2. Iron Metabolism and Storage Ahmad Sh. Silmi Staff Specialist in Haematology Medical Tech Dept, IUG 2009

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

4. Iron Element (Fe) Molecular weight 56 Abundance May be 2+ or 3+ –Ferrous (2+) “reduced” - gained an electron –Ferric (3+) “oxidised” - lost an electron Fe +++ + e -  Fe ++ Redox states allows activity passing electrons around body Redox change required for iron metabolism

5. Iron Biochemistry Fe 2+ ↔ Fe 3+ + e - Important capacity to donate (reduction) and accept (oxidation) electrons. Free intracellular Fe is dangerous, as it can catalyse production of free radicals which can then damage lipids, proteins and DNA. –Manifestations of acute toxicity (eg paediatric ingestion: Mucosal cell necrosis, altered capillary permeability, uncoupling of oxidative phosphorylation) Thus it must be bound/ carried by various proteins.

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

7. Iron Toxicity Iron can damage tissues Catalyzes the conversion of hydrogen peroxide to free-radical ions Free-radicals can attack: –cellular membranes –Proteins –DNA Iron excess possibly related to cancers, cardiac toxicity and other factors

8. 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”

9. 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%

10. Iron Distribution

11. Daily iron requirements 1.Iron is a one way element 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. 2- absorption is increased in iron deficiency and decreased when body iron stores are deleted.

12. Daily iron losses and requirements (WHO 2001)

13. Iron Storage Forms 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. Typically, 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. This is called hemosiderin; it is physiologically available.

14. 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 Ferritin Storage Molecule

15. Iron Storage Forms haemosiderin : it's not a single substance but a variety of different, amorphous, iron- protein complexes. Typically 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

16. Iron Binding Proteins Transferrin (Tf): –Long arm chromosome 3; –Single chain glycoprotein; 80kDa, hepatic synthesis. –Able to bind 2 Fe 3+ 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)

17. 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.

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

19. 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)

20. Coordinate regulation of expression of ferritin and transferrin receptor the role of the iron response element (IRE)–IRP mechanism

21. 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.

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

23. Iron Absorption 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 Only 10% of dietary iron absorbed Dietary iron usually in excess –either not absorbed, or kept in enterocytes and shed into the gut

24. Iron Absorption

25. Oral Iron intake Non Haem: –Cereals, legumes –10% bioavailability –Absorption enhanced by ascorbic acid (maintains Fe 2+ ). –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.

26. 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

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

28. Molecular pathways of iron absorption

29. 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.

30. Regulation of Iron Balance Crypt Hypothesis Hepcidin

31. 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.

32. 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. “Childhood” – Basolateral TfR– HFE complex facilitates plasma iron uptake. “Adulthood” – expresses various proteins of iron absorption (DMT1, Ferroportin, Haphaestin).

33. The Crypt Hypothesis Crypt Cells: –HFE protein MHC Class I like protein. Located on the basolateral aspect of enterocyte precursor cells, as well as hepatic macrophages. Competes with TfR for Tf binding, reducing its affinity for Tf. May facilitate Fe into the cell and contribute to labile iron pool, in turn perhaps influencing IRP/ IRE binding and transcription of absorptive machinery for the future adult cell. The absorptive set point may thus be established in the crypt, based on the amount of circulating transferrin. Recent advances have thrown doubt on this theory as the key regulatory step of iron absorption.

34. Molecular pathways of iron absorption

35. Import Luminal (absorptive) side: –DMT1 (aka Nramp2): Absorbs Non-Haem Fe 2+ Fe 3+ must be reduced by DcytB (duodenal cytochrome b1, located in brush border) to Fe 2+. Also transports other metals eg lead, zinc, copper, coupled with protons. Concentrated towards apex of villus. Downregulated during crypt progression in Fe overload, upregulated in Fe deficiency. –Haem receptors (apical haem receptor 1) Accept and absorb Haem iron Released intracellularly by Haem oxygenase

36. Export Basolateral Membrane: –Ferroportin 1 (aka Ireg1, MTP1) Single chain glycoprotein multiple membrane spanning receptor. Present only in mature enterocytes, not crypts. Also liver Kupffer cells: role in scavenging iron from RBC. Via IRP/ IRE system, upregulated by amount of available iron. –Haphaestin: Transmembrane bound ferric oxidase, converting Fe 2+ to Fe 3+ for loading to Tf. Creates a concentration/ electrochemical gradient of Fe 2+ across the basolateral membrane. May have transporter function.

37. Regulation of Iron Balance Crypt Hypothesis Hepcidin

38. 25 aa peptide 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

39. 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).

40. Copyright ©2006 American Society of Hematology. Copyright restrictions may apply. Hematology 2006;2006:505-516 Figure 1. The effect of the hepcidin-ferroportin interaction on cellular iron export

41. Hepcidin In mice, a single 50mcg dose results in 80% drop in serum iron within 1hr followed by delayed recovery. –Consistent with rapid loss of FP1 from macrophage followed by slow re-synthesis. –Recall that Tf compartment contains 3mg iron, but 20mg of iron flows through each day, largely generated by senescent red cells, recycled via macrophages. –Thus, serum iron levels can drop rapidly upon hepcidin induction.

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

43. Hepcidin regulation by anaemia Evidence that erythropoietic activity is the most potent suppressor of hepcidin synthesis, although specific mechanism unclear. –May be the bone marrow response to erythropoeisis. Drug induced anaemia stimulates hepcidin, but this was ammeliorated if marrow was irradiated preventing erythropoiesis. Suppression of hepcidin by phlebotomy was reversed if erythropoeitic response shut off by chemotherapy. Patients with thalassaemia intermedia develop iron overload even if never transfused, and have very low urinary hepcidin levels, despite systemic iron overload.

44. 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, BMP2,4,9 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

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

46. 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)

47. 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 –I n crease: high oestrogen states (pregnancy, OCP) –Decrease: malnutrition, chronic liver disease, chronic disease (eg malignancy), protein-losing states, congenital deficiency, neonates, acute phase (negative reactant).

49. 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

50. 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

51. 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

52. 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.

53. 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

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

56. Differential diagnosis of hypochromic anaemia.

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

59. 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

60. 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.

61. 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

62. 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

63. 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.

65. IRON DEFICIENCY ANEMIA

66. 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

68. IRON DEFICIENCY ANEMIA Prevalence

69. Iron Deficiency Laboratory changes: –Low iron (poor specificity) –Low ferritin (excellent specificity) –Elevated Transferrin (TIBC) –Low transferrin saturation –Hypochromia, microcytosis –Anaemia Stages –Reduced iron stores –Iron deficient erythropoiesis –Iron deficient anaemia

70. 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)

73. General effects of iron deficiency anaemia Koilonychia- Flattening or spoon of the nail. Angular stomatitis- atrophic lessions at the corner of the mouth. Glossitis- smoothed, inflamed tongue. Atrophic gastritis- inflammation of the lining of the stomach Achlorhydria- difficulty in swallowing Pica: Soil-geophagia & Ice- pagophagia

74. Koilonychia

76. BLOOD AND BONE MARROW SMEAR BLOOD: –microcytosis, hipochromia, anulocytes, anisocytosis poikilocytosis BONE MARROW –high cellularity –mild to moderate erythroid hyperplasia (25-35%; N 16 – 18%) –polychromatic and pyknotic cytoplasm of erythroblasts is vacuolated and irregular in outline (micronormoblastic erythropoiesis) –absence of stainable iron

77. IRON REPLACEMENT THERAPY Response Usually oral; usually 300-900 mg/day Requires acid environment for absorption Poorly absorbed

78. 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

79. ANEMIA OF CHRONIC DISEASE Findings Mild, non-progressive anemia (Hgb 10, Hct 30%) Other counts normal Normochromic/normocytic (30% hypochromic/microcytic) Mild aniso- & poikilocytosis Some what shortened RBC survival Normal reticulocyte count (Inappropriately low for anemia) Normal bilirubin EPO levels increased.

80. Anaemia of chronic disease Infection, inflammation, malignancy Low iron absorption Low serum iron Stainable iron stores in RE cells Hepcidin is an acute phase protein Increased hepcidin –blocks iron in gut cells –Traps iron in macrophages and liver cells Produces a functional iron deficiency –Not responsive to iron therapy

81. Anaemia of chronic disease Hard to separate from iron deficiency anaemia May co-exist Ferritin: low with pure iron deficiency but increased with acute phase response Iron: low in both conditions Transferrin: high in pure iron deficiency but decreased with acute phase response

82. IRON DEFICIENCY versus ACD Serum IronTransferrinFerritin Iron Deficiency ACD

83. H E P CIDIN I (not available in clinical practice)

84. Pathophysiology Chronic inflammation causes activation to macrophages and upregulation of surface apotransferrin receptors. Binding of significant quantities of apotransferrin to macrophage reduces TIBC. Inflammation also stimulates neutrophils to synthesis and release large quantities of apolactoferrin, which acts as iron binding protein. The apolactoferrin is bound to specific receptors on the activated macrophages and acts like a magnet for the circulating iron. Any iron that is bound to the apolactoferrin:receptor complex is internalized by the macrophage and stored as ferritin. Thus increasing tissue iron stores. Erythropoietic activity of the BM is suppressed in ACD. This most likely to be caused by the release of growth inhibitors such as IL-1, γ-interferon and tumor necrosis factor in response to the primary condition. RBC life span is also reduced.

86. Sideroblastic Anaemia The sideroblastic anaemias are heterogeneous group of disorders, which are characterized by disordered incorporation of iron within the haem in developing erythroblasts. The resulting toxic accumulation of iron in the mitochondria of erythroblast leads to the formation of iron encircling the nuclei (ringed sideroblast) and ineffective erythropoiesis ensues. However, ringed sideroblasts are not specific indicators of sideroblastic anaemia: they are frequently found in leukaemia, megaloblastic anaemia and alcoholism. The sideroblastic anaemias are classified according to their aetiology as: Hereditary Secondary or idiopathic

87. Sideroblastic anaemia. Erythroblasts showing perinuclear rings of iron (Perls’ stain).

88. Hereditary Sideroblastic Anaemia They are X - linked inherited diseases, which are mostly characteri z ed by functional deficiencies of enzymes of the haem synthetic pathway, most commonly δ - aminolaevulinic acid (δ -ALA) synthetase or ferrochelatase. Affected male have hypochromic, dimorphic anaemia with mild ineffective erythropoiesis and erythroid hyperplasia.

89. Secondary Sideroblastic Anaemia Drug-induced Siderblastic Anaemia: The most common cause of this condition is the administration of drugs such as Chloramphenicol and alcohol. These drugs inhibit the synthesis of δ A L A synthetase and ferrochelatase. The blood picture is the same as in hereditary sideroblastic anaemia.

90. Lead Poisoning Chronic lead poisoning was a relatively common condition when most drinking water was supplied via lead pipes. Lead is absorbed by inhalation or ingestion. Most absorbed lead accumulates in bone & bone marrow. In bone marrow, lead is associated with red cell precursors and more specifically with mitochondrial membranes and disrupts haem synthesis. This leads to sever sideroblastic changes. Lead also cause damages to red cell membrane and inhibits glycolytic activity. These two activities result in mild haemolysis which contributes to anaemia of chronic lead poisoning.

91. Investigation of lead poisoning Blood lead levels. The free erythrocyte protoporphyrin. (Lead particularly affects the enzymes involved in haem synthesis; thus a screening test for early lead poisoning is the measurement of haem precursors). An abdominal radiograph may show radio-opaque lead fragments in the gastrointestinal tract. Also lead lines may be seen on examination of a radiograph of bony structures because lead interferes with the growing ends of bones.

92. Genetic haemochromatosis Iron overload disease Caused by increased iron absorption Known since 1700s May affect liver, pancreas, skin, heart, joints, endocrine organs (bronze diabetes) Gradual accumulation of iron over the life of the person (positive iron balance) –Iron overload detectable in teens and 20s –Organ overload in 30s –Organ damage in 40s and 50s Cirrhosis and liver disease main cause of increased mortality

93. Genetic Haemochromatosis > 95 % defect in HFE gene (C282Y) Associated with low hepcidin Leads to overactivity of ferroportin –Increased gut absorption of iron Also other mechanisms –Increased DMT1 and DcytB activity –Not related to hepcidin Limited penetrance (1 – 50%) –May require other genes to be involved

94. Genetic Haemochromatosis HFE-Related Type 1 – HFE defects Non HFE Related Type 2a – Haemojuvelin defects Type 2b – Hepcidin defects Type 3 – Transferrin receptor defects Type 4 – Ferroportin defects

95. Other tests related to iron status Haemoglobin –Low with iron deficiency, anaemia of chronic disease Mean Cell Volume –Low with iron deficiency, thallassaemia Liver iron –High with iron overload –Better marker for GH when corrected for age –(Hepatic iron index) Bone marrow iron –Low with iron deficiency

96. Future possibilities Treatment with hepcidin for iron overload Blocking of hepcidin for anaemia of chronic disease Diagnostic tests based on hepcidin

97. Conclusions Iron related diseases are common and clinically important Recent advances have changed our understanding Groups of tests “Iron studies” are the best first line investigation New tests and therapies will follow the new understandings.

98. Reading Andrews NC. Medical Progress: disorders of iron metabolism. NEJM 1999;341:1986-95 Pietrangelo A. haemochromatosis – a new look at an old disease. NEJM 2004;350:2383- 97. Weiss G, Goodnough LT. Medical Progress: Anaemia of chronic disease. NEJM 2005;352:1011-1023 Fleming RE, Bacon BR. Orchestration of iron homeostasis. NEJM 2005;352:1741-4