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• Vitamin B12 is a coenzyme for two biochemical reactions:
  • As methyl B12 it is a cofactor for methionine synthase, the enzyme responsible for methylation of homocysteine to methionine using methyl tetrahydrofolate (methyl THF) as methyl donor.
  • As deoxyadenosyl B12 (ado B12) it assists in conversion of methylmalonyl coenzyme A (CoA) to succinyl CoA.
• Folates are needed in a variety of biochemical reactions in the body involving single carbon unit transfer, in amino acid interconversions (e.g. homocysteine conversion to methionine) and serine to glycine or in synthesis of purine precursors of DNA.

The biochemical basis of megaloblastic anaemia caused by vitamin B12 or folate deficiency. Folate is required in one of its coenzyme forms, 5,10‐methylene tetrahydrofolate (THF) polyglutamate, in the synthesis of thymidine monophosphate from its precursor deoxyuridine monophosphate. Vitamin B12 is needed to convert methyl THF, which enters the cells from plasma, to THF, from which polyglutamate forms of folate are synthesized. Dietary folates are all converted to methyl THF (a monoglutamate) by the small intestine.

Absorption:

• Iron: duodenum
• Folate: proximal jejunum
• Vitamin B12: terminal ileum

The absorption of dietary vitamin B12 after combination with intrinsic factor (IF), through the ileum. Folate absorption occurs through the duodenum and jejunum after conversion of all dietary forms to methyltetrahydrofolate (methyl THF).

ado-cbl, 5′- deoxyadenosylcobalamin; HC, haptocorrin; also called TC I or transcobalamin I; IF, intrinsic factor; methyl-cbl, methylcobalamin; TC, transcobalamin; also called TC II.

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Macrocytosis, anisopoikilocytosis, a few polychromatophilic red blood cells with rare purple Cabot rings (panel A, arrow), and coarse basophilic stripling (panels B and C).

• The earliest morphologic change in the blood is the hypersegmented granulocyte followed by macrocytosis. Individual macrocytes appear first and can be detected by reticulocyte hemoglobin and cell size measurements, followed by a gradual rise in MCH and then MCV in routine blood counts. These indices eventually cross the line into abnormality (MCV greater than 97 fL) before the hemoglobin levels fall.
  • In cobalamin deficiency, with its slower progression, macrocytosis precedes anemia by months.
  • Macroovalocytes are characteristic but are not specific.
  • Early megaloblastic changes in the bone marrow precede the macrocytosis but are easily missed.
  • Eventually, poikilocytosis becomes more pronounced with teardrop cells.
  • Nucleated red cells, Howell–Jolly bodies, and even Cabot rings appear in the blood in advanced megaloblastosis.
    • Cabot rings are thin, threadlike ring- or “figure eight”–shaped red blood cell inclusions, likely remnants from mitotic spindles. They are rarely seen in peripheral blood, and their presence indicates a defect in erythrocyte production, especially in pernicious anemia and lead poisoning.
  • As megaloblastic anemia worsens, neutropenia and thrombocytopenia develop.
• As anemia progresses, serum iron and transferrin receptor levels, sideroblast counts, and the ferritin content of erythroid precursors and macrophages increase. Erythropoietin levels correlate with the severity of anemia.
• The hypercellular marrow is characteristically megaloblastic and hypercellular.
  • The megaloblastic hallmark is nuclear “immaturity” with seemingly mature cytoplasm (nuclear-cytoplasmic dissociation), which is best appreciated in precursor cells in the bone marrow aspirate. Megaloblastic nuclei are larger than normoblastic nuclei, and their chromatin appears abnormally dispersed because of its retarded condensation. Giant band cells and metamyelocytes with large and often misshapen nuclei are typical.
  • Bone marrow hyperplasia is intense, but reticulocytosis does not occur. As megaloblastosis advances, most precursors die within the hypercellular bone marrow and are phagocytosed.

A, Pronormoblast and two normoblasts in normal bone marrow. B, Megaloblastic equivalent of the normal pronormoblast shown in picture A. C, Megaloblastic equivalents of the two smaller pronormoblasts shown in picture A. D, Red blood cells (RBC) in peripheral blood from three patients: (Left) Megaloblastic anemia (note oval macrocytes, anisocytosis, and poikilocytosis); (Middle) Normal blood smear (note size and uniformity of RBC); (Right) Liver disease (note macrocytic RBC with uniform size and shape, including target cells). E, Peripheral blood with early band forms from a patient with megaloblastic anemia; the cell on the left appears normal, and the cell on the right is larger, and its larger nucleus has “looser” chromatin. F, Megaloblastic peripheral blood smear with hypersegmented neutrophil at 3- o’clock.

a. The sensitivity and specificity estimates (very good, >90%; good, 80%-90%; poor, <70%) apply only to clinically expressed cobalamin deficiency. They do not apply to subclinical cobalamin deficiency, in which sensitivity and specificity tend to be lower than in clinical deficiency, have usually been determined only against other biochemical tests, or are unknown. b. The test is not widely available. c. Testing has largely involved comparison with methylmalonic or other tests; data from clinical settings with matching against defined patients have been limited. d. The discriminatory diagnostic power arises from including testing with vitamin additives in vitro. Abbreviations: N, normal result; ND, not determined.

• The serum and red cell folate are both low in megaloblastic anemia caused by folate deficiency.
• In the absence of B12 deficiency, however, the red cell folate may be a more accurate guide of tissue folate status than the serum folate.
• In B12 deficiency, the serum folate tends to rise but the red cell folate falls.

Differential Results of the Schilling Test in Several Diseases Associated with Cobalamin Malabsorption

• Measurement of serum methylmalonic acid is a test for B12 deficiency and measurement of homocysteine is a test for folate or B12 deficiency. These are not specific, however, and it is difficult to establish normal levels in different age groups. These tests are also not widely available.

b. Examples are polymorphisms causing mild elevations of MMA in a valine-related pathway (such as HIBCH) and mitochondrial depletion diseases (such as SUCLG1, which causes succinate CoA ligase deficiency). c. Reduction of MMA levels has been described after antibiotic therapy in patients with bacterial contamination of the small bowel. The phenomenon has been attributed to propionate metabolism by bacteria. d. The optimal cut point for MMA has not been defined. Laboratories and studies that use lower cut points than usual (eg, 210 nmol/L) identify greater numbers of subjects with “high” or “abnormal” levels.

a. Evidence of metabolically or clinically defined cobalamin deficiency or malabsorption has been found in only some cases in these categories. b. Does not apply to postmenopausal hormone replacement. c. Frequencies of “low levels” depend greatly on cut-point selection, which is often arbitrary. The impact can be great; for example, the frequency of “low levels” increases 2-fold or more following the shift of the cobalamin cut point from 200 to 300 ng/L. HC, haptocorrin.

Vit B12 Deficiency

• Neuromyelopathy is a common, dreaded feature of cobalamin deficiency, but it is not specific to it; copper deficiency, for example, produces similar findings along with macrocytosis.
• Demyelination with subsequent axonal disruption and gliosis can affect all parts of the central nervous system. Peripheral nerves, however, tend to show axonal degeneration without demyelination. The classic myelopathic syndrome is subacute combined degeneration, in which posterior and lateral column damage predominates and tends to be symmetrical; dorsal, pyramidal, and spinocerebellar tracts are affected.
• Unlike anemia, ND does not always reverse after cobalamin therapy. Residual deficits persist in 6% of patients.

• Pernicious anaemia is caused by autoimmune attack on the gastric mucosa leading to atrophy of the stomach. The wall of the stomach becomes thin, with a plasma cell and lymphoid infiltrate of the lamina propria. Intestinal metaplasia may occur. There is achlorhydria and secretion of IF is absent or almost absent. Serum gastrin levels are raised.

• Helicobater pylori infection may initiate an autoimmune gastritis which presents in younger subjects as iron deficiency and in the elderly as PA.

• PA is distinguished from simple atrophic or autoimmune gastritis by the presence of antibodies to IF. Fifty per cent of PA patients show in serum an antibody to IF which inhibits IF binding to B12.

• A second antibody blocking IF attachment to its ileal binding site is less frequent.

• IF antibodies also occur in PA in gastric juice. IF antibodies are virtually specific for PA but as they occur in the serum of only half of patients, their absence from serum does not exclude the diagnosis.

• The more common parietal cell antibody is less specific as it occurs quite commonly in older subjects (e.g. 16% of normal women over 60 years) even without PA.

• Congenital lack or abnormality of IF usually presents at approximately 2 years of age when stores of B12 that were derived from the mother in utero have been used up.

• Specific malabsorption of B12 is due to genetic mutation of the IF–B12 receptor, cubilin, or of amnionless. It usually presents in infancy or childhood and is associated with proteinuria in 90% of cases.

• Lesser degrees of B12 deficiency with borderline or low serum B12 levels, but almost always without megaloblastic anaemia, occur resulting from inadequate intake of B12 or from malabsorption of food B12, especially in the elderly with atrophic gastritis, and in association with prolonged therapy with proton pump inhibitors or metformin.

• Folate deficiency is most often a result of a poor dietary intake of folate alone or in combination with a condition of increased folate utilization or malabsorption.