Iron Metabolism: Too little, too much!

Justin Ridge & Simon Worrall School of Chemistry and Molecular Biosciences
COMMONWEALTH OF AUSTRALIA
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MEDI1012 - Biochemistry

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Iron
A metallic element Toxic but also an essential
nutrient

26
55.85

Fe

An important component of
many macromolecules

Able to undergo redox reactions (Fe2+, Fe3+, Fe4+) 35mg/kg in women and 45 mg/kg in men

MEDI1012 - Biochemistry

Iron Content of Foods

Iron is found in virtually all foods. Two types:
Non-haem fruits, vegetables, grain products Haem meat and poultry

RDA for vegetarians Males Females 40 mg 60 mg Males

RDA 8 mg 18 mg Females

Pregnant 27 mg
MEDI1012 - Biochemistry

Iron Trafficking
DIET
10 mg/ day

GUT
1 mg/ day

FAECES Immature RBC RBC RES Iron stores

Labile Iron
~70%

ECF
Plasma
Enzyme cofactor ~10%

Total Body Pools Males 3.5g Females 2.5g

MEDI1012 - Biochemistry

Ferritin Haemosiderin
~11.5% ~10%

Absorption of Dietary Iron

Two forms of Fe in diet:
Non-haem & haem

Main mechanism for non-haem is well understood.

Occurs in first part of the duodenum. Requires an acidic environment.

Mechanism for haem is as yet unknown but is very

efficient.

MEDI1012 - Biochemistry

Duodenal Absorption of Ferrous Iron

Gut Lumen H+
Fe2+

Enterocyte H+
DMT1 Fe2+ Ireg-1

Blood
Hp

Fe2+
Cp

vitamin C ferrireductase

Ferritin (Fe3+)

Fe3+

Fe2+

Fe3+ Tf

DMT1 (Nramp1): Fe, Zn, Mn, Co, Cd, Cu, Ni and Pb.
MEDI1012 - Biochemistry

Ceruloplasmin (Cp)
Plasma protein Carries Cu from liver
to periphery H2O or SH O2 or S-S-

Controls oxidation

reduction reactions of Fe, Fe transport & utilization but has a transmembrane domain at C-terminus

Cp Cu2+

Cp Cu+

Hephaestin analogous

Fe2+ Ft

Fe3+ Tf

MEDI1012 - Biochemistry

Proposed Duodenal Absorption of Haem Iron

Gut Lumen Enterocyte
inorganic Fe Metalloporphyrin (H) haemoglobin

haem

degraded by haem oxidase

Endocytosis ??? ?

globins are proteolysed

MEDI1012 - Biochemistry

Transferrin Fe transporter
Soluble protein synthesised by the liver Active as a monomer (80kDa)
Each monomer binds two Fe in ferric form Is at a large excess in plasma

Delivers Fe from the blood to all cells e.g.

erythroid precursors

MEDI1012 - Biochemistry

Transmembrane Iron Transport

1

1. Tf binds to receptor 2. Receptor endocytosed 3. Endosome acidified 4. Reduced Fe pumped out of endosome 5. Tf and TfR returned to membrane
MEDI1012 - Biochemistry

DMT1

TfR

i
endocytosis 5 2
ferric reductase Fe3+ Fe2+

i
3

4
DMT1

H+

H+

endosomes

Regulation of iron uptake

Uptake of iron affected by two
proteins:
HFE Hepcidin

MEDI1012 - Biochemistry

HFE
Originally known as HLA-H and expressed by most
cell types Binds 2-microglobulin

Also binds to the Tf receptor (TfR) with high

affinity. Binding of HFE to TfR leads to HFE stabilisation. Binding halves the capacity of TfR for Tf and
decreases the affinity for apoTf or Tf-Fe. Decreases iron retention in the cell (some types)

MEDI1012 - Biochemistry

HFE
Fe Fe

HFE

DMT 1 Fe Fe Fe

H+

HFE

HFE: 1.Decreases TfR capacity 2.Stabilises Tf binding to TfR; less Fe is released into endosome; Tf returns to cell surface carrying Fe
Based on Roy et al. J. Biol. Chem. 274:9022, 1999.

MEDI1012 - Biochemistry

Hepcidin
25 aa peptide (hec-25) synthesized by
hepatocytes Induced by dietary iron overload Expression affected by:

Plasma conc. of TfFe2 (via TfR) Hepatocyte Fe stores (indirect; changes in TfR)

Released into blood

Acts at enterocytes Acts at RE macrophages
and crypt cells

MEDI1012 - Biochemistry

Hepcidin Action on Enterocytes

Hepcidin interacts
with ferroportin (ireg1)
Causes internalisation Decreased Fe export

Fe accumulation leads
to
Decrease of expression
of DMT; Vit C ferrireductase Shedding of enterocytes

Net loss of Fe
MEDI1012 - Biochemistry

Action of Hepcidin on Crypt Cells and Macrophages

Interacts with 2-HFETfR1

Increase Fe uptake Crypt cells differentiate

with reduced expression of Fe transport proteins

Reduced Fe uptake

MEDI1012 - Biochemistry

From Fleming, R. E. & Sly, W. S. (2001) PNAS 98:81608162.

Intra-Cellular Storage of Iron

Ferritin

Apoferritin consists of a mixture 24 subunits (H or L) H chains oxidise Fe2+ Fe3+ Synthesis regulated by intracellular [Fe] Each ferritin complex can bind up to 4,500 Fe

Haemosiderin
Amorphous Fe deposited near ferritin when capacity
exceeded

Frataxin
A potential Fe storage protein in mitochondria (Fedependent function????)

MEDI1012 - Biochemistry

Regulation of Gene Expression by Iron

The expression of these human genes is known to
be controlled by intracellular [Fe] in all cells Transferrin receptor Ferritin DMT1 Ireg-1 -aminolevulinate synthase (Hb synthesis) Transferrin (in liver)

MEDI1012 - Biochemistry

Post-transcriptional Control
Iron Deficiency
Inactive IRE-BP
IRE-BP Active

ferritin mRNA

TfR mRNA

AAAAAA

AAAAAA

No translation IRE-BP

Inhibited degradation

= increased translation IRE-BP IRE-BP

5
MEDI1012 - Biochemistry

AAAAAA

AAAAAA

Congenital Diseases Associated with Iron Metabolism

Disease Haemochromatosis Friedrichs ataxia Affected Gene HFE FRDA Function Modulation of Fe transport Frataxin a putative Fe storage protein Hb synthesis Fe storage protein Fe transport

Hallervorden-Spatz Hyperferritinaemiacataract Hypotransferrin-aemia

MEDI1012 - Biochemistry

PANK2 IRE of L-ferritin mRNA Transferrin (?)

Hereditary Haemochromatosis (HH)

Hereditary disorder of small intestine
Autosomal, recessive Over-adsorption of Fe from food Organs such as pancreas, liver, and skin store the

excess Fe damage Untreated, it can result in liver disease, heart disease, and diabetes.

The most common form of Fe overload disease. Incidence: ~1 in 200 have mutations inHFE gene;
affects ~73,000 people in Australia.

MEDI1012 - Biochemistry

Hereditary Haemochromatosis (HH)

Symptoms include:
Weakness, fatigue Abdominal pain, muscle aches, finger joint pain Shortness of breath with physical exertion Increased skin pigmentation (bronze colour), loss of body hair Hepatomegaly

MEDI1012 - Biochemistry

Iron Toxicity
Fe2+ O2Fe3+ Fe2+

O2
Superoxide dismutase

OH

H2O

catalase & peroxidases

H2O2

Cellular Damage

MEDI1012 - Biochemistry

Histopathology
Brown, granular pigment indicates the presence of iron in hepatocytes.

Tissue damage induces fibrosis

MEDI1012 - Biochemistry

Hereditary Haemochromatosis (HH)

Two major hypotheses have been proposed
to explain the pathogenesis of HFE-related HH:
The hepcidin hypothesis The duodenal crypt cell programming hypothesis

MEDI1012 - Biochemistry

iron uptake

Normal iron stores

ARI

9 December 2008

15:27

Plasma Normal plasma iron

a
Intestinal enterocyte

Normal iron absorption

Normal iron uptake

The Hepcidin Hypothesis - 1

Ferroportinp mediated mediat iron export exp
Plasma

Norma iron stor

a
Intestinal enterocyte

Normal plasma sma Normal iron absorption hepcidin levels vels

Normal iron stores

. Rev. Pathol. Mech. Dis. 2009.4:489-515. Downloaded from www.annualreviews.org by University of Queensland on 09/01/10. For personal use only.

Normal iron uptake

Macrophage Basal hepcidin expression

Hepcidinmediated ferroportin Normal plasmaFerroportin sma degradation hepcidin levels vels Hepatocyte Degraded ferroportin
Iron deciency Iron overload
Increased iron uptake Decreased Plasma Reduced Plasma plasma iron

Fe Normal plasma iron Hepcidin

Ferroportinp mediated mediat iron export exp

Basal hepcidin expression Hepatocyte

H m fe de

Plasma Normal plasma iron

Iron deciency
Increased ironNormal plasma uptake sma hepcidin levels vels Plasma
Iron deciency
Increased iron uptake

ntestinal nterocyte

Ferroportinp mediated mediat iron export exp

cb
Fe enterocyte Intestinal Hepcidin enterocyte Ferroportin Degraded ferroportin Intestinal

c
Macrophage Intestinal

Macrophage

Basal hepcidinReduced expression iron stores Hepatocyte

Hepcidinmediated ferroportin degradation

iron uptake

Reduced iron stores

enterocyte Macrophageiron

De

Increased iron stores

I pl Ferro Ferroportinme mediated iron export

Reduced plasma iron

c
Macrophage Intestinal enterocyte

Iron overload

Reduced iron stores

Decreased iron uptake

Increased Macrophage iron plasma

Reduced hepcidin expression

Plasma

Lee and Beutler Annu. Rev. Pathol. Mech. Dis. 2009. 4:489515 Increased Reduced plasma iron plasma asma iron Reduced plasma Reduced

Ferro Ferroportinme mediated iron export

asma Reduced plasma Increased vels hepcidin levels iron stores

ma High plasma vels hepcidin levels

Plasma

ma High plasma vels hepcidin levels

Hepatocyte Hepatocy

Hepcidin Hepcidin-mediated ferro ferroportin degr degradation

vels hepcidin levels

MEDI1012 - Biochemistry

hepcidin Ferro Ferroportinexpression me mediated

iron export

Anemia of inammatory disease mmatory dis sease High hepcidin

e
Macrophage Intestinal enterocyte

Hepatocyte plasma HepatocyHigh ma

Intestinal Hepcidin Hepcidin-mediated Elevated enterocyte ferro Iron ferroportin uptake degr degradation

expression

Hepatocyte iron

iro

Annu. Rev. Pathol. Mech. Dis. 2009.4:489-515. Downloaded from www.annualr by University of Queensland on 09/01/10. For personal use only.

iron uptake

Normal plasma iron

asma sma levels vels

Ferroportinp mediated mediat Intestinal iron exp Increased enterocyte export iron uptake Hepcidinmediated ferroportin Basal hepcidin degradation Plasma expression

Iron deciency

Macrophage Reduced iron stores

Ferroportinp Hepatocyte Degraded mediated mediat Increased Reduced ferroportin iron export iron expplasma plasma iron HepcidinIron overload Ferroportinc Normal plasma Ferro sma mediated hepcidin levels mediated Macrophage vels me Intestinal ferroportin Decreased iron export ma Basal hepcidinHigh plasma enterocyte iron uptake vels hepcidin levels degradation expression asma Reduced plasma Reduced Increased High hepcidin
vels hepcidin levels hepcidin iron stores expression Plasma Hepatocyte Hepatocy

The Hepcidin Hypothesis - 2

Increased iron stores Plasma

Fe Hepcidin Reduced iron stores Normal Ferroportin plasma iron

Plasma Macrophage

c
Intestinal enterocyte

Normal iron stores

Decreased iron uptake

ferroportin

Iron overload
Macrophage

Fe Hepcidin Hepcidin Hepcidin-mediated

ferro ferroportin degr degradation

Ferroportin Degraded ferroportin

Hepatocyte

expression

Hepatocyte

brroportinrro

d Iron deciency

Increased plasma iron mmatory disease Anemia of inammatory dis sease

me mediated ntestinal on export nterocyte

cyte cy

Intestinal Macrophage Hepcidin Hepcidin-mediated Elevated enterocyte ferro ferroportin Macrophage Iron uptake Increased ma High plasma degr degradation iron accumulation ironhepcidin levels uptake vels High hepcidinReduced Plasma expression iron stores iron accumulation Hepatocyte Low Plasma plasma iron

Intestinal enterocyte Intestinal

e c

Hemochromatosis
High iron uptake Decreased Plasma High Plasma plasma iron

Iron overload
Macrophage

enterocyte

Macrophage
Low iron stores

iron uptake

Increased iron stores

isease is sease
Macrophage

Reduced Intestinal High iron plasmaplasma enterocytema

Hemochromatosis

iron ccumulation

me mediated Lee and Beutler Annu. Rev. Pathol. iron export Mech. Dis. 2009. 4:489515
High plasma iron

High hepcidin levels iron uptake vels High hepcidin Low expression iron Ferro Ferroportin- stores Plasma Hepatocyte

Hepcidin-mediated ferroportin Macrophage degradation

Increased ferroportinmediated Iron export

Increased High iron plasma iron

Low hepcidin expression Hepatocyte

Increased ferroportinmediated Iron export

asma Reduced plasma MEDI1012 - Reduced Biochemistry vels hepcidin levels hepcidin pcidin-mediated expression High iron ferroportin Increased 492 Lee Beutler Hepatocyte Hepatocy degradation ferroportin-

ma High plasma vels hepcidin levels

asma Low pla plasma din l l hepcidin levels

Hepcidin Hepcidin-mediated ferro ferroportin degr degradation

Increased ferroportin-

High hepcidin expression Hepatocyte

The Duodenal Crypt Cell Programming Hypothesis - 1

Villus enterocytes differentiate from crypt cells during
on the basolateral surface Program the level of Fe transport expressed on differentiation to villus absorptive enterocytes.

migration from the crypts to the apex of the villus. The crypt cells sense plasma Fe via the HFE-TfR1 complex

In HFE-related HH
Loss of functional HFE protein Decreased TfR1-mediated Fe uptake by crypt cells Relatively Fe-deficient enterocyte phenotype; increased Fe
absorption by the villus enterocytes.

MEDI1012 - Biochemistry

The Duodenal Crypt Cell Programming Hypothesis - 2

MEDI1012 - Biochemistry

Fleming, R. E. et al. (2004) Clin. Liver Dis. 8:755 73

Iron Deficiency and Anaemia

The most common cause of anaemia Characterised by small, pale RBCs and Fe depletion Major causes are:
1. 2. 3. 4. 5.
Blood loss (occult GI & menstrual bleeding) Pregnancy (loss of Fe to foetus) Rapid growth (esp. 0-2 yr) Maladsorption Dietary insufficiency

MEDI1012 - Biochemistry

What do RBCs look like?

Normal peripheral blood smear

Anaemic peripheral blood smear

MEDI1012 - Biochemistry

Pathophysiology of Anaemia
Fe loss exceeds Fe intake leading to depletion of
storage Fe.

Fe stores can no longer support needs of erythroid

marrow.

Anaemia with normal appearing RBCs. Microcytosis and then hypochromia. Fe deficiency affects all tissues.
MEDI1012 - Biochemistry

Iron Deficiency and the Immune System

Low Fe can protect against some infectious agents
e.g. plasmodia (malaria) and mycobacteria (TB)

Generally it results in immune deficits

macrophage bacteriocidal activity

neutrophil myeloperoxidase activity

T-cell number

Humoral function is spared.

MEDI1012 - Biochemistry

Other Effects of Iron Deficiency

Pregnancy
birth weight length of gestation (prematurity)

Skeletal muscle
lethargy, apathy, listlessness decreased exercise tolerance altered metabolism e.g. lactate production
MEDI1012 - Biochemistry

References
Lee and Beutler (2009) Regulation of Hepcidin and IronOverload Disease Annu. Rev. Pathol. Mech. Dis. 4:489 515 hemochromatosis. Clin. Liver Dis. 8:755 773

Fleming, R. E. et al. (2004) Pathogenesis of hereditary Roy et al. (1999) The hereditary hemochromatosis

protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells. J. Biol. Chem. 274:9022. Biochimica et Biophysica Acta 1790:309325

Garrick & Garrick (2009) Cellular Iron Transport.

MEDI1012 - Biochemistry