Iron Metabolism and Storage Ahmad Sh. Silmi Staff Specialist in Haematology Medical Tech Dept, IUG 2011

Iron in Man Biochemistry Recent advances in understanding of iron metabolism Role in disease

Iron Element (Fe) Molecular weight 56 Abundance May be 2+ or 3+ Ferrous (2+) “reduced” - gained an electron Ferric (3+) “oxidized” - lost an electron Fe+++ + e-  Fe++

Iron Biochemistry Fe2+ ↔ Fe3+ + e- Important capacity to donate (reduction) and accept (oxidation) electrons. Redox states allows activity passing electrons around body Redox change required for iron metabolism

Iron functions Oxygen carriers Oxygen storage Energy Production Other haemoglobin Oxygen storage Myoglobin Energy Production Cytochromes (oxidative phosphorylation) Krebs cycle enzymes Other Liver detoxification (cytochrome p450) An essential element

Iron Toxicity Thus it must be bound/ carried by various proteins Iron can damage tissues Catalyzes the conversion of hydrogen peroxide to free-radical ions Free-radicals can attack: cellular membranes Proteins DNA Thus it must be bound/ carried by various proteins Iron excess possibly related to cancers, cardiac toxicity and other factors

Principle Bodies require the right amount of substance Too much or too little of any required substance may be detrimental “There is no substance, which taken in sufficient excess, is not toxic to the body”

Iron Distribution 35 – 45 mg / kg iron in adult male body Total approx 4 g Red cell mass as haemoglobin - 50% Muscles as myoglobin – 7% Storage as ferritin - 30% Bone marrow (7%) Reticulo-endothelial cells (7%) Liver (25%) Other Haem proteins - 5% Cytochromes, myoglobin, others In Serum - 0.1%

Iron Distribution

Daily iron requirements Iron is a one way element 2- absorption is increased in iron deficiency and decreased when body iron stores are exceeded. 3- daily iron requirement = amount lost + amount required 4- Increased requirement is found : A- menstruating female / 30-60 ml of blood in each cycle. This contains between 15-30 mg iron/cycle B- pregnancy (1) Foetal/placental growth requirement.      (2) Expansion in maternal mother blood volume.      (3) Haemorrhage in delivery involve highly significant loss of iron.

Daily iron losses and requirements (WHO 2001)

Iron Storage Forms 1st ferritin : MW 45000. consist of  24 polypeptide sub-unit cluster together to form hollow sphere of 5 nm in diameter.  The stored iron form the central core of the sphere. Iron store in the liver and nearly all other cells. ferritin contains about 25% of iron by weight. About 2/3 of body iron stores are present as ferritin. If the capacity for storage of iron in ferritin is exceeded, a complex of iron with phosphate and hydroxide forms (hemosiderin).

Ferritin Storage Molecule Ferritin molecules store thousands of iron atoms within their mineral core. When excess dietary iron is absorbed, the body responds by producing more ferritin to facilitate iron storage

Iron Storage Forms 2nd haemosiderin : it's not a single substance but a variety of different, amorphous, iron- protein complexes. it contains about 37% of iron by weight. Haemosiderin may represent ferritin in various form of degradation.  As the body burden of iron increases beyond normal levels, excess hemosiderin is deposited in the liver and heart. This can reach the point that the function of these organs is impaired, and death.

Iron Binding Proteins Transferrin (Tf): Long arm chromosome 3; Single chain glycoprotein; 80kDa, hepatic synthesis. Able to bind 2 Fe3+ molecules with very high affinity at pH7.4, but reduced affinity in acidic conditions. Transports iron through plasma. 3mg of total body iron. Transferrin Receptor (TfR): Also located on 3q. Transmembrane glycoprotein dimer with two transferrin binding sites. Found on most cells (esp erythroid precursors, hepatocytes, placental cells)

Transferrin-TfR interactions Each TfR can bind two Tf molecules, which are endocytosed through clathrin coated pits. A proton pump generates acidity in the endosome, facilitating release of Fe from Tf. DMT-1 transporter exports Fe from endosome.

Incorporation of iron from plasma transferrin into haemoglobin in developing red cells. Uptake of transferrin iron is by receptor-mediated endocytosis.

Iron Absorption Regulation of iron stores occurs at the level of absorption. There is no capacity to increase iron excretion.

Iron Absorption The average western diet contain 10-15 mg of iron daily. Only 5-10% is absorbed. 1 – 2 mg iron are absorbed each day (in iron balance 1 – 2 mg iron leaves the body each day) Occurs in the duodenum Taken up as ionic iron or haem iron

Iron Absorption

Iron Absorption The main dietary sources are liver, red meat, green vegetables, spinach, supplemented cereals and fish. Dietary iron usually in excess either not absorbed, or kept in enterocytes and shed into the gut Dietary iron falls into one of two categories: Haem & non-haem.

Oral Iron intake Non Haem: Haem: Cereals, legumes 10% bioavailability Absorption enhanced by ascorbic acid (maintains Fe2+). Inhibited by tanins, phytates (chappatis). Haem: Meat, fish 30% bioavailability Iron released from complexes by acid, proteases Binds to mucin and travels to small bowel.

Molecular pathways of iron absorption

Haem iron absorption Haem split from globin in intestine Absorbed into enterocyte as haem Iron freed into enterocyte pool or absorbed intact Accounts for over half of iron in western diet but much less in other diets Not well understood

Iron Absorption DcytB DMT1 Ferritin Hephaestin Ferroportin Reduction Fe+++ to Fe++ DMT1 Transport into cell Ferritin Storage in cell Hephaestin Oxidises Fe++ to Fe+++ Ferroportin Transport out

Cellular Control of Iron Iron Responsive Elements (IRE): Loop configuration of nucleotides located in the 5’ or 3’ ends of mRNA coding for ferritin, TfR, DMT1, others. Iron Regulatory Proteins (IRP): Serve as a sensor of cell iron Modulate the synthesis of iron regulatory proteins by binding to the IREs. Contain an iron-sulphur cluster: low affinity for IRE when iron abundant, but higher affinity when iron absent. Binding to 5’ end reduces translation (eg for ferritin) Binding to 3’ end protects mRNA and increases translation (eg for TfR)

Cellular control of Iron In the presence of increased iron: IRP detaches from ferritin mRNA allowing more ferritin to be synthetised. IRP detaches from TfR, reducing synthesis. Effect is to reduce influx of iron into cell and facilitate storage.

FERRITIN/TRANSFERRIN REGULATION

Regulation of Iron Balance Crypt Hypothesis Hepcidin

Duodenal Iron Absorption – ‘The Crypt Hypothesis’ Precursor cells proliferate in the crypt. As they mature and differentiate, they migrate up the villus. Their apical membrane develops microvilli and absorptive transport enzymes.

The ‘Crypt Hypothesis’ Precursor cells in the crypts detect the serum iron concentration. This establishes the ‘set point’ iron absorptive capacity of that cell as it differentiates into a mature enterocyte.

Control of iron absorption mucosal block theory

Hepcidin 25 aa peptide , synthesised in the liver. Identified 2000 Antimicrobial activity Hepatic bacteriocidal protein Master iron regulatory hormone Inactivates ferroportin Stops iron getting out of gut cells Iron lost in stool when gut cells shed Leads to decreased gut iron absorption

Hepcidin 25 amino acid peptide, synthesised in the liver. Function: Binds to ferroportin and induces its internalisation and lysosomal degradation. Removal of ferroportin prevents iron efflux from enterocyte to plasma: iron is lost from body when cell is shed after 1-2 days. Ferroportin enables iron export from reticuloendothelial/ hepatic macrophages, thus hepcidin prevents transport of recycled iron to plasma. Likely that rising iron levels also secondarily influence IRE/IRP system and processing of iron protein mRNA. In Hepcidin deficient mice, DMT1 and dcytb1 were significantly increased (?primary or secondary effect).

Hepcidin 25 amino acid peptide, synthesised in the liver. Function: Binds to ferroportin and induces its internalisation and lysosomal degradation. Removal of ferroportin prevents iron efflux from enterocyte to plasma: iron is lost from body when cell is shed after 1-2 days. Ferroportin enables iron export from reticuloendothelial/ hepatic macrophages, thus hepcidin prevents transport of recycled iron to plasma. Likely that rising iron levels also secondarily influence IRE/IRP system and processing of iron protein mRNA. In Hepcidin deficient mice, DMT1 and dcytb1 were significantly increased (?primary or secondary effect).

Interplay of Key Proteins in Iron Homeostasis Interplay of Key Proteins in Iron Homeostasis. In the duodenal enterocyte, dietary iron is reduced to the ferrous state by duodenal ferric reductase (Dcytb), transported into the cell by divalent metal transporter 1 (DMT1), and released by way of ferroportin into the circulation. Hephaestin facilitates enterocyte iron release. Hepatocytes take up iron from the circulation either as free iron or transferrin-bound iron (through transferrin receptor 1 and transferrin receptor 2). Transferrin receptor 2 may serve as a sensor of circulating transferrin-bound iron, thereby influencing expression of the iron regulatory hormone hepcidin. The hepcidin response is also modulated by HFE and hemojuvelin. Hepcidin is secreted into the circulation, where it down-regulates the ferroportin-mediated release of iron from enterocytes, macrophages, and hepatocytes (dashed red lines). Fleming, R. E. et al. N Engl J Med 2005;352:1741-1744

The effect of the hepcidin-ferroportin interaction on cellular iron export The effect of the hepcidin-ferroportin interaction on cellular iron export. When hepcidin concentrations are low, cellular iron is exported into plasma through membrane-associated ferroportin (Fpn). When hepcidin concentrations are high, hepcidin binds to ferroportin, and ferroportin is internalized and degraded. As a consequence of the loss of ferroportin, cellular iron export decreases and iron accumulates in cytoplasmic ferritin. Hematology 2006;2006:505-516 Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.

IRON FLOWS ARE REGULATED BY THE HEPCIDIN/FERROPORTIN INTERACTION x x x

IRON FLOWS ARE REGULATED BY THE HEPCIDIN/FERROPORTIN INTERACTION x x x Plasma Fe

Hepcidin In mice, a single 50mcg dose results in 80% drop in serum iron within 1hr followed by delayed recovery. Thus, serum iron levels can drop rapidly upon hepcidin induction.

Regulation of Hepcidin Evidence of regulation of synthesis by: Anaemia/ Hypoxia Inflammation Iron Precise mechanisms of regulation remain unclear.

REGULATION OF HEPCIDIN PRODUCTION ANEMIA HYPOXIA INFLAMMATION IRON hepcidin

Hepcidin regulation by anaemia Evidence that erythropoietic activity is the most potent suppressor of hepcidin synthesis, although specific mechanism unclear.

REGULATION OF HEPCIDIN BY ANEMIA Pak M, Blood 2006

Hepicidin regulation by inflammation IL-6 is a potent inducer of hepcidin synthesis during acute inflammation. Thus hepcidin is an acute phase protein. In mice, IL-1, TGFB have been shown to regulate hepcidin (?in humans). Lowered serum iron is an acute host defence. Hepcidin itself may have some antimicrobial activity (probably not at physiological levels). Mediates anaemia of chronic disease

Hepcidin regulation by iron Iron loading increases Hepcidin synthesis. Molecular details unclear. Hepcidin mRNA lacks IRE.

HEPCIDIN PRODUCTION IS REGULATED BY AN IRON SIGNAL

HEPCIDIN REGULATION INFLAMMATION IRON SIGNAL ERYHTROPOIETIC SIGNAL

SUMMARY Hepcidin is an iron-regulatory hormone that maintains plasma iron levels and iron stores within normal range Hepcidin regulates the entry of iron into plasma from duodenal enterocytes, from macrophages (and from hepatocytes) Hepcidin acts by binding the receptor/iron channel ferroportin and causing its degradation Hepcidin is regulated by iron, erythropoiesis and inflammation Excess hepcidin causes the hypoferremia and anemia of inflammation Hepcidin deficiency, or resistance to hepcidin, cause hemochromatosis

Principles For any metabolic process there is a pathway (which is usually complex). For any pathway there will be a regulatory process (which may also be complex). Often diseases are due to changes in the regulation of a pathway, not due to defects in the pathway itself.

Iron Loss Physiological Pathological Cell loss: gut, desquamation Menstruation (1mg/day) Pregnancy, lactation Pathological Bleeding Gut, menorrhagia, surgery, gross haematuria

Iron Loss An unregulated process No mechanisms to up- or down-regulate iron loss from the body Over-intake cannot be matched by increased loss Under intake cannot be matched by decreased loss Thus iron homeostasis is regulated by adjusting iron intake

Iron re-use Old cells broken down in macrophages in spleen and other organs Iron transported to liver and other storage sites Red cell iron recovered from old red cells Very little iron lost in routine metabolism

Iron Scavenging Intravascular haemolysis Breakdown of red cells in the circulation Free haemoglobin binds haptoglobins -> taken up by liver Free haem binds haemopexin -> taken up by liver Haem passing through kidney resorbed Three mechanisms to conserve iron in pathological situations Historically iron deficiency is the disease we have evolved to avoid.

The liver and iron metabolism Hepcidin production by the liver controls gut iron absorption and therefore body iron stores HFE and haemojuvelin involved in hepcidin regulation

Tests of body iron burden Principle Interpretation of a “blood test” requires knowledge of all factors which affect concentration Includes Disease of interest (signal) Other conditions (noise)

Transferrin Testing A routine blood test used for iron status Also known as TIBC (total iron binding capacity) High : Low body iron stores. Low : High body iron stores. Other conditions Increase: high oestrogen states (pregnancy, OCP) Decrease: malnutrition, chronic liver disease, chronic disease (eg malignancy), protein-losing states, congenital deficiency, neonates, acute phase (negative reactant).

Transferrin Receptors Collects iron from transferrin for uptake into cells Recognises and binds transferrin Receptor + transferrin endocytosed Iron released into cell via Iron transporter (DMT1) Receptor + transferrin return to cell surface Transferrin released

Soluble Transferrin Receptors Truncated form of cell surface receptors Found in the circulation High levels with iron deficiency Low levels with iron overload Possible role in diagnosis of iron deficiency compared in setting of inflammation Not currently routinely available

Serum Iron The serum contains about 0.1% of body iron Over 95% of iron in serum bound to transferrin Serum iron is a routine blood test Measures all serum iron (not in red cells) Of limited use on its own Useful for interpretation of iron status only if grossly abnormal – eg iron poisoning Commonly combined with serum transferrin to express transferrin saturation

Serum Iron Measurement Serum iron is a routine blood test Low levels: Iron deficiency Other: Random variation; acute or chronic inflammation; pre-menstrual. High levels: Iron Overload Other: Random variation, OCP, pregnancy, recent iron ingestion.

Transferrin Saturation Percent of transferrin (TIBC) iron-binding sites which are filled with iron Combines two factors to improve sensitivity Iron overload High iron plus low transferrin High saturation (50 – 100%) Best serum marker of increased body iron Used as a screen for iron overload

Transferrin Saturation NORMAL IRON STATUS Normal iron Normal transferrin Saturation 40% IRON OVERLOAD High iron Low transferrin Saturation 80% Iron Transferrin

Differential diagnosis of hypochromic anaemia.

Principle In homeostasis - intake of any element equals loss of any element nitrogen, water, salt, iron In “steady state” intake must balance loss. Even slight imbalances over time can create excesses or deficiencies. 1% excess per day doubles content 70 days.

IRON DEFICIENCY ANEMIA

Iron Deficiency Extremely common Due to reduced intake, increased loss or increased demands Stores reduced before deficiency seen Iron deficiency is not a diagnosis A cause needs to be identified! Eg obstetric causes, low intake, malabsorption, bowel cancer, haemorrhoids, inflammatory bowel disease

IRON DEFICIENCY ANEMIA Prevalence

IRON DEFICENCY - STAGES Prelatent reduction in iron stores without reduced serum iron levels Hb (N), MCV (N), iron absorption (), transferin saturation (N), serum ferritin (), marrow iron () Latent iron stores are exhausted, but the blood hemoglobin level remains normal Hb (N), MCV (N), TIBC (), serum ferritin (), transferrin saturation (), marrow iron (absent) Iron deficiency anemia blood hemoglobin concentration falls below the lower limit of normal Hb (), MCV (), TIBC (), serum ferritin (), transferrin saturation (), marrow iron (absent)

Symptoms GLOSSITIS, STOMATITIS DYSPHAGIA (esophageal web) ATROPHIC GASTRITIS DRY, PALE SKIN SPOON SHAPED NAILS: KOILONYCHIA BLUE SCLERAE HAIR LOSS PICA (APETITE FOR NON FOOD SUBSTANCES SUCH AS AN ICE, CLAY) SPLENOMEGALY (10%)

Koilonychia

STOMATITIS

Glossitis

IDA Laboratory Findings: Microcytic/hypochromic anemia Low Hb, HCT, and RCC MCV = 55-74 fl MCHC = 22-31 g/dl MCH = 14-26 pg Hb < 10 g/dL Anisocytosis: Increased RDW Poikilocytosis: elliptocytes, target cells, pencil cells Anulocytes RPI <2.0 You may see thrombocytosis esp. when the cause is due to bleeding. BM stainable iron: ABSENT High TfR High Transferrin Low serum ferritin Low Serum iron High TIBC Low Transferrin saturation% High FEP

IDA: the CBC Shift to left

Blood & Bone Marrow BLOOD Film: Microcytosis, Hypochromia, Anulocytes, Anisocytosis, Poikilocytosis BONE MARROW Film: High cellularity. Mild to moderate erythroid hyperplasia (25-35%; N: 16 – 18%) Cytoplasm of polychromatic and pyknotic normoblasts is scanty (indicating its immaturity), vacuolated and irregular in outline. This type of erythropoiesis has been described as micronormoblastic (micronormoblastic erythropoiesis) Staining the BM aspirates for iron (hemosiderin) gives indicate that normoblast iron are absent. i.e. Absence of hemosiderin stainable iron. Usually BM iron is scored from 0 to +4 (0, +1, +2, +3, +4), in IDA it is 0.

IDA blood film: Anisocytosis, Hypochromia, Microcytosis.

IDA Anulocyte

BM in IDA show erythroid hyperplasia, but unfortunately ineffective

Prussian Blue Stain for iron of BM Iron Present No Iron Present

Serum Iron N.R. 75-175 µg/dl Serum iron concentrations shows diurnal rhythm, which is attributed to the change in release of iron from the macrophages which is highest in the morning and lowest in the evening. So we expect that erythropoiesis is active in the morning more than the evening!.

Transferrin Molecule Normal Iron Deficiency Saturation 30% T I B C Iron Deficiency Saturation 10% T I B C Hemochromatosis (iron overload) Saturation 60-75% T I B C NR transferrin levels take up 250-430 µg/dL in the lab

Transferrin vs iron binding capacity Normal Transferrin level= 240-430 mg/dL Total Iron binding capacity (TIBC): amount of iron (as a reagent, excess iron is added by you in the lab) that can be taken up by washed transferrin molecules in patient serum in the laboratory, in µg/dL, normal = 253-435 µg iron/dL Serum iron (Fe)= 75-175 µg/dL Transferrin saturation (TfS)= [Fe/TIBCx100] (%), normal 20-50%, with a mean value of 30%.

Transferrin and TIBC Transferrin = the protein for transport of serum iron; measured in mg/dL, normal = 240- 430 mg/dL Not to be confused with TIBC, measured in µg Fe/dL

%Transferrin saturation (TfS) Percent of transferrin (TIBC) iron-binding sites which are filled with iron. Normally 20-50%, with normal mean value of 30%. Calculated (%TfS)= Serum Iron/TIBC x100 In IDA it is less than 10%. Typical normal TfS is 100 µg/dL Fe ÷ TIBC 300 µg/dL = about 30% IDA shows ↑TIBC ~ 400 µg/dL and ↓Fe ~ 25 µg/dL, TfS≤ 10%

Serum Ferritin Ferritin is a molecule measured in µg/L NR 15-300 µg/L Is an indirect indication of stores of body iron. Acute phase reactant, e.g. increases in hepatitis C and B. (increased in chronic inflammation), causing spurious/erroneous increases.

Free Erythrocyte Protoporphyrin (FEP) FEP - Precursor of Iron binding Elevated in iron def. anemia Highly elevated with lead poisoning. Iron deficiency impaired heme. Synthesis and accumulation of protoporphyrin, which is: heme precursor, in erythrocytes. Measurement of FEP level is a sensitive indicator of iron deficiency.

Microcytic Hypochromic Anemia Serum Ferritin < 15 µg/L 15-300 µg/L > 300µg/L TIBC N or ↓ HIGH - + BM Fe Iron Deficiency Anemia IDA Not IDA, Other Micro Anemia

Mentzer Index Moreover RDW In additionHb-A2 The Mentzer index is used to differentiate iron def. anemia from beta thalassemia minor. Mentzer Index= MCV/RCC. If > 13 then Fe deficiency anemia If < 13 - Hb defect (i.e. thalassemia) Moreover RDW in IDA is inc., But thalassemia minor normal. In additionHb-A2 In IDA= Dec./Normal; but in B thal minor is Increased

Hookworm drinks its host’s blood!

IRON THERAPY Response Initial response takes 7-14 days Modest reticulocytosis (7-10%) Correction of anemia requires 2-3 months 6 months of therapy beyond correction of anemia needed to replete stores, assuming no further loss of blood/iron Parenteral iron possible, but problematic