Ferritin primary iron storage protein

§   Large molecule (480,000 mol wt) with a spherical shell enclosing up to 4000 iron molecules

§   Different subtypes (L in liver and spleen and H in heart and red cells)

§   Low levels of circulating ferritin contain very little iron and are predominantly L subtype)

§   Ferritin thought to be secreted by macrophages in response to changing iron levels

Haemosiderin

§   Water insoluble crystalline complex visible using Pearl’s stain

§   Higher iron to protein ratio and is probably formed by partial digestion of ferritin aggregates by lysosomal enzymes.

§   Largely found in macrophages rather than hepatocytes unless iron overload in which case found in both

Transferrin

§   Present in plasma and extravascular fluid (half life of 8-11 days)

§   Contains around 4mg iron at any one time but 30mg passes through each day

§   Transferrin receptors on the cell surface allow the uptake of transferrin (stain with CD71)

Lactoferrin

§   Similar structure to transferrin

§   Found in milk and neutrophils, also secreted onto surfaces as has bacteriostatic actions (preventing them growing by depriving them of iron)

Intracellular regulation of iron

§   Low iron levels lead to activation of the proteins IRP1 and IRP2 which bind Iron Response Element (IRE) sequences of mRNA.

§   Transferrin receptor mRNA has multiple IREs and binding by IRPs leads to increased stability and transcription by preventing degradation of the mRNA

§   Ferritin mRNA transcription inhibited by IRP binding

Iron absorption

§   Iron released from protein complexes by stomach acid – majority absorbed in duodenum

§   Reduced from Fe3+ to 2+ by Dcytb on the brush border and then transported across the membrane by DMT1.

§   Haem iron transported directly across the brush border and Fe released by haem oxygenase

§   Transported across the basolateral membrane by Ferroportin 1 and converted to Fe3+ by Hephestin

§   Ferroportin 1 activity inhibited by Hepcidin ie. hepcidin down regulates iron absorption and iron mobilisation

§   Hepcidin synthesised in the liver in response to:

§   increased iron stores ( HFE/ haeomjuvelin thought to be necessary for hepcidin synthsis.)

§   IL-6

§   Downregulated by anaemia/ hypoxia

§   Ie. hepcidin results in iron trapping within iron storage cells (macrophages) and a block in iron absorption from the gut.

§   Hepcidin deficiency (AR nonsense mutations in the HAMP gene) leads to severe juvenile haemochromatosis

Diagnositic methods for investigating iron metabolism

Serum ferritin

§        Correlates well with iron stores as assessed by quantitative phlebotomy or tissue biopsy in healthy individuals

§        Level <15 highly specific for iron deficiency

§        Spuriously increased in

§        Acute phase reaction

§        Tissue release of ferritin from iron rich organs (hepatic necrosis, chronic liver disease, splenic or bone marrow infarction eg in sickle cell)

§        Reduced in ascorbate deficiency

§        Rare hereditary disorder of ferritin synthesis (hereditary hyperferritinaemia) which is associated with normal iron levels – results in cataracts due to deposition of ferritin in the lens.

Red cell ferritin

§   Difficult to measure as extremely low levels in red cells and so not used in general practice

§   Has theoretical advantage that wouldn’t be affected by ferritin release into serum by tissue damage

Serum iron, total iron binding capacity (TIBC) and transferrin saturation

§        Labile – fluctuations mean that a single value may not reflect iron supply over a longer period

§        Iron levels have a diurnal pattern (lower in the morning than the evening)

§        Transferrin saturation = serum iron / TIBC

§        Reduces in iron deficiency due to combination of reduced iron and increased TIBC

§        Normal in anaemia of chronic disease

§        Increased hepcidin (mediated by Il-6) blocks iron release from macrophages and jejunal enterocytes

§        Sustained increase is an early change in the development of iron loading

Serum transferrin receptor concentration

§   Increased in iron deficiency

§   Concentration reflects both the number of erythroid precursors and iron supply to the bone marrow

§   Increased erythropoiesis will result in increased concentration

§   Sensitivity and specificity can be increased by using ferritin-transferrin receptor ratios

§   Assay is method specific and some consider that it adds little to information provided by ferritin assay

Red cell protophophyrin

§   Protoporphyrin accumulates in red cells when iron supply is restricted. 

§   Can measure red cell level (particularly zinc protoporphyrin)

§   Values may not reflect current iron supply

§   Levels also increased in sideroblastic anaemia and lead poisoning

Percentage of hypochomic red cells

§   Some analysers can measure this

§   Helpful in CRF in identifying iron deficiency early in patients on EPO (other measures likely to be confounded by chronic inflammation)

§   Helpful in diagnosing functional iron deficiency

Anaemia of chronic disease/ inflammation

§   Inadequate production of epo

§   Inadequate response of erythroid progenitors to epo

§   Impaired release of iron from stores secondary to increased hepcidin

§   Results in low iron and transferrin saturation, but with a high ferritin due to both stored iron which can’t be mobilised and acute phase response

§   Shortening of red cell survival

§   Treatment

§   Controversial

§   Can use erythropoietin – concerns regarding poorer overall survival/ progression free survival in cancer patients, therefore its use is restricted

§   Parenteral iron may be an alternative

Iron deficiency anaemia

1. Body stores depleted

2. Iron deficient erythropoiesis

§   Low serum transferrin saturation and ferritin

§   MCV / Hb may be normal

3. Iron deficiency anaemia

§   Microcytosis, poikilocytosis, target cells, reduced reticulocytes

§   Erythroblasts have ragged, vacuolated cytoplasm and pyknotic nuclei

§   Bone marrow macrophages show a total absence of iron

4. Tissue effects of iron deficiency

§        Severe and chronic iron deficiency causes widespread tissue changes

§        Koilonychia

§        Angular stomatitis

§        Glossitis

§        Pharyngeal webs (Patterson-Kelly syndrome)

§        Atrophic gastritis (reducing stomach acid and worsening iron absorption)

§        Very severe deficiency results in mitochondrial swelling which can cause impaired neutrophil function, mental development

Causes of iron deficiency

§        Diet

§        Vegetarians

§        Malabsorption

§        Coeliac disease

§        Achlorhydria

§        May show a response to oral iron

§        Increased requirements

§        Infancy – worse in premature babies and those with infections and delayed mixed feeding

§        Pregnancy (despite amenorrhea)

§        Blood loss

§        More than 6-8 mL/day (3-4 mg iron) becomes important

§        Menorrhagia

§        GI blood loss

§        Intravascular haemolysis leads to urinary iron loss

Treatment

1. Oral iron

Ferrous sulphate (200mg provides 67mg iron)

Ferrous gluconate (300mg provides 36mg)

Need to provide 100-200mg iron / day in adults (3mg/kg in children)

Minimum response Hb rise of 2 g every 3 weeks

Failure to respond

§   Poor compliance

§   Ongoing haemorrhage

§   Malabsorption

§   Wrong diagnosis (anaemia of chronic disease, thalassaemia trait)

2. Parenteral iron

§   Usually unnecessary

§   Can be used in those genuinely intolerant of oral iron

§   Occasionally in malabsorption

§   Sometimes used when oral replacement can’t keep up with ongoing haemorrhage (eg HHT)

§   Chronic renal failure on epo (increased demand may outstrip ability to mobilise stores – functional iron deficiency)

§        Can increase mobilisation with ascorbic acid

§   Iron taken up by macrophages of the reticuloendothelial system which then release iron to circulating transferrin

§   Deficit in iron stores calculated from degree of anaemia (usually 1-2g)

§   Should be avoided if there is a history of allergy as anaphylaxis sometimes occurs

§   Other reactions include immediate nausea, urticaria and aches and pains as well as delayed reactions including fever, arthralgia and lymphadenopathy.  May also exacerbate rheumatoid arthritis. 

§   Venofer (iron sucrose) considered to be the safest form but can be excreted in urine exacerbating UTIs

§   Cosmofer (iron dextran) – IM or as a single dose iv

Iron overload

Causes of iron overload

Severe (>5g)

§        Increased absorption

§        Hereditary haemochromatosis

§        Massive ineffective haemopoiesis (beta thal intermedia, sideroblastic anaemia, CDA)

§        Increased iron intake (sub-saharan dietary iron overload / excess iron therapy)

§        Repeated red cell transfusions (congenital / acquired anaemia)

Moderate overload (<5g)

§        Chronic liver disease (alcoholic cirrhosis)

§        Porphyria cutana tarda

§        Rare genetic abnormalities of iron metabolism

§        Atranferrinaemia

§        Aceruloplasminaemia

Focal iron overload

§   Pulmonary haemorrhage / idiopathic pulmonary haemosiderosis

§   Chronic haemoglobinuria

Chronic iron overload syndromes of genetic origin

Mean age at diagnosis = 46yrs

Pathophysiology

§   Continued absorption of iron from the small intestine, despite normal/ increased total body iron

§   Due to hepcidin deficiency (type IV due to hepcidin resistance)

§   Low iron – reduced hepcidin – increased absorption

§   Hepcidin is an acute phase protein and this results in reduced absorption and anaemia of chronic disease

Classification of genetic haemochromatosis

HFE genetics

C282Y

§   12% prevalence

§   1 in 200 homozygotes

§   90% of haemochromatosis due to homozygotes

§   Heterozygotes (for both C282Y and H63D) have slightly increased iron and transferrin saturation which may confer protection against iron deficiency

H63D

§   20% prevalence in UK population

§   C282Y/H63D heterozygotes account for 4% of haemochromatosis

§   Homozygotes may have higher ferritin and transferrin saturation than heterozygotes but significant iron accumulation is rare

S65C

§   Rare mutation that may cause mild iron accumulation in combination with C282Y or H63D

Diagnosis

1. Candidates for testing

§   European ancestry with weakness, abnormal LFTs, arthralgia, impotence, DM, cirrhosis, pigmentation

2. Initial investigations

Transferrin saturation

§   Most sensitive and specific biochemical parameter

§   Fasting sample (if raised should be repeated on a fasting sample)

§   100 x serum iron/ TIBC (>55% in men, >50% in pre-menopausal women)

§   Not specific for HFE haemochromatosis – need to check CRP to exclude inflammation

Ferritin

§        Normal in the early stages of iron accumulation

§        If transferrin saturation is raised but ferritin normal should go on to do genetic testing

§        Close correlation (>300 in men, >200 in women)

§        >1000, severe clinical complications more likely

§   Falsely elevated in alcohol, dysmetabolic syndrome and inflammation

3. Confirmation of the diagnosis

HFE testing

§   Homozygous C282Y confirms diagnosis

§   Penetrance is variable, therefore not suitable for population screening

§   Heterozygotes C282Y/ H63D – mild variant

§   5% of patients with genetic haemachomatosis don’t have the identified phenotypes

4. If evidence of iron overload but HFE testing negative

Exclude other diagnosis (alcoholic liver disease / haematological disease)

Liver biopsy

§   Not necessary unless evidence of iron accumulation and genotype negative

§   Hepatic iron index > 1.9 (iron concentration in hepatic biopsy / age) diagnositic of haemochromatosis

Quantitative phlebotomy

§   Removal of 4g iron (20 phlebotomies of 450ml demonstrates that body iron stores are compatible with genetic haemochromatosis).

4. Staging investigations

Hepatic

§   AST

§   USS – cirrhosis, hepatoma. ?role of fibroscan – can grade cirrhosis

§   Liver biopsy - Determines whether there is cirrhosis

§   MRI – iron concentration but no information on cirrhosis

Cardiac

§   ECG and ECHO

Dexa scan

Endocrine tests

§   Glu, TFTs, bone profile, LH, FSH, testosterone etc.

Differential Diagnosis

<30yrs

§   Juvenile HC

§        Massive iron overload with endocrine and cardiac complications

§        Genetic testing for hemojuvelin and hepcidin mutations

§   TFR2 HC

>30yrs

§        TFR2 HC

§        Ferroportin disease – type B

§        AD, therefore diagnosis facilitated by documenting hyperferritinaemia in 1^st degree relatives

Elevated ferritin with low transferrin saturation

§        Dysmetabolic syndrome

§        Multiple metabolic abnormalities

§        High ferritin / Normal TS

§        Mild hepatic iron excess

§        Mixed iron deposition in hepatocytes and macrophage

§        Ferroportin disease – type A

§        Macrophage iron excess

§        Hereditary aceruloplasminaemia

§        Chromosome 3

§        High ferritin / Low TS

§        Neurological syndrome

§        Undetectable levels of serum ceruloplasmin

Staging for C282Y homozygosity

Treatment

Avoid ingesting large quantities of vitamin C containing food

Venesection - Start when ferritin raised but asymptomatic (stage 2)

1. Induction

§        Weekly – 450-500mls –

§        Calculate iron removed weigh bag – 450ml blood with Hb 13.5 contains 200mg iron

§        Absorption 3mg/day = 20mg/week

§        25 weekly venesections will remove 4.5g iron

§        Aim ferritin <20 and TS <16%

§        Withold if Hb <11

2. Maintenance

§        1-4 monthly

§        Aim ferritin <50 and TS <50

§   Serum ferritin should be checked at least every 2 venesections, and Hb checked within the week prior to venesection.

Liver cirrhosis

§   AFP and USS 6 monthly (monitoring for HCC)

Family screening

Ideas for the future

§        Oral iron chelators

§        Poor venous access

§        Anaemia

§        Poor tolerance of phlebotomy

§        Patient choice

§        Study ongoing with deferasirox

§        Inhibition of cellular iron transport

§        Correction of the systemic iron regulating defect

Iron chelators

Conditions associated with transfusional iron overload

§        Thalassaemia major

§        Blackfan-diamond anaemia

§        HbSS

§        Fanconi’s anaemia

§        Aplastic anaemia

§        Severe haemolytic anaemia

§        Repeated myeloablative chemotherapy

§        MDS

§        No physiological mechanisms to excrete excess iron

Goals of chelation therapy

§        Maintain iron balance with safe tissue iron levels

§        Prevention

§        Match transfused iron with chelated iron

§        Prevent iron reaching levels where tissue damage occurs

§        Rescue

§        Remove excess iron – slow process – finite pools

§        Reverse dysfunction

§        Detoxification of labile iron – 24 hour protection

§        Wide therapeutic safety margin of regime

Combination deferioxamine and defriprone more rapidly reduce hepatic and cardiac iron than deferioxamine alone without increased toxicity

§        Patients beginning chelation with normal cardiac function

§        Defrasirox with regular assessment of iron burden to achieve correct dose

§        Patients with severe iron overload/ organ toxicity despite treatment with deferioxamine

§        Consider combining with deferiprone