Iron Toxicity and Clinical Sequelae

John B. Porter, MA, MD, FRCP

ProfessorDepartment of HaematologyUniversity College LondonLondon, United Kingdom

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.

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

Conditions Associated with Iron Overload

Transfusional Nontransfusional Age of onsetComplications

Thalassaemia major1 Type 2 haemochromatosis (rare)2 Childhood

Blackfan Diamond Anaemia1 2a hemojuvelin2 (Risks from HH)

Fanconi’s Anaemia1 2b hepcidin2

Early stroke with HbSS1

Severe haemolytic anaemias1

Aplastic anaemia1,2 Type 1 haemochromatosis1 Typically adult

Other transfusion in HbSS1 Thalassaemia intermedia1

Myelodysplasia (MDS)3

Repeated myeloablative chemotherapy1

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.

*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*

Rare Abnormalities of Iron Distribution

Aceruloplasminaemia Plasma reductase Retina RetinopathyAR1,2 Basal ganglia Extrapyramidal

Pancreas Diabetes

Hallervorden-Spatz Pantotenate kinase Basal ganglia Extrapyramidal AR3 cysteine accumulation

Neuroferritinopathy Ferritin light chain Basal ganglia ExtrapyramidalAD4 Forebrain Parkinsonian

Cerebellum

Freidrich’s Ataxia5,6 Frataxin Mitochondrial AtaxiaAR oxidative stress Sensory neurons

Spinal cordDorsal root gangliaMyocardium Cardiomyopathy

Condition Cause Iron Distribution Effects

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.

How Does Transfusional Iron Loading Develop?

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/dayOtherparenchyma

0.3 g

Hepatocytes1 g

Simplified Iron Turnover and Storage

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 estimation1

– 1 unit contains 200 mg of iron

– Adult may receive 4–10 g/y from transfusion

• More-precise method2

– 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

Parenchyma

Hepatocytes

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

50403020100

50

100

150

200

250Thalassaemia major

HH homozygote

HH heterozygote

Age (years)

Hep

atic

Iro

n (

µm

ol/

g w

et w

eig

ht)

50

40

30

20

10

0

Hep

atic

Iro

n (

mg

/g,

dry

wei

gh

t)

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.

How Do Inherited Nontransfusional Forms of Iron Loading Develop?

Effect of Hepcidin on Iron Turnover

Erythron

20–30 mg/day

1–2 mg/day

Gut

Transferrin20–30 mg/day2–3 mg/day

20–30 mg/dayHepatocytes

IL6Iron

Prohepcidin

Hepcidin

Hypoxia

+ - Macrophages

Adapted from Porter JB. Hematol/Oncol Clinics. 2005;19(suppl 1):7.

Factors Affecting Hepcidin Expression?

• TfR21

• HJV2

• Oral iron1

• Iron stores1,2

• LPS2 • IL-62

• HFE1

+ -• Erythropoiesis1 • Anaemia1

• Hypoxia1

• 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.

Type 1 Type 2A Type 2B Type 3 Type 4

Gene HFE HJV HAMP (hepcidin)

TFR2 Ireg1Ferroportin 1

Function Interact with TfR1

Unknown Regulates iron export

Iron uptake Iron export

InheritanceIncidence

RecessiveCommon

RecessiveRare

RecessiveRare

RecessiveRare

DominantRare

Tissues affected

Liver;hepatocytesmacrophages

Skeletal muscle, heart,liver

Liver hepatocyte

Liver hepatocyte

DuodenumMacrophage

Clinical presentation

Late variable Early onsetSevere

Early onsetSevere

Severe Variable

Hepcidin levels ??

With permission from Worwood M. Blood Rev. 2005;19:69.

Classification of Haemochromatosis

Why Is Iron Overload Toxic?

Fe 2+

Fe 3+

+ e- - e-

Redox Cycling of Iron

Slide courtesy of Dr. J. Porter.

Porter J. Hematol/Oncol Clinics 2005;19(suppl 1):7.

Hydroxyl Radical (HO.) Generation

Haber Weiss Reaction

O2.- + H2O2 -----> O2 + OH- + HO.

Catalysed by Iron in two steps; (Fenton reaction)

Fe3+ + O2.- -----> Fe2+ + O2

Fe2+ + H2O2 -----> Fe3+ + OH- + HO.

OO

OOH

Hydrogen abstraction ( H.)

Molecular rearrangement

Oxygen uptake

Lipid hydroperoxide

Peroxyl radical propagates peroxidation by abstracting H.

from another fatty acid

+ O2.

.

.

H2O

Decompositioneg, 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

Consequences of Iron-Mediated Toxicity

Increased free iron

Hydroxyl radical generation

Lipid peroxidation

Organelle damage

Lysosomal fragilityEnzyme leakage

Cell death

Collagensynthesis

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.

Evans R et al. J Biol Inorg Chem. 2007;13:57.

Nature of NTBI

• Nature of NTBI– Citrate iron

Polymeric Slowly chelated Oligomeric Dimeric Monomeric Rapidly chelated

• Protein-bound iron– Binds weakly to albumin

– As citrate oligomers bound to albumin

• OtherNTBI = non–transferrin-bound iron.

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 NTBIReceptors

• Divalent metal transporter (DMT1)1

– Enterocytes

– Erythron (negatively regulated by iron loading)

– ? Other

• L-type calcium-dependent channels2

– Myocardium (positively induced by iron loading)

– Anterior pituitary (positively induced by iron loading)

• T-type calcium channels3

– Hepatocytes (positively induced by iron loading)

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.

Intracellular Iron-Mediated Toxicityfrom Labile Intracellular Iron

Transferriniron

Lysosomaldegradation

LV

DC

CNon-transferriniron

Organelle damage

Ironproteins

Free-radical generation

Ferritin

LVDCC = L-type voltage-dependent calcium channel.Porter JB. Am J Hematol. 2007;82:1136.

Labile iron pool

(LIP)

Where Is Iron Toxic ?

Adapted from Porter JB. Hematol/Oncol Clinics 2005;19(suppl 1):7.

Parenchyma

Hepatocytes

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

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 stages1

• Why great variability in iron distribution in different tissues?– Liver, endocrine glands, anterior pituitary1

– Very little in brain, skeletal muscle1

– Liver iron correlates with units transfused2

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.

Liver Heart

Liver and Iron Content Postmortem in Thalassaemia Major

0–4 5–9 10–14 15–19 >20 Total

Heart disease 0 6 39 35 16 96

Infection 2 6 9 3 0 20

Liver disease 0 0 2 7 1 10

Malignancy 2 2 1 1 2 8

Endocrine disease 0 0 2 1 1 4

Accident 0 0 2 2 0 4

Thromboembolism 0 0 2 1 1 4

Anaemia 2 0 0 0 0 2

Other 0 1 1 0 1 3

Unknown 0 1 3 3 1 8

Total 6 16 61 53 23 159

Zurlo MG, et al. Lancet. 1989;2:27.

Causes of Death in Thalassaemia

n = 1078

Age (years)

131 transfused adult patients

• 101 leukaemias

• 30 other anaemias

0–25 26–50 51–75 76–100 101–200 201–3000

20

40

60

80

100

Units of Blood Transfused

Pat

ien

ts w

ith

Car

dia

c Ir

on

(%

)

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.

Blood Transfusion Predicts Heart Iron in Unchelated Patients

0

2

4

6

8

10

12

14

16

18

20

Est

imat

ed H

eart

Iro

n (

µm

ol/

g)

25 50 75 100

125

150

Blood Units Transfused

Jensen PD, et al. Blood. 2003;101:4632.

Upper Normal Limit

Slide courtesy of Dr. J. Porter.

0 20 40 60 80

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?

Shah F. Presentation at ASH Dec 2002. Blood 2002;100:668a.

-2

-1

0

1

2

3

4

5

6

7

8

Thalassaemia majorHbSS

NT

BI (

µM

)Patients Treated at UCLH

3.38 ± 2.4

0.17 ± 1.8

P = 0.0001

LIC = 4.34 LIC = 4.22

NTBI in Sickle Cell or Thalassaemia Major Matched for Liver Iron Concentration

Slide courtesy of Dr. J. Porter.

Which Forms of Iron Are Most Toxic?

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

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 survival1

• Liver iron association with cardiac death2

Of 15/53 thalassaemia major patients with cardiac disease, all had liver iron >15 mg/g dry weight3

– Association or causation?

• But– Iron in different tissues at postmortem does not

correlate with damage to those organs3

– Link of cardiac iron to damage & death not known3

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 tissues2

– Relationship between heart iron and mortality unknown2

1. Porter JB. Br J Haematol. 2001;115:239.

2. Porter J. Hematol/Oncol Clinics. 2005;19(suppl 1):7.

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

• Liver1

• Heart1

• Endocrine system1

• Cancer

• Other potential sequelae – Arthropathy2

– Hyperpigmentation2

Diabetes1Pancreas

Gonads

Cirrhosis, carcinoma1Liver

Cardiomyopathy1Heart

Hypoparathyoidism1Parathyroid

Hypothyroidism1Thyroid

Hypogonadotrophic hypogonadism1Pituitary

ConsequencesOrgan

Joints Arthropathy2

Skin Pigmentation2

Hypogonadotrophic hypogonadism1

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

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