Iron Metabolism and Iron Refractory Iron

Deficiency Anemia (IRIDA)

Presented by: Dr. Abhiraami

Moderator: Dr. Rakhee Kar
 Objectives
• History of iron deficiency anemia.
• Total body iron and iron containing compounds.
• Iron balance and iron cycle
• Dietary iron absorption.
• Plasma iron transport and uptake by erythroid precursors.
• Macrophage iron recycling and iron storage
• Regulation of iron metabolism : Systemic and intracellular
mechanisms
• Summary of iron metabolism
• Genetic defects in iron metabolism and Iron Refractory Iron
Deficiency Anemia (IRIDA)
??
 Chlorosis
• Green sickness/ Morbus virgineus
• 16th century, German physician Johannes
Lange described it as “ the disease of virgins”
• Hysterical illness due to suppressed sexuality
in virgins (adolescent girls)
• Chlorosis was coined by J. Varandal in from
the Greek chloros - green, because of the
greenish tint on the skin of his patients
 Chlorosis
• Sydenham in 17th century : "The sick must
drink some mineral water impregnated with
the Iron Mine"
• In 1936, Arthur J. Pathek and Clark W. Health
of Harvard School of Medicine concluded that
chlorosis was identical to hypochromic
anemia.
 Iron
• Essential micronutrient for life but potentially
toxic
• Free iron: ability to accept/ release electrons -
propensity to cause tissue damage.

Iron deficiency Iron maldistribution Iron excess

 Role of iron

O2
transportation
& storage
molecules

Many redox
Host
defence Iron enzymes –
electron transport
chain

Production of
various metabolic
intermediates
 Total body iron

Infants • 75 mg/kg body weight

Adult men • 50mg/kg body weight.

Adult • 35mg/kg body weight

women
 HEMOGLOBIN

FUNCTIONAL POOL
MYOGLOBIN

ENZYMES

IRON POOL TRANSPORT POOL TRANSFERRIN

FERRITIN
STORAGE POOL
HEMOSIDERIN
 Iron containing Compounds
Fe oxidation
PROTEIN FUNCTION PERCENT
state

Hemoglobin RBC O2 Transport 65 Heme (Fe 2+)

Muscle O2
Myoglobin 6 Heme (Fe 2+)
Transport
Plasma Iron
Transferrin 0.1 Fe 3+
transport
Intracellular Iron
Ferritin 13 Fe 3+
storage
Intracellular Iron
Hemosiderin 12 Fe 3+
storage

Enzymes : Catalase,
Peroxidase, Aconitase,
Heme / Fe-S
Ferrochelatase, 3.9
cluster
Cytochrome, Duodenal
cytochrome oxidase b.
 IRON INTAKE

IRON
BALANCE

ABSORPTION IRON LOSS

 Iron balance
• No active mechanism for excretion
• Primarily achieved by control of absorption.

Iron loss Iron intake

• 1-2 mg of iron should be
• 1-2 mg/day
• Loss of gastrointestinal absorbed from the diet
daily.
epithelial cells, skin
• Bio-availability: 10 to 15%
epidermis, sweat, urine
• Absorption can be
• Menstrual loss in women :
increased upto 3 to 5 fold
0.006 to 0.025 mg/kg/day
 RDA OF IRON (mg/day)
(ICMR guidelines for Indian
population)
ADULT MEN 17

ADULT WOMEN 21

PREGNANCY 35

LACTATION 25

INFANTS 5
RDA of Iron in children and adolescents
(ICMR guidelines for Indian population)
Age RDA
1-3 years 9

4-6 years 13

7-9 years 16

10-12 years 21 (boys)

27(girls)
13-15 years 32 (boys)
27(girls)
16-17 years 28 (boys)
26(girls)
 IRON CYCLE
Iron metabolism

Dietary iron absorption

Plasma iron transport

Iron delivery to erythroid

precursors

Macrophage iron recycling

Iron storage

Regulation of iron metabolism:

Systemic and cellular.
Functional body iron : 20-25 mg/day
 IRON CYCLE
Iron metabolism

Dietary iron absorption

Plasma iron transport

Iron delivery to erythroid

precursors

Macrophage iron recycling

Iron storage

Regulation of iron metabolism:

Systemic and cellular.
Functional body iron : 20-25 mg/day
 Dietary iron absorption

FERROUS
NON-HEME
DIETARY (90%)
FERRIC
IRON HEME
(10%)

• SITE : Duodenum.
 Dietary iron absorption

• Derived from meat.

• Taken up by mucosal cells.
Heme iron • High bio-availability.
• Unaffected by composition of diet

• From plant sources.

Non heme • Ferric hydroxide or bound to
iron sugars/ amino acids.
• Profoundly affected by dietary
constituents
 Factors affecting Iron Absorption
Increasing haemochromatosis
Decreasing
• Heme iron • Inorganic iron
• Ferrous form • Ferric form
• Acidic pH (HCl,Vit C) • Alkaline pH – antacids
• Solubilizing agents: • Precipitating agents
sugars, amino acids phytates, tannins
Reduced hepcidin: Increased hepcidin:
• Ineffective • Inflammation
erythropoiesis
• Hereditary
 Intestinal Iron Absorption

HEME IRON:
 Heme is released from apo proteins by gastric
ph and proteases.
 Taken up by mucosal cells.
 Heme oxygenase cleaves the porphyrin ring
and releases iron.
Absorption of Heme iron
 Absorption of Heme iron
• Heme receptor/ Heme carrier protein 1
• Heme exporter protein : FLVCR – Feline
Leukemia virus subgroup C receptor
• Heme-binding protein in plasma : Hemopexin
NON HEME IRON
 Ferroportin
• Iron exporter expressed in tissues handling
large iron fluxes. Associated multi-copper
oxidase ( Fe 2+ to Fe 3+)

• Basolateral surface of
enterocytes Hephaestin
• Placental trophoblast

• Macrophages
Ceruloplasmin
• Hepatocytes
 IRON CYCLE
Iron metabolism

Dietary iron absorption

Plasma iron transport

Iron delivery to erythroid

precursors

Macrophage iron recycling

Iron storage

Regulation of iron metabolism:

Systemic and cellular.
Functional body iron : 20-25 mg/day
Iron cannot be stored or circulated in
the body in free form
 Plasma Iron transport
Transferrin • Produced by liver inversely proportional to iron stores.
• Two ferric ions per molecule
• Uptake by erythroid cells/ lymphocytes.
• Total Iron Binding capacity (TIBC) (250 to 450 μg/dl )

Non transferrin • When Transferrin saturation > 45%

bound iron • Iron bound to citrate/ acetate/ albumin.
(NTBI) • High affinity for parenchymal cells - hepatocytes

Labile plasma • When transferrin saturation exceeds 75%

iron (LPI) • Toxic form
• Capacity to produce reactive oxygen species

Haptoglobin/ • Binds hemoglobin/ free heme released from intravascular

hemopexin hemolysis.
 IRON CYCLE
Iron metabolism

Dietary iron absorption

Plasma iron transport

Iron delivery to erythroid

precursors

Macrophage iron recycling

Iron storage

Regulation of iron metabolism:

Systemic and cellular.
Functional body iron : 20-25 mg/day
Iron delivery to erythroid precursors

TfR1

Fe-Transferrin
DMT1 interaction is pH
dependent

Siderotic granules:
aggregates of ferritin
surrounded by membrane
ABCB7
 IRON CYCLE
Iron metabolism

Dietary iron absorption

Plasma iron transport

Iron delivery to erythroid

precursors

Macrophage iron recycling

Iron storage

Regulation of iron metabolism:

Systemic and cellular.
Functional body iron : 20-25 mg/day
Macrophage iron recycling

+
+ IRPs

Ferroportin
 IRON CYCLE
Iron metabolism

Dietary iron absorption

Plasma iron transport

Iron delivery to erythroid

precursors

Macrophage iron recycling

Iron storage

Regulation of iron metabolism:

Systemic and cellular.
Functional body iron : 20-25 mg/day
 Iron storage

Hepatocytes and reticuloendothelial cells in

liver, spleen and bone marrow.

Ferritin Hemosiderin
 Ferritin
• Soluble form
• Protein shell: 24 subunits - H and L
ferritin
• Central hollow cavity : contains
Ferric-hydroxy- phosphate
polymers.
• Upto 4500 iron atoms per molecule
• Cytosolic poly(rC)-binding protein 1
(PCBP1) delivers Fe 2+ to ferritin.

Plasma ferritin : Reflects iron stores

Normal : 15 – 300 ng/mL
 Hemosiderin
• Denatured form of ferritin: partly degraded
protein shell
• Less soluble in aqueous solution
• High iron : protein ratio
• More stable and less available storage form
 IRON CYCLE
Iron metabolism

Dietary iron absorption

Plasma iron transport

Iron delivery to erythroid

precursors

Macrophage iron recycling

Iron storage

Regulation of iron metabolism:

Systemic and cellular.
Functional body iron : 20-25 mg/day
Iron regulation : at the level of absorption

Systemic - Hepcidin Cellular level

• Erythroid
• Iron regulatory
regulator
proteins 1 and 2
• Stores regulator
(IRP) and HIF alpha
• Inflammation
• Post transcriptional
• HIF-alpha and
regulation
growth factors
 Hepcidin
• Peptide produced in liver and excreted by
kidneys.
• Stores regulator : Intracellular iron in
hepatocytes and plasma diferric transferrin
increase hepcidin synthesis
• Erythroid regulator : Increased rate/
ineffective erythropoiesis inhibit hepcidin
synthesis.
Systemic iron regulation by hepcidin

HIF alpha
Hepcidin mediated iron regulation
Hepcidin regulation: Molecular mechanism
High affinity for
BMP2/6 TfR-1

Neogenin

Hepcidin
HAMP gene
Hepcidin mediated regulation

IL-6

Effective: Erythroferrone
Ineffective: GDF15
 Intracellular iron regulation – Iron
regulatory proteins
IRON IRON REPLETE STATE IRON DEFICIENT STATE
REGULATORY
PROTEIN (IRP)

• Iron-sulphur cluster saturated with • Binds to iron regulatory

iron elements (IRE) on mRNA
IRP1 • Cytosolic aconitase activity
• Citrate to isocitrate in TCA cycle

• Iron sensitive ubiquitin ligase • Binds to iron regulatory

• Ubiquitination and proteosomal elements (IRE) on mRNA
IRP2 degradation
 Iron Regulatory Elements (IRE)
• Stem loop structures located in 5’ or 3’
untranslated regions of different mRNAs.

Binding of IRPs to 3’ IRE Binding of IRPs to 5’IRE

Stabilizes mRNA. Blocks translation of

Promotes translation of Ferritin
DMT1 Ferroportin
TfR1 ALAS2
 Iron Regulatory Elements

5’IRE 3’IRE
• Translation happens in the • Translation happens in the
absence of IRPs presence of IRPs
• Ferritin, ferroportin, ALAS2 • DMT1 and TfR1.
Regulation of iron metabolism by
intracellular iron
Fe deficient
state

Binding of IRPs to IRE.

5’IRE
3’IRE
ferritin,
DMT1
ferroportin
TfR1
and ALAS2

Iron importing phenotype

Regulation of Iron metabolism by
intracellular iron
Fe replete
state

IRPs do not bind to IRE.

5’IRE
3’IRE
ferritin,
DMT1
ferroportin
TfR1
and ALAS2

Iron storing phenotype

Summary of iron metabolism
 Disorders of Iron metabolism
Iron refractory Iron
Iron overload without Iron overload with deficiency anemia
anemia anemia (IRIDA)

Hereditary
hemochromatosis Defect in
• TMPRSS6
• HJV, HFE, TfR2 mutations • Iron recycling
mutation – codes
• Gain of function • Iron transport/
for Matriptase-2
mutation in ferroportin – uptake by RBCs.
(MT2)
Hepcidin resistance • Erythroid
• Iron deficiency-
• Loss of function mitochondrial iron
No overload
mutation in ferroportin utilization
– Ferroportin disease
Hereditary hemochromatosis
Iron refractory microcytic hypochromic anemia
due to defects in iron metabolism
 IRON REFRACTORY IRON DEFICIENCY
ANEMIA (IRIDA) - OBJECTIVES

History of IRIDA

Definition

Pathogenesis

When to suspect IRIDA ? Clinical and laboratory features.

Approach to diagnosis of IRIDA

Differential diagnosis

Treatment and natural history

 Iron Refractory Iron Deficiency Anemia
(IRIDA)
First described by Buchanan and Sheehan in 1981 in 3
siblings who presented with :

Iron deficiency anemia despite adequate nutritional intake.

No rise in Hb following oral iron therapy.

Partial slow response to parenteral iron treatment.

No longterm growth or development related problems.

Iron Refractory Iron Deficiency Anemia
• Finberg et al in 2008 classified them as Iron
Refractory Iron deficiency anemia and
described the pathogenic mutation in
TMPRSS6 gene and key abnormalities.
 DEFINITION OF ANEMIA
WHO CDC
Children
 0.5-4.9yrs <11g/dl
Men <13g/dl  5-12yrs
Women <12g/dl <11.5g/dl
Pregnant <11g/dl Men <13d/dl
Women <12g/dl
Pregnant
 1st and 3rd trimester <11g/dl
 2nd trimester <10.5g/dl
 Definition
Iron deficiency anemia:
• Defined as Hb < 110 gm/L with a ferritin <12
microg/l in children <5 years
• Hb < 115 gm/L with a ferritin <15 microg/l in
children 5 to 12 years
Refractoriness to oral iron therapy
• Less than 1 g/dl Hb increase after 4 weeks of
oral iron therapy (3mg/kg in divided doses).
• Provided patient compliance is adequate and
acquired forms of GI disorders have been
ruled out .
Iron Refractory Iron Deficiency anemia
• Hereditary autosomal recessive condition.
• Microcytic hypochromic anemia refractory to
oral iron therapy.
• Slow and partial response to parenteral iron
therapy.
• Caused due to mutation in TMPRSS6 gene
encoding Matriptase-2 protein.
 Pathogenesis
• TMPRSS6 gene encodes matriptase-2, a
transmembrane serine protease expressed by the
liver.
• Synthesised as inactive membrane bound
polypeptide. Undergoes series of proteolytic
cleavage during zymogen activation.
Pathogenesis
TMPRSS6
gene
 Pathogenesis
Hyperhepcidinemia
Failure to suppress hepcidin in
response to low iron

Ferroportin internalization

Enterocytes
• Defective basolateral Macrophages
export of iron • Defective iron recycling
• Iron accumulates in due to defective iron
enterocytes and lost export
while shedding

Refractoriness to oral iron Partial response to

parenteral iron
 Why partial response to parenteral iron
therapy?
• Parenteral iron : Iron carbohydrate complex
must be phagocytosed by macrophages before
iron is released to plasma transferrin for
erythropoiesis
• Defect in macrophage iron recycling :
Incomplete utilization of parenteral iron
formulations.
 Mutations in TMPRSS6 gene
• Long arm of chromosome22
• Nonsense/ missense/ frame-shift mutations in
exon : usually affecting catalytic domain.
• Intronic/promoter region mutations, affecting
splicing.
 Genotype-phenotype correlation
• Biallelic nonsense mutations – most severe
phenotype
• SNPs and mutations affecting single alleles
(heterozygosity) – milder phenotype modified
by environmental factors.
 When to suspect IRIDA?
Clinical features
• Onset of anemia : infancy or early childhood
• Normal growth and development. Absence of
other iron deficiency stigmata like
koilonychia/skin/ hair changes
• Presence of history of anemia in other siblings
or an elder sibling being chronically treated for
iron deficiency without much improvement.
• Oral iron refractoriness as per standard criteria
for evaluation of response to oral iron
 When to suspect IRIDA?
Laboratory parameters
• Microcytosis and hypochromia extreme
compared to degree of anemia (Very low MCV
and MCH)
• Low-normal to normal serum ferritin with
extremely low serum iron and transferrin
saturation (<5%)
• Inappropriately high serum hepcidin levels
compared to degree of anemia
• High RBC count and low reticulocyte count
 Approach to diagnosis of IRIDA
• All other causes of congenital/ acquired
microcytic hypochromic anemia and
refractoriness to iron therapy should be ruled
out before making a diagnosis of IRIDA
• Orphan condition. TMPRSS6 gene sequencing
is cumbersome and difficult.
Structured approach to diagnosis of IRIDA
Serum iron studies
 Laboratory diagnosis of IRIDA
• No raise in serum Iron following oral iron
challenge : indicates a intestinal iron
absorption defect.
• High serum hepcidin/ TSAT ratio – highly
suggestive of IRIDA.
• No FDA approved hepcidin assay available for
clinical use yet.
 Gene analysis
• TMPRSS 6 gene analysis is confirmatory
• PCR based DNA sequencing
• Examines all coding regions (exons and
intron/exon boundaries).
IRIDA vs true iron deficiency anemia
IRIDA True iron deficiency
anemia
No skin/ hair/ nail changes Koilonychia, hair loss, angular
stomatitis.

Normal growth and development, Retarded growth and

Similar history in siblings. development

Low-normal to normal serum Markedly low serum ferritin

ferritin ( <15ng/mL)

High RBC count Low RBC count

High hepcidin Low hepcidin

IRIDA vs. anemia of chronic disease
• Elevated hepcidin causing iron retention in
macrophages.
• Low serum iron/ TSAT with high serum ferritin
• Normocytic to moderately microcytic anemia
IRIDA vs. Beta thalasssemia trait
Microcytic hypochromic anemia
with high RBC count

Beta thalassemia
IRIDA
trait
• Low retic
• High retic count
count • Normal to high
• Low serum
serum iron and
iron and TSAT
TSAT
• Normal HbA2
• Elevated HbA2
IRIDA vs other defects in iron metabolism

IRIDA Other genetic defects

Low serum iron/ TSAT High serum iron/ TSAT.

( Low in aceruloplasminemia)

Low-normal ferritin High serum ferritin.

Ringed sideroblasts in bone
marrow in case of sideroblastic
anemias.
Neonatal/ early infancy onset. Congenital onset
MT-2 not required for in-utero In-utero iron transfer is
iron transfer. affected.
 Treatment of IRIDA
• Limited experience with long-term
management.
• Trial of oral iron therapy with ascorbic acid for
6 to 8 weeks may be beneficial in subset of
patients.
 Parenteral iron therapy
• Parenteral Iron Dextran therapy – slow and
partial correction.
• Following iron infusion serum ferritin
increases while serum iron and TSAT remains
low.
• Need repeated infusions.
• May develop reticuloendothelial pattern of
iron overload (haemosiderosis)
 Newer therapy
• Anti hepcidin antibody
• Anticalin : Modified lipocalin targeting
hepcidin with high affinity
• PEGylated anti-hepcidin L RNA oligonucleotide
• Degradation of HAMP mRNA by siRNA ( small
interfering RNA)
 Screening of siblings
• Initially screened for presence of anemia
• If present genetic testing targeting the
mutation present in other sibling is offered.
 Natural history
• Correction of anemia in adulthood – No or
reduced iron requirements.
• Microcytic hypochromic indices, low serum
iron and TSAT persists.
• Due to decrease in iron demands for body
growth, limited iron that is available is used
for erythropoiesis.
Summary
 References
• Lawrence T. Iron deficiency and related
disorders. In: Greer JP, Arber DA, Glader B, List
AF, Means RT, Foerster J editors. Wintrobes
clinical hematology. Thirteenth ed. Vol 2.
Philadelphia: Lippincott Williams and Wilkins;
2014:617-61.
• Hoffbrand AV, Paul AH. Hoffbrand’s Essential
Haemtology. Seventh ed. John Wiley and Sons
Ltd;2016:28-40.
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