This document discusses iron homeostasis in the human body. It covers the following key points: 1. Dietary iron is absorbed in the duodenum and upper jejunum. It is transported across intestinal cells via DMT1 and exported into circulation by ferroportin. 2. In the circulation, iron is carried by the protein transferrin and delivered to cells via transferrin receptors that undergo endocytosis. 3. Iron is either used in cellular processes or stored in ferritin complexes in tissues. Iron is also recycled from senescent red blood cells by macrophages. 4. Tight regulation of iron absorption, transport, and storage is needed as iron is essential but also toxic in excess

1. IRON HOMEOSTASIS
1

2. 1. Systemic iron homeostasis.Tomas Ganz. physiol rev 2013; 93:
1721-41.
(review article)
2. Regulation of iron transport and the role of transferrin.
Gkouvatsos K, Papanikolaou G, Pantopoulos K. Biochimica et
biophysica acta 2012; 1820: 188-202.
(review article)
3. Molecular control of iron transport.Tomas Ganz. J am soc
nephrol 2007; 18: 394-400.
4. Murray RK, Jacob M, Varghese J. Plasma proteins and
immunoglobulins. In Michael Weitz, Brian Kearns.Harper’s
illustrated biochemistry (29th ed). New York: McGraw-Hill
2012: 629-49.
5. Trefor Higgins, James C Barton. Hemoglobin, iron and
bilirubin.In Burtis CA, Ashwood ER, Bruns DE.Tietz textbook
of clinical chemistry and molecular diagnostics(5th ed).New
Delhi:Elsevier 2012: 985-1030
2

3. ABOUT IRON:
- Has unique ability to serve as both electron donor and acceptor.
- Because it is highly reactive and toxic, in biological organisms,
its chemical reactivity is constrained and directed by its
association with prosthetic groups and proteins.
- Iron containing proteins (FEW EXAMPLES)
• carry or store oxygen (Hb and Myoglobin)
• Produce energy (cytochromes and cytochrome oxidases etc)
• Host defense: Lactoferrin, siderocalin, NGAL (Neutrophil
Gelatinase-associated lipocalin) etc.
• NADPH oxidase, NO synthase, ribosome nucleotide reductase,
Prolyl/lysyl hydroxylase , myeloperoxidase etc..
3

4. Distribution of iron in body:
In an average 70 kg male:
- Total: 3-4 grams
- >2 grams in haemoglobin
- About 1 grams stored as ferritin (mostly liver)(in females
100-400mg)
- about 3-4 mg as transferrin
- About 300 mg in myoglobin and other enzymes
- Daily absorption : about 1mg/day (average American diet
contains >10mg/day of iron)
- Daily loss : about 1mg/day (more in females 1.5-2)
4

5. THE MAJOR IRON FLOWS:
5

6. DIETARY ABSORPTION OF IRON:
• Absorbed either as a heme-iron or non-heme iron.
NON HEME IRON:
- About 90% of dietary iron
- Absorbed primarily in proximal duodenum.
- Also in upper jejunum.
- Iron absorbed is almost equal to iron lost per day
[loss through intestinal epithelial exfoliation, epidermal
sloughing, bleeding and ,in females, menstruation]
6

7. ABSORPTION CONT’D
- Dietary iron usually presents in ferric form.
- At epithelial surface, it is reduced to ferrous form by
Ferric reductase [ one major example being
duodenal cytochrome called dCytB]
- In several studies, disruption of dcytb, however, didn’t
show significant effect in body iron stores…So, the
existence of alternative mechanisms for iron reduction
have been suggested.
- Vitamin C is one of the important electron donor for
iron reduction, suggesting its vital role in iron
absorption.
- Gastric acids and number of other reducing agents
also play major role.
7

8. - After reduction it gets transported as Fe2+ by Divalent
metal transporter 1 (DMT1).
- DMT1 is a member of SLC family of membrane
transport proteins and also known as SLC11A2.
- Experiments have shown that DMT1 is indispensable
for dietary iron absorption (unlike dcytb for reduction).
- DMT1 also transports Mn2+, Co2+, Zn2+,Cu2+ and Pb2+ .
- Once inside the enterocytes, iron is either stored as
ferritin , or transferred across basolateral membrane
into the circulation via Ferroportin(FPN1).
- Ferroportin also belongs to SLC transport family, and
also known as SLC40A1.
ABSORPTION CONT’D
8

9. - To transport, iron must be in ferric form.
- So, ferroportin mediated absorption occurs in conjunction
with Hephaestin,which is a membrane bound copper
containing Ferroxidase, analogous to Cerruloplasmin (which
also acts as ferroxidase, alpha 2 globulin, 160kd, synthesized
by liver).
-Now, in plasma, ferric iron is transported by transferrin.
[Apotransferrin when bound to iron is called transferrin
or holotransferrin(Tf-Fe)]
- Excess iron stored as ferritin is lost when enterocytes are
sloughed off into gut lumen.
[ Short life span of enterocytes ensure that the iron that is not
transferred to plasma is shed into fecal stream]
[Under stress condition that may exceed oxidative capacity of
Hephestin, cerruloplasmin accomplishes the oxidation of
ferrous iron to ferric] ]
ABSORPTION CONT’D
9

10. • transferrin
is
expressed
predomin-
antly in
liver.
lower
amounts
also in
brain,
testis,
kidney
etc.
10

11. HEME IRON:
- Heme iron absorption in the enterocyte is independent of the
above mentioned mechanism.
- Dietary heme is taken up by apical heme transporters(e.g.
HCP1, heme carrier protein1, which is predominantly a
folate transporter).
- In enterocytes, it is broken up by heme oxygenase, to
release iron.
- This iron is either stored as ferritin or transported into
circulation by ferroportin.
- Sometimes, heme may also be exported as an intact
molecule across the basolateral membrane to plasma via
heme exporter FLVCR (Feline leukemia virus, subgroup
C, receptor), and then scavenged by circulating Hemopexin
(Hpx).
ABSORPTION CONT’D
11

12. TRANSFERRIN:
- It is a plasma protein (a glycoprotein, beta globulin..mol
wt 76 kilo dalton) that transports ferric iron in plasma.
- Free iron in plasma is extremely reactive, as illustrated
by the following Fenton reaction:
Fe2+ + H2O2 Fe3+ + OH. + OH-
[the free radical can oxidize cellular macromolecules
resulting in tissue damage]
- So, it needs a transporter transferrin (Tf).
- Tf has 2 high affinity binding sites for ferric iron.
12

13. - Plasma concentration of Tf is 300 mg/dl.
- This amount can bind approx. 300 µg/dl of iron.
- This represents Total Iron Binding Capacity(TIBC) of
plasma.
- Normally, Tf is 30% saturated.
- Saturation decreases to about 16% during severe iron
deficiency.
- Saturation is more than 45% during iron overload.
- During congenital disorders of glycosylation and
chronic alcoholism Tf fails to be glycosylated, resulting in
increased amount of CDT (Carbohydrate Deficient
Transferrin).
- [NOTE: - CDT is also an important marker for chronic
alcoholism]
TRANSFERRIN CONT’D:
13

14. Tf- TfR1 cycle/ Transferrin cycle:
14

15. - Transferrin Receptor 1 (TfR1) is present on the
surface of almost all cell, esp. erythroid precursors
and in the bone marrow.[high TfR1 also in
intestinal epithelial cells, placental
syncitiotrophoblast and neoplastic cells]
- TfR1 is a transmembrane glycoprotein, which forms
a disulfide bonded homodimer, which can bind 1 Tf
molecule at each of its subunit.
- When iron is added to Tf its affinity to TfR1
increases.
[ Diferric Tf has 30 times more affinity to TfR1 than
monoferric Tf…AND 500 times more than apo-Tf]
Transferrin cycle CONT’D:
15

16. - After holo-Tf binds to TfR1, the complex undergoes
endocytosis via Clathrin coated pits.
- Early endosome matures to form late endosome.
- The acidic pH inside late endosome (by proton pump
ATPase to pH5.5) causes iron to dissociate from Tf.
- Tf still remains bound to TfR1 inside endosome.
- DMT1 present on endosomal membrane transports the
free iron [ After it is reduced to ferrous form by ferric
reductase STEAP3(Six Transmembrane Epithelial
Antigen of prostrate3)] to cytoplasm.
- TfR1-apoTf complex is recycled back to cell
membrane.
- ApoTf is released from TfR1 .
Transferrin cycle CONT’D:
16

17. 17

18. Minor TfR1 independent mechanisms of holoTf
endocytosis have also been found
- In PCT via membrane receptor Cubilin.
- TfR2 has been expressed, mainly on hepatocytes.
However its low tissue distribution (and low affinity
to Tf compared to TfR1) doesn’t support its role in
iron uptake.
[MORE OF TfR2 later]
- GADPH (Glyceraldehyde 3 Phosphate
dehydrogenase) and proteoglycans have also been
reported to mediate endocytosis of holoTf in
macrophages and hepatocytes
[NOTE: -TfR2 not expressed in intestinal crypt cells]
18

19. Transferrin independent mechanisms in Macrophages
- Erythrocytes have normal life span of 120 days
- Iron in Senescent or damaged Erythrocytes is recycled
by macrophages.
- After lysis of RBC, Hb or free heme are released.
- Haptoglobin (synthesized by liver) binds to free Hb
and promotes endocytosis in macrophages (of RES) upon
recognition by CD163 receptor.
- Free heme is scavenged by hemopexin and the resulting
complex is endocytosed via CD91 receptor( in
macrophages , hepatocytes.)
- RBC may directly be endocytosed too.
19

20. - Heme oxygenase converts heme to biliverdin, releasing
iron.
- Iron released from heme is exported from phagocytic
vesicle in macrophage by NRAMP1(Natural resistance
associated macrophage protein1), a transporter
homologous to DMT1.
- Then, by ferroportin in the macrophage membrane, the
iron releases to circulation.
- For oxidation of Fe2+ , Cerruloplasmin is required.
- Then , in circulation,to transferrin.
This is actually the major source of iron in the body (25
mg/day), rather than intestinal absorption (1-
2mg/day)
Transferrin independent mechanisms in Macrophages
CONT’D
20

21. 21

22. ABOUT FERRITIN :
• Under normal circumstances, iron is stored as ferritin in
various tissues and constitutes approx. 1g of total body
iron content.
• Also stored as hemosiderin (a partially degraded
ferritin), but more about it later.
• Ferritin consists of a protein shell apoferritin(440 kDa)
that surrounds an interior ferric oxyhydroxide
crystalline core (FeOOH)x .
• The apoferritin shell contains 24 ferritin chains that that
may be classified as L (for light) or H (heavy) chains.
• The diameter of the shell is 12-13mm, and its interior 7-
8mm. 22

23. - Ferritin only takes ferrous iron, which is oxidized to
ferric form by a catalytic site on the H chain.
- H chains also contain small intra sub unit channels that
facilitate entry of iron into storage cavity of the
molecule.
- The exact composition of FeOOH core crystal varies
according to species, and may also contain some amount
of phosphates.[3000-4500 ferric atoms in 1 ferritin]
- In humans it is ferrihydrite (Fe2O3.9H2O).
- Function of L chain isn’t properly known (proposed to
play a role in ferritin nucleation and stability).
ABOUT FERRITIN CONT’D:
23

24. - Release of iron from ferritin is probably nonenzymatic and
may involve reduction by reduced flavin nucleotide or
others.
- Ferritin is present in nearly all cells and provide a reserve of
iron that is readily available for formation of Hb and other
proteins.
Besides this, some amounts of ferritin (50-200µg/dl) are also
found in plasma.
Such ferritin contains mostly L chains, and is iron poor
{mostly apoferritin}
They increase in amount during liver injury and other infections
not associated with iron overload.
However, their amount in plasma proportionate the total iron
stores in body, and thus considered to be an indicator of
body iron stores.
ABOUT FERRITIN CONT’D:
24

25. - It is an aggregated, partially deproteinized ferritin that is
formed when ferritin is partially degraded in secondary
lysosomes.
- While ferritin is soluble, it is insoluble in aqueous
solutions [which forms traditional basis of
distinguishing these 2 proteins]
- From hemosiderin, iron is only slowly released.
- Detected in tissues under condtions of iron overload
(hemosiderosis), by histological stains (e.g. prussian
blue).
HEMOSIDERIN:
25

26. IRON homeostasis in a nutshell
• We may broadly classify it into Cellular and Systemic.
• If free iron is more inside the cell, it is stored as ferritin or
exported through ferroportin.
• If Heme containing iron is more, it would either get degraded
into free form and stored as ferritin, or exported from
ferroportin or directly exported through heme exporter like
FLCVR.
• In systemic circulation, it is transported by transferrin.
• If there is iron deficiency inside the cell, transferrin(through
TfR1) imports it inside the cell.
• And there is iron absorption from the intestine too, as needed.
But this seemingly simple mechanism is very very delicately
regulated.
26

27. CELLULAR IRON METABOLISM
- Let’s begin at a point where iron is endocytosed through
Tf-TfR1 cycle inside the cell.
- From endosome the iron (ferrous) is transported to
cytosol through DMT1.
- This forms a transient pool of redox-active iron called
labile iron pool. (LIP)
- It may be associated with chelates like citrate,
ATP,AMP, pyrophosphate etc
- This represents only a fraction (3-5%) of total cellular
iron, but it is important to note that this LIP represents
the cellular iron status.
- This LIP level is sensed by intracellular censors
triggering homeostatic adaptive responses.
27

28. - Now, the iron enters mitochondria.[Where it will be
utilized in various enzymatic processes of cellular
metabolism]
- Mitochondrial entry is by yet another transporter,
Mitoferrin[Mfrn, also a member of SLC transporter
…AKA SLC25A37]
- Several experiments in hemoglobin synthesizing
erythroid cells have also provided evidence that iron can
be directly transported from endosome to the
mitochondria via ‘kiss and run’ mechanism, where 2
organelles exchange iron when in direct contact.
CELLULAR IRON METABOLISM CONT’D:
28

29. - Cellular iron not required immediately for metabolic
purposes are sequestered in the cytosol as ferritin.
- or they may be transported through ferroportin, to
transferrin and then to other cells, if required.
- Heme iron may also export via FLVCR if the cell
expresses this transporter.
- In some cells, a distinct isoform of ferritin has also been
found in mitochondria.
- Their function is to detoxify excessive iron accumulated
in these organelles under pathological conditions like
sideroblastic anemia.
CELLULAR IRON METABOLISM CONT’D:
29

30. CELLULAR REGULATION BY IRE/IRP SYSTEM:
• Iron Responsive Element (IRE) are 30 nucleotides RNA
motifs that forms special stem loop (hairpin) structures.
• Several proteins of iron metabolism are encoded by
mRNAs that contain IREs.
• IREs may be on 5’ or 3’ UTR (un translated region) of
such mRNAs that encode the proteins of iron
metabolism.
• IREs on 5’ UTR are present in mRNAs encoding :
- H and L ferritin, ALAS2 (erythroid specific5
aminolevulenic synthase2, key enzyme of heme
synthesis), HIF2α(hypoxia inducible factor 2α) etc
• IREs on 3’ UTR are present in mNAs encoding:
- TfR1, DMT1
30

31. - IRP (Iron regulatory protein) binds with IREs thus
creating a vital IRE/IRP system.
- IRE/IRP interaction on 5’UTR (e.g. ferritin encoding
mRNA) inhibits the translation of mRNA…thus no
proteins are formed.
- IRE/IRP interaction on 3’UTR (e.g.mRNA encoding
TfR1, DMT1) stabilizes the mRNAs, thus inducing
translation.
We’ll take a specific examples of Ferritin and TfR1
mRNAs and see how it works:
CELLULAR REGULATION BY IRE/IRP SYSTEM CONT’d:
31

32. - Ferritin mRNA has IRE on 5’UTR.
- There are normally 2 IRPS present, IRP1 and IRP2.
- IRP1 is abundant, IRP2 is less abundant and has its
strongest expression in intestinal and brain cells.
- When there is excessive iron supply, there is no binding
of IRP to IRE (So, NO IRE/IRP interaction)
……………………………………………………………
……………………………………………….………….
- TfR1 mRNA has IRE on 3’UTR.
- When there is excessive iron supply, there is no
IRE/IRP interaction.
CELLULAR REGULATION BY IRE/IRP SYSTEM CONT’d:
32

33. - Under such circumstances ( i.e. when no is no IRE/IRP
interaction) mRNAs of ferritin can be translated, and
hence ferritin is synthesized.
- Excess iron is now stored in the cytoplasm.
- In case of TfR1, the mRNA where IRE is not bound to
IRP is degraded….so, no TfR1 synthesis..no entry of
systemic iron into cell.
CELLULAR REGULATION BY IRE/IRP SYSTEM CONT’d:
33

34. 34

35. HOW IRON EXCESS PREVENTS IRE/IRP
INTERACTION??
- In case of IRP1, it involves insertion of 4Fe-4S cluster
that converts the protein (IRP1) into a cytosolic
aconitase(cAcn)
- When cells lack iron, iron sulfur cluster is not formed,
the protein again acts as IRP1.
- Thus iron excess promotes cAcn, iron deficiency
promotes IRP1.
- In case of IRP2 ,it undergoes iron and oxygen
dependant degradation following ubiquitination by
FBXL5 (F-Box and leucine rich repeat protein 5, an E3
ubiquitin ligase, that senses iron levels via Fe-O-Fe
center within its hemyrythrin domain.)
35

36. IRPs and iron independent signals:
- Both IRP1 and IRP2 can be induced (besides low Fe)
upon exposure of cells to H2O2 or NO, stimulating
TfR1 expression and iron uptake via DMT1.
- Hypoxia leads to IRP2 stabilization, but decreases IRE
binding activity of IRP1.
THIS PROVIDES SOME LINK BETWEEN IRON
METABOLISM, INFLAMMATION AND
HYPOXIC RESPONSES….and there are more to
come
36

37. HEPCIDIN AND SYSTEMIC IRON HOMEOSTASIS:
- Regulations by hepcidin
- Regulations of hepcidin
37

38. THESE 2 FACTS ARE ENOUGH TO ELUCIDATE
THE IMPORTANCE OF HEPCIDIN IN IRON
METABOLISM:
1. Injection of synthetic hepcidin into mice induced
profound hyperferrimia within 1 hour.
2. Complete deficiency of hepcidin caused juvenile
hemochromatosis.
38

39. What is hepcidin??
• It is a 25amino acid cationic peptide that contains 4
sulfide bonds.
• Encoded as a 84 amino acid prepropeptide and is
synthesized, processed and secreted predominantly by
hepatocytes.
• hepcidin gene is called HAMP.
39

40. WHAT DOES HEPCIDIN DO?
• It binds to ferroportin and promotes its
phosphorylation, internalization and lysosomal
degradation.
• We know, ferroportin is the unique cellular iron
exporter in mammals, and its expression in enterocytes
and macrophages determines the degree of intestinal
iron absorption and reticuloendothelial iron release.
• Thus hepcidin acts as a negative regulator of iron
absorption and release.
40

41. 41

42. 42

43. REGULATION OF HEPCIDIN:
• Hepcidin levels are mediated by various processes,
some of which are iron dependant and some are not.
• We shall talk in brief about both types of regulation of
hepcidin.
Fig. below shows several molecular pahways regulating
hepcidin transcription:
44

44. There are basically 2 ways of Hepcidin mRNA
transcription:
1. BMPR- SMAD way
2. IL6R- JAK-STAT way.
All of the other regulators(iron dependant or independent) control
hepcidin transcription from either of these 2 pathways..
BMPR- Bone Morphogenic Protein receptor,
IL6R- Interleukin 6 receptor,
JAK-Janus Kinase
STAT- Signal Transducer & Activator of Transcription
SMAD – homologous to both drosophilla protein Mothers against
decapentaplegic (MAD) and C. elegans protein SMA(hence
SMAD) 45

45. An important example of HFE regulation of hepcidin:
- Abbreviation for High Fe [AKA HUMAN
HEMOCHROMATOSIS PROTEIN].
- It is a Major Histocompatibility class 1 (MHC class1) like
molecule that is expressed on the cell surface, bound to β2
microglobulin and TfR1.
- Mice with severe disruption of either HFE or TfR2 have
been found to develop iron overload due to hepcidin
suppression.
- HFE hemochromatosis is actually the most prevalent form
of hereditary hemochromatosis.
So, there should be some interplay between HFE, TfR1,
TfR2 and hepcidin.
46

46. - HFE competes with holotransferrin(Tf-Fe) for TfR1.
- When there is excessive iron overload, HFE is freed from
TfR1.
- The displaced HFE now binds to TfR2.
[NOTE: TfR2 may bind with both HFE and holo Tf at once.
Holo-Tf (and not apo-Tf) actually stabilizes TfR2.]
- HFE- TfR2 interaction complex activates signaling to
hepcidin expression .
[NOTE:-However, some independent effects of HFE and TfR2
on hepcidin regulation (besides these) have also been
suggested because Mice with deficient HFE and TfR2
developed more severe iron overload than mice with either
HFE or TfR2 deficiency.]
An important example of HFE regulation of hepcidin
CONT’D:
47

47. - Thus the function of TfR1 in this mechanism is actually
to prevent binding of HFE to TfR2.
- HFE-TfR2 complex now activates intracellular signaling
cascade.
- ERK/MAPK pathway (Extracellular signal regulated
kinase/ mitogen activated protein kinase) is found to
be activated by HFE/TfR2.
- HFE- TfR2 complex has also found to activate
BMP6/SMAD signalling.
- It is thought that there is a possible cross talk
between ERK/MAPK and BMP6/ SMAd
pathway..which finally induces transcription of
HAMP gene and finally hepcidin.
An important example of HFE regulation of hepcidin
CONT’D:
48

48. Displaced HFE from TfR1 binds to TfR2, along with holo
Tf(Tf-Fe), to signal via the ERK/MAPK pathway to induce
hepcidin.
49

49. BONE MORPHOGENIC PROTEIN (BMP, esp. BMP6) AND
HEPCIDIN REGULATION
• BMP6/SMAD pathway of hepcidin regulation is a
major pathway of hepcidin regulation.
• Increased hepatic iron is thought to induce hepcidin
expression via this pathway.
• HFE-TfR2-holoTf is also thought to enter the
intermediate of this pathway for hepcidin regulation.
The core thing is the binding of BMP to BMPR leading
to phosphorylation of SMAD(intracellular signalling
protein)
[Hemojuvelin (HJV) acts as the co-receptor of BMPR and
genetic mutation to form this protein forms the basis for
iron overload condition called HJV Hemochromatosis]
50

50. BMP binds to BMPR and HJV (co receptor) to activate R-SMAD.R-SMAD
dimerizes with SMAD 4,translocates to nucleus where it binds to BMP-
RE.This results in transcriptional activation of hepcidin.
51

51. 52

52. Inflammation and hepcidin expression:
• Till now we studied hepcidin regulation pathway that
were somehow related to iron content in body (liver or
serum).
• But iron independent hepcidin regulation is also
prevalent in many cases like inflammation, hypoxia,
liver injury etc.
• Then there is also erythropoesis related regulation of
hepcidin expression.
First we shall talk about Anemia of inflammation/anemia
of chronic disease.
53

53. • Anemia of inflammation or Anemia associated with
chronic inflammation is probably due to upregulation of
hepcidin by inflammatory condition.
• IL-6, an inflammatory cytokine, signals through JAK-
STAT (Janus Kinase-Signal Transducer and Activator of
Transcription) pathway to mediate this effect.
• IL-6 promotes phosphorylation of STAT3, which
translocates into nucleus and activates hepcidin
transcription upon binding to a proximal promoter
element.
Inflammation and hepcidin expression CONT’D:
54

54. 55

55. - It is thought that hepcidin up regulation in
inflammatory condition is a mode by which our body
makes availability of iron less to the
microorganisms(which require iron for their growth).
- Even during maliganancies, hepcidin is up regulated
(p53 tumor suppressor protein acts within the
hepcidin gene promoter region resulting in its up
regulation), and hence is possibly a part of defence
mechanism against cancer, through iron deprivation
of cancer cells.
56

56. HYPOXIA AND HEPCIDIN EXPRESSION:
- Hypoxia and oxidative stress are further signals that decrease
hepcidin expression.
- Studies in mice with liver specific ablation of VHL (Von Hippel
Lindau) factor, a tumor suppressor that regulates
HIFα(Hypoxia inducing factor alpha) subunit levels, proposed
a key role for HIF1/HIF2 in the hypoxic pathway of hepcidin
expression regulation.
- Biochemical data, however, didn’t support a possible direct
transcriptional functions of HIFs on hepcidin promoter…so,
binding of HIF to hepcidin promoter is still controversial.
However, hypoxia is known to promote erythropoetin(EPO)
synthesis, which consequently decreases expression of
hepcidin…Thus there is at least indirect relation between
hepcidin expression and hypoxia.
57

57. ERYTHROPOESIS AND HEPCIDIN EXPRESSION:
• Anemia induced erythropoesis requires an increase in iron
absorption.
• Injection of EPO(erythropoetin) in mice resulted in a dose
dependent decrease in hepcidin levels.
• Treatment of human volunteers with recombinant EPO
reduced urinary hepcidin considerably.
• However, patients with aplastic anemia do not increase iron
absorption despite high serum EPO levels.
• Also when erythropoesis in mice was blocked with
carboplatin, and exogenous EPO was given, there was no
response of hepcidin to EPO.
• Furthermore when erythropoesis in mice was suppressed
with irradiation, hepcidin mRNA level increased… but post
irradiation EPO didn’t suppress hepcidin level.
58

58. - Thus, it can be fairly concluded that hepcidin down
regulation is rather triggered by erythropoetic activity and
increase iron demand by erythroid precursor cells than
directly by EPO.
- However, it is to be noted that EPO can modulate iron
homeostasis by inducing TfR1 expression, cellular iron
uptake and subsequently heme biosynthesis in erythroid
precursor cells.
o In persons with β-thallasemia major, hepcidin levels
were found to be decreased.
o 2 molecules secreted by erythroblasts, Growth
differentiation factor 15(GDF15) and Twisted
Gastrulation 1(TWSG1), have been shown to inhibit
hepcidin expression in β-thallasemia.
ERYTHROPOESIS AND HEPCIDIN EXPRESSION CONT’D:
59

59. IRON DEFICIENCY:
- Extremely common.
- Major causes include dietary deficiency,
malabsorption, intestinal bleeding, episodic blood loss
(such as menstruation) and increase demand (e.g.
during pregnancy).
- Persistence iron deficiency finally leads to anemia.
- Failure of intestinal iron absorption, leads to negative
iron balance.
- This leads to progressive depletion of iron stores as
they become mobilized to meet requirement.
- At this stage lab tests are normal, except for a low
serum ferritin, a biomarker of body iron stores.
60

60. • If serum ferritin falls below 15µg/dl, transferrin level
increases, thus a rise in Total iron binding
capacity(TIBC).
• Transferrin saturation level ,however, decreases.
• Upon reaching 20% or below, hemoglobin synthesis
will be impaired, resulting in iron deficient
erythropoesis.
• If still not corrected, haemoglobin level in blood will
gradually fall, leading to iron deficiency anemia.
• Patients typically present a hypochromic, microcytic
blood picture accompanied by fatigue, pallor and
reduced exercise capacity.
IRON DEFICIENCY CONT’D:
61

61. • Erythrocytes of persons suffering from iron deficiency
anemia display increased levels of surface TfR1 and
deficits on ferrochelatase catalyzed incorporation of
iron into protoporphyrin IX.
• This results in rise in levels of Soluble transferrin
receptor protein(sTfR) in plasma, resulting from partial
proteolysis of cell surface transferrin receptors.
• This and the resultant accumulation of red-cell
protoporphyrin serve as a diagnostic biomarkers for
iron deficiency anemia.
• Especially, serum sTfR is particularly used to
differentiate it from anemia due to chronic inflammation.
IRON DEFICIENCY CONT’D:
62

62. VARIOUS LABORATORY TESTS USED TO ASSESS IRON DEFICIENCY
ANEMIA
63

63. HEMOCHROMATOSIS OR IRON OVERLOAD :
• Presence of stainable iron in tissues, hemosiderosis, is
characteristic of persons suffering from iron overload or
hemochromatosis.
• Hereditary causes are the major one.
• The most common hereditary hemochromatosis is the
mutation of HFE gene (HFE hemochromatosis).
• Other causes may be mutation in genes encoding
hepcidin, TfR2,HJV or ferroportin, leading to
hyperabsorption of iron by intestines.
.
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64. • Secondary iron overload is usually associated with
ineffective erythropoesis, as in thallassemia
syndromes.
• Repeated blood transfusions also may lead to
progressive iron overload.
• In either case, accumulation of iron in heart, liver,
pancreas, joints and skin can lead to generation of toxic
level of reactive oxygen species.
• It may take years to occur.
• Common complications are liver cirrhosis, diabetes,
arthritis and dermatitis.
HEMOCHROMATOSIS OR IRON OVERLOAD CONT’D:
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65. IRON OVERLOAD CONDITIONS:
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66. 67

67. Additional notes on Hemachromatosis
from
“CLINICAL BIOCHEMISTRY”
by
WILLIAM J MARSHALL et al
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68. • The term hemachromatosis refers to the group of
genetic disorders in which, as a result of excessive
absorption of dietary iron and long-term positive
iron balance, iron déposition causes tissue
damage, particularly to the liver, pancreas, heart,
anterior pituitary and joints.
• Haemosiderosis implies iron overload without
tissue damage, often an early stage of iron
accumulation, while secondary
haemochromatosis occurs in conditions requiring
multiple blood transfusions and in some other
haematological disorders.
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69. • Although previously considered a single gene disorder, it is
now known that haemochromatosis can be caused by
mutations in several genes that appear to have different
functions in iron metabolism.
• The most common form of haemochromatosis, found
almost exclusively in people of northern European
descent, is caused by homozygosity for a low penetrant
mutation, C282Y, in the hereditary haemochromatosis
gene, HFE.
• This condition affects mainly men and is characterized by
the insidious accumulation of iron, with the onset of
symptoms and signs of iron overload delayed until the
fourth or fifth decades of life.
• Heterozygotes for the C282Y mutation do not develop
iron overload, although minor abnormalities in plasma
iron and ferritin concentrations occur in approximately
15% of such individuals.
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70. • Adult onset haemochromatosis is very rarely associated
with mutations in TfR2, the gene encoding transferrin
receptor 2, a cell surface glycoprotein involved in iron
transport and uptake by cells including hepatocytes.
• A contrasting group of disorders, in which the iron loading
process is very rapid, presenting by the second or third
decades and affecting males and females equally, is known
as juvenile haemochromatosis.
• Although liver disease is invariably present, usually as
cirrhosis, the clinical presentation in this form of genetic
iron overload is with cardiac and endocrine failure.
• Juvenile haemochromatosis is caused by mutations in HJV
and HAMP, genes that encode, respectively, hemojuvelin
and hepcidin.
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71. • The iron loading process resulting from the
digenic inheritance of mutations in HFE and TfR2,
genes normally associated with adult onset
haemochromatosis, can be so rapid as to lead to
the phenotype of juvenile haemochromatosis.
• The recent identification of the iron regulatory
hormone hepcidin provides a link between HFE,
TfR2 and HJV, since mutations in these genes,
and in HAMP itself, cause loss of hepcidin
production by the liver.
• This peptide acts by inhibiting dietary iron
absorption and iron release from recycling and
storage sites.
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72. • All these forms of iron overload are characterized by
hepcidin deficiency and it appears that the proteins
encoded by HFE, TfR2 and HJV function as sensors of iron
status on the hepatocyte membrane acting upstream of
HAMP.
• Genotyping for mutations in HFE is now an essential part
of diagnosis and screening for haemochromatosis.
• Mutations in the other genes are so rare that routine
genetic analysis for these is not undertaken.
• A further variant of haemochromatosis is associated with
mutations in ferroportin, a gene that encodes the hepcidin
receptor. 73

73. • The penetrance of the homozygous C282Y genotype
in HFE-related haemochromatosis, defined in terms
of severe iron overload with tissue damage
manifesting as cirrhosis and type 1 diabetes, is low,
probably in the order of ~1–2%.
• Biochemical penetrance, defined as an increase in
transferrin saturation of >60% and a minimally raised
plasma ferritin concentration, probably occurs in 20–
50% of homozygous individuals.
• Other undetermined genetic loci and possibly
environmental factors are likely to determine
penetrance, but these are currently uncharacterized.
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74. • A clinical diagnosis of haemochromatosis in a
severely iron loaded subject may be made on
the basis of signs of liver disease, glycosuria
and a slate-grey appearance of the skin, but
confirmation of the diagnosis is entirely
dependent on genetic and laboratory tests.
• The crucial investigations are plasma iron and
ferritin concentration and transferrin
saturation.
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75. • In a patient with full gene penetrance, the
fasting plasma iron concentration is usually
>40 μmol/L (reference range 10– 30 μmol/L)
and transferrin is usually >60% saturated.
• Plasma ferritin concentration, which is
approximately proportional to the iron excess,
is usually >1000 μg/L (upper limit of reference
range 200–300 μg/L) and starts to rise when
hepatic iron stores exceed twice the reference
range.
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76. • If these tests suggest iron overload and liver function tests
are abnormal, a liver biopsy should be undertaken to assess
the degree of liver damage.
• Histological staining for iron (using Perls stain) gives a
semiquantitative assessment of the degree of iron overload,
but the amount of iron in a biopsy can also be quantitated
directly by inductively coupled mass spectrophotometry
(ICP-MS).
• The reference range is up to about 20 μmol/g dry liver
weight and >40 μmol/g dry liver weight is found in patients
with haemochromatosis.
• In less severe disease, when the patient is <40 years of age,
plasma ferritin is <1000 μg/L, LFTs are normal and
genotyping confirms the homozygous C282Y mutation, then
liver biopsy is not indicated as cirrhosis is unlikely to have
developed.
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77. • Treatment involves weekly venesection until the patient
starts to develop iron deficiency anaemia and the plasma
ferritin concentration falls to ~50 μg/L.
• Since 500 mL of blood contains about 250 mg of iron, the
total body iron at the time of diagnosis can be calculated
from the total volume removed.
• In an iron loaded individual, it is usually in the order of 10–
25 g compared with less than 4 g in the normal individual.
• The same figures can be obtained from knowledge of the
plasma ferritin concentration, which can also be used to
monitor the progress of treatment (1 μg/L of ferritin is
equivalent to approximately 8 mg of iron).
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78. • Unless cirrhosis has already occurred, phlebotomy is
successful in preventing progressive liver disease.
• The high risk of development of hepatocellular
carcinoma remains in treated patients with cirrhosis.
• It is also important to screen the patient’s relatives
for haemochromatosis by HFE genotyping so that
treatment can be instituted if appropriate before
liver damage occurs.
• Plasma ferritin and transferrin saturation are the
most sensitive biochemical tests for
presymptomatic detection in population studies.
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79. THANKS...
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