Iron regulation and determination of iron stores Sean Lynch Eastern Virginia Medical School USA

Function and toxicity Selected Functions Oxygen transport and storage Oxidative metabolism Cellular growth and proliferation Neuroreceptor function Toxicity Free radical formation, particularly OH· Increased pathogen virulence Red cells: 2300 mg Functional tissues: 500 mg Store: 1000 mg Excretion: 1 mg Absorption: 1 mg

Ferroportin Ferroportin, a transmembrane iron transporter, is theFerroportin, a transmembrane iron transporter, is the cornerstone of the internal iron circuit cornerstone of the internal iron circuit It exports iron into the plasma from the duodenum (control of absorption), macrophages and hepatocytes (control of release of iron recovered from red blood cell catabolism and from stores); the iron is then bound to transferrin It regulates the plasma iron concentration so that transferrin is ~ 35% saturated with iron (average early morning value)

Hepcidin Hepcidin, a 25 amino acid peptide produced primarily in hepatocytes, is the key regulator of iron homeostasisHepcidin, a 25 amino acid peptide produced primarily in hepatocytes, is the key regulator of iron homeostasis It is a negative regulator of iron release from macrophages (red cell processing and iron recovery, iron store), hepatocytes (iron store) and enterocytes (iron absorption) cell processing and iron recovery, iron store), hepatocytes (iron store) and enterocytes (iron absorption) It binds to cell-surface ferroportin, triggering tyrosine phosphorylation and ubiquitin-mediated degradation in lysosomes; iron transport to circulating transferrin is reduced It binds to cell-surface ferroportin, triggering tyrosine phosphorylation and ubiquitin-mediated degradation in lysosomes; iron transport to circulating transferrin is reduced Hepcidin synthesis is induced independently by increasing storage iron, and inflammation (IL 6)Hepcidin synthesis is induced independently by increasing storage iron, and inflammation (IL 6) Hepcidin synthesis is suppressed by iron deficiency, anemia, hypoxemia and accelerated ineffective eythropoiesisHepcidin synthesis is suppressed by iron deficiency, anemia, hypoxemia and accelerated ineffective eythropoiesis

FPN Plasma Fe-Tf Spleen Liver Duodenum FPN Bone marrow Erythrocytes Tf R HEPCIDIN FPN: Ferroportin Tf: Transferrin TfR: Transferrin receptor Iron transport TfR

Cellular iron uptake Plasma iron bound to transferrin is delivered to all cells where transferrin binds to specific transferrin receptors and then transports the iron into the cell; the apotransferrin returns to the cell surface to be released back into the plasma and reutilizedPlasma iron bound to transferrin is delivered to all cells where transferrin binds to specific transferrin receptors and then transports the iron into the cell; the apotransferrin returns to the cell surface to be released back into the plasma and reutilized The rate of iron uptake is again regulated at the cellular level; the number of transferrin receptors on the cell surface is determined by the individual cell’s requirementsThe rate of iron uptake is again regulated at the cellular level; the number of transferrin receptors on the cell surface is determined by the individual cell’s requirements

- Tf - Fe TfR Tf-Fe-TfR Tf-TfR Tf Fe IRP Functional iron Ferritin IRP-Fe Iron sufficient cell

- Tf - Fe TfR Tf-Fe-TfR Tf-TfR Tf Fe IRP Functional iron Ferritin IRP IRP-IRE Iron deficient cell

Absorption, transfer across the placenta and uptake by the nervous system are more complicated processes.

Iron absorption Fe Heme DMT 1 – DCytb ? Ferritin Ferroportin Hephaestin Transferrin IRP-Fe IRP-IRE Duodenal enterocyteLumen Hepcidin Heme oxygenase

Systemic disorders of iron metabolism (balance and distribution) With the exception of iron deficiency (most often nutrtional or due to chronic blood loss), the common disorders of iron metabolism are all linked to hepcidin or ferroportinWith the exception of iron deficiency (most often nutrtional or due to chronic blood loss), the common disorders of iron metabolism are all linked to hepcidin or ferroportin Hepcidin “insufficiency” and ferrportin abnormalities cause iron Hepcidin “insufficiency” and ferrportin abnormalities cause iron overload: overload: Primary: HFE hemochromatosis Hemochromatosis types 2 and 3 Hemochromatosis types 2 and 3 Ferroportin disease Ferroportin disease Secondary: Iron overload associated with hemolysis, thalassemias Secondary: Iron overload associated with hemolysis, thalassemias Hepcidin “excess”Hepcidin “excess” Anemia of inflammation (anemia of chronic disease)

Evaluation of body iron status Iron overloadIron overload Serum iron concentration, percentage transferrin saturation Serum ferritin concentration Bone marrow iron Computed tomography (CT scan) Magnetic resonance imaging (MRI) Liver biopsy Magnetic susceptometry employing the superconducting quantum interference device (SQUID) quantum interference device (SQUID) Iron deficiencyIron deficiency Most of the laboratory tests are related to red blood cell production

Iron deficiency indicators  Storage iron depletion Stainable bone marrow iron Serum ferritin concentration Increased total iron binding capacity (TIBC)  Early functional iron deficiency (Iron deficient erythropoiesis) Reduced serum iron, transferrin saturation, raisedTIBC Increased serum transferrin receptor level Increased zinc protoporphyrin level Reticulocyte hemoglobin, proportion of hypochromic red cells  Established functional iron deficiency (Iron deficiency anemia) Hemoglobin, hematocrit, erythrocyte count Red cell indices (MCV, MCH, MCHC, RDW) Red cell morphology (hypochromia, microcytosis, anisocytosis) Red cell morphology (hypochromia, microcytosis, anisocytosis)

HgbMCVSTfRSFZPPBMIIron storeErythron iron NNNNNN TFS N Normal iron status Storage iron depletion Early functional iron deficiency Established functional iron deficiency NNNNN NN

Calculated iron store Transferrin receptor / ferritin ratioTransferrin receptor / ferritin ratio  Provides a measure of iron status over the full spectrum from iron overload to iron deficiency spectrum from iron overload to iron deficiency  Other biomarkers such as hemoglobin are not required to establish the severity of iron deficiency  Standardization of the transferrin receptor assay is pending

Unresolved issues Serum ferritin is the most commonly employed indicator of the size of iron storesSerum ferritin is the most commonly employed indicator of the size of iron stores 1 ug/L serum ferritin = ~ 8 mg storage iron in normal adults and those with uncomplicated iron deficiency or iron overload Serum ferritin <12 ug/L is specific for iron deficiency (absent iron stores) Serum ferritin values in the normal and above normal ranges are a measure of the size of iron stores, but are also affected (increased) by inflammationSerum ferritin values in the normal and above normal ranges are a measure of the size of iron stores, but are also affected (increased) by inflammation There is no satisfactory method for correcting serum ferritin values for the effect of inflammationThere is no satisfactory method for correcting serum ferritin values for the effect of inflammation In field studies C-reactive protein is often measured concurrently and samples with high values excluded from the serum ferrtin analysisIn field studies C-reactive protein is often measured concurrently and samples with high values excluded from the serum ferrtin analysis

Unresolved issues (cont) Serum ferritin values are:  Higher in diabetics  Directly correlated with markers of insulin resistance  Directly correlated with body mass index

Unresolved issues (cont) Indicators of functional iron deficiency are related to erythropoiesis  It is possible that the supply to other tissues may be suboptimal despite an adequate supply to the bone marrow or in situations in which bone marrow demand is high  All tissues may not compete equally for available iron  Physical performance may be reduced by iron deficiency in the absence of anemia  Increased erythropoieses due to hypoxia is associated with down- regulation of myoglobin in skeletal muscle in short term studies (Robach et al., Blood, 109:4724, 2007)  Mental, motor and emotional development of infants may be impaired in the presence of iron deficiency without anemia