1. Iron Toxicity and Clinical Sequelae John B. Porter, MA, MD, FRCP Professor Department of Haematology University College London London, United Kingdom

2. Learning Objectives Analyze the mechanisms contributing to the development of iron overload and the clinical consequences of iron overload on the liver, heart, and endocrine system. Utilizing an understanding of the factors contributing to the development of iron overload, identify patients at risk in the practice setting.

3. Topics Causes of iron overload Mechanisms of iron-overload–mediated toxicity –Molecular level –Non–transferrin-bound iron—extracellular –Labile iron pool—intracellular –Free radical formation  Microscopic level  Macroscopic level Clinical impact consequences of iron overload

4. Conditions Associated with Iron Overload TransfusionalNontransfusionalAge of onset Complications Thalassaemia major 1 Type 2 haemochromatosis (rare) 2 Childhood Blackfan Diamond Anaemia 1 2a hemojuvelin 2 (Risks from HH) Fanconi’s Anaemia 1 2b hepcidin 2 Early stroke with HbSS 1 Severe haemolytic anaemias 1 Aplastic anaemia 1,2 Type 1 haemochromatosis 1 Typically adult Other transfusion in HbSS 1 Thalassaemia intermedia 1 Myelodysplasia (MDS) 3 Repeated myeloablative chemotherapy 1 1. Porter JB. Br J Haematol. 2001;115:239. 2. Brittenham G. In Hoffman R, et al, ed. Hematology: Basic Principles and Practice, 4th ed. Philadelphia, PA: Churchill Livingstone, 2004. 3. Taher A, et al. Semin Hematol. 2007;44:S2. Slide courtesy of Dr. J. Porter.

5. *Dietary and hereditary components. Bacon BR. In Goldman L, ed. Cecil’s Textbook of Medicine, 23rd ed. Philadelphia, PA: Saunders- Elsevier, 2008. Acquired, Nontransfusional Forms of Iron Overload Chronic liver disease –Hepatitis C –Alcoholic liver disease –Nonalcoholic steatohepatitis Porphyria cutanea tarda Portacaval shunting Inappropriately high dietary intake –Latrogenic (eg, treatment of microcytosis) –African (Bantu) siderosis*

6. Rare Abnormalities of Iron Distribution AceruloplasminaemiaPlasma reductaseRetinaRetinopathy AR 1,2 Basal gangliaExtrapyramidal PancreasDiabetes Hallervorden-Spatz Pantotenate kinaseBasal gangliaExtrapyramidal AR 3 cysteine accumulation NeuroferritinopathyFerritin light chainBasal gangliaExtrapyramidal AD 4 ForebrainParkinsonian Cerebellum Freidrich’s Ataxia 5,6 FrataxinMitochondrialAtaxia AR oxidative stressSensory neurons Spinal cord Dorsal root ganglia MyocardiumCardiomyopathy ConditionCauseIron DistributionEffects 1. Mariani R, et al. Gut. 2004;53:756-8. 2. Hellman NE, et al. Gut. 2000;47:858-60. 3. Hayflick SJ. Curr Opin Pediatr. 2003;15:572-7. 4. Crompton DE, et al. Blood Cells Mol Dis. 2002;29:522-31. 5. Koepen A, et al. Acta Neuropahtol. 2007;114:163-73. 6. Michael, et al. Cerebellum. 2007;5:257-67. AR = autosomal recessive; AD = autosomal dominant.

7. How Does Transfusional Iron Loading Develop?

8. Porter J. Hematol/Oncol Clinics. 2005;19(suppl 1):7. Red Erythron 2g 20–30 mg/day Macrophages 0.6 g 1–2 mg/day Gut Transferrin 20–30 mg/day 2–3 mg/day 20–30 mg/day Other parenchyma 0.3 g Hepatocytes 1 g Simplified Iron Turnover and Storage

9. 1.Taher A, et al. Semin Hematol. 2007;44:S2. 2.Porter JB. Br J Haematol. 2001;115:239. Rate of Iron Loading from Transfusion Simple estimation 1 –1 unit contains 200 mg of iron –Adult may receive 4–10 g/y from transfusion More-precise method 2 –Volume of blood transfused x mean haematocrit of processed blood obtained from the transfusion centre x 1.08 For exchange transfusion need to know –Volume and haematocrit transfused –Volume and haematocrit removed

10. Parenchyma Hepatocytes Parenchyma Red Erythron Macrophages Gut Transfusion 20–40 mg/day (0.3–0.7 mg/kg/day) Transferrin Adapted from Porter JB. Hematol/Oncol Clinics. 2005;19(suppl 1):7-12. NTBI NTBI = non–transferrin-bound iron. Transfusional Iron Overload

11. 50403020100 50 100 150 200 250 Thalassaemia major HH homozygote HH heterozygote Age (years) Hepatic Iron (µmol/g wet weight) 50 40 30 20 10 0 Hepatic Iron (mg/g, dry weight) Threshold for cardiac disease and early death Increased risk of complications Normal Olivieri N, Brittenham G. Blood. 1997;89:739. Liver Iron and Risk from Iron Overload 0 HH = hereditary haemochromatosis.

12. How Do Inherited Nontransfusional Forms of Iron Loading Develop?

13. Effect of Hepcidin on Iron Turnover Erythron 20–30 mg/day 1–2 mg/day Gut Transferrin 20–30 mg/day2–3 mg/day 20–30 mg/day Hepatocytes IL6 Iron Prohepcidin Hepcidin Hypoxia + - Macrophages Adapted from Porter JB. Hematol/Oncol Clinics. 2005;19(suppl 1):7.

14. Factors Affecting Hepcidin Expression? TfR2 1 HJV 2 Oral iron 1 Iron stores 1,2 LPS 2 IL-6 2 HFE 1 + - Erythropoiesis 1 Anaemia 1 Hypoxia 1 NTBI? Hepcidin Tf = transferrin; TfR = transferrin receptor; HJV = hemojuvelin; LPS = lipopolysaccharide; IL = interleukin; NTBI = non–transferrin-bound iron. 1. Leong W, Lönnerdal B. J Nutr. 2004;134:1. 2. Lee P, et al. Proc Natl Acad Sci U S A. 2004;101:9263.

15. Type 1Type 2AType 2BType 3Type 4 GeneHFEHJVHAMP (hepcidin) TFR2Ireg1 Ferroportin 1 FunctionInteract with TfR1 UnknownRegulates iron export Iron uptakeIron export Inheritance Incidence Recessive Common Recessive Rare Recessive Rare Recessive Rare Dominant Rare Tissues affectedLiver; hepatocytes macrophages Skeletal muscle, heart, liver Liver hepatocyte Duodenum Macrophage Clinical presentation Late variableEarly onset Severe Early onset Severe Variable Hepcidin levels?? With permission from Worwood M. Blood Rev. 2005;19:69. Classification of Haemochromatosis

16. Why Is Iron Overload Toxic?

17. Fe 2+ Fe 3+ + e - - e - Redox Cycling of Iron Slide courtesy of Dr. J. Porter.

18. Porter J. Hematol/Oncol Clinics 2005;19(suppl 1):7. Hydroxyl Radical (HO. ) Generation Haber Weiss Reaction O 2. - + H 2 O 2 -----> O 2 + OH - + HO. Catalysed by Iron in two steps; (Fenton reaction) Fe 3+ + O 2. - -----> Fe 2+ + O 2 Fe 2+ + H 2 O 2 -----> Fe 3+ + OH - + HO.

19. OOOO OOHOOH Hydrogen abstraction ( H. ) Molecular rearrangement Oxygen uptake Lipid hydroperoxide Peroxyl radical propagates peroxidation by abstracting H. from another fatty acid + O 2... H2OH2O Decomposition eg, to MDA Porter J. Hematol/Oncol Clinics 2005;19(suppl 1):7. With permission from Gutteridge JM, Halliwell B. Baillieries Clin Haematol. 1989;2:195. Lipid Peroxidation by HO.

20. 20 Consequences of Iron-Mediated Toxicity Increased free iron Hydroxyl radical generation Lipid peroxidation Organelle damage Lysosomal fragility Enzyme leakage Cell death Collagen synthesis TGF-b1 Fibrosis Gutteridge JMC, Halliwell B. Bailleres Clin Haematol. 1989;2:195-256. Bacon BR, et al, J Clin Invest. 1983;71:429-439. Myers BM, et al. J Clin Invest. 1991;88:1207-1215. Tsakamota H, et al. J Clin Invest. 1995;96:620-630. Houglum K, et al. Hepatology.1997;26:605-610.

21. Evans R et al. J Biol Inorg Chem. 2007;13:57. Nature of NTBI –Citrate iron  PolymericSlowly chelated  Oligomeric  Dimeric  MonomericRapidly chelated Protein-bound iron –Binds weakly to albumin –As citrate oligomers bound to albumin Other NTBI = non–transferrin-bound iron.

22. 1.Bacon BR. In Goldman L, ed. Cecil’s Textbook of Medicine, 23rd ed. Philadelphia, PA: Saunders- Elsevier, 2008. 2. Oudit GY, et al. Circulation. 2004;109:1877. 3. Rafique et al. Blood. 2006;108:1542a. Uptake of NTBI Receptors Divalent metal transporter (DMT1) 1 –Enterocytes –Erythron (negatively regulated by iron loading) –? Other L-type calcium-dependent channels 2 –Myocardium (positively induced by iron loading) –Anterior pituitary (positively induced by iron loading) T-type calcium channels 3 –Hepatocytes (positively induced by iron loading)

23. Antioxidant Capacity in Iron Overload 806040200 Vitamin C B-carotene Vitamin A Vitamin E Ubiquinone Ubiquinol Lycopene % Decrease of Control 48 thalassaemia major (age 11–22 years) Vitamin E and NTBI negatively correlate (r = -0.81) No correlation with serum ferritin De Luca C, et al. Free Radic Res. 1999;30:453. Slide courtesy of Dr. J. Porter.

24. Intracellular Iron-Mediated Toxicity from Labile Intracellular Iron Transferrin iron Lysosomal degradation LVDCC Non- transferrin iron Organelle damage Iron proteins Free-radical generation Ferritin LVDCC = L-type voltage-dependent calcium channel. Porter JB. Am J Hematol. 2007;82:1136. Labile iron pool (LIP)

25. Where Is Iron Toxic ?

26. Adapted from Porter JB. Hematol/Oncol Clinics 2005;19(suppl 1):7. Parenchyma Hepatocytes Parenchyma Erythron Macrophages Gut Transfusion 20–40 mg/day (0.3–0.7 mg/kg/day) Transferrin NTBI NTBI = non–transferrin-bound iron. Transfusional Iron Overload

27. 1. Porter JB. Hematol/Oncol Clinics 2005;19(suppl 1):7. 2. Taher A, et al. Semin Hematol. 2007;44:S2. Iron Distribution in Transfusional Overload Transfusional overload distribution differs from absorption distribution at early stages 1 Why great variability in iron distribution in different tissues? –Liver, endocrine glands, anterior pituitary 1 –Very little in brain, skeletal muscle 1 –Liver iron correlates with units transfused 2

28. Adapted from Modell B, Mathews R. Birth Defects Orig Artic Ser. 1976;12:13. 86420 Fe % d.w. Minimum Maximum Skeletal muscle Testes Kidney Heart Adrenal Salivary gland Thyroid Pancreas Liver Parathyroid Distribution of Body Iron at Postmortem in TM in Prechelation Era Tm = thalassaemia major; d.w. = dry weight.

29. Liver Heart Liver and Iron Content Postmortem in Thalassaemia Major

30. 0–45–910–1415–19>20Total Heart disease063935 1696 Infection2693 020 Liver disease0027 110 Malignancy2211 28 Endocrine disease0021 14 Accident0022 04 Thromboembolism0021 14 Anaemia2000 02 Other0110 13 Unknown0133 18 Total6166153 23159 Zurlo MG, et al. Lancet. 1989;2:27. Causes of Death in Thalassaemia n = 1078 Age (years)

31. 131 transfused adult patients 101 leukaemias 30 other anaemias 0–25 26–5051–7576–100101–200201–300 0 20 40 60 80 100 Units of Blood Transfused Patients with Cardiac Iron (%) Buja LM, Roberts WC. Am J Med. 1971;51:209. Blood Transfusion and Cardiac Iron Deposits at Postmortem in the Prechelation Era Slide courtesy of Dr. J. Porter.

32. Blood Transfusion Predicts Heart Iron in Unchelated Patients 0 2 4 6 8 10 12 14 16 18 20 Estimated Heart Iron (µmol/g) 255075 100125150 Blood Units Transfused Jensen PD, et al. Blood. 2003;101:4632. Upper Normal Limit Slide courtesy of Dr. J. Porter.

33. 020406080 Thalassaemia major (n = 108) Thalassaemia intermedia (n = 23) Congenital sideroblastic (n = 4) PK deficiency (n = 9) Diamond Blackfan (n = 7) Myelodysplasia (n = 7) Sickle (n = 37) Patients (%, n) with T2* < 20 ms UCLH patients with cardiac MRI Glanvillle J, et al. Presented at ASH 2006. Blood. 2006;108:abstract 1553. Is the Heart Equally at Risk of Iron Loading in All Forms of Transfusional Iron?

34. Shah F. Presentation at ASH Dec 2002. Blood 2002;100:668a. -2 0 1 2 3 4 5 6 7 8 Thalassaemia major HbSS NTBI (µM) Patients Treated at UCLH 3.38 ± 2.4 0.17 ± 1.8 P = 0.0001 LIC = 4.34LIC = 4.22 NTBI in Sickle Cell or Thalassaemia Major Matched for Liver Iron Concentration Slide courtesy of Dr. J. Porter.

35. Which Forms of Iron Are Most Toxic?

36. Porter J. Hematol/Oncol Clinics. 2005;(suppl 1):S7. Labile Toxic Iron Pools? NTBI in plasma? –Correlates with antioxidant depletion –Promotion of lipid peroxidation in vitro –BUT which species? Labile iron pools (LIP) in cells? –In vitro: clear evidence linking free iron to lipid peroxidation and organelle damage Clinical evidence? –Improvement in cardiac performance with intravenous desferrioxamine precedes changes in cardiac iron –BUT direct link of NTBI or LIP to clinical damage not established

37. 1.Olivieri NF, et al. N Engl J Med. 1994;331:574. 2.Brittenham GM, et al. N Engl J Med. 1994;331:567. 3.Porter JB. Hematol/Oncol Clinics. 2005;19(suppl 1):S7. Absolute Tissue Levels? Evidence (serum ferritin) >2500 µg/L & cardiac disease-free survival 1 Liver iron association with cardiac death 2  Of 15/53 thalassaemia major patients with cardiac disease, all had liver iron >15 mg/g dry weight 3 – Association or causation? But –Iron in different tissues at postmortem does not correlate with damage to those organs 3 –Link of cardiac iron to damage & death not known 3

38. Intracellular Iron Levels and Toxicity Concepts –“Safe iron”  No toxicity in heterozygotes of hereditary haemochromatosis where liver levels < 7mg/g dry weight. 1 –“Dangerous iron”  High risk of cardiac death if liver >15 mg/g dry weight. 1 Limitations –Uneven distribution within and between tissues 2 –Relationship between heart iron and mortality unknown 2 1. Porter JB. Br J Haematol. 2001;115:239. 2. Porter J. Hematol/Oncol Clinics. 2005;19(suppl 1):7.

39. 1.Porter JB. Hematol/Oncol Clinics. 2005;19(suppl 1):S7. 2.Brittenham G. In Hoffman R, et al, ed. Hematology: Basic Principles and Practice, 4th ed. Philadelphia, PA: Churchill Livingstone, 2004. Functional Consequences of Transfusional Iron Overload Liver 1 Heart 1 Endocrine system 1 Cancer Other potential sequelae –Arthropathy 2 –Hyperpigmentation 2

40. Diabetes 1 Pancreas Gonads Cirrhosis, carcinoma 1 Liver Cardiomyopathy 1 Heart Hypoparathyoidism 1 Parathyroid Hypothyroidism 1 Thyroid Hypogonadotrophic hypogonadism 1 Pituitary ConsequencesOrgan Joints Arthropathy 2 Skin Pigmentation 2 Hypogonadotrophic hypogonadism 1 1.Taher A, et al. Semin Hematol. 2007;44:S2. 2.Brittenham G. In Hoffman R, et al, ed. Hematology: Basic Principles and Practice, 4th ed. Philadelphia, PA: Churchill Livingstone, 2004. Organ Systems Affected by Iron Overload

41. Conclusions Conditions associated with iron overload include transfusional iron overload as well as hereditary and acquired nontransfusional iron overload Because the body has no mechanism for excretion of excess iron, iron can accumulate Iron accumulation results in –Increased free iron –Hydroxyl radical generation –Lipid peroxidation This results in cell death and fibrosis, with impact on a variety of organ systems and functional consequences –Heart –Liver –Endocrine system