BY

Azza Mostafa Elkady Assistant lecturer of clinical pathology

Assiut universityA.R.E.

Distribution of Iron

3-5 gm of iron is in the body(total)

2-2.5 g of which is in the hemoglobin.(66%)

Some (about 130 mg) are in myoglobin (oxygen carrier in tissues).(4%)

Little (about 8 mg) is bound to enzymes like peroxidases, cytochromes and other enzymes involved in the Krebs Cycle.

Some are stored in ferritin and hemosiderin.(29%)

Little (3-5 mg) is in plasma in transferrin.(0.1%)

Requirements

15-20 mg /day, only 1 mg is absorbed normally

Increased physiological iron requirements:

Infancy,It is aggravated by prematurity, infections and delay in mixed feeding.

It is also frequent in adolescence, in females and in pregnancy;

The fetus acquires about 280 mg of iron.further 400 – 500 mg is required for the

temporary expansion of maternal red cell mass. Another 200 mg of iron is lost with the placenta

and with bleeding at delivery.

Although iron absorption increases throughout pregnancy and increased requirements are partly offset by amenorrhoea, this may not be sufficient to meet the resultanet maternal outlay of over 600 mg iron.

Iron is better absorbed from animal than vegetable sources.

Iron is released from protein complexes by acid and proteolytic enzymes in the stomach

and small intestine..Iron is maximally absorbed from the

duodenum and less well from the jejunum, probably because the increasingly alkaline environment leads to the formation of insoluble ferric hydroxide Acid complexes.

Physical State (bioavailability(

heme > Fe2+ > Fe3+

Inhibitors

phytates, tannins, soil clay, laundry starch, iron overload, antacids

Competitorslead, cobalt, strontium, manganese, zinc

Facilitatorsascorbate, citrate, amino acids, iron deficiency

Factors That Influence Iron Absorption:

Therapeutic ferrous iron salts are well absorbed on an empty stomach, but when taken with a meal absorption is reduced as a result of the same ligand - binding processes that affect dietary non - haem iron;

Molecular pathways of iron absorption. the diagram refers to iron absorption by the villous epithelial cell. DMT1, divalent metal transporter 1; FPN, ferroportin; Hp, hephaestin; TF, transferrin; TFR, transferrin receptor.

•Non – haem iron is released from food as Fe 3 + and reduced by duodenal cytochrome b1 DCytb) to Fe 2+.

•This is transported across the brush border membrane by DMT1, which is upregulated in iron deficiency.

•It is assumed that iron enters the labile pool and some may be incorporated into ferritin and lost when the cells are exfoliated.

•Iron destined for retention by the body is transported across the serosal membrane by ferroportin before uptake by transferrin as Fe 3 + .

•Hephaestin is a (copper - containing ferroxidase) expressed predominantly in villous cells of the small intestine that converts Fe 2 + to Fe 3 + in the basolateral transfer step of iron absorption.

•Haem iron is initially bound by haem receptors at the brush border membrane and released intracellularly by haem oxygenase before entering the labile iron pool and following a common pathway with iron of non - haem origin.

Proteins important in iron metabolism

HaemoglobinDivalent metal transporter 1Ferroportin (SLC40A1)HepcidinGrowth differentiation factor and twisted

gastrulation protein Ferroportin (SLC40A1)Matriptase - 2 (TMPRSS6)Transferrin and transferrin receptorsFerritin and haemosidern

Divalent metal transporter 1(DMT)1 (DMT)1 is an electrogenic pump that requires proton

cotransport in order to transfer Fe across cell membranes.

The intestinal DMT is produced by different mRNA splicing from that which produces endosomal DMT1.

DMT1 expression is upregulated in iron deficiency and may be involved in absorption of other divalent metal cations including Mn 2+ ,Co 2+ , Zn 2+ , Cu 2+ and Pb 2+ , although this is not established as a major function.

Ferroportin (SLC40A1)This transmembrane domain protein is the

basolateral transporter of iron, essential for iron release from macrophages.

It is also present in intracellular compartments.Caeruloplasmin is required for the cell surface

localization of ferroportin, whose concentration is controlled by hepcidin.

Hepcidin:Hepcidin has a central role in the regulation of iron

metabolism and absorption . A product of the HAMP gene, it is a small peptide with

several isoforms. It is predominantly expressed in the liver. It regulates iron homeostasis by binding to cell – surfac

ferroportin, causing it’s tyrosine phosphorylation, internalization, ubiquitination and degradation in lysosomes. It therefore acts to inhibit iron absorption, iron release from macrophages and iron transport across the placenta.

It is bound in plasma to α 2 – macroglobulin and the major route of clearance is the kidney.

Hepcidin can be measured in serum or urine by ELISA or mass spectrometry - based techniques.

These have shown low or undetectable levels in iron deficiency and extremely high levels in inflammatory conditions, with inappropriately low levels in haemochromatosis and iron - loading anaemias.

Regulation of hepcidin expressionI.The regulation of hepcidin expression is

transcriptional. Expression depends on signalling through the BMP/SMAD pathway.

(A) HJV: is a member of the repulsive guidance molecules

(RGM) family that is highly expressed in liver, skeletal muscles and the heart.

It is either associated with cell membranes or released as a soluble form.

Membrane - bound HJV participates in the pathway regulating hepcidin expression as a BMP coreceptor, whereas soluble HJV antagonizes BMP - 6. BMP - 6 is the master hepcidin activator in vivo .

HJV binds to BMP, then phosphorylates SMADs to form an SMAD- 1/- 5/- 8– SMAD- 4 complex, which translocates to the nucleus and stimulates hepcidin production by activating its promoter.

(B)HFE and TFR2 are also involved in hepcidin expression.

HFE is able to bind TFR1 and TFR2. During low or basal serum iron conditions,

HFE and TFR1 exist as a complex.TFR1 serving to sequester HFE to silence its

activity.

Increased serum transferrin saturation therefore results in dissociation of HFE from TFR1. Acting as an iron sensor, HFE then binds to TFR2 and conveys the Fe 2+ - TF status to the signal transduction effector complex.

Simulatory and inhibitory signals of hepcidin regulation High plasma iron and inflammation stimulate hepcidin synthesis. This is mediated by SMADs and STAT3 respectively.

Conversely, low plasma iron, increased rates of erythropoiesis (including ineffective erythropoiesis) and hypoxia inhibit hepcidin production. This is mediated by matriptase, growth differentiation factor (GDF) - 15, twisted gastrulation protein (TWSG1) and HIF1 – α respectively.

Hepcidin binds ferroportin (FPN), causing its destruction and so inhibits iron absorption and iron release from macrophages into plasma and from intracellular compartments. BMP,bone morphogenetic protein.

Genetic mutations of HFE, TFR2, HJV and

hepcidin all result in haemochromatosis with low serum hepcidin levels.

II. A second type of transcriptional hepcidin regulation occurs in inflammation. Interleukin (IL) - 6 and IL - 1 β induce transcription of the hepcidin gene by activating STAT3 (signal transducer and activator of transcription 3) and its binding to a regulatory element in the hepcidin promoter.

III. Other studies indicate that the von Hippel – Lindau hypoxia inducible factors (HIFs), which stimulate erythropoietin synthesis, control iron homeostasis by the downregulation of hepcidin, repressing its promoter with the upregulation of ferroportin.

Under normal conditions, HIFs play a useful roll by mobilizing iron and supporting erythrocyte production in response to anaemia/hypoxia.

However, the same mechanism may contribute to the harmful accumulation of iron in response to chronic anaemia associated with ineffective erythropoiesis in thalassaemia and other dyserythropoietic anaemias.

The hepcidin response is remarkably rapid. In humans, iron ingestion results in a sharp increase in urinary hepcidin excretion within 12 – 24 hours of starting treatment. Likewise, infusion of recombinant IL - 6 results in significant increase in urinary hepcidin and decreased serum iron and transferrin saturation within 2 hours of infusion.

These observations imply that hepcidin expression is directly controlled by serum iron (probably by transferrin saturation) and IL - 6 and not by long - term gradual accumulation of iron in tissues.

Growth differentiation factor and twisted gastrulation protein

Growth differentiation factor (GDF) - 15 is a member of the transforming growth factor (TGF) - β superfamily of proteins.

GDF - 15 exerts different functions according to the cell context, inhibiting hepcidin synthesis, macrophage activation, proliferation of immature haemopoietic progenitors and growth of tumour cell lines.

It is inducible by iron depletion and by hypoxia but is independent of hypoxia - inducible transcription factors (HIFs).

A second erythroid regulator of hepcidin expression, twisted gastrulation protein (TWSG1), has been identified.

Matriptase - 2 (TMPRSS6)This is a type 2 member of the transmembrane

serine protease family mainly expressed in the liver.

Membrane – bound matriptase - 2 regulates hepcidin expression by cleaving membrane bound HJV, releasing soluble HJV fragments.

Mutations in mice and humans result in marked upregulation of hepcidin and blockade of intestinal and macrophage iron transport into plasma, leading to a refractory hypochromic microcytic anaemia.

Transferrin and transferrin receptors:Transferrin is a single - chain polypeptide present in

plasma (1.8 – 2.6 g/L) and extravascular fluid.

The protein is synthesized predominantly by the liver, synthesis being inversely related to iron stores.

Two atoms of ferric iron bind to each molecule. Although transferrin contains only about 4 mg of body

iron at any time, it is vital to iron transport, with over 30 mg iron passing through this compartment each day .

The transferrin receptor:The uptake of iron from transferrin requires that

the protein is attached to specifc receptors on the

cell surface.The transferrin receptor gene (TFRC) codes for

TFR1, a transmembrane protein (identified as CD71).

A second receptor, TFR2, also binds transferrin.Through their binding with HFE, TFR1 and TFR2

are involved in regulating hepcidin synthesis.

Lactoferrin:Lactoferrin is a glycoprotein that is

structurally related to transferrin. It is found in milk and other secretions and in

neutrophils. It is thought to have a bacteriostatic action at secreting surfaces by depriving microorganisms of the iron needed for their growth.

2 forms of storage iron:

Ferritin:Ferritin is the primary iron storage protein . It

is water-soluble complex consists of an approximately spherical apo-protein shell enclosing a core of ferric hydroxyphosphate.

Human ferritin is made up from 24 subunits of two immunologically distinct types: H and L.

Variation in the proportion of H to L subunits explains the heterogeneity of ferritin from different tissues on isoelectric focusing: L - rich ferritins (from spleen and liver) are more basic than H - rich ferritins (from heart and red cells). The small amount of ferritin normally present in serum contains little iron and consists almost exclusively of L subunits.

a positive acute phase reactant/protein – increases in inflammation.

Synthesis controlled at 2 levels:DNA transcription via its promotor.mRNA translation via interactions with iron

regulatory proteins.

Ferritin in erythroid precursors may be of special importance in haem synthesis especially at beginning of Hb accumulation, when Tf-TfR pathway still in sufficient.

When ferritin accumulates, it aggregates, proteolyzed by lysosomal enzymes, , then converted to iron-rich, poorly characterised haemosiderin, which releases iron slowly.

M-ferritin – present in mitochondria. Expression correlated with tissues that have high mitochondrial number, rather than those involved in iron storage.

Hemosiderin:Haemosiderin, unlike ferritin, is a water -

insoluble, crystalline,protein – iron complex that is visible by light microscopy when stained by

the Prussian blue (Perl’s) reaction.

It has an amorphous structure, with a higher iron/protein ratio than ferritin, and is probably

formed by the partial digestion of ferritin aggregates by lysosomal enzymes.

In normal subjects, majority of storage iron is present as (ferritin, and haemosiderin) is

predominantly found in macrophages rather than hepatocytes.

In iron overload, the proportion present as haemosiderin increases considerably in both

cell types.