IRON and it's metabolism and it's disorder.pptx

Introduction
Iron stores
Iron absorption
Mucosal block theory
Iron excretion

1. IRON BY DR. PRIYANKA I YEAR POSTGRADUATE BY DR. PRIYANKA I YEAR POSTGRADUATE

2. INTRODUCTION IRON DISTRIBUTION IN THE BODY FUNCTIONS OF IRON IRON ABSORPTION REGULATION OF IRON STORES IRON TRANSPORT IRON STORAGE EXCRETION OF IRON IRON METABOLISM DISORDERS ANALYTICAL METHODS FOR IRON ESTIMATION

3. INTRODUCTION Iron (Fe) is fourth most abundant element in the earth's crust and the most abundant transition metal Iron is classified as a trace element in the body. Iron ions are able to participate in redox chemistry in both the ferrous [Fe(+2)] and ferric [Fe(+3)] states Free iron ions in the body participate in destructive chemistry, primarily in catalyzing the formation of toxic free radicals

4. IRON DISTRIBUTION IN THE BODY Total body iron is 3 – 5 grams 60 – 70% - Hemoglobin 15 – 30% - stored in liver and reticuloendothelial system 4% - Myoglobin 1% - Iron containing enzymes <0.2% - Blood plasma

5. Fe is mostly locked into Fe protoporphyrin ( heme ) and Fe– sulphur clusters, which serve as enzyme cofactors Iron is availabe in two forms Functional forms Non functional forms

6. Functional forms or Haemoproteins Hemoglobin Myoglobin Cytochromes Catalase Peroxidase Tryptophan pyrrolase Nitric oxide synthase

7. Iron sulphur complex Complex III – Fe-S in mitochondria Xanthine oxidase in Liver Succinate dehydrogenase in liver

8. Non fuctional forms Ferritin Transferrin Haemosiderin Aconitase Phenyl alanine hydroxylase

9. Recommended Daily A llowances Adult men - 10mg/day P ostmeopausal women - 10mg/day Premenopausal women - 15 – 20 mg/day Pregnant women -30 – 60mg/day Children -20 – 30mg/day

10. Dietary sources Majority of iron is received from cereals & pulses Rich sources : Green leafy vegetables, liver, meat Good sources: Jaggery Poor sources: Milk

11. Biochemical functions Synthesis of hemoglobin, myoglobin , cytochromes , catalase and peroxidase Cytochromes and iron sulphur proteins are necessary for electron transport chain and oxidative phosphorylation Peroxidase is required for phagocytosis Hemoglobin - Transport, storage and utilization of oxygen Effective immuno -competence of the body Catalase is involved in free radical scavenging system

12. Iron homeostasis Duodenal enterocytes , hepatocytes , macrophages and erythroid precursors are involved in Iron regulation

13. Absorption of iron Iron is a one way element because it is regulated by absorption and utilisation , not by excretion Iron is absorbed by upper part of duodenum and proximal jejunum

14. Factors favoring iron absorption Reduced form of iron Acids- Hydrochloric acid, Ascorbic acid and citric acid Cysteine , glutathione and –SH groups of proteins

15. Factors reducing iron absorption Phytates Phosphates Oxalates Tannates minerals – calcium, copper, lead and zinc

16. Mucosal block theory Major iron regulation is at the level of absorption When iron stores are depleted, absorption is enhanced When adequate quantity of iron is stored, absorption is decreased

17. 3 phases of iron absorption Luminal phase Mucosal cellular phase Basolateral phase

18. In the luminal phase, Fe is solubilized and converted from Fe3+ into Fe2+ by duodenal cytochrome B During the mucosal phase, Fe is bound to the brush border and transported into the mucosal cell by the Fe transporter divalent metal transporter (DMT1 ) In the cellular phase, Fe is either stored in cellular ferritin or transported directly to the opposite side of the mucosal side.

19. In the basolateral phase of Fe absorption, Fe2+ is released into the portal circulation by the basolateral cellular exporter ferroportin Ferroportin requires hephaestin , a multicopper oxidase homologous to ceruloplasmin , which oxidises Fe2+ to Fe3+ for loading into transferrin This cellular efflux of Fe is inhibited by the peptide hormone hepcidin by binding to ferroportin and subsequent degradation of the ferroportin-hepcidin complex.

20. Regulation of iron release in to bloodstream Iron-response proteins that bind to iron-responsive elements in mRNAs regulate the expression of proteins that are involved in iron homeostasis L iver secretes the peptide hormone hepcidin , which controls iron transport in the body

21. Hepcidin Hepcidin is a 25-amino acid peptide with molecular weight:2789.4 Dalton Subsequent amino-terminal processing of the 25-amino acid form can result in the appearance of smaller hepcidin forms of 24, 23, 22, and 20 amino acids These hepcidin peptides form a hairpin structure, with four intramolecular disulfide bridges

22. Hepcidin inhibits the release of iron into the bloodstream from intestinal epithelial cells, macrophages in the spleen and bone marrow and Kupffer cells in the liver Inflammation and high iron stores in the liver stimulate hepcidin secretion and thus diminish the concentration of iron in the blood In contrast, ineffective erythropoiesis , anemia, and hypoxia minimize hepcidin secretion from the liver and enhance the release of iron from the intestine, spleen or liver into the blood

23. Regulation of Hepcidin Synthesis Four functionally defined hepcidin regulatory pathways are Erythropoiesis Iron status Oxygen (O2) tension and Inflammation.

24. Erythropoiesis Increased erythropoiesis is associated with decreased hepcidin expression by signaling molecules from the marrow include Growth differentiation factor-15 (GDF-15) Twisted gastrulation protein homolog 1 (TWSG1), and Erythroferrone (ERFE).

25. Iron status Increased body iron status increases hepcidin expression through two mechanisms: Circulating iron signal provided by ferri-transferrin and Cellular iron stores signal provided by bone morphogenetic protein 6 (BMP-6)

26. Ferri-transferrin signal acts through transferrin receptors 1 and2 and is modulated by the hemochromatosis protein (HFE) BMP-6 signal acts through its receptor and is modulated by the BMP co-receptor hemojuvelin (HJV) and by neogenin

27. Oxygen tension Decreased O2 tension leads to decreased hepcidin expression by increasing the transcription of two genes i.e. matriptase-2 and furin , which are responsive to hypoxia-inducible factor (HIF) Matriptase-2 cleaves Hemojuvelin from the cell surface, preventing its function as a co-receptor Furin cleaves Hemojuvelin during processing to produce a soluble form that serves as a BMP-6 decoy.

28. Inflammation Infections and other forms of inflammation increase hepcidin expression by the cytokine interleukin (IL)-6

29. Intracellular Iron Regulation The expression of proteins involved in Fe uptake, storage and release of Fe from the cell is determined by the Fe need of the cell and regulated at the posttranscriptional level by the Fe responsive protein and Fe regulatory element (IRP/IRE) network In the cell, the IRP1 and IRP2 interact with the IRE on the mRNA and affect the translation to protein In conditions of high cellular Fe levels, the IRPs are inactive.

30. In contrast, low cellular Fe levels increase IRP activity. IRPs bind to the IRE and inhibit mRNA translation (in the case of ferritin and ferroportin ) or increase mRNA stability (in the case of cellular Fe importers, such as TfR1 and DMT1). The result is that more Fe enters the cell, and less Fe is stored as ferritin or leaves the cell via ferroportin In conditions of low cellular Fe, there is also less synthesis of ALAS-2, the first enzyme in the heme synthesis and less production of hypoxia-inducible factor (HIF)-2α in the kidney fibroblasts, resulting in a decrease in erythropoetin (EPO) production

31. As such, the body decreases the heme and RBC synthesis in case of Fe scarcity, which results in microcytic anemia Key aspects of systemic Fe metabolism are regulated transcriptionally ( hepcidin expression) and post translationally ( ferroportin function by hepcidin ) Intracellular Fe homeostasis is largely controlled by a posttranscriptional mechanism involving the IRP1/IRP2 network

32. Iron transport Iron (Fe2+) entering the mucosal cells by absorption is oxidised to ferric state (Fe3+) by enzyme ferroxidase activity by ceruloplasmin Fe3+ combines with apoferritin to form ferritin which is the temporary storage form of iron Ferric iron binds with specific iron binding protein transferrin or siderophilin Transferrin is a glycoprotein synthesised in liver

33. Iron storage Iron is stored in Liver, spleen & bone marrow in the form of ferritin In the mucosal cells, ferritin is the temporary storage form Ferritin contains 23% of iron Serum ferritin level is elevated in iron overload

34. Ferritin level in blood is an index of body iron stores Ferritin is an acute phase reactant protein elevated in inflammatory conditions Hemosiderin is an another storage protein, can hold about 33% of iron by weight Hemosiderin accumulates when iron levels are increased

35. Iron excretion Normal iron excretion is 1mg/day Unabsorbed iron is excreted through feces Iron is not excreted in urine, but in nephrotic syndrome, loss of transferrin may lead to increased loss of iron in urine Women upto menopause will lose iron at a rate of 1mg/day Men will lose <0.5 mg/day

36. Iron metabolism disorders Iron deficiency, Fe overload and Anemia of Chronic disease are the most prevalent disorders of Iron metabolism Iron disorders may be classified (1) according to pathophysiology (2) heritability (genetic or acquired), or (3) as being a “primary” ( eg . resulting from a defect in an Fe metabolism–related protein)or “secondary” ( eg . the consequences of defects in other proteins).

37. Iron deficiency anemia Iron deficiency and Iron deficiency anemia is defined when anemia and Fe deficiency coexist These are global health problems and common medical conditions seen in everyday clinical practice Iron deficiency is a disorder of children and premenopausal women in developing and in developed countries.

38. In children, Fe deficiency is frequently caused by the increased physiologic needs for dietary iron for growth and development In adults, and especially premenopausal women, Fe deficiency is almost always the result of chronic blood loss or childbearing

39. Stages of iron deficiency

40. Causes of Iron deficiency Increased demand Rapid growth in children and adolescent Pregnancy Erythropoietin therapy Increased iron loss Chronic blood loss Menses Blood donation Phlebotomy as treatment for polycythemia Decresed iron intake or absorption Inadequate diet Malabsorption Hook worm infestation Acute and chronic inflammation

41. Clinical features Fatiguability , apathy and poor scholastic performance are the characteristic feature Signs: pallor Koilonychia Glossitis Cheilosis Angular stomatitis Achlorhydria

42. Laboratory findings Hemoglobin < 10 g% Microcytic hypochromic anemia Serum ferritin - < 12 μg / dL Serum iron – decreased Serum TIBC - increased Serum transferrin - increased Transferrin Saturation % - decreased

43. Treatment Red cell transfusion Oral iron supplementation along with vitamin C Parenteral iron therapy

44. Anemia of chronic disease Anemia of chronic disease, also named anemia of inflammation is an Iron distribution disorder It is observed in patients with infectious and inflammatory diseases, patients with chronic kidney disease, inflammatory bowel disease, chronic heart failure, malignancies and hepatic diseases

45. Pathogenesis Comprises three principal abnormalities: shortened erythrocyte survival impaired marrow response and disturbances in Fe metabolism

46. Laboratory findings Normocytic normochromic anemia Hemoglobin < 10 g% Serum ferritin – normal or elevated Serum iron – decreased Serum TIBC - increased Serum transferrin - decreased Transferrin Saturation % - decreased Hepcidin - increased sTfR concentration - normal

47. Iron overload disorders Fe overload may also develop as a consequence of multiple RBC transfusions and parenteral Fe supplementation. Fe overload disorders can be categorized as Pathophysiological defect is in the hepcidin ferroportin axis Defective Erythroid maturation Defective iron transport Consequences of repeated blood transfusion Overdose of parenteral iron supplementation

48. Disorders of the Hepcidin-Ferroportin Axis.

49. HFE-Related Hereditary Hemochromatosis • HFE-HH is an autosomal recessive inherited Fe overload disorder • HFE-HH is associated with low hepcidin levels that lead to increased intestinal Fe absorption • Fe overload in patients with HFE-HH is characterised by elevation of TSAT percentage and ferritin levels.

50. Pathogenesis

51. Clinical features In Early stages fatigue and joint pain. In later stages, disease manifestations may include Diabetes mellitus Hypogonadism Liver cirrhosis Cardiomyopathy Skin pigmentation and Increased susceptibility of liver cancer

52. Treatment Phlebotomy remains the mainstay of treatment for Heriditary hemochromatosis One unit of blood of 500 mL contains approximately 200 to 250 mg Fe, depending on the Hb concentration, and should be removed once per week Each phlebotomy should be preceded by measurement of the Hct or Hb to avoid anemia

53. Serum ferritin is recommended to monitor treatment, and its analysisshould be performed after every 10 to 12 phlebotomies (≈3months) in the initial stages of treatment Standard practice is to maintain the serum ferritin at 50 to 100 μg /L Fe overload is treated with chelation with desferrioxamine which forms ferroxamine , excreted in urine

54. Disorders of Erythroid Maturation This class of disorders represents forms of secondary Fe overload, and includes the Fe-loading anemias thalassemia syndromes (especially the β- thalassemias ) sideroblastic anemias and congenital dyserythropoietic anemias

55. Pathogenesis These diseases are characterized by ineffective erythropoiesis ( ie , by apoptosis of erythroid precursors) failure of erythroid maturation and consequent expansion of the number of erythroid precursor cells in the bone marrow

56. Laboratory findings Hepcidin – low Transferrrin saturation% - increased Serum ferritin - increased

57. Sideroblastic anemias sideroblastic anemias are heterogeneous disorders of heme and Fe-sulfur cluster synthesis It is caused by mutation in genes encoding for proteins involved in the heme or Fe-sulfur cluster synthesis in the mitochondria. Fe incorporated in end products & accumulates in the mitochondria produces the characteristic ring sideroblasts , which is a ring around the nucleus of the erythroid precursor cells in the bone marrow.

58. congenital dyserythropoietic anemias It is a group of hereditary disorders characterized by congenital anemia, ineffective erythropoiesis with distinct morphological featuresin the bone marrow late erythroblasts and the development of secondary hemochromatosis Patients usually present with macrocytic or normocytic anemia, jaundice, splenomegaly and low reticulocyte

59. Treatment Management may require repeated transfusions. Fe overload is treated with chelation with desferrioxamine which forms ferroxamine , excreted in urine

60. Disorders of Iron Transport The pathophysiologic feature is insufficient delivery of transferrin -bound Fe to the bone marrow for heme synthesis despite Fe stores The resulting Fe-restrictive erythropoiesis , anemia and hypoxia all contribute to low hepcidin -induced Fe overload.

61. Hypotransferrinemia is a rare autosomal recessive disease in which transferrin levels are severely reduced and insufficient to bind all the Fe that enters the plasma The consequent levels of Non- Transferrin -Bound-Iron cannot be used for heme synthesis and cannot increase hepcidin synthesis

62. Other Forms of Iron Overload Neonatal hemochromatosis It is a severe form of an acquired and secondary severe Fe overload associated with newborn liver failure It is allo -immune–mediated caused by transplacental maternal immunoglobulin-G directed against unidentified fetal liver antigen

63. Friedreich ataxia Mutations in the frataxin gene are responsible for Friedreich ataxia, the most common form of inherited ataxias Frataxin appears to be required for normal mitochondrial Fe export The Fe-mediated mitochondrial injury results in neurologic and cardiac manifestations

64. Hemosiderosis Acquisition of Fe from non dietary sources in amounts that exceed the body’s limited excretory capacity can cause acquired and secondary forms of Fe overload Accumulation of iron as hemosiderin without associated tissue injury Common in patients receiving repeated blood transfusion

65. Bantu siderosis Bantus is a tribal people in south africa , who cooks food in iron utensils and mostly ingest large quantities of beer prepared in iron pots or drums Serum ferritin increased with normal transferrin

66. Analytical methods

67. Serum iron: (1) iron is released from transferrin by decreasing the pH of the serum (2) reduced from Fe3+ to Fe2+, and (3) complexed with a chromogen such as bathophenanthroline or ferrozine . Such Fe- chromogen complexes have an extremely high absorbance in the visible region that is proportional to Fe concentration Normal range is 70 to 200 μ g/ dL

68. Transferrin : Transferrin can be measured directly using immunological techniques like automated immunoturbidimetric or immunonephelometric procedures Alternatively, transferrin can be quantified in terms of the amount of Fe it will bind called total Iron-binding capacity (TIBC) Normal range is 200 – 300 mg/dl Transferrin (g/L) = 0.007 × TIBC( μ g/L)

69. Total iron binding capacity: TIBC can be calculated after the measurement of unsaturated Fe binding capacity (UIBC), by adding UIBC to Fe concentration 1.Addition of supraphysiological amounts of Fe(III)-chloride to saturate the free binding sites on transferrin 2.Unbound excess Fe is removed by adsorption onto solid magnesium carbonate, charcoal, or by an ion exchange Resin 3.Colorimetric determination of Fe that is dissociated from transferrin at acidic pH

70. TIBC ( μ g/ dL ) = transferrin (g/L) x 25.2 Normal range of TIBC is 250 to 435 μ g/ dL Transferrin saturation%: TSAT % = Serum Fe ( μ g/ dL ) x 70.9 transferrin (mg/ dL ) TSAT % = Serum Fe ( μ g/ dL ) x 100 TIBC ( μ g/ dL )

71. Serum ferritin : enzyme-linked immunoassays (ELISAs) that use colorimetric and fluorescent substrates or by antibodies with chemiluminescent labels Normal range is 12 to 300 μg / dL Values < 12 to 30 μg /L identify absolute Fe deficiency Values > 200 and 300 μg /L are often applied for women and men respectively, to define Fe overload Ferritin is an acute-phase reactant increased in inflammation

72. Hepcidin : Serum and urine hepcidin assays that represent two main methodologies: (1) mass spectrometry and (2) immunochemical assays which include c-radioimmunoassay using antibody mimetics Immunochemical assays measure total hepcidin , without distinguishing the full-length hepcidin-25 from the smaller isoforms (hep20, hep22, hep23, and hep24).

73. Hepcidin concentrations are lower in premenopausal women than in postmenopausal women Normal range in males is 6 – 46 ng /ml females is 3 – 25 ng /ml Hepcidin are in the diagnosis of IRIDA, in the differentiation between ACD and IDA in the guidance of Fe supplementation therapy, and in assessing indication and monitoring of therapy with hepcidin agonists or antagonists

74. Serum Transferrin Receptor Cell membranes of developing erythroid cells, especially the erythroblasts, in bone marrow are rich in TfRs (TfR1)to which the Fe- transferrin complex binds. The number of transferrin receptors increases in the presence of high erythroid proliferation rates and low Fe supplies and decreases in bone marrow hypoplasia and suppression

75. ELISAs and Immunoenzymatic assays( nephelometry and turbidimetry ) Fluoroimmunoassays Immunofluorometric assays Uses: Investigate the pathophysiology of anemia Distinguish IDA from ACD Detect functional iron deficiency

76. Erythrocyte Zinc Protoporphyrin Metal-free protoporphyrin in erythrocytes has been associated with lead poisoning and Fe deficiency In the last step of the heme biosynthetic pathway , the enzyme ferrochelatase catalyzes Fe and zinc chelation to protoporpyhrin This reaction occurs to a trace extent in the bone marrow during normal heme biosynthesis and cell maturation, whereas enhanced ZnPP accumulation appears in circulating erythrocytes during states of Fe deficiency in the marrow

77. Normal range is 30 to 80 ZnPP mmol /mol heme The ZnPP ratio of the erythrocytes reflects Fe availability and use in the bone marrow Free erythrocyte protoporphyrin or ZnPP increased in Fe deficiency, ineffective erythropoiesis ( thalassemia syndromes), sideroblastic anemias , ACDs and lead poisoning

78. Red Blood Cell Analytical Parameters RBCs are defined by three quantitative values: MCV - average volume of RBCs, expressed in femtoliters (80-100 fl) MCH - measure of the average Hb content per red cell and is expressed in picograms (27-31pg/cell) MCHC - is the average concentration of Hb in a given red cell volume and is expressed in grams of Hb per deciliter (32-36g/dl)

79. Clinical Relevance: Hemoglobin, MCV, MCH and MCHC are (slightly) elevated in many persons with HFE hemochromatosis MCV, MCH, and reticulocyte Hb content are reduced in Iron deficiency anemia

80. Bone Marrow Examination. Gold Standard Assessment of Body Iron The absence of stainable bone marrow Fe is the most commonly accepted reference standard index of Fe deficiency Normal marrow is graded 1+ to 3+ In Fe deficiency, marrow hemosiderin is absent In anemia of chronic disorders, marrow iron is graded 2+ or 3+ Fe stores are greatly increased (5 to 6+) in Thalassemia Minor and in sideroblastic anemias

81. Liver biopsy Chemical analysis of liver iron biopsy is considered the gold standard method used for the analysis of liver Fe concentration Liver biopsy also has a role in assessing liver fibrosis and cirrhosis in patients with iron overload.

82. References Tietz textbook of clinical chemistry Harrisons principle of internal medicine Bishop textbook of clinical chemistry Vasudevan textbook of biochemistry Devlin textbook of biochemistry