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MODERN INSIGHTS INTO ANEMIA

Hepcidin,the key regulator of iron metabolism Dr.Hossein Ghaziasgar

IRON

An essential element ,must be precisely regulated

Intestinal absorption is essential for the iron balance ,but the precise mechanism of its regulation was unknown

Conservation of iron

Recycling from Hb .(about 20 mg/day) Iron absorption in the duodenum Deposition in the liver (hepatocytes)

The coordination is essential and vital

BUT

Humans and other mammals lack effective mechanisms to excrete excess iron (the daily loss of iron from the body is 1-2mg/day)

Intestinal iron absorption is the sole means of iron balance

Excess iron

Is deposited in the liver, endocrine glands ,the heart and the skin

Tissue damage (in hereditary hemochromatosis)

Question

Who is playing the central role in “orchestration” on iron metabolism?

?

Answer

Hepcidin ,the key regulator of iron metabolism

What is Hepcidin?

Was first identified in human urine and plasma in June 2000

A 25-amino acid disulfide-rich peptide Molecular weight about 2 KDa Is highly folded and cationic amphipathic

What is Hepcidin?

It was recognized as an antimicrobial peptide ( Defensin-like) and is produced in the liver and excreted in urine.

Of course recent studies detected Hepcidin synthesis in bacteria-activated neutrophils and macrophages (at a lower level)

What is Hepcidin?

In addition to the 25-amino acid form ,the urine also contains 20- and 22-amino acid forms (but the role is not identified)

Hence the bioactive form is the 25-amino acid form which contains 4 disulfide bands (Hepcidin 20- has been found in serum too, in 2007 )

Hepcidin gene

Is named HAMP Contains 3 exons : produces 84-amino acid preprohepcidin

60-amino acid prohepcidin

25-amino acid hepcidin Has also been identified in other

vertebrates like mice , rats , pigs and several species of fish.

Hepcidin

Controls extracellular iron by regulating:

1. Intestinal absorption

2. Recycling by macrophages

3. Releasing from stores

4. Placental transport

The Mechanism

The major mechanism of Hepcidin function is “the regulation of transmembrane iron transport”.

HOW?

Hepcidin binds to its receptor, protein FERROPORTIN , which serves as a transmembrane iron channel enabling iron efflux from cells.

THEN

The Hepcidin-Ferroportin complex is degraded in lysosomes and iron is locked inside the cells (mainly enterocytes, hepatocytes and macrophages)

So

Hepcidin lowers iron absorption in the intestine ,lowers iron releasing from hepatocytes and macrophages

Serum iron is decreased.

Ferroportin

The sole protein exporter is located on:

1. the intestinal cells

2. hepatocytes

3. macrophages

4. placental cells

The second role

Hepcidin is an acute phase protein type 2 and is increased in inflammation.

Hormonal activity of Hepcidin

Lack of Hepcidin results in Hemochromatosis (iron deposition in the liver ,pancreas and macrophages)

Hepcidin excess results in severe iron –deficiency (blocking intestinal iron uptake and iron releasing from hepatocytes and macrophages and inhibiting the placental transport of iron too)

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Injected synthetic 25-amino acid Hepcidin causes 75% decrease in serum iron levels within 1 hour persisting for more than 2 days.

By a decrease in Hepcidin level , erythroid regulation can increase iron uptake 40mg/day(normal 1-2 mg/day).

Regulation of Hepcidin synthesis by iron

Dietary iron induces Hepcidin synthesis Urinary Hepcidin concentrations are

greatly increased within less than 1 day after iron ingestion.

But

The mechanism is not known completely yet:

In the liver the proteins:

1. HFE

2. Transferrin receptor 2

3. Hemojuvelin

may be involved in mediating this signal

Regulation of Hepcidin synthesis by anemia and hypoxia

Oxygen Hepcidin

Uptake of diet iron

Iron release from hepatocytes

Iron release from macrophages

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Erythropoietic signal Hepcidin Tissue iron differic transferrin Hepcidin

Regulation of Hepcidin synthesis by inflammation Interleukin-6 Hepcidin

iron anemia of chronic disease

The role of Hepcidin in hereditary hemochromatosis

Hereditary hemochromatosis:

-excessive intestinal iron absorption

-Saturation of transferrin

-Iron deposition in vital organs

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Mutations in:

-HFE gene: most common form

-TfR2 gene: much rarer

-HAMP gene: Severe phenotype

-HJV gene: Severe phenotype

The role of Hepcidin in anemia of inflammation This anemia results from:

-Chronic infections

-Noninfectious generalized inflammatory disorders

-Some cancers

-Sepsis

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IL-6 Hepcidin Hypoferremia

anemia of inflammation

The role of Hepcidin in iron-loading anemias

Ineffective erythropoiesis Increased intestinal iron absorption Increased ferritin Decreased Hepcidin

Hepcidin in differential diagnosis

Increased HepcidinDecreased Hepcidin

High iron stores

Anemia of chronic disease

Ferroportin disease(?)

Iron deficiency

Increased and/or ineffective erythropoiesis

Classical, ,juvenile and TfR2 HH

Hepcidin assays

Immunoassay (urine ,UCLA) Mass spectrometry (urine ,serum) Elisa (for Prohepcidin)

Hepcidin assays

Serum Prohepcidin:50-150 ng /mL Serum Hepcidin :1-500 ng/mL

(based on 2007 findings)

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Hepcidin in urine is affected by multiple freeze/thaw cycles. Serum is more stable.

High diurnal variation of especially serum Hepcidin.

Use of internal standard:Hepcidin-24 to control for matrix influences and instrumental settings.

Overall summary

Hepcidin

-Is a recently discovered liver produced 25 amino-acid peptide

-Is a regulator of iron metabolism that controls iron absorption and macrophage iron release.

-Is regulated by erythropoietic needs( ) ,body iron stores( ) and inflammation( )

Overall summary

MS Hepcidin assay for urine and serum:

-Precludes the need for Hepcidin specific antibodies

-Will provide:

-information on etiology of iron metabolism disorders

-leads for new therapeutic strategies

-novel diagnostic approaches