Lecture presentation

1. IRON KINETICS
By
Josiah Bimabam

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

3. Iron
• Element (Fe)
• Molecular weight 56
• Abundance
• May be 2+ or 3+
1. –Ferrous (2+) “reduced” - gained an electron
2. –Ferric (3+) “oxidised” - lost an electron
• Fe+++ + e- Fe++
• Redox states allows activity passing electrons around
body
• Redox change required for iron metabolism

4. Iron Biochemistry
Fe2+ ↔ Fe3+ + e-
• Important capacity to donate (reduction) and accept
(oxidation) electrons.
• Free intracellular Fe is dangerous, as it can catalyse
production of free radicals which can then damage
lipids, proteins and DNA.
• Manifestations of acute toxicity (eg paediatric ingestion: Mucosal cell necrosis, altered
capillary permeability, uncoupling of oxidative phosphorylation)
• Thus it must be bound/ carried by various proteins.

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

6. Iron Toxicity
• Iron can damage tissues
• Catalyzes the conversion of hydrogen peroxide to
free-radical ions
• Free-radicals can attack:
–cellular membranes
–Proteins
–DNA
• Iron excess possibly related to cancers, cardiac
toxicity and other factors

7. 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”

8. 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. Daily Iron Requirements
• 1. Iron is a one way element
• 2. Absorption is increased in iron deficiency and decreased
when the iron stores are depleted
• 3- daily iron requirement = amount lost + amount required
• 4- Increased requirement is found :
A- menstruating female / 30-60 ml of blood in each cycle. This
contains between 15-30 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.

11. Daily Iron Requirements
Daily iron losses and requirements (WHO 2001)

12. Iron Storage Forms
• 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. Typically, 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. This is called hemosiderin; it is
physiologically available.

14. Iron Storage Forms
• haemosiderin : it's not a single substance but a
variety of different, amorphous, iron- protein
complexes. Typically 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

15. Iron Binding Proteins
• Transferrin (Tf):
–Long arm chromosome 3;
–Single chain glycoprotein; 80kDa, hepatic synthesis.
–Able to bind 2 Fe 3+ 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)

16. Transferrin- TfR interactions

19. 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)

21. Cellular Control of Iron
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.

22. Systemic Iron Regulation
• Iron is absorbed through the
enterocyte of the duodenum
and into the plasma via the
portal circulation.
• There it binds to
apotransferrin for transport
to cells, such as the
developing red blood cells.
• In red blood cells, the iron is
used in hemoglobin that
circulates with the cell until
it becomes aged and is
ingested by a macrophage.

23. Systemic Iron Regulation
• There the iron is
removed from the
hemoglobin and can be
recycled into the plasma
for use by other cells.
• The level of stored and
circulating body iron is
detected by the
hepatocyte, which is able
to produce a protein,
hepcidin, when iron
levels get too high.

24. Systemic Iron Regulation
• Hepcidin will inactivate
the absorption and
recycling of iron by acting
on enterocytes,
macrophages, and
hepatocytes.
• When body iron
decreases, hepcidin will
also decrease so that
absorption and recycling
are again activated.

25. Iron transport
• Iron exported from the enterocyte into the blood is ferrous
and must be converted to the ferric form for transport in
the blood.
• Hephaestin, a protein on the basolaminal enterocyte
membrane, is able to oxidize iron as it exits the
enterocyte.
• Once oxidized, the iron is ready for plasma transport,
carried by a specific protein, apotransferrin (ApoTf).
• Once iron binds, the molecule is known as transferrin (Tf).
• Apotransferrin binds two molecules of ferric iron.

26. Cellular Iron Absorption and disposition
• Iron exported
•

27. Laboratory Assessment of Body Iron Status
• Disease occurs when body iron levels are either too
low or too high.
• The tests used to assess body iron status are able to
detect both conditions.
• They include the traditional or classic iron studies:
serum iron (SI), total iron-binding capacity (TIBC),
percent transferrin saturation, and Prussian blue
staining of tissues.
• More recently, ferritin assays have been included
among the routine tests. For special circumstances

28. Laboratory Assessment of Body Iron Status
• For special circumstances when the results of routine
assays are equivocal or too invasive, newer assays
include the soluble transferrin receptor (sTfR) and
hemoglobin content of reticulocytes.
• The results of these measured parameters can be
combined to calculate an sTfR/log ferritin ratio or graph a
Thomas plot.
• Finally, zinc protoporphyrin is another assay with special
application in sideroblastic anemia.
• Diagnostically, the tests can be organized to assess each
of the iron compartments as indicated in the table below

29. Laboratory Assessment of Body Iron Status

30. Serum Iron
• Serum iron can be measured colorimetrically using
any of several reagents such as ferrozine.
• The iron is first released from transferrin by acid, and
then the reagent is allowed to react with the freed iron,
forming a colored complex that can be detected
spectrophotometrically.
• Reference intervals are reported separately for men,
women, and children, and will vary from laboratory to
laboratory and from method to method

31. Serum Iron
• The serum iron level has limited utility on its own
because of its high within-day and between-day
variability; it also increases after recent ingestion of
iron-containing foods and supplements.
• To avoid the apparent diurnal variation, the standard
practice has been to collect the specimen fasting and
early in the morning when levels are expected to be
highest

32. Total Iron Binding Capacity (TIBC)
• The amount of iron in plasma or serum will be limited
by the amount of transferrin that is available to carry it.
• To assess this, transferrin is maximally saturated by
addition of excess ferric iron to the specimen.
• Any unbound iron is removed by precipitation with
magnesium carbonate powder.
• Then the basic iron method as described above is
performed on the absorbed serum, beginning with the
release of the iron from transferrin.

33. Total Iron Binding Capacity (TIBC)
• The amount of iron detected represents all the binding
sites available on transferrin—that is, the total iron-
binding capacity (TIBC).
• It is expressed as an iron value, although it is actually
an indirect measure of transferrin.

34. Percent Transferrin Saturation
• Since the TIBC represents the total number of sites for
iron binding and the SI represents the number bound with
iron, the degree to which the available sites are occupied
by iron can be calculated.
• The percent of transferrin saturated with iron is calculated
as: SI/TIBC x 100%= % Trans. Saturation
• It is important that both the SI and TIBC be expressed in
the same units, but it does not matter which units are
used in the calculation.
• A convenient rule of thumb evident from the table is that
about one third (1⁄3) of transferrin is typically saturated
with iron

35. Prussian Blue Staining
• Prussian blue is actually a chemical compound with
the formula Fe7(CN)18.
• The compound forms during the staining process,
which uses acidic potassium ferrocyanide as the
reagent/stain.
• The ferric iron in the tissue reacts with the reagent,
forming the Prussian blue compound that is readily
seen microscopically as dark blue dots

36. Prussian Blue Staining
• Tissues can be graded or scored semiquantitatively by
the amount of stain that is observed.
• Prussian blue stain is considered the gold standard for
assessment of body iron.
• Staining is conducted routinely when bone marrow or
liver biopsies are taken for other purposes.
• Although ferric iron reacts with the reagent, ferritin is
not detected, likely due to the intact protein cage.
• However, hemosiderin stains readily.

37. Ferritin
• As mentioned above, until the development of serum
ferritin assays, the only way to truly assess body iron
stores was to take a sample of bone marrow and stain it
with Prussian blue.
• Such an invasive procedure prevented regular
assessment of body iron.
• The development of the serum immunoassay for ferritin
provided a convenient assessment of body iron stores.
• Though ferritin is an intracellular protein, it is secreted by
macrophages into plasma for reasons that are not yet
understood.

38. Ferritin
• The level of serum ferritin has been shown to correlate
highly with stored iron as indicated by Prussian blue
stains of bone marrow.
• There is a significant drawback in the interpretation of
serum ferritin results.
• Ferritin is an acute phase protein or acute phase
reactant (APR).
• The APRs are proteins that are produced, mostly by
the liver, during the acute (i.e., initial) phase of
inflammation, especially during infections.

39. Ferritin
• They include cytokines that are nonspecific, but also other
proteins with the apparent intent to suppress bacteria.
• Since bacteria need iron, the body’s production of ferritin
during the acute phase seems to be an attempt to
sequester the iron away from the bacteria.
• Thus increases in ferritin can be induced without an
increase in the amount of systemic body iron.
• These rises may not be outside the reference interval but
still high enough to elevate a patient’s ferritin above what
it would otherwise be.

40. Ferritin
• Ferritin values between 20 and 100 ng/mL are most
equivocal, making it difficult to recognize true iron
deficiency when an inflammatory condition is also
present.
• Therefore, the predictive value of a ferritin result within
the reference interval is weak.
• However, only a decreased level of stored body iron
can lower ferritin levels below the reference interval,
so the predictive value of a low ferritin result is high for
iron deficiency.