MEDI1012_Iron(1)

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Iron Metabolism: Too little, too much!

Justin Ridge & Simon Worrall School of Chemistry and Molecular Biosciences

MEDI1012 - Biochemistry

Iron   A metallic element

  Toxic but also an essential nutrient

  An important component of many macromolecules

  Able to undergo redox reactions (Fe2+, Fe3+, Fe4+)

  35mg/kg in women and 45 mg/kg in men


Fe 26

55.85

Iron Content of Foods   Iron is found in virtually all foods. Two types:

  Non-haem – fruits, vegetables, grain products

  Haem – meat and poultry


RDA

Males 8 mg

Females 18 mg

Pregnant 27 mg

RDA for vegetarians

Males 40 mg

Females 60 mg

Iron Trafficking


Total Body Pools Males 3.5g Females 2.5g

DIET 10 mg/

day

GUT

Labile Iron

ECF

FAECES

Plasma

1 mg/day

Ferritin

~11.5%

RES

RBC

~70%

Immature RBC

Enzyme cofactor ~10%

Haemosiderin

~10%

Iron stores

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.


Duodenal Absorption of Ferrous Iron


Gut Lumen Blood Enterocyte

Fe3+ Fe2+

Tf

vitamin C ferrireductase

Fe2+ Fe2+

H+

DMT1 H+

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

Fe3+

Cp Ferritin (Fe3+)

Fe2+

Hp

Ireg-1

Ceruloplasmin (Cp)   Plasma protein

  Carries Cu from liver to periphery

  Controls oxidation reduction reactions of Fe, Fe transport & utilization

  Hephaestin analogous but has a trans-membrane domain at C-terminus

MEDI1012 - Biochemistry Ft

O2 or –S-S-

H2O or –SH

Cp Cu2+ Cp Cu+

Fe2+ Fe3+

Tf

Proposed Duodenal Absorption of Haem Iron


Gut Lumen Enterocyte

haemoglobin

haem

globins are proteolysed

inorganic Fe

Metallo-porphyrin (H)

degraded by haem oxidase

Endocytosis ????

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


Transmembrane Iron Transport


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

i

TfR DMT1

5

1

endocytosis 2

i

endosomes H+

3

i

4

DMT1

ferric reductase

Fe3+ → Fe2+

H+

Regulation of iron uptake

 Uptake of iron affected by two proteins:

 HFE

 Hepcidin


HFE   Originally known as HLA-H and expressed by most

cell types   Binds β2-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)


HFE

MEDI1012 - Biochemistry Based on Roy et al. J. Biol. Chem. 274:9022, 1999.

HFE

Fe Fe

Fe

DMT 1

H+

HFE: 1. Decreases TfR capacity 2. Stabilises Tf binding to TfR; less Fe is released into endosome; Tf returns to cell surface carrying Fe HFE

Fe Fe

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


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


Action of Hepcidin on Crypt Cells and Macrophages

  Interacts with β2-HFE-TfR1

  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 98:8160–8162.

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 (Fe-

dependent function????)


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

  γ-aminolevulinate synthase (Hb synthesis)   Transferrin (in liver)


Post-transcriptional Control


Iron Deficiency

IRE-BP Inactive Active

5’ AAAAAA

Inhibited degradation

5’ AAAAAA

TfR mRNA

IRE-BP

= increased translation

IRE-BP

ferritin mRNA

No translation

5’ AAAAAA

IRE-BP

5’ AAAAAA

IRE-BP

Congenital Diseases Associated with Iron Metabolism


Disease Affected Gene Function

Haemochromatosis HFE Modulation of Fe transport

Friedrich’s ataxia FRDA Frataxin – a putative Fe storage protein

Hallervorden-Spatz PANK2 Hb synthesis

Hyperferritinaemia-cataract

IRE of L-ferritin mRNA

Fe storage protein

Hypotransferrin-aemia Transferrin (?) Fe transport

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 in HFE gene; affects ~73,000 people in Australia.



  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


Iron Toxicity


Fe2+ Fe3+

O2 O2- Fe2+ OH

H2O2 H2O Cellular Damage catalase &

peroxidases

Superoxide dismutase

Tissue damage induces fibrosis

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

Histopathology



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

  The duodenal crypt cell programming hypothesis


The Hepcidin Hypothesis - 1


ANRV368-PM04-20 ARI 9 December 2008 15:27

Macrophage

Plasma

Hepatocyte

Intestinalenterocyte

Ferroportin-mediated

iron export

Reducedplasma iron

Increasediron uptake

Reduced plasmahepcidin levels

Ferroportin

HepcidinFe

Degradedferroportin

Reducediron stores

Hepatocy

Ferrome

iron

asmavels

Reduced hepcidin

expression

Iron deficiency

Macrophage

Plasma



iron export

Normalplasma iron

Normal plasmahepcidin levels

Hepcidin-mediated

ferroportindegradation

Macrophage

Normaliron stores

Hepatocyte

pmediat

iron exp

smavels

Basal hepcidinexpression

Normal iron absorption

Macrophage

Plasma

Hepatocyte


Lowplasma iron

High plasmahepcidin levels

Hepatocyte

mavels

High hepcidinexpression

Anemia of inflammatory disease

Macrophage

Plasma

Hepatocyte


Increasedplasma iron


Hepcidin-mediatedferroportin

degradation

Macrophage

Increasediron stores

Hepatocyte

mavels

Hepcidinferro

degr


Iron overload

Macrophage

Plasma

Hepatocyte


Increasedferroportin-

mediatedIron export


mediatedIron export

Highplasma iron

Low plasmahepcidin levels

Macrophage

Lowiron stores

pladin levels

Low hepcidinexpression

Hemochromatosis

a

b c

d e

Decreasediron uptake

Highiron uptake

Plasma

enterocyte

Lowplasma iron

Elevatedon uptaIr ke

iron accumulation

Macrophage

seasemmatory dis

ironaccumulation


degradation

Highplasma iron

asmal l

High iron

Normaliron uptake

492 Lee · Beutler

Ann

u. R

ev. P

atho

l. M

ech.

Dis

. 200

9.4:

489-

515.

Dow

nloa

ded

from

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Uni

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For

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Macrophage

Plasma

Hepatocyte



iron export

Reducedplasma iron



Ferroportin

HepcidinFe

Degradedferroportin

Reducediron stores

Hepatocy

Ferrome

iron

asmavels

Reduced hepcidin

expression

Iron deficiency

Macrophage

Plasma



iron export

Normalplasma iron


Hepcidin-mediated


Macrophage

Normaliron stores

Hepatocyte

pmediat

iron exp

smavels



Macrophage

Plasma

Hepatocyte


Lowplasma iron


Hepatocyte

mavels



Macrophage

Plasma

Hepatocyte





degradation

Macrophage


Hepatocyte

mavels

Hepcidinferro

degr


Iron overload

Macrophage

Plasma

Hepatocyte



mediatedIron export


mediatedIron export

Highplasma iron


Macrophage

Lowiron stores

pladin levels


Hemochromatosis

a

b c

d e


Highiron uptake

Plasma

enterocyte

Lowplasma iron


iron accumulation

Macrophage

seasemmatory dis

ironaccumulation


degradation

Highplasma iron

asmal l

High iron

Normaliron uptake

492 Lee · Beutler

Ann

u. R

ev. P

atho

l. M

ech.

Dis

. 200

9.4:

489-

515.

Dow

nloa

ded

from

ww

w.a

nnua

lrevi

ews.o

rgby

Uni

vers

ity o

f Que

ensl

and

on 0

9/01

/10.

For

per

sona

l use

onl

y.


Macrophage

Plasma

Hepatocyte



iron export

Reducedplasma iron



Ferroportin

HepcidinFe

Degradedferroportin

Reducediron stores

Hepatocy

Ferrome

iron

asmavels

Reduced hepcidin

expression

Iron deficiency

Macrophage

Plasma



iron export

Normalplasma iron


Hepcidin-mediated


Macrophage

Normaliron stores

Hepatocyte

pmediat

iron exp

smavels



Macrophage

Plasma

Hepatocyte


Lowplasma iron


Hepatocyte

mavels



Macrophage

Plasma

Hepatocyte





degradation

Macrophage


Hepatocyte

mavels

Hepcidinferro

degr


Iron overload

Macrophage

Plasma

Hepatocyte



mediatedIron export


mediatedIron export

Highplasma iron


Macrophage

Lowiron stores

pladin levels


Hemochromatosis

a

b c

d e


Highiron uptake

Plasma

enterocyte

Lowplasma iron


iron accumulation

Macrophage

seasemmatory dis

ironaccumulation


degradation

Highplasma iron

asmal l

High iron

Normaliron uptake

492 Lee · Beutler

Ann

u. R

ev. P

atho

l. M

ech.

Dis

. 200

9.4:

489-

515.

Dow

nloa

ded

from

ww

w.a

nnua

lrevi

ews.o

rgby

Uni

vers

ity o

f Que

ensl

and

on 0

9/01

/10.

For

per

sona

l use

onl

y.

Lee and Beutler Annu. Rev. Pathol. Mech. Dis. 2009. 4:489–515

The Hepcidin Hypothesis - 2


Lee and Beutler Annu. Rev. Pathol. Mech. Dis. 2009. 4:489–515


Macrophage

Plasma

Hepatocyte



iron export

Reducedplasma iron



Ferroportin

HepcidinFe

Degradedferroportin

Reducediron stores

Hepatocy

Ferrome

iron

asmavels

Reduced hepcidin

expression

Iron deficiency

Macrophage

Plasma



iron export

Normalplasma iron


Hepcidin-mediated


Macrophage

Normaliron stores

Hepatocyte

pmediat

iron exp

smavels



Macrophage

Plasma

Hepatocyte


Lowplasma iron


Hepatocyte

mavels



Macrophage

Plasma

Hepatocyte





degradation

Macrophage


Hepatocyte

mavels

Hepcidinferro

degr


Iron overload

Macrophage

Plasma

Hepatocyte



mediatedIron export


mediatedIron export

Highplasma iron


Macrophage

Lowiron stores

pladin levels


Hemochromatosis

a

b c

d e


Highiron uptake

Plasma

enterocyte

Lowplasma iron


iron accumulation

Macrophage

seasemmatory dis

ironaccumulation


degradation

Highplasma iron

asmal l

High iron

Normaliron uptake

492 Lee · Beutler

Ann

u. R

ev. P

atho

l. M

ech.

Dis

. 200

9.4:

489-

515.

Dow

nloa

ded

from

ww

w.a

nnua

lrevi

ews.o

rgby

Uni

vers

ity o

f Que

ensl

and

on 0

9/01

/10.

For

per

sona

l use

onl

y.


Macrophage

Plasma

Hepatocyte



iron export

Reducedplasma iron



Ferroportin

HepcidinFe

Degradedferroportin

Reducediron stores

Hepatocy

Ferrome

iron

asmavels

Reduced hepcidin

expression

Iron deficiency

Macrophage

Plasma



iron export

Normalplasma iron


Hepcidin-mediated


Macrophage

Normaliron stores

Hepatocyte

pmediat

iron exp

smavels



Macrophage

Plasma

Hepatocyte


Lowplasma iron


Hepatocyte

mavels



Macrophage

Plasma

Hepatocyte





degradation

Macrophage


Hepatocyte

mavels

Hepcidinferro

degr


Iron overload

Macrophage

Plasma

Hepatocyte



mediatedIron export


mediatedIron export

Highplasma iron


Macrophage

Lowiron stores

pladin levels


Hemochromatosis

a

b c

d e


Highiron uptake

Plasma

enterocyte

Lowplasma iron


iron accumulation

Macrophage

seasemmatory dis

ironaccumulation


degradation

Highplasma iron

asmal l

High iron

Normaliron uptake

492 Lee · Beutler

Ann

u. R

ev. P

atho

l. M

ech.

Dis

. 200

9.4:

489-

515.

Dow

nloa

ded

from

ww

w.a

nnua

lrevi

ews.o

rgby

Uni

vers

ity o

f Que

ensl

and

on 0

9/01

/10.

For

per

sona

l use

onl

y.


Macrophage

Plasma

Hepatocyte



iron export

Reducedplasma iron



Ferroportin

HepcidinFe

Degradedferroportin

Reducediron stores

Hepatocy

Ferrome

iron

asmavels

Reduced hepcidin

expression

Iron deficiency

Macrophage

Plasma



iron export

Normalplasma iron


Hepcidin-mediated


Macrophage

Normaliron stores

Hepatocyte

pmediat

iron exp

smavels



Macrophage

Plasma

Hepatocyte


Lowplasma iron


Hepatocyte

mavels



Macrophage

Plasma

Hepatocyte





degradation

Macrophage


Hepatocyte

mavels

Hepcidinferro

degr


Iron overload

Macrophage

Plasma

Hepatocyte



mediatedIron export


mediatedIron export

Highplasma iron


Macrophage

Lowiron stores

pladin levels


Hemochromatosis

a

b c

d e


Highiron uptake

Plasma

enterocyte

Lowplasma iron


iron accumulation

Macrophage

seasemmatory dis

ironaccumulation


degradation

Highplasma iron

asmal l

High iron

Normaliron uptake

492 Lee · Beutler

Ann

u. R

ev. P

atho

l. M

ech.

Dis

. 200

9.4:

489-

515.

Dow

nloa

ded

from

ww

w.a

nnua

lrevi

ews.o

rgby

Uni

vers

ity o

f Que

ensl

and

on 0

9/01

/10.

For

per

sona

l use

onl

y.

The Duodenal Crypt Cell Programming Hypothesis - 1

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

on the basolateral surface

  Program the level of Fe transport expressed on differentiation to villus absorptive enterocytes.

  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.


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.  Blood loss (occult GI & menstrual bleeding) 2.  Pregnancy (loss of Fe to foetus)

3.  Rapid growth (esp. 0-2 yr) 4.  Maladsorption

5.  Dietary insufficiency


What do RBCs look like?


Normal peripheral blood smear

Anaemic peripheral blood smear

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.


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.


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


References   Lee and Beutler (2009) Regulation of Hepcidin and Iron-

Overload Disease Annu. Rev. Pathol. Mech. Dis. 4:489–515

  Fleming, R. E. et al. (2004) Pathogenesis of hereditary hemochromatosis. Clin. Liver Dis. 8:755– 773

  Roy et al. (1999) The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells. J. Biol. Chem. 274:9022.

  Garrick & Garrick (2009) Cellular Iron Transport. Biochimica et Biophysica Acta 1790:309–325


MEDI1012_Iron(1)

Documents

reduced iron

increased

iron uptake

iron deficiency

crypt cells

iron deciency

iron overloadmacrophage

aintestinal