Megaloblastic Anemia Notes

Megaloblastic Anemia

Learning Outcomes

• Explain the etiology, pathophysiology, clinical, and laboratory features of megaloblastic anemia.
• Identify megaloblastic anemia given clinical and laboratory data.

Classification of Anemia

• Microcytic, Hypochromic (MCV < 80 fL, MCH < 27 pg):
  • Iron deficiency
  • Thalassemia
  • Anemia of chronic disease (some cases)
  • Lead poisoning
  • Sideroblastic anemia (some cases)
• Normocytic, Normochromic (MCV 80-95 fL, MCH ≥ 27 pg):
  • Many hemolytic anemias
  • Anemia of chronic disease (some cases)
  • After acute blood loss
  • Renal disease
• Macrocytic (MCV > 95 fL):
  • Megaloblastic:
    • Vitamin B12 or folate deficiency
  • Non-megaloblastic:
    • Alcohol
    • Liver disease
    • Myelodysplasia
    • Aplastic anemia

Megaloblastic Anemias

• A group of anemias/disorders in which erythroblasts show characteristic abnormality where:
  • Maturation of the nucleus is delayed relative to that of the cytoplasm.
  • Due to defects in DNA synthesis caused by dietary deficiency of folic acid or vitamin B12 (cobalamin).

Vitamin B12 (Cobalamin)

• Physiology:
  • Also known as Cobalamin.
  • Elucidated in 1948.
  • Largest of all vitamins – MW 1355 Da.
  • Composed of:
    • A ring of 4 pyrrole units.
    • An atom of cobalt at its center.
  • Other active vitamin B12 forms:
    • Methylcobalamin – in human plasma.
    • Deoxyadenosylcobalamin – in human tissue.
    • Hydroxocobalamin – used in treatment.
  • Daily requirement: 1 $μg$/day.
  • Absent in the Plant kingdom: synthesized in nature by bacteria (i.e., Streptomyces species).
  • Acquired from food of animal origin, i.e., meat & dairy products.

Absorption of Vitamin B12

• Vitamin B12:
  • Extracted from food by proteolytic enzyme pepsin and acid stomach environment.
  • First binds to haptocorrin (HC).
  • In the duodenum, B12 is released from HC by proteolytic action of pancreatic trypsin.
  • Then binds to intrinsic factor (IF) (synthesized by gastric parietal cells) in the duodenum.
  • IF-B12 complex is then absorbed in the distal ileum where the complex binds to the IF receptor (cubulin).
  • In blood, Vitamin B12 binds to transcobalamin II (TCII).
  • TCII takes B12 to the bone marrow and tissues.
  • Stored in the liver in sufficient amounts to last 6-12 months.

Biochemical Functions of B12

• Methyl B12:
  • Acts as a cofactor of Methionine synthase (an enzyme responsible for methylation of homocysteine to methionine using methyl THF as a methyl donor).
  • $THF \longrightarrow Methyl THF$
  • $Homocysteine \longrightarrow Methionine$
  • Methylation of DNA.
  • Synthesis of myelin, amines, proteins, etc.
• Deoxyadenosyl B12 (Ado B12):
  • Acts as a cofactor of Methylmalonic mutase in the conversion of Methylmalonyl CoA to Succinyl CoA.
  • Propionyl CoA $\longrightarrow$ Methylmalonyl CoA $\longrightarrow$ Succinyl CoA
  • Leads to the generation of thymidine, required for DNA synthesis.
  • $ALA \longrightarrow HAEM$

B12 Deficiency

• Nutritional: strict veganism.
• Malabsorption:
  • Gastric causes: pernicious anemia, gastrectomy, congenital lack/abnormality of IF, use of proton pump inhibitors.
  • Intestinal causes: tropical sprue, fish tapeworm, ileal disease, bacterial overgrowth.
  • Pancreatic insufficiency.

Folate

• Folic (pteroylglutamic) acid.
• Obtained from diet:
  • Fruit.
  • Vegetables (dark green leafy vegetables).
  • Dairy products.
  • Eggs, etc.
• Requirements: 200-300 $μg$/day.

Folate Absorption and Function

• Folate absorption:
  • Dietary folates are converted to methyl tetrahydrofolate (THF) during absorption primarily through the duodenum and jejunum.
  • In cell converted to folate polyglutamate
  • Dietary folates $\longrightarrow$ Methyl THF (in plasma and small intestines)
  • Methyl THF $\longrightarrow$ THF polyglutamate (in cell)

Folate Deficiency

• Causes:
  • Nutritional – especially old age, poverty, etc.
  • Malabsorption – tropical sprue, gluten-induced enteropathy.
  • Increased requirements caused by cell proliferation (pregnancy, infancy, chronic lymphocytic leukemia, malignancy, psoriasis).
  • Drugs – alcohol, anticonvulsants.
  • Vitamin B12 deficiency.

Megaloblastic Anemia (Reiterated)

• A group of anemias/disorders in which erythroblasts show characteristic abnormality where:
  • Maturation of the nucleus is delayed relative to that of the cytoplasm.
  • Due to defects in DNA synthesis caused by dietary deficiency of folic acid or vitamin B12 (cobalamin).

Biochemical Basis of Megaloblastic Anemia

• $dUMP \longrightarrow dTMP \longrightarrow dTDP \longrightarrow dTTP \longrightarrow DNA$
• Folate coenzyme form 5,10-methylene THF polyglutamate is needed in the synthesis of dTMP from dUMP.
• Folate deficiency inhibits thymidylate synthesis needed for dTMP synthesis.
• B12 plays an indirect role assisting in the conversion of Methyl THF to THF
• B12 Deficiency leads to reduced supply of folate coenzymes required in dTMP synthesis.
• The diagram shows:
  • Dietary folates being converted to Methyl THF in the plasma and small intestines.
  • Methyl THF being converted to THF polyglutamate in the cell.
  • THF polyglutamate being converted to 5,10-methylene THF polyglutamate.
  • 5,10-methylene THF polyglutamate being used in the synthesis of dTMP from dUMP.
  • Vitamin B12's role in converting Methyl THF to THF.

Biochemical Basis of B12 & Folate Deficiency

• DNA Synthesis:
  • B12/folate deficiency affects dTTP synthesis.
  • Causes macrocytic cells, hypersegmented neutrophil, epithelial changes.
• Myelin Synthesis:
  • B12 deficiency affects the conversion of s-adenosylmethionine to s-adenosylhomocysteine.
  • Results in peripheral neuropathy.

Clinical Features

• Severe B12 Deficiency:
  • Peripheral neuropathy (tingling of the toes and feet).
  • Jaundice
  • Glossitis: beefy-red sore tongue.
  • Angular cheilosis
• Neural tube defect (spina bifida) results from Folate or B12 deficiency in the mother.

Laboratory Findings

• Full Blood Count:
  • Reduced hemoglobin, increased MCV, possibly also in the MCH and MCHC.
• Blood Film:
  • Presence of macrocytic red cells, anisocytosis, occasional nucleated red cells.
  • Increased number of hyperchromic red blood cells.
  • Hypersegmented neutrophils.
• Bone Marrow:
  • Presence of megaloblasts (thereby defining megaloblastic anemia).
• Serology:
  • Presence of autoantibodies to gastric parietal cells and to intrinsic factor (if present, these define pernicious anemia).
  • Low levels of serum vitamin B12, often with reduced serum and red cell folate.

Additional Lab Findings

• MCV > 95 fl - macrocytic (usually oval).
• Normal or increased MCH.
• Hypersegmented neutrophils.

Macrocytic Anemia - Microscopic Observations

• Megaloblasts: large, oval-shaped erythroblasts.
• Abnormal giant metamyelocytes and band forms.

Differential Diagnosis of Macrocytic Anemias

• Vitamin B12/Folate deficiency – Serum Vit. B12 & folate and red cell folate.
• Alcoholism – especially if the patient is not anemic; check alcohol use history.
• Liver dysfunction – liver function tests.
• Hypothyroidism – thyroid function tests.
• Myelodysplasia – bone marrow examination.
• Certain drugs, i.e., cyclophosphamide (chemotherapy), trimethoprim (antimicrobial), primidone (anticonvulsants); check drug history.

Laboratory Findings of B12 and Folate Deficiencies

Macrocytosis Mechanism

• The number of mitotic divisions during erythropoiesis determines red cell size.
• Defects in DNA synthesis due to misincorporation of dUTP instead of dTTP.
• Reduced mitotic division.
• Failure to progress from G to M phase during the cell cycle.
• Cell grows without division, resulting in larger than normal cells - macrocytes and blasts (immature cells).

Pernicious Anemia

• Autoimmune – attack IF-producing parietal cells of the gastric mucosa.
• Reduced or no secretion of IF = defective B12 absorption.
• Achlorhydria.
• Increased serum gastrin.
• Affects more females than males.
• Presence of serum antibodies against gastric H+/K+-ATPase and IF.

Case Studies

• Case Study A: 30-year-old woman with lassitude, koilonychia, angular cheilitis, pallor, and menorrhagia (likely iron deficiency).
• Case Study B: 58-year-old man with jaundice, swollen painful tongue, and mild peripheral neuropathy (likely megaloblastic anemia).