Download Iron: the iron man and the iron deficient woman

Iron Metabolism and Storage
Ahmad Sh. Silmi
Staff Specialist in Haematology
Medical Tech Dept, IUG
2011
Iron in Man
• Biochemistry
• Recent advances in understanding of iron
metabolism
• Role in disease
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++
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
Iron functions
• Oxygen carriers
– haemoglobin
• Oxygen storage
– Myoglobin
• Energy Production
– Cytochromes (oxidative phosphorylation)
– Krebs cycle enzymes
• Other
– Liver detoxification (cytochrome p450)
• An essential element
Iron Toxicity
• 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
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”
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%
Iron Distribution
Daily iron requirements
1.
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 / 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.
Daily iron losses and requirements
(WHO 2001)
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).
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
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.
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)
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.
Incorporation of iron from plasma transferrin into haemoglobin in developing red
cells. Uptake of transferrin iron is by receptor-mediated endocytosis.
Iron Absorption
• Regulation of iron stores occurs at the level of
absorption.
• There is no capacity to increase iron
excretion.
Iron Absorption
• The average western diet contain 10-15 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
Iron Absorption
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.
Oral Iron intake
• Non 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.
Molecular pathways of iron absorption
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
Iron Absorption
• DcytB
– Reduction Fe+++ to Fe++
• DMT1
– Transport into cell
• Ferritin
– Storage in cell
• Hephaestin
– Oxidises Fe++ to Fe+++
• Ferroportin
– Transport out
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)
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.
FERRITIN/TRANSFERRIN REGULATION
Regulation of Iron Balance
• Crypt Hypothesis
• Hepcidin
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.
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.
Control of iron absorption mucosal block theory
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
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).
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).
Interplay of Key Proteins in Iron Homeostasis
Fleming, R. E. et al. N Engl J Med 2005;352:1741-1744
The effect of the hepcidin-ferroportin interaction on cellular iron export
Hematology 2006;2006:505-516
Copyright ©2006 American Society of Hematology. Copyright restrictions may apply.
IRON FLOWS ARE REGULATED BY THE
HEPCIDIN/FERROPORTIN INTERACTION
x
x
x
IRON FLOWS ARE REGULATED BY THE
HEPCIDIN/FERROPORTIN INTERACTION
x
x
x
Plasma
Fe
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.
Regulation of Hepcidin
• Evidence of regulation of synthesis by:
– Anaemia/ Hypoxia
– Inflammation
– Iron
• Precise mechanisms of regulation remain
unclear.
REGULATION OF HEPCIDIN PRODUCTION
INFLAMMATION
ANEMIA
HYPOXIA
IRON
hepcidin
Hepcidin regulation by anaemia
• Evidence that erythropoietic activity is
the most potent suppressor of hepcidin
synthesis, although specific mechanism
unclear.
REGULATION OF HEPCIDIN BY ANEMIA
Pak M, Blood 2006
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
Hepcidin regulation by iron
• Iron loading increases Hepcidin synthesis.
– Molecular details unclear.
– Hepcidin mRNA lacks IRE.
HEPCIDIN PRODUCTION IS
REGULATED BY AN IRON SIGNAL
HEPCIDIN REGULATION
INFLAMMATION
IRON SIGNAL
ERYHTROPOIETIC SIGNAL
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
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.
Iron Loss
• Physiological
– Cell loss: gut, desquamation
– Menstruation (1mg/day)
– Pregnancy, lactation
• Pathological
– Bleeding
– Gut, menorrhagia, surgery, gross haematuria
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
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
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.
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
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)
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).
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
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
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
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.
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
Transferrin Saturation
NORMAL IRON STATUS
Normal iron
Normal transferrin
Saturation 40%
IRON OVERLOAD
High iron
Low transferrin
Transferrin
Saturation 80%
Iron
Differential diagnosis of hypochromic anaemia.
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.
IRON DEFICIENCY ANEMIA
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
IRON DEFICIENCY ANEMIA
Prevalence
Country
Men (%) Women
(%)
Pregnant
Women (%)
S. India
N. India
Latin America
Israel
Poland
Sweden
USA
6
56
80
38
47
22
4
14
35
64
17
29
1
7
13
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)
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%)
Koilonychia
STOMATITIS
Glossitis
IDA Laboratory Findings:
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Microcytic/hypochromic anemia
Low Hb, HCT, and RCC
MCV = 55-74 fl
MCHC = 22-31 g/dl
MCH = 14-26 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
IDA: the CBC
Shift to left
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.
IDA blood film:
Anisocytosis, Hypochromia, Microcytosis.
IDA
Anulocyte
BM in IDA show erythroid hyperplasia, but
unfortunately ineffective
Prussian Blue Stain for iron of BM
Iron Present
No Iron Present
Serum Iron
• N.R. 75-175 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!.
Transferrin Molecule
Normal
Saturation
30%
TIBC
Iron Deficiency
Saturation
10%
TIBC
Hemochromatosis (iron overload)
Saturation
60-75%
TIBC
NR transferrin levels take up 250-430 µg/dL in the lab
Transferrin vs iron binding capacity
• Normal Transferrin level= 240-430 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 =
253-435 µg iron/dL
• Serum iron (Fe)= 75-175 µg/dL
• Transferrin saturation (TfS)= [Fe/TIBCx100]
(%), normal 20-50%, with a mean value of
30%.
Transferrin and TIBC
• Transferrin = the protein for
transport of serum iron; measured
in mg/dL, normal = 240- 430 mg/dL
• Not to be confused with TIBC,
measured in µg Fe/dL
%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%
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.
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.
Microcytic Hypochromic Anemia
Serum Ferritin
< 15 µg/L
> 300µg/L
15-300 µg/L
TIBC
N or ↓
HIGH
-
BM Fe
Iron Deficiency Anemia IDA
+
Not IDA, Other
Micro Anemia
Mentzer Index
• 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 < 13 - 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
Hookworm drinks its host’s blood!
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