1. به نام یگانه هستی بخش

2. MODERN INSIGHTS INTO ANEMIA

3. Hepcidin,the key regulator of iron metabolism  Dr.Hossein Ghaziasgar

4. IRON  An essential element,must be precisely regulated  Intestinal absorption is essential for the iron balance,but the precise mechanism of its regulation was unknown

5. Conservation of iron  Recycling from Hb.(about 20 mg/day)  Iron absorption in the duodenum  Deposition in the liver (hepatocytes) The coordination is essential and vital

6. BUT  Humans and other mammals lack effective mechanisms to excrete excess iron (the daily loss of iron from the body is 1-2mg/day) Intestinal iron absorption is the sole means of iron balance

7. Excess iron  Is deposited in the liver, endocrine glands,the heart and the skin Tissue damage (in hereditary hemochromatosis)

8. Question  Who is playing the central role in “orchestration” on iron metabolism? ?

9. Answer  Hepcidin, the key regulator of iron metabolism

10. What is Hepcidin?  Was first identified in human urine and plasma in June 2000  A 25-amino acid disulfide-rich peptide  Molecular weight about 2 KDa  Is highly folded and cationic amphipathic

11. What is Hepcidin?  It was recognized as an antimicrobial peptide ( Defensin-like) and is produced in the liver and excreted in urine.  Of course recent studies detected Hepcidin synthesis in bacteria-activated neutrophils and macrophages (at a lower level)

12. What is Hepcidin?  In addition to the 25-amino acid form,the urine also contains 20- and 22-amino acid forms (but the role is not identified)  Hence the bioactive form is the 25-amino acid form which contains 4 disulfide bands (Hepcidin 20- has been found in serum too, in 2007 )

13. Hepcidin gene  Is named HAMP  Contains 3 exons :  produces 84-amino acid preprohepcidin 60-amino acid prohepcidin 25-amino acid hepcidin  Has also been identified in other vertebrates like mice, rats, pigs and several species of fish.

15. Hepcidin  Controls extracellular iron by regulating: 1. Intestinal absorption 2. Recycling by macrophages 3. Releasing from stores 4. Placental transport

16. The Mechanism The major mechanism of Hepcidin function is “the regulation of transmembrane iron transport”.

17. HOW?  Hepcidin binds to its receptor, protein FERROPORTIN, which serves as a transmembrane iron channel enabling iron efflux from cells.

18. THEN  The Hepcidin-Ferroportin complex is degraded in lysosomes and iron is locked inside the cells (mainly enterocytes, hepatocytes and macrophages)

19. So  Hepcidin lowers iron absorption in the intestine,lowers iron releasing from hepatocytes and macrophages Serum iron is decreased.

20. Ferroportin  The sole protein exporter is located on: 1. the intestinal cells 2. hepatocytes 3. macrophages 4. placental cells

21. The second role  Hepcidin is an acute phase protein type 2 and is increased in inflammation.

22. Hormonal activity of Hepcidin  Lack of Hepcidin results in Hemochromatosis (iron deposition in the liver,pancreas and macrophages)  Hepcidin excess results in severe iron – deficiency (blocking intestinal iron uptake and iron releasing from hepatocytes and macrophages and inhibiting the placental transport of iron too)

23. Continue  Injected synthetic 25-amino acid Hepcidin causes 75% decrease in serum iron levels within 1 hour persisting for more than 2 days.  By a decrease in Hepcidin level, erythroid regulation can increase iron uptake 40mg/day(normal 1-2 mg/day).

24. Regulation of Hepcidin synthesis by iron  Dietary iron induces Hepcidin synthesis  Urinary Hepcidin concentrations are greatly increased within less than 1 day after iron ingestion.

25. But  The mechanism is not known completely yet:  In the liver the proteins: 1. HFE 2. Transferrin receptor 2 3. Hemojuvelin may be involved in mediating this signal

26. Regulation of Hepcidin synthesis by anemia and hypoxia  Oxygen Hepcidin Uptake of diet iron Iron release from hepatocytes Iron release from macrophages

27. Continue  Erythropoietic signal Hepcidin  Tissue iron differic transferrin Hepcidin

28. Regulation of Hepcidin synthesis by inflammation  Interleukin-6 Hepcidin iron anemia of chronic disease

29. The role of Hepcidin in hereditary hemochromatosis  Hereditary hemochromatosis: -excessive intestinal iron absorption -Saturation of transferrin -Iron deposition in vital organs

30. Continue  Mutations in: -HFE gene: most common form -TfR2 gene: much rarer -HAMP gene: Severe phenotype -HJV gene: Severe phenotype

31. The role of Hepcidin in anemia of inflammation  This anemia results from: -Chronic infections -Noninfectious generalized inflammatory disorders -Some cancers -Sepsis

32. Continue IL-6 Hepcidin Hypoferremia anemia of inflammation

33. The role of Hepcidin in iron- loading anemias  Ineffective erythropoiesis  Increased intestinal iron absorption  Increased ferritin  Decreased Hepcidin

34. Hepcidin in differential diagnosis Decreased HepcidinIncreased Hepcidin Iron deficiency Increased and/or ineffective erythropoiesis Classical,,juvenile and TfR2 HH High iron stores Anemia of chronic disease Ferroportin disease(?)

35. Hepcidin assays  Immunoassay (urine,UCLA)  Mass spectrometry (urine,serum)  Elisa (for Prohepcidin)

36. Hepcidin assays  Serum Prohepcidin:50-150 ng /mL  Serum Hepcidin :1-500 ng/mL (based on 2007 findings)

37. Continue  Hepcidin in urine is affected by multiple freeze/thaw cycles. Serum is more stable.  High diurnal variation of especially serum Hepcidin.  Use of internal standard:Hepcidin-24 to control for matrix influences and instrumental settings.

38. Overall summary  Hepcidin - Is a recently discovered liver produced 25 amino- acid peptide -Is a regulator of iron metabolism that controls iron absorption and macrophage iron release. - Is regulated by erythropoietic needs( ),body iron stores( ) and inflammation( )

39. Overall summary  MS Hepcidin assay for urine and serum: -Precludes the need for Hepcidin specific antibodies -Will provide: -information on etiology of iron metabolism disorders -leads for new therapeutic strategies -novel diagnostic approaches