2. Iron Metabolism and Storage
Ahmad Sh. Silmi
Staff Specialist in Haematology
Medical Tech Dept, IUG
2011

3. Iron in Man Biochemistry
Recent advances in understanding of iron metabolism
Role in disease

4. Iron Element (Fe) Molecular weight 56 Abundance May be 2+ or 3+
Ferrous (2+) “reduced” - gained an electron
Ferric (3+) “oxidized” - lost an electron
Fe e-  Fe++

5. Iron Biochemistry Fe2+ ↔ Fe3+ + e-
Important capacity to donate (reduction) and accept (oxidation) electrons.
Redox states allows activity passing electrons around body
Redox change required for iron metabolism

6. Iron functions Oxygen carriers Oxygen storage Energy Production Other
haemoglobin
Oxygen storage
Myoglobin
Energy Production
Cytochromes (oxidative phosphorylation)
Krebs cycle enzymes
Other
Liver detoxification (cytochrome p450)
An essential element

7. Iron Toxicity Thus it must be bound/ carried by various proteins
Iron can damage tissues
Catalyzes the conversion of hydrogen peroxide to free-radical ions
Free-radicals can attack:
cellular membranes
Proteins
DNA
Thus it must be bound/ carried by various proteins
Iron excess possibly related to cancers, cardiac toxicity and other factors

8. Principle Bodies require the right amount of substance
Too much or too little of any required substance may be detrimental
“There is no substance, which taken in sufficient excess, is not toxic to the body”

9. Iron Distribution 35 – 45 mg / kg iron in adult male body
Total approx 4 g
Red cell mass as haemoglobin - 50%
Muscles as myoglobin – 7%
Storage as ferritin - 30%
Bone marrow (7%)
Reticulo-endothelial cells (7%)
Liver (25%)
Other Haem proteins - 5%
Cytochromes, myoglobin, others
In Serum - 0.1%

10. Iron Distribution

11. Daily iron requirements
Iron is a one way element
2- absorption is increased in iron deficiency and decreased
when body iron stores are exceeded.
3- daily iron requirement = amount lost + amount required
4- Increased requirement is found :
A- menstruating female / ml of blood in each cycle. This contains between mg iron/cycle
B- pregnancy
(1) Foetal/placental growth requirement.
     (2) Expansion in maternal mother blood volume.
     (3) Haemorrhage in delivery involve highly significant loss of
iron.

12. Daily iron losses and requirements (WHO 2001)

13. Iron Storage Forms 1st ferritin : MW 45000.
consist of  24 polypeptide sub-unit cluster together to form hollow sphere of 5 nm in diameter. 
The stored iron form the central core of the sphere.
Iron store in the liver and nearly all other cells.
ferritin contains about 25% of iron by weight.
About 2/3 of body iron stores are present as ferritin.
If the capacity for storage of iron in ferritin is exceeded, a complex of iron with phosphate and hydroxide forms (hemosiderin).

14. Ferritin Storage Molecule
Ferritin molecules store thousands of iron atoms within their mineral core. When excess dietary iron is absorbed, the body responds by producing more ferritin to facilitate iron storage

15. Iron Storage Forms 2nd haemosiderin :
it's not a single substance but a variety of different, amorphous, iron- protein complexes.
it contains about 37% of iron by weight.
Haemosiderin may represent ferritin in various form of degradation. 
As the body burden of iron increases beyond normal levels, excess hemosiderin is deposited in the liver and heart. This can reach the point that the function of these organs is impaired, and death.

16. Iron Binding Proteins Transferrin (Tf): Long arm chromosome 3;
Single chain glycoprotein; 80kDa, hepatic synthesis.
Able to bind 2 Fe3+ molecules with very high affinity at pH7.4, but reduced affinity in acidic conditions.
Transports iron through plasma.
3mg of total body iron.
Transferrin Receptor (TfR):
Also located on 3q.
Transmembrane glycoprotein dimer with two transferrin binding sites.
Found on most cells (esp erythroid precursors, hepatocytes, placental cells)

17. Transferrin-TfR interactions
Each TfR can bind two Tf molecules, which are endocytosed through clathrin coated pits.
A proton pump generates acidity in the endosome, facilitating release of Fe from Tf.
DMT-1 transporter exports Fe from endosome.

18. Incorporation of iron from plasma transferrin into haemoglobin in developing red cells. Uptake of transferrin iron is by receptor-mediated endocytosis.

19. Iron Absorption
Regulation of iron stores occurs at the level of absorption.
There is no capacity to increase iron excretion.

20. Iron Absorption
The average western diet contain mg of iron daily. Only 5-10% is absorbed.
1 – 2 mg iron are absorbed each day
(in iron balance 1 – 2 mg iron leaves the body each day)
Occurs in the duodenum
Taken up as ionic iron or haem iron

21. Iron Absorption

22. Iron Absorption
The main dietary sources are liver, red meat, green vegetables, spinach, supplemented cereals and fish.
Dietary iron usually in excess
either not absorbed, or kept in enterocytes and shed into the gut
Dietary iron falls into one of two categories:
Haem & non-haem.

23. Oral Iron intake Non Haem: Haem:
Cereals, legumes
10% bioavailability
Absorption enhanced by ascorbic acid (maintains Fe2+).
Inhibited by tanins, phytates (chappatis).
Haem:
Meat, fish
30% bioavailability
Iron released from complexes by acid, proteases
Binds to mucin and travels to small bowel.

24. Molecular pathways of iron absorption

26. Haem iron absorption Haem split from globin in intestine
Absorbed into enterocyte as haem
Iron freed into enterocyte pool or absorbed intact
Accounts for over half of iron in western diet but much less in other diets
Not well understood

27. Iron Absorption DcytB DMT1 Ferritin Hephaestin Ferroportin
Reduction Fe+++ to Fe++
DMT1
Transport into cell
Ferritin
Storage in cell
Hephaestin
Oxidises Fe++ to Fe+++
Ferroportin
Transport out

28. Cellular Control of Iron
Iron Responsive Elements (IRE):
Loop configuration of nucleotides located in the 5’ or 3’ ends of mRNA coding for ferritin, TfR, DMT1, others.
Iron Regulatory Proteins (IRP):
Serve as a sensor of cell iron
Modulate the synthesis of iron regulatory proteins by binding to the IREs.
Contain an iron-sulphur cluster: low affinity for IRE when iron abundant, but higher affinity when iron absent.
Binding to 5’ end reduces translation (eg for ferritin)
Binding to 3’ end protects mRNA and increases translation (eg for TfR)

29. Cellular control of Iron
In the presence of increased iron:
IRP detaches from ferritin mRNA allowing more ferritin to be synthetised.
IRP detaches from TfR, reducing synthesis.
Effect is to reduce influx of iron into cell and facilitate storage.

30. FERRITIN/TRANSFERRIN REGULATION

32. Regulation of Iron Balance
Crypt Hypothesis
Hepcidin

33. Duodenal Iron Absorption – ‘The Crypt Hypothesis’
Precursor cells proliferate in the crypt.
As they mature and differentiate, they migrate up the villus.
Their apical membrane develops microvilli and absorptive transport enzymes.

34. The ‘Crypt Hypothesis’
Precursor cells in the crypts detect the serum iron concentration.
This establishes the ‘set point’ iron absorptive capacity of that cell as it differentiates into a mature enterocyte.

35. Control of iron absorption mucosal block theory

36. Hepcidin 25 aa peptide , synthesised in the liver. Identified 2000
Antimicrobial activity
Hepatic bacteriocidal protein
Master iron regulatory hormone
Inactivates ferroportin
Stops iron getting out of gut cells
Iron lost in stool when gut cells shed
Leads to decreased gut iron absorption

37. Hepcidin 25 amino acid peptide, synthesised in the liver. Function:
Binds to ferroportin and induces its internalisation and lysosomal degradation.
Removal of ferroportin prevents iron efflux from enterocyte to plasma: iron is lost from body when cell is shed after 1-2 days.
Ferroportin enables iron export from reticuloendothelial/ hepatic macrophages, thus hepcidin prevents transport of recycled iron to plasma.
Likely that rising iron levels also secondarily influence IRE/IRP system and processing of iron protein mRNA.
In Hepcidin deficient mice, DMT1 and dcytb1 were significantly increased (?primary or secondary effect).

38. Hepcidin 25 amino acid peptide, synthesised in the liver. Function:
Binds to ferroportin and induces its internalisation and lysosomal degradation.
Removal of ferroportin prevents iron efflux from enterocyte to plasma: iron is lost from body when cell is shed after 1-2 days.
Ferroportin enables iron export from reticuloendothelial/ hepatic macrophages, thus hepcidin prevents transport of recycled iron to plasma.
Likely that rising iron levels also secondarily influence IRE/IRP system and processing of iron protein mRNA.
In Hepcidin deficient mice, DMT1 and dcytb1 were significantly increased (?primary or secondary effect).

39. Interplay of Key Proteins in Iron Homeostasis
Interplay of Key Proteins in Iron Homeostasis. In the duodenal enterocyte, dietary iron is reduced to the ferrous state by duodenal ferric reductase (Dcytb), transported into the cell by divalent metal transporter 1 (DMT1), and released by way of ferroportin into the circulation. Hephaestin facilitates enterocyte iron release. Hepatocytes take up iron from the circulation either as free iron or transferrin-bound iron (through transferrin receptor 1 and transferrin receptor 2). Transferrin receptor 2 may serve as a sensor of circulating transferrin-bound iron, thereby influencing expression of the iron regulatory hormone hepcidin. The hepcidin response is also modulated by HFE and hemojuvelin. Hepcidin is secreted into the circulation, where it down-regulates the ferroportin-mediated release of iron from enterocytes, macrophages, and hepatocytes (dashed red lines).
Fleming, R. E. et al. N Engl J Med 2005;352:

40. The effect of the hepcidin-ferroportin interaction on cellular iron export
The effect of the hepcidin-ferroportin interaction on cellular iron export. When hepcidin concentrations are low, cellular iron is exported into plasma through membrane-associated ferroportin (Fpn). When hepcidin concentrations are high, hepcidin binds to ferroportin, and ferroportin is internalized and degraded. As a consequence of the loss of ferroportin, cellular iron export decreases and iron accumulates in cytoplasmic ferritin.
Hematology 2006;2006:
Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.

41. IRON FLOWS ARE REGULATED BY THE HEPCIDIN/FERROPORTIN INTERACTIONx x x

42. IRON FLOWS ARE REGULATED BY THE HEPCIDIN/FERROPORTIN INTERACTIONx x x
Plasma Fe

43. Hepcidin
In mice, a single 50mcg dose results in 80% drop in serum iron within 1hr followed by delayed recovery.
Thus, serum iron levels can drop rapidly upon hepcidin induction.

44. Regulation of Hepcidin
Evidence of regulation of synthesis by:
Anaemia/ Hypoxia
Inflammation
Iron
Precise mechanisms of regulation remain unclear.

45. REGULATION OF HEPCIDIN PRODUCTION
ANEMIA
HYPOXIA
INFLAMMATION
IRON
hepcidin

46. Hepcidin regulation by anaemia
Evidence that erythropoietic activity is the most potent suppressor of hepcidin synthesis, although specific mechanism unclear.

47. REGULATION OF HEPCIDIN BY ANEMIA
Pak M, Blood 2006

48. Hepicidin regulation by inflammation
IL-6 is a potent inducer of hepcidin synthesis during acute inflammation.
Thus hepcidin is an acute phase protein.
In mice, IL-1, TGFB have been shown to regulate hepcidin (?in humans).
Lowered serum iron is an acute host defence.
Hepcidin itself may have some antimicrobial activity (probably not at physiological levels).
Mediates anaemia of chronic disease

49. Hepcidin regulation by iron
Iron loading increases Hepcidin synthesis.
Molecular details unclear.
Hepcidin mRNA lacks IRE.

50. HEPCIDIN PRODUCTION IS REGULATED BY AN IRON SIGNAL

51. HEPCIDIN REGULATION
INFLAMMATION
IRON SIGNAL
ERYHTROPOIETIC SIGNAL

52. SUMMARY
Hepcidin is an iron-regulatory hormone that maintains plasma iron levels and iron stores within normal range
Hepcidin regulates the entry of iron into plasma from duodenal enterocytes, from macrophages (and from hepatocytes)
Hepcidin acts by binding the receptor/iron channel ferroportin and causing its degradation
Hepcidin is regulated by iron, erythropoiesis and inflammation
Excess hepcidin causes the hypoferremia and anemia of inflammation
Hepcidin deficiency, or resistance to hepcidin, cause hemochromatosis

53. Principles
For any metabolic process there is a pathway (which is usually complex).
For any pathway there will be a regulatory process (which may also be complex).
Often diseases are due to changes in the regulation of a pathway, not due to defects in the pathway itself.

54. Iron Loss Physiological Pathological Cell loss: gut, desquamation
Menstruation (1mg/day)
Pregnancy, lactation
Pathological
Bleeding
Gut, menorrhagia, surgery, gross haematuria

55. Iron Loss An unregulated process
No mechanisms to up- or down-regulate iron loss from the body
Over-intake cannot be matched by increased loss
Under intake cannot be matched by decreased loss
Thus iron homeostasis is regulated by adjusting iron intake

56. Iron re-use
Old cells broken down in macrophages in spleen and other organs
Iron transported to liver and other storage sites
Red cell iron recovered from old red cells
Very little iron lost in routine metabolism

57. Iron Scavenging Intravascular haemolysis
Breakdown of red cells in the circulation
Free haemoglobin binds haptoglobins -> taken up by liver
Free haem binds haemopexin -> taken up by liver
Haem passing through kidney resorbed
Three mechanisms to conserve iron in pathological situations
Historically iron deficiency is the disease we have evolved to avoid.

58. The liver and iron metabolism
Hepcidin production by the liver controls gut iron absorption and therefore body iron stores
HFE and haemojuvelin involved in hepcidin regulation

59. Tests of body iron burden
Principle
Interpretation of a “blood test” requires knowledge of all factors which affect concentration
Includes
Disease of interest (signal)
Other conditions (noise)

60. Transferrin Testing A routine blood test used for iron status
Also known as TIBC (total iron binding capacity)
High :
Low body iron stores.
Low :
High body iron stores.
Other conditions
Increase: high oestrogen states (pregnancy, OCP)
Decrease: malnutrition, chronic liver disease, chronic disease (eg malignancy), protein-losing states, congenital deficiency, neonates, acute phase (negative reactant).

62. Transferrin Receptors
Collects iron from transferrin for uptake into cells
Recognises and binds transferrin
Receptor + transferrin endocytosed
Iron released into cell via Iron transporter (DMT1)
Receptor + transferrin return to cell surface
Transferrin released

63. Soluble Transferrin Receptors
Truncated form of cell surface receptors
Found in the circulation
High levels with iron deficiency
Low levels with iron overload
Possible role in diagnosis of iron deficiency compared in setting of inflammation
Not currently routinely available

64. Serum Iron The serum contains about 0.1% of body iron
Over 95% of iron in serum bound to transferrin
Serum iron is a routine blood test
Measures all serum iron (not in red cells)
Of limited use on its own
Useful for interpretation of iron status only if grossly abnormal – eg iron poisoning
Commonly combined with serum transferrin to express transferrin saturation

65. Serum Iron Measurement
Serum iron is a routine blood test
Low levels:
Iron deficiency
Other: Random variation; acute or chronic inflammation; pre-menstrual.
High levels:
Iron Overload
Other: Random variation, OCP, pregnancy, recent iron ingestion.

66. Transferrin Saturation
Percent of transferrin (TIBC) iron-binding sites which are filled with iron
Combines two factors to improve sensitivity
Iron overload
High iron plus low transferrin
High saturation (50 – 100%)
Best serum marker of increased body iron
Used as a screen for iron overload

67. Transferrin Saturation
NORMAL IRON STATUS
Normal iron Normal transferrin Saturation 40%
IRON OVERLOAD
High iron Low transferrin Saturation 80%
Iron
Transferrin

69. Differential diagnosis of hypochromic anaemia.

71. Principle
In homeostasis - intake of any element equals loss of any element
nitrogen, water, salt, iron
In “steady state” intake must balance loss.
Even slight imbalances over time can create excesses or deficiencies.
1% excess per day doubles content 70 days.

72. IRON DEFICIENCY ANEMIA

73. Iron Deficiency Extremely common
Due to reduced intake, increased loss or increased demands
Stores reduced before deficiency seen
Iron deficiency is not a diagnosis
A cause needs to be identified!
Eg obstetric causes, low intake, malabsorption, bowel cancer, haemorrhoids, inflammatory bowel disease

75. IRON DEFICIENCY ANEMIA Prevalence

76. IRON DEFICENCY - STAGES
Prelatent
reduction in iron stores without reduced serum iron levels
Hb (N), MCV (N), iron absorption (), transferin saturation (N), serum ferritin (), marrow iron ()
Latent
iron stores are exhausted, but the blood hemoglobin level remains normal
Hb (N), MCV (N), TIBC (), serum ferritin (), transferrin saturation (), marrow iron (absent)
Iron deficiency anemia
blood hemoglobin concentration falls below the lower limit of normal
Hb (), MCV (), TIBC (), serum ferritin (), transferrin saturation (), marrow iron (absent)

78. Symptoms GLOSSITIS, STOMATITIS DYSPHAGIA (esophageal web)
ATROPHIC GASTRITIS
DRY, PALE SKIN
SPOON SHAPED NAILS: KOILONYCHIA
BLUE SCLERAE
HAIR LOSS
PICA (APETITE FOR NON FOOD SUBSTANCES SUCH AS AN ICE, CLAY)
SPLENOMEGALY (10%)

79. Koilonychia

81. STOMATITIS

82. Glossitis

83. IDA Laboratory Findings:
Microcytic/hypochromic anemia
Low Hb, HCT, and RCC
MCV = fl
MCHC = g/dl
MCH = pg
Hb < 10 g/dL
Anisocytosis: Increased RDW
Poikilocytosis: elliptocytes, target cells, pencil cells
Anulocytes
RPI <2.0
You may see thrombocytosis esp. when the cause is due to bleeding.
BM stainable iron: ABSENT
High TfR
High Transferrin
Low serum ferritin
Low Serum iron
High TIBC
Low Transferrin saturation%
High FEP

84. IDA: the CBC
Shift to left

85. Blood & Bone Marrow BLOOD Film:
Microcytosis, Hypochromia, Anulocytes, Anisocytosis, Poikilocytosis
BONE MARROW Film:
High cellularity.
Mild to moderate erythroid hyperplasia (25-35%; N: 16 – 18%)
Cytoplasm of polychromatic and pyknotic normoblasts is scanty (indicating its immaturity), vacuolated and irregular in outline. This type of erythropoiesis has been described as micronormoblastic (micronormoblastic erythropoiesis)
Staining the BM aspirates for iron (hemosiderin) gives indicate that normoblast iron are absent. i.e. Absence of hemosiderin stainable iron. Usually BM iron is scored from 0 to +4 (0, +1, +2, +3, +4), in IDA it is 0.

86. IDA blood film: Anisocytosis, Hypochromia, Microcytosis.

87. IDA
Anulocyte

88. BM in IDA show erythroid hyperplasia, but unfortunately ineffective

89. Prussian Blue Stain for iron of BM
Iron Present
No Iron Present

90. Serum Iron
N.R µg/dl
Serum iron concentrations shows diurnal rhythm, which is attributed to the change in release of iron from the macrophages which is highest in the morning and lowest in the evening.
So we expect that erythropoiesis is active in the morning more than the evening!.

91. Transferrin Molecule Normal Iron Deficiency
Saturation
30%
T I B C
Iron Deficiency
Saturation
10%
T I B C
Hemochromatosis (iron overload)
Saturation
60-75%
T I B C
NR transferrin levels take up µg/dL in the lab

92. Transferrin vs iron binding capacity
Normal Transferrin level= mg/dL
Total Iron binding capacity (TIBC): amount of iron (as a reagent, excess iron is added by you in the lab) that can be taken up by washed transferrin molecules in patient serum in the laboratory, in µg/dL, normal = µg iron/dL
Serum iron (Fe)= µg/dL
Transferrin saturation (TfS)= [Fe/TIBCx100] (%), normal 20-50%, with a mean value of 30%.

93. Transferrin and TIBC
Transferrin = the protein for transport of serum iron; measured in mg/dL, normal = mg/dL
Not to be confused with TIBC, measured in µg Fe/dL

94. %Transferrin saturation (TfS)
Percent of transferrin (TIBC) iron-binding sites which are filled with iron.
Normally 20-50%, with normal mean value of 30%.
Calculated (%TfS)= Serum Iron/TIBC x100
In IDA it is less than 10%.
Typical normal TfS is 100 µg/dL Fe ÷ TIBC 300 µg/dL = about 30%
IDA shows ↑TIBC ~ 400 µg/dL and ↓Fe ~ 25 µg/dL, TfS≤ 10%

95. Serum Ferritin Ferritin is a molecule measured in µg/L NR 15-300 µg/L
Is an indirect indication of stores of body iron.
Acute phase reactant, e.g. increases in hepatitis C and B.
(increased in chronic inflammation), causing spurious/erroneous increases.

96. Free Erythrocyte Protoporphyrin (FEP)
FEP - Precursor of Iron binding
Elevated in iron def. anemia
Highly elevated with lead poisoning.
Iron deficiency impaired heme.
Synthesis and accumulation of protoporphyrin, which is:
heme precursor, in erythrocytes.
Measurement of FEP level is a sensitive indicator of iron deficiency.

97. Microcytic Hypochromic Anemia
Serum Ferritin
< 15 µg/L
µg/L
> 300µg/L
TIBC
N or ↓
HIGH - +
BM Fe
Iron Deficiency Anemia IDA
Not IDA, Other Micro Anemia

98. Mentzer Index Moreover RDW In additionHb-A2
The Mentzer index is used to differentiate iron def. anemia from beta thalassemia minor.
Mentzer Index= MCV/RCC.
If > 13 then Fe deficiency anemia
If < Hb defect (i.e. thalassemia)
Moreover RDW
in IDA is inc., But thalassemia minor normal.
In additionHb-A2
In IDA= Dec./Normal; but in B thal minor is Increased

99. Hookworm drinks its host’s blood!

100. IRON THERAPY Response Initial response takes 7-14 days
Modest reticulocytosis (7-10%)
Correction of anemia requires 2-3 months
6 months of therapy beyond correction of anemia needed to replete stores, assuming no further loss of blood/iron
Parenteral iron possible, but problematic