Vitamin B12 (cyanocobalamin) is necessary for adequate DNA synthesis through its role in folic acid metabolism. Vitamin B12 transforms metabolically inactive 5-methyl-tetrahydrofolate to tetrahydrofolate, which can then be converted to an active coenzyme. Vitamin B12 also acts as a single-carbon transfer agent in certain other metabolic pathways, such as metabolism of propionic acid. Most vitamin B12 comes from food of animal origin, primarily meat, eggs, or dairy products. In the stomach, the B12 is detached from binding proteins with the help of gastric acid. Vitamin B12 is absorbed through the ileum with the aid of “intrinsic factor” (IF) produced by parietal cells in the fundus of the stomach.

Vitamin B12 deficiency may be produced in several ways. The most common cause is inability to absorb B12 due to deficiency of intrinsic factor (pernicious anemia). Another cause is failure to release B12 from binding proteins caused by severe deficiency of gastric acid. A third cause is other malabsorption syndromes involving the ileum mucosa or B12 absorption by the ileum. Besides direct damage to ileal mucosa, various causes of vitamin B12 malabsorption include bacterial overgrowth in the intestine (blind loop syndrome), infestation by the fish tapeworm, severe pancreatic disease, and interference by certain medications. Finally, there is dietary B12 deficiency, which is rare and found mainly in strict vegetarians.

Vitamin B12 assay. Vitamin B12 deficiency can usually be proved by serum B12 assay. Therefore, a therapeutic trial of oral B12 is seldom needed. In patients with severe anemia of unknown etiology it is often useful to freeze a serum specimen before blood transfusion or other therapy begins, since vitamin B12 and folic acid measurements (or other tests) may be desired later. Chloral hydrate is reported to increase B12 levels in some patients using certain assay kits but not others. Pregnancy, large doses of vitamin C, and folic acid deficiency may be associated with reduced B12 assay levels. Achlorhydria is reported to produce decreased release or utilization of vitamin B12 from food, although intestinal absorption of crystalline vitamin B12 used in the Schilling Test dose or in oral therapeutic B12 is not affected. Vitamin B12 deficit may be accompanied by folic acid deficiency in some conditions and by chronic iron deficiency.

Vitamin B12 is transported in serum bound to several serum proteins, among which the most important are transcobalamin and “R-protein.” Severe liver disease or myeloproliferative disorders with high white blood cell (WBC) counts produce elevated vitamin B12-binding protein levels and falsely raise total serum B12 values. In 1978, reports were published demonstrating that certain metabolically inactive analogues of B12 were present in serum bound to R-protein. It was found that commercial vitamin B12 assay kits contained varying amounts of R-protein in the material used to segregate patient B12 for measurement and that the B12 analogues could bind to the reagent R-protein and be included with active B12 in the measurement, thereby falsely increasing the apparent B12 value. A low vitamin B12 value could be elevated into the reference range. Most manufacturers have redesigned their B12 assay kits to eliminate the effects of R-protein. Nevertheless, case reports of patients with symptoms of B12 deficiency but serum B12 assay results within reference range continue to appear. In these cases some additional test, such as intrinsic factor antibodies or a therapeutic trial with B12, may be desirable.

A decreased B12 level does not guarantee that actual B12 deficiency exists. Several investigators have reported that less than 50% of their patients with decreased serum B12 levels had proven B12 deficiency.

Megaloblastic changes. Deficiency of either vitamin B12 or folic acid eventually leads to development of megaloblastic anemia. Vitamin B12 deficiency may take 1 to 2 years for the MCV to become elevated and megaloblastic changes to appear. The RBC precursors in the marrow become slightly enlarged, and the nuclear chromatin develops a peculiar sievelike appearance, referred to as megaloblastic change. This affects all stages of the precursors. The bone marrow typically shows considerable erythroid hyperplasia as well as megaloblastic change. Not only RBCs are affected by folate or B12 deficiency but also WBCs and platelets. Actual anemia develops 6 to 18 months later (2 to 3 years after the original disease onset). In far-advanced megaloblastic anemia there are peripheral blood leukopenia and thrombocytopenia in addition to anemia; in early cases there may be anemia only. The bone marrow shows abnormally large metamyelocytes and band neutrophils. Macrocytes are usually present in the peripheral blood, along with considerable anisocytosis and poikilocytosis. Hypersegmented polymorphonuclear neutrophils are characteristically found in the peripheral smear, though their number may be few or the degree of hypersegmentation (five lobes or more) may be difficult to distinguish from normal variation. The reticulocyte count is normal.

Methylmalonic acid assay. In patients with borderline serum B12 values or clinical suspicion of B12 deficiency but serum B12 within reference range, serum or urine methylmalonic acid assay may be helpful. B12 is a necessary cofactor in the reaction converting methylmalonate to succinate. If insufficient B12 is available, the reaction is partially blocked and methylmalonic acid (MMA) accumulates in serum and is increased in urine. Both serum and urine MMA are said to be increased in 95% of patients with B12 deficiency, even when serum B12 assay is within population reference range. One study found that only 60% of patients with increased urine MMA has decreased serum B12 values. MMA assay usually has to be sent to a reference laboratory.

Bone marrow examination. For a long time, bone marrow aspiration was the classic way to confirm the diagnosis of megaloblastic anemia. Now that B12 and folate assays are available, most physicians no longer obtain bone marrow aspiration. The major difficulty occurs if megaloblastic anemia is strongly suspected and the B12 and folate assay results are within normal limits. To be useful, a bone marrow aspiration should be performed as early as possible and definitely before blood transfusion. Blood transfusion, hospital diet, or vitamin B12 administered as part of a Schilling test may considerably reduce or eliminate diagnosable megaloblastic change from the bone marrow in as little as 24 hours’ time, even though the underlying body deficiency state is not cured by the small amount of B12 and folate in blood transfusion.

Megaloblastic changes in the bone marrow are not diagnostic of folic acid or vitamin B12 deficiency. Some cytologic features of megaloblastic change (“megaloblastoid” or “incomplete megaloblastic” change) may appear whenever intense marrow erythroid hyperplasia takes place, such as occurs in severe hemolytic anemia. Similar changes may also be found in chronic myelofibrosis, in sideroblastic anemias, in certain myelodysplasia syndromes, in erythroleukemia (formally called Di Guglielmo’s syndrome), in some patients with neoplasia, cirrhosis, and uremia, and in association with certain drugs, such as phenytoin (Dilantin) and primidone, methotrexate and folic acid antagonists, and alcohol (substances affecting folate metabolism); colchicine and neomycin (affecting absorption); and antineoplastic drugs such as 5-fluorouracil and 6-mercaptopurine (that interfere with DNA synthesis).

Red blood cell indices. The MCV in megaloblastic anemia is typically elevated. However, 15%-30% of patients (0%-96%) with folic acid or B12 deficiency, more often folic acid deficiency, had an MCV in the upper half of the reference range. In some patients normal MCV was probably due to early or mild disease. In others, these findings were explained on the basis of a coexisting condition that produced microcytosis (most commonly chronic iron deficiency, thalassemia minor, and anemia of infection, and less commonly malnutrition. In one study, 21% of patients with pernicious anemia had a significant degree of iron deficiency. If chronic iron deficiency coexists, therapy for megaloblastic anemia alone may only partially (or not at all) correct the anemia but will unmask the chronic iron deficiency picture. If the iron deficiency alone is treated, the megaloblastic defect is unmasked (unless it is a deficiency that is inadvertently treated by hospital diet or vitamins).

The MCV is less likely to be elevated in B12 deficiency without anemia. In one study, only 4% of nonanemic patients with decreased serum B12 during one time period had elevated MCV. On the other hand, the MCV may become elevated before there is hemoglobin decrease to the level of anemia; therefore, an elevated MCV, with or without anemia, should not be ignored.

Serum lactic dehydrogenase. Serum lactic dehydrogenase (LDH) levels are elevated in most patients with hemolytic anemia and in 85% or more of patients with megaloblastic anemia. The electrophoretically fast-migrating LDH fraction 1 is found in RBCs, myocardial muscle cells, and renal cortex cells. Megaloblastic change induces increased destruction of RBCs within bone marrow, which is reflected by the increase in serum LDH levels. If the LDH is included in patient admission chemistry screening panels, an LDH elevation might provide a clue to the type of anemia present. In hemolytic and megaloblastic anemia, LDH isoenzyme fractionation typically displays elevated LDH fraction 1 with fraction 1 greater than fraction 2 (reversal of the LDH-1/LDH-2 ratio). However, the usefulness of serum LDH or LDH isoenzymes is limited by their nonspecificity (since LDH fraction 1 is not specific for RBCs, and other LDH isoenzymes are present in other tissues such as liver) and by the fact that the degree of LDH change is roughly proportional to the severity of anemia, so that patients with mild cases may have total LDH or fraction 1 values still within reference range.

Neutrophil hypersegmentation. Hypersegmentation is most often defined as a segmented neutrophil with five or more lobes (although some restrict the definition to six or more lobes). An elevated neutrophil lobe count (defined as five or more lobes in more than 5% of all neutrophils) is reported to be more sensitive and reliable than elevated MCV or peripheral smear macrocytosis in detecting megaloblastic anemia and also is not affected by coexisting iron deficiency.