Non-regenerative Anaemia and Iron metabolism

Question 1

List factors that can inhibit the normal bone marrow response to anaemia from haemolysis or haemorrhage

Answer 1

• Iron deficiency
• Iron sequestration - typically from chronic inflammation
• Decreased erythrocyte half life
• Concurrent bone marrow disease (including targeting by the immune system of RBC precursors
• Co-morbidities

Question 2

Describe the major pathophysiological mechanisms leading to non-regenerative anaemia

Answer 2

* Decreased erythrocyte lifespan * Decreased or ineffective erythropoiesis * Bone marrow failure

Question 3

List general causes of decreased erythrocyte lifespan

Answer 3

(Normal - 90-100 days in dogs, 72 days in healthy cats) * Normal removal of RBC by the mononuclear phagocytic cells in the spleen, liver, and lymph nodes

1. Increased oxidative stress
2. Mechanical stress
3. Complement-induced injury
4. Rearrangement of membrane phospholipids
5. Contact with cationic proteins - from activated neutrophils
6. Heinz body formation
7. Haemotropic parasites

Question 4

List causes of decreased or ineffective erythropoiesis

Answer 4

1. Relative or absolute reduction in EPO production in the kidney
2. Reduced BM response to EPO
3. Deficiency in nutrients crucial to haemoglobin biosynthesis
   
   • Eg. iron
4. Chronic inflammation
   
   • Cytokine abnormalities - IL-1, IL-6, TNFa, INF-gamma: all reduce endogenous EPO production and reduce marrow responsiveness to EPO.
5. Increased hepcidin levels (responsible for iron homeostasis)
6. Copper deficiency (decreased Hb synthesis)
7. Vitamin B 12 deficiency (inhibits purine synthesis - erythroblast apoptosis)
8. Hypoalbuminaemia and elevated PTH
9. Aluminium toxicosis
10. ACEI use

Question 5

Discuss tests to differentiate iron deficiency anaemia and anaemia of chronic disease (iron sequestration)

Answer 5

Differentiation may be difficult and both conditions can occur concurrently

• Serum iron - low in both conditions (and with PSVA)
• Ferritin (soluble storage form of iron) is a positive acute phase protein - low with iron deficiency, increased with inflammatory disease
• Transferritin - measured indirectly and reported as total iron binding capacity. Transferritin is a negative acute phase protein (decreased with inflammation). TIBC normal to increased with iron deficiency
• Reticulocyte indicies if available

Question 6

Describe the production and regulation of EPO

Answer 6

• Renal hypoxia is the main driving force of EPO production
• Produced in the peritubular interstitial cells in the inner renal cortex and outer medulla
• renal hypoxia –> inhibition of hypoxia inducible factor 1 degradation
• HIF-1 binds to hypoxia response elements of oxygen regulated genes
• EPO production is reduced in both acute and chronic renal disease
• EPO also produced in extra-renal sites, especially the liver
• EPO production from extra-renal sites cannot adequately compensate for decreased renal production.

Question 7

List bone marrow disease that cause non-regenerative anaemia

Answer 7

• Pure Red Cell Aplasia (may be severe form of IMHA)
• IMHA targeting precursor cells
• Myelodysplastic syndromes
• Myeloproliferative neoplasia
• Myelofibrosis
• Aplastic anaemia / pancytopenia

Question 8

List secondary (extra-medullary) causes of bone marrow failure

Answer 8

1. Myelophthisis - secondary to neoplasia outside the marrow
2. Pyruvate kinase deficiency (causes osteosclerosis /myelofibrosis and marrow loss)
3. Oestorgen toxicosis (causes myelofibrosis)
4. Phenobarbitol - causes dysmyelopoiesis
5. Secondary myelodysplasia - chemotherapy or radiation induced.

Question 9

Discuss symptomatic therapeutic options for Non-Regenerative Anaemia

Answer 9

Blood transfusion is the only general symptomatic therapy of note for dogs with non-regenrative anaemia. Blood transfusion is only indicated with severe disease and must be based on clinical parameters. In general, dogs and especially cats with chronic, slowly progressive anaemia can adequately compensate even with very low RCC.

Treatment directed at the extra-medullary causes of non-regenerative anaemia is more important.

Question 10

List the specific therapeutic options for management of non-regenerative anaemia.

Answer 10

• Identifying the promary cause of the problem is most important initially.

1. Iron deficiency anaemia can be managed with iron supplements
2. Erythropoietin supplementation - can be effective for causes other than myelodysplasia
3. Immunosuppression for medullary causes such as IMHA

Question 11

List the various body systems to which IRON contributes

Answer 11

1. Haemoglobin and myoglobin production
2. Enzyme systems - essential cofactor
3. Neurotrasmitter production
4. Myelin production
5. Collagen formation
6. Immune system function
7. Energy metabolism
8. DNA and RNA synthesis

Question 12

Describe the three pools of iron within the body

Answer 12

1. Transport pool
   
   • < 1% of total body iron
   • Bound to transferritin
2. Functional pool
   
   • Haemoglobin
   • Myoglobin
   • Enzymes
   • > 2/3 of total body iron is within haemoglobin
   • 10-15% is within muscles and enzymes (cytochromes)
3. Storage pool
   
   • liver and macrophages

Question 13

Discuss iron absorption within the duodenum

Answer 13

• No excretion pathway for excess iron, so absorption is tightly controlled
• Heme iron enters body through the apical membrane of the duodenal enterocytes
• Non-heme iron (Fe³⁺) insoluble at pH>3 - cytochrome reductase B highly expressed at the enterocyte brush border catalyses reduction of Fe³⁺ to Fe²⁺
• Fe²⁺ transported through divalent metal transporter 1
• Fe²⁺ atoms are stored within the enterocyte within ferritin
• Fe2+ can be actively transported out of the cell into plasma via ferroportin
• Ferritin is lost as the enterocytes slough helping regulate total body stores

Question 14

Discuss Iron trasport through the plasma

Answer 14

• Iron is transported through the basolateral membrane as Fe²⁺
• Fe²⁺ is oxidised to Fe³⁺ by hephaestin (a multicopper iron oxidase)
• Fe³⁺ binds to transferrin within the plasma
• Apotransferrin - iron-free transferrin has two iron binding lobes and is produced primarily by the liver (+ Sertoli cells and various cells in the brain)
• Transferrin is normally 20-60% saturated in the plasma
• Iron is transported into cells via transferrin receptor 1

Question 15

Discuss Iron Uptake from the plasma

Answer 15

• Transferrin receptor 1 (TFR-1) is expressed on all cells to varying degrees
  
  • TFR-1 is most abundant on erythrocyte precursors, rapidly dividing cells, activated lymphocytes and hepatocytes
• TFR-1 binds to divalent transferrin –> stimulates receptor mediated endocytosis –> membrane contains proton pump and DMT-1.
• H+ ions pumped into endosome –> decreased pH leads to Fe³⁺ release from transferrin –> Fe³⁺ reduced to Fe²⁺ by ferrireductase –> Fe2+ transported out of the endosome by DMT-1
• Apotransferrin and TFR-1 are recycled back to the cell membrane

Question 16

Discuss Iron Storage

Answer 16

• Iron is primarily stored within the cytoplasm of hepatocytes
• Ferritin is a water-soluble protein that stores retrievable iron.
• Ferritin is a cage like protein that can store up to 4500 iron atoms
• Iron enters as ferrous iron, is oxidised to ferric iron and polymerised to ferric oxyhydroxide polymer
• Stored as ferric form to ensure it is non-reactive
• Ferritin is the precursor to haemosiderin an insoluble, non-retrievable aggregate of non-reactive iron in the cytoplasm
• ~95% of iron stored in the liver is within ferritin.

Question 17

Discuss theories surrounding systemic iron regulation

Answer 17

• Dietary regulator
  
  • iron absorption is decreased in the days after a dietary iron bolus
• Stores regulator
  
  • Probably works at the level of the intestinal crypt cells to ‘program’ future enterocytes
• Erythropoietic regulator
  
  • Most influencial - adjusts absorption based on erythropoietic demands.
• Humoral Hypoxia regulator
  
  • increased iron uptake with acute hypoxia - may be directly linked to erythropoietic regulator
• Inflammatory regulator
  
  • Results in cellular iron retention in the setting of inflammation or infection.
  • May help withhold iron from invading organisms

Question 18

Discuss the role of hepcidin in iron metabolism

Answer 18

• Primary regulator of iron homeostasis
• Produced by cleaving a precursor molecule
• Pimarily produced in the liver
  
  • plus small amount from inflammatory macrophages
• Small size (~2.8 kDa) so freely filtered from the kidney
• Regulated primarily at the level of production
• Binds to ferroportin on the cell membrane, causing internalisation and degradation in lysosomes
• Net result is retention of iron within enterocytes, macrophages and hepatocytes
• Also inhibits DMT-1 production
  
  • Reduced iron uptake into enterocyte
  • Reduced release of iron from endosomes
• Increased production with iron overload, inflammation, infection
• Decreased production in anaemia and with hypoxia

Question 19

List major mechanisms by which chronic inflammatory disease contributes to non-regenerative anaemia

Answer 19

1. Production of cytokines by activated T cells alters iron metabolism
2. LPS and IL-6 upregulate hepcidin production
3. IFN-g, LPS, TNA-a up-regulate DMT-1 on macrophages - leads to iron sequestration
   
   • Factors above also down-regulate ferroportin expression, inhibiting iron release from cells
4. IL-10 increases transferrin expression - increased iron uptake into cells
5. TNA-a, IL-1, IL-6, IL-10 increase ferritin expression promoting iron storage
6. INF-a, b, g, TNA-a, IL-1: inhibit differentiation and proliferation of erythrocyte precursors
7. RBC lifespan may be reduced
8. Inflammatory cytokines can alter EPO, interfere with EPO signal transduction and down-regulate EPO receptor production