Anemia & DNA Metabolism: Vitamin B12 & Folate

• Anemia caused by defects in DNA Metabolism

Physiologic Roles of Vitamin B12 and Folate

• Vitamin B12 (Cobalamin) is a coenzyme involved in two biochemical reactions in humans
  • Isomerization of Methylmalonyl CoA
  • Transfer of Methyl group to 5-methyl THF
• Vitamin B9 (Folate), also known as folic acid (pteridine ring) plays a role in DNA synthesis

Roles of Vitamins

• Folate, a general term for folic acid, transfers carbon units in the form of methyl groups
• Folate is important for the metabolism of amino acids and nucleotides
• Deficiency in either vitamin B12 or folate impairs thymidine nucleotide production, which is needed for DNA synthesis

Megaloblastic Anemia

• Deficiency in vitamin B12 and folic acid, as well as certain drugs, can cause megaloblastic anemia
• Results in depleted stores of DNA precursors trimidine triphosphate
• Impaired DNA synthesis results in very large cells in the BM, due to reduction in the number of cell divisions

Defects Causing Megaloblastic Anemia

• Defects in megaloblastic anemia are caused by deficiency in folate and vitamin B12
• These vitamins have an indirect effect preventing methylation of dUMP
• Results in diminished thymidine availability, causing uridine to be incorporated into DNA
• Cell division is halted, causing either cell lysis or apoptosis

Pernicious Anemia

• Pernicious anemia, also called megaloblastic madness, is due to ↑ megalocyte or macrocytes
• It is autoimmune, with 25 new cases per 100,000 persons
• Pernicious anemia results in impaired absorption of vit B12 due to intrinsic factor deficiency
• Patients at high risk of gastric tumors
• Commonly seen during sixth decade of life (60 years old)
• Reduced the amount of Intrinsic factor release

Vitamin B12 Deficiency

• Inadequate intake of sources like strict vegetarian
• Increased need in instances like Pregnancy and lactation
• Impaired absorption of R protein or transcobalamin 1 (aided by pepsin); binding of intrinsic factor

Other Conditions Caused by Vitamin B12 Deficiency

• Gastrectomy
• Dietary deficiency of vitamin B12
• Blind loop syndrome
• Reversible malabsorption of vitamin B12
• Immerslund's syndrome
• Zollinger-Ellison syndrome
• Patient on hemodialysis
• Crohn's disease
• Presence of D. latum
• Megaloblastic anemia

Impaired Absorption

• Failure to separate vitamin B12 from food proteins in the stomach (Food cobalamin malabsorption)
• Failure to separate vitamin B12 from haptocorrin in the intestine (Chronic Pancreatic disease)
• Lack of intrinsic factor (Pernicious anemia) (H.pylori infection) Malabsorption (celiac disease)
• Inherited errors of Vitamin B12 absorption and transport ( Imerlund Grasbeck syndrome)
• Competition for available vitamin B12. (Blind loop)

Folate Deficiency

• Inadequate intake of food sources like green leafy vegetable, dried beans, liver
• Increased need instances like Pregnancy and lactation
• Impaired absorption: deficiency of a folate transporter protein
• Sprue (steatorrhea is malabsorption)(tropical sprue, nontropical sprue (idiopathic stearorrhea), Celiac disease (gluten-sensitive enteropathies)
• celiac disease intolerance to gluten)
• Impaired use of Folate:
• Antineoplastic, antibacterial, and antiseizure agent (macrocytosis with frank megaloblastic anemia) Supplementation with folic acid or reduced folic acid (in the form of folinic acid) is sufficient to override the impairment
• Excessive loss of Folate: Kidney (Dialysis)

Other Causes of Megaloblastosis

• Dysplastic erythroid precursors (MDS)- refractory anemia
• Myelodysplastic anemia such as refractory anemia
• Congenital dyserythropoietic anemia (CDA)
• Type 1
• Type 3
• Type 2- HEMPAS
• Acute erythroid leukemia (M6) associated with DiGuglielmo syndrome

Disorders Affecting DNA Synthesis

• Deficiency in:
• Transcobalamin II
• Foramino-transferase
• N-methyl tetrahydrofolate reductase
• Dihydrofolate reductase
• Orotic aciduria: presentce of orotic crystal in urine
• Lesch-Nyhan syndrome: orange diaper; increased UA
• Used of chemotherapeutic drugs (target cells)

Disruption of the Folate Cycle in Cancer Chemotherapy

• Folate deficiency leads to DNA strand breaks, which leave the DNA vulnerable to mutation
• The central role of folate in cell division also makes it a target for chemotherapeutic drugs used to treat cancer
• Methotrexate, used in treatment of leukemia and arthritis, enters the folate cycle in preference to tetrahydrofolate, and the folate cycle is blocked by the drug
• Methotrexate treatment typically is followed by what is known as leucovorin rescue, which is a folic acid derivative that can be administered to counteract the effects of methotrexate or other folate antagonists

Lab Diagnosis

• Screening Test: Complete Blood Count
• Macrocytic, normochromic; MCV of 120 fl
• MCH is elevated by the increased volume of the cells, but the MCHC is usually within reference interval because hemoglobin production is unaffected. The RDW is also elevated.
• Peripheral findings are oval macrocytes and hypersegmented neutrophils

Lab Assays

• Assays for Folate, Vitamin B12, Methylmalonic Acid, and Homocysteine
• immunoassay
• chemiluminescence
• Gastric Analysis and Serum Gastrin used to confirm achlorhydria
• Markedly elevated result of serum gastrin
• Antibody Assays indirect fluorescent antibody techniques or enzyme-linked immunosorbent assays
• Holotranscobalamin Assay detect vitamin B12 deficiency and recommend its use in screening for metabolic vitamin B12 deficiency
• Stool Analysis for Parasites D. latum identification

Diagnostic Tests for Vitamin and Folate Deficiency

• Complete blood count
• Manual differential count
• Absolute reticulocyte count
• Serum total and indirect bilirubin
• Serum lactate dehydrogenase
• Bone marrow examination
• Serum vitamin B12
• Serum folate
• RBC folate
• Serum methylmalonic acid
• Serum/plasma homocysteine
• Antibodies to intrinsic factor and gastric parietal cells
• Serum gastrin
• Gastric analysis, Holotranscobalamin assay
• Stool analysis for parasites

Sequence in Development of Megaloblastic Anemia

• Decrease in vitamin levels
• Hypersegmentation of neutrophils in peripheral blood
• Oval macrocytes in peripheral blood
• Megaloblastosis in bone marrow
• Anemia

Treatment

• High-dose oral vitamin B12 (Pernicious anemia)
• Folic acid