Chapter 8: Folate, Cobalamin, and Megaloblastic Anemias

• These disorders are caused by impaired synthesis of DNA.

• They most commonly result from folate or cobalamin (vitamin B₁₂) deficiency.

• Characteristics are megaloblastic cells, typically present in the erythroid series as large cells with immature-appearing nuclei but with increasing hemoglobinization of the cytoplasm—often referred to as nuclear-cytoplasmic asynchrony.

• Megaloblastic granulocytic cells have large size. Giant band neutrophils are a feature in the marrow with hypersegmented neutrophils in the marrow and blood. Megakaryocytes may be abnormally large with nuclear abnormalities.

• Table 8–1 lists causes of megaloblastic anemia.

• By far the most common causes worldwide are folate deficiency and cobalamin deficiency.

• The underlying defect is impaired DNA synthesis because of failure of conversion of dUMP to dTMP.

• Intramedullary destruction of red cell precursors (ineffective erythropoiesis) is a major feature of megaloblastic anemia. Ineffective granulopoiesis and thrombopoiesis are also present and can result in neutropenia and thrombocytopenia. Ineffective hematopoiesis is characterized by marked hyperplasia of precursor cells (hypercellular marrow) with exaggerated apoptosis of late precursors, which results in blood cytopenias.

• Mild hemolysis also occurs; the red cell life span is reduced by about 40%.

• Anemia develops gradually, and patients can adapt to very low hemoglobin levels. Eventually, as it progresses, the presenting symptoms are those of anemia with weakness, palpitation, fatigue, light-headedness, and shortness of breath.

• The condition may present initially with neurologic manifestations without anemia.

• Folic acid deficiency and cobalamin deficiency have indistinguishable blood and marrow changes (megaloblastosis), but the former deficiency is not associated with neuropathology and the latter characteristically is (see “Pernicious Anemia” below).

• All cell lines are affected.

• Erythrocytes show marked anisocytosis and poikilocytosis, with many oval macrocytes and, in severe cases, basophilic stippling, Howell-Jolly bodies, and Cabot rings. Erythrocytes with megaloblastic nuclei may be present in the blood (Figure 8–1).

• Absolute reticulocyte count is low.

• Anemia is usually macrocytic, with a mean cell volume (MCV)of 100 to 150 fL or more, but coexisting iron deficiency, thalassemia trait, or inflammation may prevent macrocytosis.

• Leukopenia and thrombocytopenia are frequently present.

• Hypersegmented neutrophils are an early sign of megaloblastosis. Typically, the nuclei of more than 5% of the cells have more than five lobes. Normal blood has less than 1% five-lobed neutrophils.

• Platelets are smaller than usual and vary more widely in size. Platelets are functionally abnormal in severe megaloblastic anemia.

• Marrow cells show erythroid hyperplasia with striking megaloblastic changes. Promegaloblasts with mitotic figures are abundant in severe cases. The number of sideroblasts is increased, and macrophage iron content may also be increased.

• Coexisting iron deficiency may reduce the megaloblastic erythroid morphologic changes, but hypersegmented neutrophils are still present in the blood, and giant metamyelocytes and bands persist in the marrow.

• Treatment of a patient with folic acid or cobalamin more than 12 hours before marrow biopsy may mask the megaloblastic changes.

• Serum bilirubin, iron, and ferritin levels are increased.

• Serum lactic dehydrogenase-1 and -2 and muramidase (lysozyme) levels are markedly elevated.

• See “Laboratory Diagnosis” below for measurement of cobalamin and folate tissue deficiency.

• Macrocytosis occurs without megaloblastic anemia in patients with liver disease, hypothyroidism, aplastic anemia, myelodysplasia, pregnancy, and anemias with reticulocytosis, but in these settings, the MCV rarely exceeds 110 fL.

• Pancytopenia with reticulocytopenia, which is often present in severe megaloblastic anemia, should be distinguished from aplastic anemia (markedly hypocellular marrow without megaloblastic morphologic changes), myelodysplastic syndrome (often blasts in blood or marrow, dysmorphic neutrophils eg, acquired Pelger-Huet cells, hypogranular cells) and platelets (eg, abnormal size and granulation), and acute myelogenous leukemia (evident leukemic myeloblasts in marrow and usually blood).

• Certain chemotherapeutic drugs, especially folate antagonists (eg, methotrexate), hydroxyurea, and antiretroviral agents, may induce megaloblastic marrow and blood cell changes.

Cobalamin Deficiency

• Table 8–1 presents the causes of cobalamin deficiency.

• Cobalamin deficiency usually results from impaired absorption, most often as a consequence of a deficiency in gastric intrinsic factor (pernicious anemia).

• Less common causes include gastric and ileum resection syndromes, Zollinger-Ellison and “blind loop” syndromes, intestinal parasites, pancreatic disease, and dietary deficiencies.

Pernicious Anemia

• This disease of later life, usually after age 40 years, is caused by failure of secretion of intrinsic factor by the gastric mucosa.

• This form of anemia is an autoimmune disease in which there is immune destruction of the acid- and pepsin-secreting cells of the stomach.

• Antibodies to intrinsic factor are found in up to 70% of patients and are highly specific for pernicious anemia. Serum parietal cell antibodies are present in 90% of patients but are not specific.

• Concordance with several other autoimmune diseases (eg, immune thyroid diseases, type 1 diabetes mellitus, Addison disease, and others) is found.

• A family history is common, and dominant inheritance with low penetrance has been proposed. Pernicious anemia is more common in persons of Northern European or African descent and less common in those of Asian descent.

• Gastric atrophy and achlorhydria occur in all patients. Absence of achlorhydria is incompatible with diagnosis of pernicious anemia.

• The skin often assumes a lemon-yellow hue because of pallor combined with slight hyperbilirubinemia.

• Lingual papillary atrophy (smooth, beefy red tongue) is seen in advanced disease.

• The clinical features of cobalamin deficiency are those of megaloblastic anemia generally, plus neurologic abnormalities specifically caused by cobalamin deficiency.

• Neurologic abnormalities may occur before the onset of anemia and may be irreversible. The neurologic disorder usually begins with paresthesias of the fingers and toes and disturbances of vibration and position sense. The earliest signs may be loss of position sense in the second toe and loss of vibration sense to 256 Hz but not to 128 Hz. If untreated, the disorder progresses to spastic ataxia because of demyelination of the posterior and lateral columns of the spinal cord, referred to as combined system disease.

• Cobalamin deficiency also affects the brain, and patients may develop somnolence and perversion of taste, smell, and vision, sometimes with optic atrophy. Dementia or frank psychosis may occur, the latter sometimes referred to as “megaloblastic madness.” Magnetic resonance imaging can confirm cobalamin deficiency affecting the brain by detecting demyelinization as T2-weighted hyperintensity of the white matter.

• Because neurologic complications may develop in patients with cobalamin deficiency treated with folic acid, a trial with folic acid is not recommended as a diagnostic test.

• Because of the possible development of neurologic complications in untreated patients with cobalamin deficiency, it is important to evaluate all patients with macrocytic anemia for both cobalamin and folic acid deficiency.

Gastrectomy and Ileal Resection Syndromes

• Cobalamin deficiency develops within 5 to 6 years of total gastrectomy or resection of the terminal ileum as a result of loss of secretion of intrinsic factor from the stomach or failure to absorb cobalamin-intrinsic factor complexes in the ileum. The delay in onset of the anemia reflects the time required to exhaust cobalamin stores after absorption ceases. Diseases or injury to the terminal ileum may also lead to impaired cobalamin absorption and megaloblastic anemia (eg, regional ileitis, radiation, sprue).

• Cobalamin absorption may also be impaired after subtotal gastrectomy.

Zollinger-Ellison Syndrome

• Gastrin-secreting tumor, usually in the pancreas, stimulates gastric mucosa to elaborate immense amounts of hydrochloric acid.

• Sufficient acid may be secreted to inactivate pancreatic proteases and to prevent release of cobalamin from its binder, preventing its attachment to intrinsic factor; both are necessary for cobalamin absorption.

“Blind Loop” Syndrome

• Intestinal stasis from anatomic lesions or impaired motility may lead to intestinal colonization with bacteria that bind cobalamin before it can be absorbed.

Diphyllobothrium latum Infestation

• These intestinal parasites, usually ingested in raw fish, bind cobalamin and prevent absorption. Only about 3% of people infested with the parasites become anemic. It is most prevalent in the Baltic Sea region, Canada, and Alaska where raw or undercooked fish is consumed. Diagnosis is made by identification of tapeworm ova in the feces.

Pancreatic Disease

• Pancreatic exocrine insufficiency leads to deficiency of pancreatic proteases necessary for cobalamin absorption. Clinically significant deficiency of cobalamin is rare.

Dietary Cobalamin Deficiency

• This type of megaloblastic anemia occurs rarely, usually in strict vegetarians who also avoid dairy products and eggs (“vegans”).

• Symptomatic cobalamin deficiency can take decades to appear because of enterohepatic reabsorption of cobalamin, conserving body stores.

• Breast-fed infants of vegan mothers may also develop cobalamin deficiency.

Folic Acid Deficiency

• Table 8–1 summarizes the causes of folic acid deficiency.

• An inadequate diet is the principal cause of folic acid deficiency. Folic acid reserves are small, and deficiency can develop rapidly.

• Alcohol use can depress absorption and serum folate levels and can accelerate the appearance of megaloblastic anemia in people with early folate deficiency.

Megaloblastic Anemia Caused by Drugs

• Table 8–2 presents a partial list of drugs that cause megaloblastic anemia.

• Methotrexate is almost structurally identical to folic acid and acts by inhibiting dihydrofolate reductase, the enzyme which reduces folic acid to the active, tetrahydro form. Methotrexate toxicity is treated with folinic acid, which is already fully reduced, and therefore can bypass the inhibited dihydrofolate reductase.

Acute Megaloblastic Anemia

• Acute megaloblastic anemia refers to a syndrome of rapidly developing thrombocytopenia and/or leukopenia, with very little change in the hemoglobin level. The marrow is floridly megaloblastic.

• The most common cause is nitrous oxide anesthesia. Nitrous oxide destroys methylcobalamin, inducing cobalamin deficiency. The marrow becomes megaloblastic within 12 to 24 hours. Hypersegmented neutrophils appear in the blood after 5 days.

• Serum cobalamin levels are low in most affected patients. Cobalamin levels are usually normal in cobalamin deficiency resulting from exposure to nitrous oxide and in some of the inherited abnormalities of cobalamin metabolism (see below).

• The effects of nitrous oxide disappear in a few days. Administration of folinic acid or cobalamin accelerates recovery.

• Fatal megaloblastic anemia has occurred in patients with tetanus who were treated with nitrous oxide for weeks.

• Acute megaloblastic anemia may also occur in seriously ill patients in intensive care units, patients transfused extensively, patients on dialysis or total parenteral nutrition, or patients receiving weak folic acid antagonists. The diagnosis is made from finding a megaloblastic marrow. Treatment is with both parenteral cobalamin (1 mg) and folic acid (5 mg).

Megaloblastic Anemia in Childhood

• Cobalamin malabsorption occurs in the presence of normal intrinsic factor in an inherited disorder of childhood (selective malabsorption of cobalamin, or Imerslund-Graesbeck disease). There is associated albuminuria. Anemia usually develops before age 2 years. Treatment is with parenteral cobalamin.

• Congenital intrinsic factor deficiency is an autosomal recessive disorder in which parietal cells fail to produce intrinsic factor. The disease presents at 6 to 24 months of age. Treatment is with parenteral cobalamin.

• Transcobalamin II deficiency is an autosomal recessive disorder that leads to megaloblastic anemia in early infancy. Serum cobalamin levels are normal, but there is severe tissue cobalamin deficiency because transcobalamin II mediates transport of cobalamins into the tissues. The diagnosis is made by measuring serum transcobalamin II concentration. Treatment is with sufficiently large doses of cobalamin to override the deficient transport.

• True juvenile pernicious anemia is an extremely rare disorder that usually presents in adolescence. The diagnosis and treatment are the same as for the adult disease.

Other Megaloblastic Anemias and Changes

• Megaloblastic anemia may occur in some patients with inborn errors of cobalamin metabolism, inborn errors of folate metabolism, hereditary orotic aciduria, and the Lesch-Nyhan syndrome. A thiamine responsive megaloblastic anemia has also been reported.

• Anemia with megaloblastic-like red cell morphology (“megaloblastoid”) may occur in some patients with congenital dyserythropoietic anemias, myelodysplastic syndromes, and erythroleukemia.

Cobalamin Tissue Deficiency

• Serum cobalamin levels are low in most affected patients but may be normal because of nitrous oxide inhalation and some of the inherited abnormalities of cobalamin metabolism.

• Serum cobalamin levels may be low with normal tissue levels in vegetarians, older persons, the chronically ill, people taking megadoses of vitamin C, pregnancy (25%), transcobalamin I deficiency, or folate deficiency (30%).

• Transcobalamin-bound cobalamin represents about 25% of the total plasma cobalamin and is the functionally important fraction. Assays permit measurement of this more relevant, transcobalamin-bound cobalamin level.

• Methylmalonic aciduria and elevated serum levels of methylmalonic acid are reliable indicators of tissue cobalamin deficiency (except in the presence of severe renal insufficiency). They are the earliest changes and precede anemia or morphologic blood cell changes. If normal, they argue strongly against tissue deficiency even if serum levels of the vitamin are low.

• Elevated serum homocysteine can indicate tissue cobalamin deficiency but, unlike abnormalities in methylmalonic acid noted above, it can also be elevated in folic acid deficiency, pyridoxine deficiency, and hypothyroidism.

• In patients were pernicious anemia, serum intrinsic factor antibodies are present in 7% of patients and are specific for the diagnosis.

Folic Acid Deficiency

• Serum folate levels are reduced, but a low level may merely reflect reduced oral intake in the few days preceding the test.

• The red cell folic acid level is a more accurate reflection of tissue folate because it is not affected by recent dietary intake or drugs.

• Both red cell and serum folate are decreased in folic acid deficiency. In cobalamin deficiency, red cell folate may be low but serum folate is normal or elevated. Thus, both measurements are required to assess tissue folate levels.

Cobalamin Deficiency

• Treatment consists of parenteral administration of cyanocobalamin (vitamin B₁₂) or hydroxycobalamin in doses sufficient to replete tissue stores and provide daily requirements.

• Vitamin B₁₂ has no toxicity per se, but parenteral cobalamin doses larger than 100 μg saturate the transport proteins and much is lost in the urine.

• A typical treatment schedule consists of 1000 μg of vitamin B₁₂ intramuscularly daily for 2 weeks, then weekly until the hemoglobin level is normal, and then monthly for life.

• It has been recommended that after initial therapy, to return the hematocrit to normal, patients with neurologic abnormalities should receive 1000 μg intramuscularly every 2 weeks for 6 months.

• About 1% of an oral dose of vitamin B₁₂ is absorbed even in the absence of intrinsic factor. Therefore, patients with pernicious anemia can be successfully treated with oral vitamin B₁₂ in doses of 1,000 μg/day. Patients receiving such therapy should be carefully monitored to ensure compliance and a response.

• Infection can interfere with the response to vitamin B₁₂ therapy.

• Transfusion may be required if the clinical picture requires prompt alleviation of anemia. Most patients, however, have adapted to severe anemia and can be treated with vitamin replacement therapy.

• Following initiation of cobalamin therapy, there is often a prompt improvement in the sense of well being.

• Marrow erythropoiesis converts from megaloblastic to normoblastic beginning about 12 hours after treatment is started.

• Reticulocytosis appears on days 3 to 5 and reaches a peak on days 4 to 10. The hemoglobin concentration should become normal within 1 to 2 months.

• Leukocyte and platelet counts normalize promptly, although neutrophil hypersegmentation persists for 10 to 14 days.

• Elevated serum bilirubin, serum iron, and lactic dehydrogenase levels fall to normal rapidly.

• Severe hypokalemia may develop after cobalamin therapy, and death from hypokalemia has occurred. Potassium levels must be monitored and appropriate replacement given.

• Cobalamin therapy should be administered to all patients after total gastrectomy or resection of the terminal ileum. After partial gastrectomy, patients should be monitored carefully for the development of anemia.

• The anemia of the “blind loop” syndrome will respond to parenteral cobalamin therapy, but it also responds to oral antibiotic therapy or successful correction of an anatomic abnormality.

• Pregnant women at risk for cobalamin deficiency, such as strict vegetarians, may also be given vitamin B₁₂, 1 mg parenterally every 3 months, during pregnancy.

Folic Acid Deficiency

• Folic acid deficiency responds to physiologic doses of folic acid (200 μg/day), but cobalamin deficiency responds only to folic acid doses of 5 mg/day.

• Folic acid is administered orally at a dose of 1 to 5 mg daily. At this dosage, patients with malabsorption usually respond.

• Pregnant women should receive 1 mg of folic acid daily.

• In megaloblastic anemia with laboratory evidence of folic acid deficiency, a full response to physiologic doses of folic acid should occur. If a question of absorptive limitations is present, the folate should be administered intramuscularly.