IRON

Dr.Riddhi H PatelDr.Khushbu Soni

3rd year Residents Biochemistry

● Iron is very important in the human body because of its occurance in many hemoproteins such as hemoglobin, myoglobin and cytochromes.

● Other enzymes such as aconitase and ferredoxin have iron co-ordinated with sulfur, forms “Iron sulfur cluster”.

● Enzymes of the krebs cycle like Aconitase, Isocitrate dehydrogenase, Succinate dehydrogenase and citrate synthase modulated by iron.

● Iron modulates the expression of the critical citric acid cycle enzyme aconitase via a translational mechanism involving iron regulatory proteins.

● Iron supplementation results in increased formation of reducing equivalents (NADH) by the citric acid cycle, and thus in increased mitochondrial oxygen consumption and ATP formation via oxidative phosphorylation.

Distribution of Iron

● Total body iron in healthy adult male: 3-4 gm

Requirement

● For Adult : 20 mg/day (Only 1 mg is absorbed)

● For Children : 20-30 mg/day

● Pregnant women : 40 mg/day

Sources of Iron

● Leafy vegetables

● Pulses

● Cereals

● Liver and Meat

● Jaggery

● Cooking in Iron utensil

Absorption

● Iron is ingested in diet either as nonheme or heme iron.

● Heme Iron Absorption:

- Heme carrier protein present on microvillous surface of absorptive enterocyte.

● Non heme iron absorption:

Transferrin

● A glycoprotein playing important role in shuttle of iron between various tissues.

● It is beta-1 globulin.

● Each molecule binds with 2 Fe+3 molecule.

● Glycosylation of transferrin is impaired in :

- Congenital disorder of glycosylation

- Chronic alcoholism.

● TIBC

● Transferrin saturation

Transferrin Cycle

Recycling of Iron by macrophage

Factors influencing Iron absorption

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

Storage of Iron

● Iron is stored as a ferritin

Serum ferritin

● In serum minute quantities of ferritin present in concentrations proportional to total body stored iron.

● Serum ferritin is glycosylated.

● Contains mostly L- chain.

● Poor in iron.

● So mostly apoferritin.

● Hemosiderin :

- Aggregated, partially deproteinized ferritin.

- Formed when ferritin is partially degraded in secondary lysosomes.

-Insoluble in aqueous solution.

- Found predominantly in cells of liver, spleen and bone marrow.

- iron is released slowly from aggregates of hemosiderin.

Intracellular Iron Regulation

Systemic iron regulation

● Hepcidin is the chief regulator.

● Hepcidin decreases iron absorption in the intestine (Mucosal Block)and also prevents the recycling of iron from macrophages.

Hepcidin regulation

● Iron Sensing Complex :

-HFE Protein

-TfR1

-TfR2

-HJV● Bone Morphogenetic Proteins

● Erythropoietic Signals

● Inflammation : Interleukin-6

● Hypoxia

Excretion of Iron

● Iron is one way element and very little of it is excreted.

● Any type of bleeding will cause loss of iron.

● Women up to menopause will loss iron at a rate of about 1 mg/day.

● Male : <0.5 mg/day.

● Almost no iron is excreted through urine.

● Considerable fecal loss.

● Some amount from skin.

Clinical Significance

Major disorders of iron metabolism

– Iron deficiency -Iron overload● Abnormal iron distribution

● Abnormal production of iron related proteins

-Hereditary Hyperferritinemia-cataract syndrome

-Aceruloplasminemia

-Neuroferritinopathy

-Atransferrinemia

Iron Deficiency

● One of the most prevalent nutritional deficiency disorder.

● 70% of indians and 85% of pregnant women suffer from it.

● Anemia: a reduction in the oxygen-carrying capacity of the blood caused by a diminished erythrocyte mass.

Pathophysiology

Increase demands of iron

Increase iron loss

Decrease iron intake

Stages of Development

● 3 Stages:

1) Negative Iron balance

2) Iron deficient erythropoiesis

3) Iron deficiency Anaemia

1) Negative Iron balance

● Intestinal iron absorption is insufficient.

● Body's iron stores being mobilized to meet requirements.

● So progressive depletion of iron stores.

2)Iron deficient erythropoiesis

● Hemoglobin synthesis is impaired.

3) Iron deficiency Anaemia

Clinical Presentation

● Iron-deficiency anemia can cause:

brittle nails

cracks in the sides of the mouth

Extreme fatigue (tiredness)

chest pain

● Pale skin

● Dizziness or lightheadedness

● Fast heart rate

● Headache

● An enlarged spleen

● Cold hands and feet

● frequent infections.

● Irritability

● shortness of breath

● swelling or soreness of the tongue

Changes in Various laboratory tests

Parameters Normal Negative iron balance Iron deficient erythropoiesis

Anaemia

Serum Ferritin (ug/dl) 50-200 <20 <15 <15

TIBC (ug/dl) 300-360 >360 >380 >400

Serum Iron (ug/dl) 50-150 Normal <50 <30

Transferrin saturation (%)

30-50 Normal <20 <10

RBC Protoporphyrin (ug/dl)

30-50 Normal Increased Increased

Soluble transferrin receptor (ug/l)

4-9 Increased Increased Increased

RBC morphology Normal Normal Normal Microcytic hypochromic

● Staining of Bone marrow iron with perls' prussian blue to allow visualization of ferric ion is probably most reliable but impractical.

● It may be misleading due to:

-Insufficient sample size

-Patient treated with parentral iron suppliments

-Stainable iron may be present in the phase of deficiency

-Patients with myeloproliferative disorders

TREATMENT

IRON SUPPLEMENT

Iron overload conditions

Hemochromatosis

● Is a Disorder of iron overload :

-Hereditary hemochromatosis (HH)

-Acquired hemochromatosis

Hereditary hemochromatosis

● HH: genetic defect in iron metabolism

-Excess iron absorbed from the gut

-Symptoms due to pathologic deposition of iron in body tissue = iron overload

● Classic Triad:

-Cirrhosis (hepatic damage)

-Diabetes (type II) (pancreatic damage)

-Bronzing of skin (hyperpigmentation)

Non-Specific Symptoms and Signs

● Liver: hepatomegaly, elevated liver enzymes

● Cardiac: myocardial infarction, cardiomyopathy

● Endocrine: impotence/amenorrhea, diabetes

● Musculoskeletal: arthritis/arthralgia

● Fatigue: unexplained, severe and chronic

● Generally not evident until 40-60 years of age

● Some patients may present earlier

HFE hemochromatosis

● HFE– associated Hemochromatosis accounts for > 90% of cases and is the most common adult onset form:

● Autosomal recessive inheritance

● C282Y mutation

● Carrier rate 1 in 7 - 10 Caucasians

● Incidence 1 in 200 - 400

● Penetrance is high

Bb Bb

BB Bb Bb bb

Unaffected carrier

Unaffected

Autosomal Recessive Inheritance

Unaffectedcarrier

Susceptible genotype for Hemochromatosis

Unaffectedcarrier

Unaffected Carrier

Legend

B: Normal HFE gene

b: HFE gene with mutation

● HFE gene on chromosome 6p● Involved in iron homeostasis

● HFE protein normally limits amount of iron uptake by gut and regulates amount of iron stored in the tissues

● Mutations in HFE:

C282Y allele

H63D allele

S65C allele

● HFE gene mutations produce altered HFE protein unable to properly regulate iron metabolism - results in an excess of iron storage in tissues

HJV Hemochromatosis

● Rare disorder

● Early age of onset

● Severe multi organ iron overload

● Most common presentation :

-Testicular Atrophy

-Ammenorhea● Autosomal Recessive

● Mutation : Of HJV on chromosome 1q

● Most common deleterious mutation : G320V

HAMP Hemochromatosis

● Mutation: HAMP gene of chromosome 19q13.

● Usually associated with HFE C282Y mutation.

TFR2 Hemochromatosis

● Rare Autosomal Recessive disorder.

● Mutations : TFR2 Y250X and R455Q

● TFR2: encodes Transferrin receptor 2

- It mediates uptake of transferrin bound iron by the liver after the classical TfR1 is down reguated by iron overload .

Ferroportin(SLC40A1) Hemochromatosis

● Present on the surface of the cell responsible for iron absorption and recycling of iron from macrophage.

● Mutation : SLC40A1 gene

● Autosomal Dominant

African Iron overload and African american iron overload

● Type of iron overload due to ingestion of large quantities of iron contained in traditional beer.

Iron overload due to Anaemia with ineffective erythropoiesis

● In this, there is increased GDF 15 expression by erythroblast.

● Lead to down regulation of hepcidin expression.

● Thus increase iron absorption.

● Anemias with increased GDF 15 expression:

-beta-thalassemia major

-Pyruvate kinase deficiency

-Congenital dyserythropoietic anemia

Secondary Iron Overload

● Acquisition of iron from non dietary sources in amounts that exceeds the body's limited excretory capacity can cause iron overload.

● Causes : Chronic erythrocyte transfusion

-Excessive iron supplements (I.V or I.M)

Analytical methods

● For measurement of Serum Iron:

1) Iron is released from transferrin by decreasing pH of serum.

2) Reduced from Fe+3 to Fe+2.

3) Complexed with a chromogen like ferrozine.

● Such iron chromogen complexes have an extremely high absorbance in the visible region that is proportional to iron concentration.

● For measurement of Serum TIBC:

1) Addition of Sufficient Fe+3 to saturate empty iron binding site of transferrin.

2) Excess Fe+3 is removed by adsorption with MgCO3, silica column or ion exchange resin.

3) Assay for iron content is repeated.

● For measurement of Serum Transferrin Saturation :

Transferrin Saturation (%) = (100 X serum iron) / TIBC

● For measurement of Serum UIBC:

1) Add known excess concentration of iron to the serum.

2) pH should be neutral.

3) Add iron binding chromogen.

4) Measure the absorbance

5) Measure blank with no added serum.

● UIBC = O.D of serum free iron blank – O.D of sample

● Advantage: Can be fully automated

less cumbersome

Reference Interval

● Differs as much as 35% between commercial methods.

● A generic reference interval is not valid.

Factors affecting Serum Iron

● Hemolysis has very little effect on serum iron result because Hb iron is not released from heme by acid treatment.

● Iron is ubiquitous in the environment so care is necessary to ensure that glassware, water and reagents do not become contaminated with extraneous iron.

serum transferrin receptor

● Under normal conditions TfR1 is highly expressed on the surface of erythroid cells.

● Certain proportion is released into the circulation by proteolytic cleavage. Referred to as “Soluble TfR”.

● Number of transferrin receptor increases in iron deficiency and decreases in iron excess.

● Developing erythroid cells in bone marrow are reach in transferrin receptor.

● Variation in transferrin receptor quantity are reflected in soluble serum TfR.

● Measured by standard immunoassay technique.

● Measurement of STfR is usefull to distinguish between anemia due to iron deficiency and anemia due to chronic disease.

Red Cell protoporphyrin

● Its level is increased in Iron deficiency.

● The presence of protoporphyrin in erythrocyte reflects impaired ferrochelatase catalyzed incorporation of iron into protoporphyrin IX ring.