WelcomeHaematology joint seminar

14/11/2012

Dr Kishore & Dr Ashutosh

15-year-old male 

WBC 4.5 x 109/L, Hgb 8.6 g/dl, PLT 350 x 109/L with

MCV of 76 fl. 

Hypochromic, microcytic RBCs

No GI symptoms

Serum Fe of 15, transferrin saturation of 5 percent.

Given oral iron supplementation – No use.

IV iron supplementation –subsequent partial

improvement in Hgb

Two cousins have similar history..

In the next 30 mins lets try to understand what's happening in him…

Iron Metabolism

Kishore Kumar

Basic facts

Absorption

Transport & Recycling

Transferrin cycle & storage

Systemic Homeostasis

Cellular level regulation

Pathogenesis of IDA

Outline

Historical aspect:

It was depicted in many paintings by Dutch masters .

A disease believed to be Iron Deficiency Anemia was

described in 1554 by Johannes Lange.

It was termed cholorsis or green sickness and iron salt was used for its treatment.

Almost exclusively in adolescent girls between 14 and 17.

Most common element on earth.

Still iron deficiency is the most common nutritional disorder in

the world.

Thanks to low bioavailability and complex metabolic

pathways..

Apart from the commonly known overload states, is also

involved in neurodegenerative diseases, Sideroblastic anemia ,

MDS etc..

Some basic facts..

May be 2+ or 3+

◦ Ferrous (2+) “reduced” - gained an electron

◦ Ferric (3+) “oxidised” - lost an electron

Fe+++ + e- Fe++

Redox states allows activity passing electrons around

body

Redox change required for iron metabolism

Basic facts..

Stable state of iron in most of its biological complexes

is Fe(III).

Reduction needed for transport.

These properties render it potentially toxic.

Haber-Weis-Fenton´s reaction.

Double distress in living organisms..

Oxygen transporting Heme containing proteins (Hemoglobin ,

myoglobin)

Heme containing enzymes like cytochromes, catalases and

peroxidases.

Iron-Sulfur containing proteins like Aconitase , ferrochelatase.

Iron transport proteins

Iron storage proteins.

Key Iron containing proteins in humans

In a 70 kg male=3.5gms In females= 250-300mg less than males

Normal Iron Balance

Full term infants

75mg/kg

Adult males 50mg/kg

Adult females 35mg/kg

DMT1(Nramp, DCT1) – apical membrane of

enterocytes & endosomes.

Ferroprotein (IREG1) – Export of ferrous iron.

HCP-1 – Heme transporter in enterocytes.

Integrin- Mobilferrin complex – Ferric iron transport

TfR1 – Binds Tf-Fe2 & initiates Transferrin cycle

Proteins involved in Transport

DcytB – Ferrireductase on enterocyte surface

Hephaestin – Ferro-oxidase in basolateral membrane

Ceruloplasmin – oxidase in other tissues

Heme Oxygenase-1 – release of iron from heme

Proteins assisting in iron transport

Lactoferrin – free iron scavenger in body fluids

Siderocalcin – Acute phase reactant

Hemopexin

Haptoglobin

Iron binding proteins

No known regulated pathway of iron excretion

Intestinal mucosa responds to changes in body iron

stores, tissue hypoxia, and demand for iron, and it alters

absorption accordingly.

Duodenum and jejunum – excess Ferroprotein conc.

For optimal nutrition a daily intake of 8-10 mg of iron is

required.

Intestinal iron absorption

Acidic pH, vitamin C and some low - molecular -

weight chelates (e.g. sugars, amino acids) enhance

absorption.

Therapeutic ferrous iron salts are well absorbed on an

empty stomach.

Phytates, tannates in tea and bran inhibit absorption.

Understanding Heme Transport - N.C Andrews - New England Journal of Medicine:353;23 - 2508

DMT1

DMT1 is not specific for iron transport but also

mediates transport of other divalent metal cations

expressed on the membrane of endosomes

DMT-1 seems to have a role in transport of non

transferrin bounded iron (NTBI)

Murine microcytic anemia

While ferrous iron uses DMT-1, ferric uses integrin-

mobilferrin pathway (IMT) that solely transports

ferric iron.

In the cell cytosol these proteins are integrated into a

large protein complex called paraferritin

Contains beta-2-microglobulin and DMT-1

Is IRON absorbed in ferric state?

Transport of iron associated with some proteins (chaperones)

or transcytosis

Sequestrated as ferritin or transported into circulation

Enterocytes shed in two days

Transport of iron by ferroportin decides whether to keep or

discard

These cells also express transferrin receptors type 1 (TfR1)

and iron from the plasma can enter.

Intracellular iron transport

Ferroportin represents the only known iron exporter

Basolateral membranes of duodenal enterocytes,

RES macrophages, hepatocytes and placental cells

Requires change of redox state by ferroxidase -

hephaestin in the duodenum and ceruloplasmin

elsewhere in the body

Type IV hemochromatosis

Basolateral iron transport

Intestinal iron absorption increases withDecreased iron storesIncreased erythropoietic activityIneffective erythropoiesisAnaemiaHypoxia

Intestinal iron absorption decreases in inflammation

Iron absorption regulations

Two models have been proposed to explain

how the absorption of iron is regulated

◦Crypt programming model

◦Hepcidin model

Increased iron in plasma

Increased iron in

enterocyte

Forms a complex

with IRP/IRE

Translocates to nucleus

Inhibits Transcription of DMT1,hephestin, ferroportin and

increases ferritin

Crypt programming model

Iron Absorption DcytB

◦Reduction Fe+++ to Fe++

DMT1◦Transport into cell

Ferritin◦Storage in cell

Hephaestin◦Oxidises Fe++ to Fe+++

Ferroportin◦Transport out

Iron transport in plasma 

Fleming, R. E. et al. N Engl J Med 2009;352:1741-1744

Transferrin provides solubility, reduced reactivity and thus provides

a safe and controlled delivery of iron to all cells in the body.

Due to high affinity all non-heme iron in circulation is bounded to

transferrin

NTBI refers to all forms binding to ligands other than transferrin.

Capable of free entry into cells. very reactive and could enter

Fenton reaction.

Transferrin has two iron binding sites which binds one iron

atom each

Transferrin exists in 3 forms-apotransferrin, monoferric,

diferric

30-40% of these sites are occupied under normal

physiological conditions.

Contains only about 3 mg of body iron at any time, it is vital

to iron transport, with over 20 mg iron passing

through this compartment each day

Iron uptake in the tissues - Transferrin cycle

TfR1 andTfR2.

TfR1 is expressed by all iron-requiring cells but level of

expression varies.

Diferric transferrin has a higher affinity for TfR1

Soluble transferrin receptor (sTfR) is released by proteolytic

cleavage of the protein C-terminal end and regulated by

transferrin

The level of sTfR reflects the availability of functional iron.

Transferrin receptors

TfR2 - liver, hematopoetic cells, duodenal crypt cells.

TfR2 binds to HFE and transferrin in different

domains

It is assumed that TfR2/HFE complex is required for

transcriptional regulation of hepcidin production

Pool of iron complexed with low affinity ligands (citrate, ATP,

amino acids, ascorbic acid or by unidentified chaperones)

LIP represents < 5% of the total cellular iron.

Supplies iron to the mitochondrion, synthesis of iron-containing

proteins in cytosol thereby controlling numerous metabolic

reactions.

LIP is catalytically active and capable of initiating free radical

reactions

Labile iron pool (LIP)

Ferritin has got dual function of iron detoxification and reserve.

The protein shell is constructed of 24 molecules of two distinct ferritin subunits, designated H (for heavy or heart) and L (for light or liver). ◦H chains contain a ferroxidase - oxidize iron &

acquire iron more rapidly. ◦L chains are more stable and resistant to denaturation.◦Theoretically 4500 atoms but usually 2000 atoms

Body iron stores- Ferritin and Hemosiderin

Dia-13nmCentral core-6nm6 channels

Small quantities are present in nucleus and mitochondria

Biosynthesis of heme and enzymes that contain Fe-S group

Very small amount enters into circulation - lysosomal secretory pathway

Non-ferous, and its exact biologic purpose is still unknown

Plasma ferritin concentration of 1 µg/L corresponds to 8-10 mg tissue iron stores

Incompletely degraded ferritin

Conglomerate of iron, ferritin proteins,lipids, sialic

acids and porphyrin in small amounts

Less soluble

Stains with prussian blue

More stable and less available form of storage iron .

Hemosiderin

ONE WORD TO REMEMBER

HEPCIDIN

Regulation of systemic iron homeostasis

25 aa peptide secreted from liver.

Antimicrobial activity & “Hypoferremic hormone”

In 2000,by accident, investigators studying gluconeogenesis in

infections silenced the gene for hepcidin in the mouse –

Unusual increase in PARENCHYMAL IRON. Decreased iron stores

Increased erythropoietic activity

Anemia

Hypoxia

Hepatic bacteriocidal protein

1.Regulation by iron status, dietary iron and iron

stores.

2. Regulation by inflammation.

3. Regulation by hypoxia/anemia.

4. Regulation by erythroid factors.

What regulates HEPCIDIN

HFE/TfR2/Tf Regulation of Hepcidin Transcription

TfR1 is hypothesized to sequester HFE.

Tf and HFE compete for binding to TfR1

TFR2 is predominantly expressed in hepatocytes

Tf induced release of HFE from TfR1 - to increase the

association of HFE with TfR2 and to stimulate

hepcidin transcription.

Hepcidin regulation by iron status

HJV is a GPI-linked membrane protein encoded by the gene,

HFE2

Homozygous or compound heterozygous mutations in HFE2

result in a juvenile form of HH.

HJV is a co-receptor for BMP2, BMP4, BMP5, and BMP6

and enhances hepatic hepcidin expression by enhancing BMP

signaling

HJV, BMP6, and Matriptase-2

The HJV-BMP ligand-BMP receptors complex induces an

intracellular BMP signalling pathway which in turn activates

the SMAD4 signalling pathway, which translocates from the

cytoplasm to the nucleus to regulate gene expression

Two potential BMP-responsive elements critical for BMP6

and HJV responsiveness are present in both the distal and

the proximal regions of the hepcidin promoter

Essential component of a pathway that detects

iron deficiency

Cleaves membrane bound HJV increasing

sHJV that competitively impairs BMP

signaling.

Matriptase-2 (type II transmembrane serine proteinase; TMPRSS6)

Hepcidin regulation by inflammation, hypoxia/anemia and erythroid factors

Each cell has the capacity to regulate its own

utilisation of iron

Cells replete in iron, ferritin TFRC

In contrast, iron-depleted cells, TFRC, Ferritin

The IRE/IRP Regulatory System

IRP act as the cell sensor to iron availability

Regulation of cell iron homeostasis

IRP1, when saturated with iron, acts as a cytosolic aconitase

and catalyzes the conversion of citrate to isocitrate .

IRP2 is less abundant and does not have an identified enzyme

function.

IRE at 5´-UTR mRNA   ferritin, ferroportin - Not repressed

IRE at 3´-UTR mRNA   TfR1, DMT1 - Unstable

DcytB Hemochromatosis(HFE)

IRP1

DMT 1 STEAP3 IRP2

Hemojuvelin Transferrin IRE

FLVCR TFR1 TMPRSS6

Ferroprotein TFR2 GDF-1

Hepcidin Ferritin (H) TWSG

Hephaestin Ferritin (L) EPO

Let's try to consolidate…

Some future directions to this understanding.. Directly or indirectly modulate hepcidin for ACD

Cancer cells have a high iron demands - clinical studies

using iron chelators as anticancer therapy

Recombinant lactoferrin for treating bacterial and viral

infections

Iron chelators - neuroprotective and neurorestorative

effects

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 DEFICENCY - STAGES

Prelatent

◦ Reduction in iron stores without reduced serum iron levels

detected by a low serum ferritin measurement

Latent

◦ Iron stores are exhausted, but the blood hemoglobin level

remains normal

Iron deficiency anemia

◦ Blood hemoglobin concentration falls below the lower limit of

normal

Increased iron requirements •Blood loss •Rapid growth in body size between 2 and 36 months of age •Pregnancy and lactation

Inadequate iron supply •Poor nutritional intake in children (not a common independent mechanism in adults but often a contributing factor)

Malabsorption • Gastric bypass surgery for ulcers or obesity • Achlorhydria from gastritis or drug therapy • Severe malabsorption (for example, celiac disease [nontropical sprue]) • Abnormal transferrin function • Autoantibodies to transferrin receptors

Causes

IRON DEFICIENCY ANEMIA GENERAL ANEMIA’S SYMPTOMS:

Fatigability

Dizzeness

Headache

Irritability

Palpitation

CHARACTERISTICS SYMPTOMS

Glossitis, stomatitis DYSPHAGIA ( Plummer-Vinson syndrome)

Atrophic gastritis

Dry, pale skin

Spoon shaped nails, koilonychia,

Blue sclerae

Hair loss

Pica (apetite for non food substances such as an clay)

Increased platelet count

Thanks for your attention..

Suggested Reading…

Now we have to manage the case…

Now we have to manage the case..