heme iron and non-heme (ionic) iron. Nonheme (ionic) iron is present in fruits and vegetables, while heme iron is present in meats. Ionic non-heme iron is primarily ferric (Fe3+), while heme iron is ferrous (Fe2+). Acid digestion of food in the stomach releases non-heme iron and heme. In the duodenum ( Fig. 4.1 ), ferric iron (Fe3+) is converted to ferrous iron (Fe2+) by duodenal cytochrome B and vitamin C ferrireductase and absorbed at the luminal surface of villus enterocytes through the divalent metal transporter (DMT1) . Heme bound to heme carrier protein (HCP) is absorbed by villus enterocytes and transported to endosomes for acid and heme oxygenase (HO) mediated release of ferrous iron. Ferrous iron, bound to ICP, is transported to the basolateral surface of the enterocyte and released to the blood through the iron transporter ferroportin where it is rapidly re-oxidized to ferric iron by membrane-bound hephaestin (HAE) or extracellular ceruloplasmin , and bound to the iron transporter transferrin for release to the bloodstream.

Duodenal crypt cells differentiate into absorptive villus epithelium within 2 to 3 days. In crypt cells, the level of plasma iron, as detected by the TFR1–HFE complex (transferrin receptor–high iron [Fe] protein), programs the level of expression of iron transport proteins DMT1 and ferroportin in villus cells. Low plasma iron detected by crypt cells leads to increased expression of DMT1 and ferroportin by villus cells and increased duodenal iron uptake. In contrast, high plasma iron detected by crypt cells leads to decreased expression of DMT-1 and ferroportin by villus cells and decreased duodenal iron uptake.

Iron storage and regulation: Hepatocytes

Hepatocytes play two roles in iron metabolism, as a site of iron storage and as a regulator of iron uptake and release ( Fig. 4.2 ). Transferrin-bound iron in plasma binds to TFR1 and enters hepatocytes by receptor-mediated endocytosis. Within acidic endosomes, TF-TFR complexes are disassembled, ferric iron (Fe3+) and converted to ferrous iron (Fe2+) by ferric reductase (STEAP3) . Ferrous iron exits the endosome through the divalent metal transporter (DMT1), binds ICP, and enters the LIP to meet immediate needs. Surplus iron (ferric) is stored as ferritin and hemosiderin. Ferritin is composed of an outer shell of apoferritin protein enclosing a dense core of crystalline ferrihydrite. Hemosiderin, formed by lysosomal degradation of the apoferritin shell, is composed almost entirely of ferric iron.

The transferrin iron saturation of plasma is monitored by the TFR1–HFE complex on hepatocytes. HFE, the plasma iron sensor, competes with transferrin for binding to TFR1. With normal to low transferrin iron saturation, HFE (high iron (Fe) protein) binds to TFR1, reducing iron uptake. With high transferrin iron saturation, transferrin outcompetes HFE for TFR1 binding, leading to increased iron uptake. The released HFE binds instead to TFR2 , triggering hepcidin production. Hepcidin blocks iron transport by irreversible inactivation of ferroportin expressed by villus enterocytes, hepatocytes, and macrophages.

In a setting of chronic inflammation or infection, release of the liver hormone hepcidin blocks systemic iron uptake and release by inactivating ferroportin, the major iron exporter. Plasma iron (transferrin) saturation is monitored by the hepatocyte TFR1–HFE complex. As a counterbalance to hepcidin, the hormone HFE (high Fe) produced by GI tract, macrophages, and granulocytes blocks transferrin receptor and blocks hepcidin production in the liver. When iron saturation is high, HFE translocates from TFR1 to TFR-2, triggering hepcidin release. Inactivating HFE mutations are the most common cause of hereditary hemochromatosis (HH) . Less common HH-related mutations include those of TFR-2 and HAMP (hepcidin).

The level of intracellular iron is regulated at both transcriptional and translational levels. Iron binds to iron-responsive proteins , which bind to iron-responsive elements in iron-related genes. Hypoxia increases expression of hypoxia-inducible factor , which binds to hypoxia-responsive elements in iron-related genes. High iron triggers increased ferritin and ferroportin and decreased TFR1 and DMT1. Low iron triggers decreased ferritin and increased TFR1 and DMT1. Low oxygen (hypoxia) triggers increased TFR1.

Iron storage and recycling: Macrophages

Splenic macrophages are responsible for the removal of senescent and damaged red blood cells (RBCs) from the blood circulation ( Fig. 4.3 ). Given their critical role in oxygen transport, RBCs are naturally subject to irreparable and progressive free radical damage. Because of increased rigidity, senescent RBCs are trapped in the splenic cords and by virtue of phosphatidylserine (PS) expression are recognized and engulfed by PS receptor– bearing macrophages. In normal RBCs, the enzyme flippase restricts PS expression to the inner leaflet of the RBC plasma membrane, unrecognized by macrophages. In contrast, the enzyme scramblase leads to expression of PS on the RBC outer leaflet and recognized by the macrophage PS receptor. Ingested RBCs are confined to membrane-bound phagosomes that fuse with lysosomes to form phagolysosomes . In the acidic environment of the phagolysosome, hemoglobin is degraded to ferric iron, heme, and globin. The iron is stored as ferritin and hemosiderin. Heme is converted to biliverdin , and globin is converted to amino acids. Biliverdin is reduced to bilirubin, transported to the liver for conjugation to glucuronic acid (to increase solubility), and excreted in bile, urine, and feces. In hemolytic anemia, free heme and hemoglobin bind to carrier proteins hemopexin (HPX) and haptoglobin (HPG) , respectively, and absorbed by HPX/HPG receptor-bearing macrophages.

Bone marrow macrophages (nurse cells) provide iron to developing erythroid precursors for production of hemoglobin. Ferritin released from iron-laden macrophages is directly transferred to small groups of developing erythroid cells (erythroid islands) by the process of pinocytosis . Erythroid precursors in marrow also acquire iron by binding of plasma transferrin to RBC transferrin receptors ( Fig. 4.4 ).

Cellular iron that is not immediately used for heme synthesis or enzymatic reactions or as low-molecular-weight chelates is primarily stored in the cytoplasm as ferritin. Ferritin is composed of a core of crystalline ferrihydrite (ferric iron) enclosed within an apoferritin protein shell. Under normal circumstances, nearly all ferritin is found in the cytoplasmic compartment of cells. A very small amount of ferritin is present in the blood. In most circumstances, the serum ferritin level in blood correlates closely with the level of intracellular ferritin, thus providing a useful measure of total body iron stores. However, as an acute-phase reactant , serum ferritin is elevated in chronic inflammatory states and thus may not accurately reflect low storage iron in patients with both iron deficiency and chronic inflammation. In the marrow, most storage iron is present in macrophages as both ferritin and hemosiderin, an aggregated, iron-rich ferritin degradation product with very low apoferritin content. Approximately 20% to 50% of normal marrow erythroid precursors are sideroblasts , nucleated erythroid cells that contain a few small, ferritin-rich lysosomes called siderosomes . Storage iron distribution in macrophages and sideroblasts can be visualized by staining of marrow smears or biopsy sections with Prussian Blue, a dye that stains aggregates of ferritin and hemosiderin ( Fig. 4.5 ). Even without Prussian blue staining, golden-brown refractile aggregates of hemosiderin can often be seen by light microscopy ( Fig. 4.6 ).