Williams Hematology



General Considerations


Etiology and Pathogenesis

Clinical Features

Laboratory Features
Folic Acid Deficiency

Etiology and Pathogenesis

Clinical Features

Laboratory Features

Differential Diagnosis

Nonhematologic Effects of Folate Deficiency

Therapy, Course, and Prognosis
Cobalamin Deficiency

Etiology and Pathogenesis

Clinical Features

Laboratory Features

Differential Diagnosis

Therapy, Course, and Prognosis
Acute Megaloblastic Anemia
Megaloblastic Anemia Caused by Drugs
Megaloblastic Anemia in Childhood

Malabsorption of Cobalamin

Selective Malabsorption of Cobalamin (Imerslund-GrÄsbeck Disease)

Congenital Intrinsic Factor Deficiency

Transcobalamin II (TC-II) Deficiency

Inborn Errors of Cobalamin Metabolism

Inborn Errors of Folate Metabolism

Other Inborn Errors
Other Causes of Megaloblastic Anemia

Congenital Dyserythropoietic Anemia

Refractory Megaloblastic Anemia

Erythroleukemia (Di Guglielmo Syndrome)
Chapter References

Megaloblastic anemia is most commonly due to a deficiency of folate or cobalamin (vitamin B12). Folate deficiency is usually nutritional in origin and may be seen in alcoholics and the elderly poor but also in patients on hyperalimentation or hemodialysis. In pregnancy, even a mild folate deficiency is associated with defects in neural tube closure in the fetus, so folate supplements should always be given to pregnant women. Diagnosis is based on measurements of folate in serum, which furnishes information about the current level of folate, and in red cells, which provides data on folate levels over the preceding 6 weeks. Nutritional folate deficiency is treated with folic acid by mouth.
Folate deficiency due to malabsorption occurs in tropical and nontropical sprue. Folate deficiency due to tropical sprue is treated with folate supplements plus antibiotics; in nontropical sprue, the treatment is folate plus a gluten-free diet.
The commonest cause of cobalamin deficiency is pernicious anemia, a condition in which the portion of gastric mucosa that contains the parietal cells is destroyed through an autoimmune mechanism. The parietal cells secrete intrinsic factor, which is essential for cobalamin absorption, and without intrinsic factor a state of cobalamin deficiency develops over the course of years. Cobalamin deficiency leads not only to megaloblastic anemia but also to a demyelinating disease that manifests itself as peripheral neuropathy, spastic paralysis with ataxia (so-called combined system disease of the spinal cord), dementia, psychosis, or a combination of the foregoing. “Subtle” cobalamin deficiency, manifested as neurologic symptoms without anemia, appears to be relatively widespread among the elderly. The incidence of gastric cancer is increased by a factor of 2 to 3 in patients with pernicious anemia. Other causes of cobalamin deficiency are gastric resection; stasis of the small intestinal contents due to blind loops, strictures, or hypomotility (seen, for example, in amyloid); or disease or resection of the terminal ileum. Patients on a vegan diet will also become cobalamin deficient. The diagnosis of cobalamin deficiency is made by measuring the level of the vitamin in the blood or by measuring serum methylmalonic acid, which accumulates in the blood stream in patients with cobalamin deficiency. The cause of the cobalamin deficiency can often be determined by the Schilling test, a measure of cobalamin absorption. In patients with nutritional megaloblastic anemia, it is of great importance to establish whether the anemia is due to folate deficiency or cobalamin deficiency, because if a patient with cobalamin deficiency is treated with folic acid, the anemia will be corrected but the neurologic abnormalities will progress. Patients with cobalamin deficiency are usually treated with parenteral cobalamin.

Acronyms and abbreviations that appear in this chapter include: BFU–E, burst forming units–erythroid; CNS, central nervous system; dTMP, deoxythymidine monophosphate; dU, deoxyuracil; dUMP, deoxyuridine monophosphate; HIV, human immunodeficiency virus; IM, intramuscular; LDH, lactate dehydrogenase; MCV, mean corpuscular volume; MRI, magnetic resonance imaging; MTHFR, methylenetetrahydrofolate reductase; PA, pernicious anemia; PDW, platelet distribution width; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; TC, transcobalamin; UTP, uridine triphosphate.

Megaloblastic anemia can develop as an acute disorder with rapid development of leukopenia and/or thrombocytopenia. Nitrous oxide anesthesia is responsible for some cases of acute megaloblastic anemia. It is also seen in patients with a marginal folate status in intensive care units. The condition resembles an immune cytopenia, but this can be ruled out by examining the bone marrow, which exhibits a floridly megaloblastic picture.
Other causes of megaloblastic anemia include drugs (e.g., hydroxyurea, nucleoside analogs) and certain inborn errors of metabolism. Of these inherited conditions, transcobalamin II deficiency is singled out because it causes a severe megaloblastic anemia in infants that responds completely to high-dose cobalamin; if it is not detected in time, however, irreversible neurologic complications will supervene. Finally, megaloblastic anemia is seen in refractory megaloblastic anemia, a form of the myelodysplastic syndrome, and in early stages of acute myeloblastic leukemia of the M6 type (Di Guglielmo’s syndrome). The anemia of refractory megaloblastic anemia sometimes responds to pyridoxine in very high doses.
Megaloblastic anemias are disorders caused by impaired DNA synthesis and characterized by the presence of megaloblastic cells, the morphologic hallmark of this group of anemias. Megaloblastic red cell precursors are larger than normal and have more cytoplasm relative to the size of the nucleus. Promegaloblasts show a blue granule-free cytoplasm and a granular chromatin that contrasts with the ground-glass texture of its normal counterpart (see Color Plate VI-4). As the cell differentiates, the chromatin condenses more slowly than normal into dark aggregates that coalesce to give the nucleus a characteristic fenestrated appearance. Condensation of the chromatin to a homogeneous mass either fails or is delayed. As the cytoplasm acquires hemoglobin, its growing maturity contrasts with the immature-looking nucleus, a feature termed nuclear-cytoplasmic asynchrony.
Megaloblastic granulocyte precursors are also larger than normal and show nuclear-cytoplasmic asynchrony, with cytoplasm that looks less mature than that of their normal counterparts. A characteristic cell is the giant metamyelocyte, with a large horseshoe-shaped nucleus, sometimes irregular in shape, containing ragged chromatin.
Megaloblastic megakaryocytes may be abnormally large, with deficient granulation of the cytoplasm. In severe megaloblastosis, the nucleus may show unattached lobes.
The causes of megaloblastic anemia are listed in Table 37-1. By far the most common of these are folate deficiency and cobalamin deficiency. Megaloblastic cells have much more cytoplasm and RNA than do their normal counterparts but have a relatively normal amount of DNA,1,2 suggesting that cytoplasmic constituents (RNA and protein) are synthesized faster than is DNA. Supporting this conclusion is evidence that in megaloblastic precursors maturation is retarded,1,3 DNA synthesis is impaired,4,5 and 6 migration of the DNA replication fork and the joining of Okazaki fragments are delayed,7 and S phase is prolonged.6


The slowing of DNA replication in the megaloblastic anemias of folate and cobalamin deficiency appears to arise from a failure of the folate-dependent conversion of dUMP to dTMP (see Chap. 34). Because of this failure, and the fact that DNA polymerase has difficulty distinguishing dUTP from dTTP, dUTP instead of dTTP is incorporated into the DNA of folate-deficient cells.8,9 Recognizing the mistake, the cells try to repair the DNA by replacing uridine with thymidine, but these attempts at repair tend to fail for the same reason that UTP was incorporated into the DNA in the first place. The result is a frustrated effort at DNA repair leading ultimately to DNA fragmentation followed by cell death.
dU normally inhibits the incorporation of tritiated thymidine into DNA, probably because it is converted via dUMP®dTMP to unlabeled dTTP that competes with the tritiated thymidine. In megaloblastic cells, this effect of dU is greatly diminished. This finding is consistent with an impairment in the dUMP®dTMP reaction in the megaloblastic cells and is the basis for the dU suppression test described below. This model also explains the chromosome breaks and other abnormalities that occur in megaloblastic cells.10,11,12 and 13
There is a curious group of findings suggesting that the megaloblastic line may arise from a more “primitive” precursor than is the case for the normoblastic line. Megaloblasts contain high concentrations of fetal hemoglobin14 and the fetal isozyme of thymidine kinase.15 Like megaloblasts, BFU–E (see Chap. 38) grown with monocyte-conditioned medium are rich in g-globin chains and appear megaloblastic, but BFU–E from the same source but grown with T lymphocytes appear normal and contain the usual proportion of g-globin chains.16,17 The relationship between these observations and the pathogenesis of the nutritional megaloblastic anemias remains to be determined.
All megaloblastic anemias share certain general clinical features. Because the anemia develops slowly, it produces few symptoms until the hematocrit is severely depressed. When symptoms appear, they are those of anemia: weakness, palpitation, fatigue, light-headedness, and shortness of breath. Severe pallor and slight jaundice combine to produce a telltale lemon-yellow skin. Leukocyte and platelet counts may also be low but rarely cause clinical problems. Details of the clinical manifestations are given in the sections on the specific forms of megaloblastic anemia later in this chapter.
All cell lines are affected. Erythrocytes vary markedly in size and shape, are often large and oval, and in severe cases can show basophilic stippling and nuclear remnants (Howell-Jolly bodies, Cabot rings). The reticulocyte count is low. The more severe the anemia, the more pronounced the morphologic changes in the red cells. When the hematocrit is less than 20 percent, red cells with megaloblastic nuclei, including an occasional promegaloblast, may appear in the blood. The anemia is macrocytic (MCV = 100 to 150 fl or more), although coexisting iron deficiency, thalassemia trait, or inflammation can prevent macrocytosis.18 Slight macrocytosis is often the earliest sign of megaloblastic anemia.
Neutrophil nuclei often have more than the usual three to five lobes19 (Fig. 37-1). Typically, more than 5 percent of the neutrophils have five lobes, and cells may appear containing six or more lobes, morphology never seen in normal neutrophils. In nutritional megaloblastic anemias, hypersegmented neutrophils are an early sign of megaloblastosis19,20 and persist in the blood for many days after treatment.21 Chromosomes are elongated and broken.4,10,22 Specific therapy corrects these abnormalities, usually within 2 days, though some take months to disappear.10 Platelets are slightly smaller than normal and vary more widely in size (increased PDW).23

FIGURE 37-1 Megaloblastic hypersegmented neutrophil. ×1500.

Aspirated marrow is cellular and shows striking megaloblastic changes, especially in the erythroid series. Sideroblasts are increased in number and contain increased numbers of iron granules. The ratio of myeloid to erythroid precursors (M/E ratio) falls to 1:1 or lower, and granulocyte reserves may be decreased.24 In severe cases, promegaloblasts containing an unusually large number of mitotic figures are plentiful. Macrophage iron content is often increased.
Atypical Morphology in Megaloblastic Anemia Under certain circumstances, megaloblastic anemia may be overlooked because its characteristic morphology is imperfectly expressed.
Coexisting Microcytic Anemia. When combined with a microcytic anemia, many features of megaloblastic anemia may be masked.25 The anemia could be normocytic or even microcytic, while the blood film may show both microcytes and macro-ovalocytes (a “dimorphic anemia”) or microcytes alone if the microcytic component is sufficiently severe. The marrow may contain “intermediate” megaloblasts26 that are smaller and less “megaloblastic”-looking than usual. In this kind of mixed anemia, the microcytic component is usually iron-deficiency anemia,18 but it may be thalassemia minor or the anemia of chronic disease.18,27 Even when masked by a severe microcytic anemia, however, a megaloblastic anemia will usually show hypersegmented neutrophils in the blood and giant metamyelocytes and bands in the marrow, and neutrophil myeloperoxidase levels will be high.28
The megaloblastic component of a mixed iron-deficiency anemia can sometimes be overlooked, and the patient may be treated only with iron. In this case the anemia will respond only partly to therapy, and megaloblastic features will emerge as iron stores fill.
Incomplete Megaloblastic Anemia. If a patient with a full-blown megaloblastic anemia receives cobalamin or folate before marrow aspiration, the anemia will persist but the megaloblastic changes may be obscured. Attenuated megaloblastic changes are also seen in patients with early megaloblastic anemia, in patients with coexisting infection, or after transfusion.
Megaloblastic Anemia Misdiagnosed as Acute Leukemia. Occasionally, very severe megaloblastic anemia may produce marrow morphology so bizarre as to be mistaken for acute leukemia, especially if the marrow lacks classical megaloblasts and displays as its principal cell type the bizarre megaloblastic white cell precursors that in a more typical morphologic background would support the diagnosis of a megaloblastic anemia. In some cases, there is no maturation of the erythroid series, and the megaloblastic pronormoblast dominates the marrow, raising the possibility of erythroid leukemia.
In most forms of megaloblastic anemia, cytologic abnormalities resembling megaloblastosis may appear in other proliferating cells. Epithelial cells from the mouth, stomach, small intestine, and cervix uteri may look megaloblastic, appearing larger than their normal counterparts and containing atypical immature-looking nuclei.28,29 It is sometimes difficult to distinguish these “megaloblastic” changes from those of malignancy.
Plasma bilirubin, iron, and ferritin levels are somewhat increased.30 Serum LDH-1 and LDH-2 are markedly elevated, increasing with the severity of the anemia.31 In megaloblastic anemia LDH-1 is greater than LDH-2, while in other anemias LDH-2 is greater than LDH-1.32 Serum muramidase (lysozyme) levels are also high,33 while serum glutamic oxaloacetic transaminase is normal.34 Erythropoietin levels rise, but less than in other anemias of similar severity.35 Surprisingly, the elevated erythropoietin levels fall sharply within 1 day of the beginning of treatment, an interval too short to affect the hematocrit.
Megaloblastic anemia is associated with two pathophysiologic abnormalities: ineffective erythropoiesis and hemolysis. Ineffective erythropoiesis causes increases in the red cell precursor/reticulocyte ratio, plasma iron turnover,36 LDH-1 and LDH-2,37 and “early-labeled” bilirubin.38 Extramedullary hemolysis also occurs in megaloblastic anemia, with red cell life span decreased by 30 to 50 percent.39
Increased serum muramidase in megaloblastic anemia may be caused by increased granulocyte turnover,33 possibly induced by the disintegration of granulocyte precursors in the marrow (ineffective granulopoiesis). In cobalamin deficiency, platelet production is only 10 percent of that expected from the megakaryocyte mass,40 perhaps reflecting ineffective thrombopoiesis. Platelets in severe cobalamin deficiency are functionally abnormal.41
Folate deficiency is caused by (1) dietary deficiency, (2) impaired absorption, and (3) increased requirements (see Table 37-1).
An inadequate diet is the major cause of folate deficiency. Because folate reserves are small, deficiency develops rapidly in malnourished persons, typically the old, the poor, and the alcoholic. Folate deficiency can also occur during hyperalimentation42 or hemodialysis, where folate is lost in the dialysis fluid.43 Subclinical folate deficiency has been reported in subtotal gastrectomy.44 Folate deficiency can occur in premature infants, especially with infection, diarrhea, or hemolytic anemia45; in children on a synthetic diet for inborn errors46; and in infants raised on goat’s milk, which is folate-poor.47 Destruction of folate through excessive cooking can aggravate folate deficiency (see Chap. 30).
In alcoholic cirrhosis, megaloblastic anemia is usually caused by folate deficiency.21 In addition, alcohol may acutely depress the serum folate, even if folate stores are full,48 and will accelerate the development of megaloblastic anemia in someone with early folate deficiency.49,50 and 51 Alcohol causes acute marrow suppression, with declines in reticulocyte, platelet, and granulocyte levels49,52,53; reversible vacuolation of erythroid and myeloid precursors54; and dysfunction of granulocytes.54 These changes occur even if large doses of folate are given with the alcohol.55
Nontropical Sprue Nontropical sprue (celiac disease in children) is related to the ingestion of wheat gluten.56 Pathologically, nontropical sprue shows atrophy and chronic inflammation of the small intestinal mucosa, most severe proximally. Findings may include weight loss, glossitis (typical of folate deficiency), and other signs of a generalized vitamin deficiency, diarrhea, and the passage of light-colored, bulky stools with an unusually foul odor. Iron deficiency, hypocalcemia, osteoporosis, and osteomalacia may also occur.
Folate malabsorption occurs in most patients with this disorder.57,58 Serum folate levels are low,59,60 and megaloblastic anemia occurs frequently.
Tropical Sprue Tropical sprue is endemic in the West Indies, southern India, parts of southern Africa, and Southeast Asia. It can be acquired by travelers to those regions and persists for many years after they return.61 Tropical sprue is rapidly corrected by folate therapy, even though folate deficiency does not give rise to the disease. The etiology of tropical sprue is unknown, although infection is suggested by the response of the disease to antibiotics.62,63
Clinically and pathologically, tropical sprue is like nontropical sprue, except that tropical sprue is more severe in the distal small intestine.64 Therefore, tropical sprue usually leads to cobalamin deficiency65 and should be strongly considered as a cause of cobalamin deficiency in former residents of the tropics, even though they have been away from the tropics for 20 years or more. Folate malabsorption may also occur,66 possibly because the diseased intestine fails to deconjugate folate polyglutamates.67 Megaloblastic anemia is therefore very common in this disease68 and may be due to both folate and cobalamin deficiency.
Other Intestinal Disorders Malabsorption of folic acid commonly occurs in regional enteritis,68 after extensive resections of the small intestine,69 in lymphomatous or leukemic infiltration of the small intestine,70 in Whipple disease,70 in scleroderma and amyloidosis,71 and in diabetes mellitus.72 Systemic bacterial infections also impair folate absorption.73
Pregnancy During pregnancy, folate requirements increase five- to tenfold because of transfer of folate to the growing fetus,75 which will draw down maternal folate stores even in the face of severe maternal folate deficiency.76 Further increases may result from the presence of multiple fetuses, a poor diet, infection, coexisting hemolytic anemia, or anticonvulsant medication. Lactation aggravates folate deficiency.77 Consequently, folate deficiency is very common in pregnancy78 and is the major cause of the megaloblastic anemia of pregnancy.79
Folate deficiency, however, is difficult to diagnose in pregnancy because its signs are obscured by the normal hematologic changes of pregnancy. During pregnancy, a physiologic “anemia” develops because of an increase in plasma volume that is only partly offset by an accompanying increase in red cell mass (Fig. 37-2); hemoglobin levels may fall to 10 g/dl. This anemia is associated with a physiologic macrocytosis; rises in the MCV of 20 fl occur, although the average at term is 4 fl.80 Serum and red cell folate levels fall steadily during pregnancy even in well-nourished women.81 All these changes are false clues that suggest folate deficiency even when folate levels are normal. Conversely, hypersegmented neutrophils, usually a reliable clue to early megaloblastic anemia, are missing in early megaloblastic anemia of pregnancy.82 In many cases, the only finding that can reliably distinguish the physiologic anemia of pregnancy from folate deficiency is a megaloblastic marrow.

FIGURE 37-2 Hemoglobin and serum folate levels during pregnancy and the postpartum period. (Adapted from Shojania.74)

Increased Cell Turnover Because of increased marrow cell turnover, the folate requirement rises sharply in chronic hemolytic anemia.83,84 During the bouts of acute hemolysis that may occur in these anemias, the marrow may become megaloblastic within days. Sometimes folate therapy is successful only if unusually large doses (up to 25 mg/day) are given.83,84
Folic acid deficiency may arise in chronic exfoliative dermatitis, in which folate losses of 5 to 20 µg/day may occur.85 Patients with psoriasis who are treated with methotrexate have an added reason for developing signs of folate deficiency. Pretreating such patients with folate may prevent these signs without impairing the therapeutic effect of methotrexate.85
The clinical picture of folate deficiency includes all the nonspecific manifestations of megaloblastic anemia described above plus the following specific features: (1) a history and laboratory studies indicating folate deficiency, (2) absence of the neurologic signs of cobalamin deficiency (see “Cobalamin Deficiency”),* and (3) a full response to physiologic doses of folate.
The earliest indicator of folate deficiency is a low serum folate. Serum folate follows folate intake closely, so a low serum folate (below about 3 ng/ml) may indicate only a drop in folate intake over the preceding few days.20 Similarly, a low serum folate will rise quickly on refeeding, so serum should be frozen early in case a later assay is necessary.
A better indicator of the tissue folate status is the red cell folate,86 which remains relatively unchanged while a red cell is circulating and thus reflects folate turnover over the preceding 2 to 3 months. Red cell folate is usually quite low in folate-deficient megaloblastic anemia. It is also low, however, in more than 50 percent of patients with cobalamin-deficient megaloblastic anemia87 and therefore cannot be used to distinguish between these two deficiencies. Conversely, red cell folate may be normal in the megaloblastic state that occurs, often with little accompanying anemia, in rapidly developing folate deficiency (see “Acute Megaloblastic Anemia” later in this chapter).88
The dU suppression test, discussed in full in “The dU Suppression Test” later in this chapter, is used in research on pathogenetic mechanisms in megaloblastic states. It adds little to the clinical evaluation of a megaloblastic anemia.
Macrocytosis occurs in alcoholism without megaloblastic anemia, liver disease, hypothyroidism, aplastic anemia, certain forms of myelodysplasia, pregnancy, and any condition associated with a reticulocytosis (e.g., autoimmune hemolytic anemia), but in these conditions the MCV rarely exceeds 110 fl.
A full hematologic response to physiologic doses of folate (i.e., 200 µg daily) distinguishes folate deficiency from cobalamin deficiency, in which a response occurs only at pharmacologic doses of folate (e.g., 5 mg daily).29 This is not recommended as a diagnostic test, because neurologic problems may develop in cobalamin-deficient patients treated with folate alone. Cobalamin may produce a partial response in folate deficiency.89
The diagnosis of nontropical sprue rests on (1) the demonstration of malabsorption, (2) a jejunal biopsy showing villus atrophy, and (3) the response to a gluten-free diet. In 80 percent of patients, a gluten-free diet will gradually reverse the functional disorder, correcting folate malabsorption.57
The hematologic problems associated with folate deficiency have been recognized for decades. Recently, however, folate deficiency has been related to a number of serious disorders not involving the hematopoietic system. These disorders, moreover, are seen at folate levels usually regarded as low-normal. They include congenital anomalies, fragile site syndromes, and, most common of all, atherosclerosis.
There is a close association between mild folate deficiency and congenital anomalies of the fetus, most notably defects in neural tube closure, but also abnormalities involving the heart, urinary tract, limbs, and other sites.74,90,91,92,93 and 94 Mutations affecting enzymes of folate metabolism, especially the common 677C®T mutation of the MTHFR gene,95,96 also predispose to congenital anomalies.
A mildly elevated homocysteine level is a major risk factor for atherosclerosis and venous thrombosis, possibly due to an effect on the vascular endothelium.97,98 and 99 Homocysteine levels can be decreased with folate, cobalamin, and pyridoxine supplements, possibly reducing the risk of vascular disease.100,101 It is curious that the 677C®T MTHFR mutation, which gives rise to a thermolabile enzyme, has no effect on the incidence of vascular disease.102,103
A large study of U.S. nurses indicated that supplementation with folate at more than 400 µg per day reduces the incidence of colon cancer by 31 percent.104 This finding has important implications for public health. Individuals homozygous for the 677C®T methylenetetrahydrofolate reductase mutation also have a decreased incidence for colon cancer compared with 677C®T heterozygotes and normal controls.13
Folate is usually given orally at 1 to 5 mg daily, although 1 mg is usually enough. At this dose, anemia is usually corrected even in patients with malabsorption. A parenteral preparation containing 5 mg/ml of folate is also available.
Treatment for tropical sprue consists of the usual doses of folate, plus cobalamin if indicated. To prevent relapse, treatment should be maintained for at least 2 years. Broad-spectrum antibiotics are helpful adjuncts, although antibiotics alone fail to correct the condition.
It is essential that pregnant women be given at least 400 µg of folate per day.105,106 and 107 As to the possibility of overlooking cobalamin deficiency due to folate administration, pernicious anemia in pregnancy is rare,108 and in pregnant women at risk for cobalamin deficiency (e.g., strict vegetarians or patients with malabsorption) the deficiency is easily prevented with 1 mg of vitamin B12 given parenterally every 3 months during the pregnancy.
Therapeutic doses of folate will partly correct the hematologic abnormalities in cobalamin deficiency, but the neurologic manifestations can progress, with disastrous results.109 It is therefore essential to evaluate both folate status and cobalamin status early in the workup of a megaloblastic anemia. If treatment is urgent and the nature of the deficiency is unclear, both folate and cobalamin can be given after one has obtained samples for assay.
Patients who receive low-dose methotrexate therapy as an immunosuppressant may develop side effects, the worst of which is hepatotoxicity. The incidence of side effects, including hepatotoxicity, has been correlated with reduced folate levels.110,111 These can be prevented by administration of folic or folinic acid without any reduction in the therapeutic effect of the low-dose methotrexate.
Disorders that lead to cobalamin deficiency are listed in Table 37-1.
Cobalamin deficiency is most often a result of defective absorption, most commonly PA, a condition in which intrinsic factor production fails. There are many other causes of defective cobalamin absorption, involving the stomach, pancreas, or small intestine.
Gastric Disorders Pernicious Anemia.*112 Pernicious anemia is a disease of insidious onset that generally begins in middle age or later (usually after age 40). In this condition, intrinsic factor secretion fails because of gastric mucosal atrophy. Pernicious anemia is an autoimmune disease. The gastric atrophy of PA probably results from immune destruction of the acid- and pepsin-secreting portion of the gastric mucosa.
In patients with pernicious anemia, antibodies occur that recognize the H+/K+-ATPase, which resides in the secretory membrane of the parietal cell. These antiparietal cell antibodies occur in about 60 percent of patients with simple atrophic gastritis and 90 percent of patients with PA but in only 5 percent of a random 30- to 60-year-old population.113,114 and 115 Antiparietal cell antibodies also occur in a significant percentage of patients with thyroid disease.116 Conversely, patients with PA have a higher than expected incidence of antibodies against thyroid epithelium, lymphocytes, and renal collecting duct cells.117,118
Antibodies to intrinsic factor (“type I,” or “blocking,” antibodies) or the intrinsic factor–Cbl complex (“type II,” or “binding,” antibodies) are highly specific to PA patients.118 Blocking antibodies, which prevent the formation of the intrinsic factor–Cbl complex, are found in 70 percent of PA sera,119 while binding antibodies, which prevent the intrinsic factor–Cbl complex from binding to its ileal receptors, are found in about half the sera that contain blocking antibody. In the second part of the Schilling test (discussed below) these antibodies may produce a false-positive result by interfering with the action of exogenous intrinsic factor.120
The antiparietal cell antibodies, however, are not thought to be responsible for the pathogenesis of PA, because they have no access to the H+/K+-ATPase, which is located on the luminal side of the parietal cell. Rather, studies in mice have suggested that the gastric atrophy in PA anemia is caused by CD4+ T cells whose receptors recognize the H+/K+-ATPase. Thus, thymectomized BALB/c mice develop an autoimmune atrophic gastritis similar to that seen in PA patients, and CD4+ T cells from these mice produce atrophic gastritis when injected into nude mice.121,122
Some findings in humans support the idea that T cells are responsible for the gastric atrophy in PA. First, lymphocytes from patients with PA are hyperresponsive to gastric antigens.123 Second, the correlation between antiparietal cell antibodies and PA is not perfect.114,124 Finally, the incidence of PA is higher than expected in patients with agammaglobulinemia, even though their sera contain none of the antibodies typical of PA.125
Other Autoimmune Diseases. Antiparietal cell antibodies and PA are unexpectedly frequent in patients with other autoimmune diseases,126 including autoimmune thyroid disorders (thyrotoxicosis, hypothyroidism, and Hashimoto thyroiditis),127 type I diabetes mellitus, hypoparathyroidism,128 Addison disease, postpartum hypophysitis,129 ulcerative colitis,130 vitiligo,131 acquired agammaglobulinemia,125 infertility in female patients under age 40, 132 and hypospermia and infertility in males.133 The coexistence of these diseases and PA is further evidence that PA is an autoimmune disease.
Inherited Predisposition to Pernicious Anemia. A predisposition to PA can be inherited. The disease is associated with HLA types A2, A3, B7, and B12134 and with blood group A.135 PA and antiparietal cell antibodies occur more frequently than expected in the families of PA patients.136 In one study, gastric atrophy was found in more than 30 percent of the relatives of patients with PA; of these relatives, 65 percent had antiparietal cell antibodies and 22 percent had anti-intrinsic factor antibodies.137 PA occurs relatively frequently in northern Europeans (especially Scandinavians)137 and African Americans124,138 but is uncommon in Asians. In African Americans, the disease tends to begin early, occurs with high frequency in women, and is often severe.124,138
The Stomach and Intestine in Pernicious Anemia. Gastric manifestations in PA include achlorhydria, acquired intrinsic factor deficiency demonstrable by the Schilling test, and increases in the incidence of certain malignancies: about a twofold increase in the incidence of gastric cancer, similar increases in the incidence of certain hematologic malignancies, and an increase in the incidence of gastric carcinoid.139,140 Achlorhydria may precede the loss of intrinsic factor secretion and the development of PA by many years.141 It is present if the pH of gastric juice after stimulation with pentagastrin (6 µg/kg subcutaneously) remains above 3.5 and does not decrease by more than 1 pH unit. The absence of achlorhydria excludes the diagnosis of PA. However, measurement of gastric acid secretion has been supplanted by serum cobalamin and methylmalonic acid levels and the Schilling test.142
Helicobacter pylori, a microorganism that infects the gastric mucosa, has been identified as a major cause of gastritis and peptic ulcers. Evidence is conflicting, however, regarding the role of H. pylori in PA. In two studies, cultures of gastric biopsies showed a very low incidence of H. pylori infection in PA patients.143,144 In one it was reported that anti-H. pylori antibodies were found in only a small fraction of the sera from these patients; in the other, however, these antibodies were present in most of the PA sera, indicating that most of the patients described in that study had been infected previously. Whether H. pylori participates in the pathogenesis of PA is at present an open question.
Fasting plasma gastrin levels are high in most patients with PA, while somatostatin levels are low.145,146 In biopsies from PA stomachs, however, fundal gastrin and somatostatin were high, correlating with increases in argyrophilic cells in the basal crypts; antral gastrin and somatostatin were normal. Gastrin levels are also high in simple achlorhydria without PA.147
The stomach shows characteristic histologic abnormalities in PA (Fig. 37-3). The mucosa of the cardia and fundus is atrophic, containing few chief (i.e., pepsin-secreting) or parietal cells. The withered mucosa is infiltrated with lymphocytes148 and plasma cells. In contrast, the antral and pyloric mucosa is normal. The gastric atrophy is partly reversible by glucocorticoid treatment, with some regeneration and return of intrinsic factor secretion, further evidence for the autoimmune nature of PA.149

FIGURE 37-3 Gastric histology in pernicious anemia. Left, normal fundus. The thick mucosa is packed with gastric glands composed for the most part of chief cells and parietal cells. The mucus-secreting cells are concentrated in the necks of the glands. Right, fundus in pernicious anemia. Gastric glands in the atrophic mucosa are sparse and consist mainly of mucus-secreting cells; the mucosa is densely infiltrated by lymphocytes.

Megaloblastic changes reversible by cobalamin are seen in the gastrointestinal epithelium. Cells recovered by lavage are large150 and show atypical nuclei resembling early malignant change.151 Small intestinal biopsy shows decreased mitoses in crypts, shortening of villi, megaloblastic changes in epithelial cells, and infiltration in the lamina propria.152 These changes may account for the occasional malabsorption of D-xylose and carotene in PA.153
Gastrectomy Syndromes. Gastric surgery often leads to anemia. Most common is iron-deficiency anemia, but cobalamin deficiency with megaloblastic anemia may also occur. After total gastrectomy, cobalamin deficiency will develop within 5 or 6 years because the operation removes the source of intrinsic factor.154 The delay between surgery and the onset of cobalamin deficiency reflects the time needed to exhaust cobalamin stores after cobalamin absorption ceases.
After partial gastrectomy, few patients show frank cobalamin deficiency, but about 5 percent have intermediate megaloblastosis, approximately 25 to 50 percent have low serum cobalamin levels, and many have decreased intrinsic factor secretion (Schilling test).155,156 and 157 Achlorhydria not present before surgery often develops some years after gastrectomy. Postgastrectomy patients with low serum cobalamin levels usually have low serum iron levels as well,158 in contrast to the high iron levels typical of cobalamin deficiency.
Cobalamin deficiency after partial gastrectomy can be caused by mucosal atrophy in the unresected remnant of the stomach159 or, if a gastrojejunostomy was performed, by bacterial overgrowth in the afferent loop (see “Blind Loop Syndrome”). Postgastrectomy folate deficiency from malabsorption or reduced dietary intake actually accounts for more cases of postgastrectomy megaloblastic anemia than does cobalamin deficiency. Often the two deficiencies occur together.
Zollinger-Ellison Syndrome. In the Zollinger-Ellison syndrome, a gastrin-producing tumor, usually in the pancreas, stimulates the gastric mucosa to secrete immense amounts of HCl. The major clinical problem is a severe ulcer diathesis. Malabsorption of cobalamin occurs when the vast quantities of HCl secreted by the overactive gastric mucosa cannot be completely neutralized by the pancreatic secretions. The resulting acidification of the duodenal contents inactivates pancreatic proteases, preventing the transfer of Cbl from R binder to intrinsic factor.160
Intestinal Disorders Intestinal Diseases. A number of intestinal disorders can lead to a deficiency of cobalamin. These include (1) extensive resection of the ileum,161 (2) regional ileitis162 or another disease affecting the ileum (e.g., lymphoma, radiation damage163), (3) cobalamin malabsorption associated with hypothyroidism164 or certain drugs (see below), (4) the effects of cobalamin deficiency itself,155 and (5) sprue, either tropical or, less often, nontropical.65 In each of these, exogenous intrinsic factor fails to correct an abnormal Schilling test.
Competing Intestinal Flora and Fauna: “Blind Loop Syndrome.” The blind loop syndrome is a state of cobalamin malabsorption with megaloblastic anemia caused by intestinal stasis from anatomic lesions (strictures, diverticula, anastomoses, surgical blind loops) or impaired motility (scleroderma, amyloid).165 Serum cobalamin is low, but intrinsic factor secretion is normal, and the cobalamin malabsorption is not corrected by exogenous intrinsic factor. The defect in cobalamin absorption is caused by colonization of the diseased small intestine by bacteria that take up ingested cobalamin before it can be absorbed from the intestine.166,167,168 and 169 Steatorrhea is also seen in the blind loop syndrome.
Another cause of cobalamin deficiency is infestation with the fish tapeworm, Diphyllobothrium latum. Prevalence is highest near the Baltic Sea and in Canada and Alaska. The life cycle of the worm is illustrated in Fig. 37-4. Humans are infected by eating undercooked fish or fish roe. Once ingested, the sparaganum larva becomes an adult in 5 to 6 weeks and may live for years.

FIGURE 37-4 Life cycle of Diphyllobothrium latum. Inner circle represents developmental stages of parasite: (1) adult worm; (2) eggs; (3) embryonated egg (coracidium); (4) procercoid larva; (5) plerocercoid larva. Outside circle shows first intermediate host (Cyclops), second intermediate host (fish), and definitive host (man). (Adapted from Wirth, Farrow, Human sparganosis. JAMA 177:76, 1961.)

Cobalamin deficiency is caused by competition between the worm and the host for ingested cobalamin.170 The clinical picture of D. latum infestation ranges from no symptoms to a full-blown megaloblastic anemia with neurologic changes. Only about 3 percent of persons harboring the parasite become anemic, however.171 The infestation is diagnosed by finding tapeworm ova in the feces.
Pancreatic Disease In 50 to 70 percent of patients with exocrine pancreatic insufficiency, cobalamin malabsorption can be demonstrated by the Schilling test.172,173 Cobalamin malabsorption in pancreatic insufficiency is caused by a deficiency in pancreatic proteases, resulting in a partial failure to destroy R binder-Cbl complexes, whose destruction is a prerequisite for the transfer of cobalamin to intrinsic factor. The defect in cobalamin absorption in chronic pancreatitis is corrected by oral trypsin or by presaturating the R binder with cobinamide, a cobalamin analog that is taken up by R binder but not by intrinsic factor.174,175 Despite the high incidence of abnormal Schilling tests in pancreatic insufficiency, this disorder almost never causes clinically significant cobalamin deficiency.176
Dietary cobalamin deficiency is very unusual. It occurs mainly in vegetarians who also avoid dairy products and eggs (vegans).177,178 and 179 Low serum cobalamin levels occur in 50 to 60 percent of this group.180 Breast-fed infants of vegan mothers may also develop cobalamin deficiency.181,182,183 and 184 Cobalamin deficiency in vegans presents with mild megaloblastic anemia, glossitis, and neurologic disturbances.
Cobalamin deficiency may occur in severe general malnutrition. A megaloblastic anemia not related to cobalamin deficiency may accompany kwashiorkor or marasmus.185,186
Formerly, the neurologic abnormalities of cobalamin deficiency were attributed to disordered metabolism of myelin lipids caused by an impaired methylmalonyl CoA mutase reaction.187 Similar neurologic abnormalities do not occur in patients with inherited methylmalonyl CoA mutase deficiency,115 however, while authentic combined system disease has occurred in a patient with nutritional folate deficiency188 and in another with N5, N10-methylene FH4 reductase deficiency.189 The latter reports suggest that the neurologic lesions of cobalamin deficiency may be due to deranged methyl group metabolism. Animal studies support this hypothesis. Neurologic disorders closely resembling combined system disease develop in cobalamin-deficient pigs, fruit bats, and monkeys.190,191 The development of these disorders is prevented by methionine, which is produced in a cobalamin-dependent reaction and is the precursor of the biological methylating reagent S-adenosylmethionine (SAM). Further support for a methylation defect is the finding that brains from cobalamin-deficient pigs contain increased levels of S-adenosylhomocysteine (SAH),190 a powerful methylation inhibitor produced in SAM-dependent methylation reactions:
Against the methylation defect hypothesis, however, are the findings that cobalamin deficiency had no effect on S-adenosylmethionine, S-adenosylhomocysteine, or the methylation of phospholipids or myelin basic protein192,193 and 194 in the brains of fruit bats. Are humans more like pigs or fruit bats? Possibly pigs.195,196 and 197
Clinical Features
The clinical picture of cobalamin deficiency includes the nonspecific manifestations of megaloblastosis—anemia, weight loss, etc.—plus specific features caused by the lack of cobalamin, chiefly neurologic abnormalities. Because cobalamin reserves are large, years may pass between the cessation of cobalamin absorption and the appearance of deficiency symptoms. This interval is shortened in patients whose enterohepatic cobalamin cycle has been interrupted.
Cobalamin deficiency causes a neurologic syndrome that is particularly dangerous because it can develop in isolation, with no megaloblastic anemia to suggest a lack of cobalamin,200 and because when sufficiently far advanced, it cannot be reversed by treatment. The syndrome usually begins with paresthesias in feet and fingers due to early peripheral neuropathy, together with disturbances of vibratory sense and proprioception. The earliest signs, said to precede other neurologic findings by months, are loss of position sense in the second toe and loss of vibration sense for a 256-Hz but not a 128-Hz tuning fork.201 If untreated, the neurologic disorder progresses to spastic ataxia resulting from demyelination of the dorsal and lateral columns of the spinal cord: so-called combined system disease (Fig. 37-5).202,203

FIGURE 37-5 Degeneration of spinal cord in combined system disease. (From Harris, Kellermeyer, The Red Cell: Production, Metabolism, Destruction: Normal and Abnormal, rev ed, Harvard University Press, Cambridge, 1970.)

Besides the peripheral nerves and the spinal cord, the brain is affected by cobalamin deficiency. Somnolence and perversion of taste, smell, and vision with occasional optic atrophy are accompanied by slow waves on the electroencephalogram. A dementia mimicking Alzheimer disease can develop.204 Psychological derangements, including psychotic depression and paranoid schizophrenia, may also occur.201,205 Frank psychosis in cobalamin deficiency has been termed “megaloblastic madness.”206
The neurologic lesions of cobalamin deficiency can be detected by MRI. Demyelination appears as T2-weighted hyperintensity of the white matter. MRI is particularly useful for confirming the diagnosis of a neurologic disorder due to cobalamin deficiency. It has also been used to follow the progress of the neurologic abnormalities in the course of treating cobalamin-deficient patients.207,208 and 209
Some observations suggest the existence of a large group of patients who are hematologically normal, with a normal hematocrit and MCV, but have cobalamin-responsive neuropsychiatric disease,210,211,212,213,214,215,216 and 217 but there are conflicting views.218,219 Neuropsychiatric findings include peripheral neuropathy, gait disturbance, memory loss, and psychiatric symptoms, often with abnormal evoked potentials. Serum cobalamin may be normal, borderline, or low, but tissue cobalamin deficiency is suggested by consistently high levels of serum methylmalonic acid and/or homocysteine,220,221,222,223 and 224 by very high levels of methylmalonic acid in the cerebrospinal fluid, and by an abnormal dU suppression test. Most of the neuropsychiatric abnormalities appear to respond to cobalamin therapy.
Serum cobalamin is low in most but not all patients with cobalamin deficiency224 (see Chap. 25). Cobalamin levels are normal, however, in cobalamin deficiency due to N2O, TC-II deficiency, and inborn errors of cobalamin metabolism (see below); levels also may be normal in cobalamin-deficient patients with high TC-I levels due to myeloproliferative diseases.224,225 Conversely, serum cobalamin levels may be low in the presence of normal tissue cobalamins in vegetarians, in subjects taking megadoses of ascorbic acid,226 in pregnancy (25 percent), in the presence of TC-I deficiency,227 and in megaloblastic anemia due to folate deficiency (30 percent).225 Serum folate may be higher than expected in cobalamin deficiency; patients deficient in both cobalamin and folate may show normal serum folate levels.
Except when caused by an inborn error (see below), methylmalonic aciduria is a reliable indicator of cobalamin deficiency.228 Normal subjects excrete only traces of methylmalonate (0 to 3.4 mg/day); in cobalamin deficiency, however, urine methylmalonate is usually elevated. Cobalamin therapy restores excretion to normal in a few days.
Elevated serum methylmalonic acid and homocysteine levels are indicators of tissue cobalamin deficiency. Their levels are high in more than 90 percent of cobalamin-deficient patients and rise before the serum cobalamin falls to subnormal levels.220,221,222 and 223 Elevated serum methylmalonic acid and/or elevated homocysteine are probably the most reliable indicators of cobalamin deficiency in patients without a congenital disorder in their metabolism.
Spinal fluid methylmalonic acid levels are markedly elevated in cobalamin deficiency.214
The Schilling test assays cobalamin absorption by measuring urinary radioactivity after an oral dose of radioactive cobalamin. The test can be performed even after cobalamin deficiency has been treated. After voiding, a fasting patient drinks 0.5 µCi (0.5 to 2.0 µg) of radioactive cyanocobalamin in water, and a 24-h urine collection is begun. At 2 h, 1 mg of unlabeled cyanocobalamin is given IM to saturate the circulating cobalamin-binding proteins, after which the patient may eat. The amount of radioactivity in the 24-h urine is measured. Normal subjects excrete ³7 percent of the administered radioactivity in the first 24 h.
If excretion of radioactivity is low, the second part of the Schilling test is performed after a 5-day delay to allow intestinal megaloblastosis to be corrected by the unlabeled cobalamin given in the first part of the test.229,230 The procedure is the same except that 60 mg of active hog intrinsic factor (equivalent of 1 national formulary unit) is given orally with the radioactive cobalamin. If poor excretion in the first part was due to intrinsic factor deficiency, excretion in the second part will be normal. Intrinsic factor will not correct cobalamin malabsorption due to other causes.
The Schilling test will give a false-negative result in patients who absorb free cobalamin but fail to release the vitamin from food (e.g., after partial gastrectomy231 and vagotomy,232 in those with a gastric ulcer,233 and during cimetidine therapy234). Failure to absorb food cobalamin can be established by a modified Schilling test in which the source of the labeled vitamin is an omelet of eggs obtained from a chicken fed on radioactive cobalamin.223 Cobalamin deficiency occasionally results from malabsorption of protein-bound cobalamin only.235,236
The major source of error in the Schilling test is incomplete urine collection. Completeness of collection may be assessed by measuring the creatinine in the specimen (normal >15 mg/kg per day). Renal disease may delay excretion of radioactivity, giving a false-positive Schilling test237; whole-body counting can be used to measure cobalamin absorption in severe renal insufficiency.238 Other causes of a false-positive result are inadequate saturation of cobalamin-binding proteins by unlabeled cobalamin (first part of test), inactive intrinsic factor or neutralization of intrinsic factor by anti-intrinsic factor antibodies in the stomach120 (second part), and malabsorption due to megaloblastic changes in the ileum229,239 (second part). A false negative may be caused by isotope given with an earlier Schilling or other test.
The dU suppression test is based on the finding that unlabeled dU can suppress the uptake of [3H]thymidine ([3H]Thd) into the DNA of cultured lymphocytes or marrow cells.240 Thymidine enters DNA through the dTMP pool, into which it is fed by thymidine kinase (Fig. 37-6). Deoxyuridine also enters DNA through the dTMP pool, first being phosphorylated to dUMP by thymidine kinase, then being methylated to dTMP by thymidylate synthetase. The theory of the dU suppression test is that treating normal cells with dU loads them with unlabeled dTMP, which competes for uptake into DNA with the labeled dTMP formed during a later incubation with [3H]Thd; dU thereby suppresses the uptake of [3H]Thd into DNA. If thymidylate synthetase activity is low, the conversion of dU into dTMP will be slowed, and the suppressive effect of dU on [3H]Thd uptake into DNA will be diminished. Because thymidylate synthetase uses N5, N10-methylene FH4 as a methylating agent, its activity depends directly on folate and indirectly on cobalamin (see Chap. 30). Failure of dU suppression therefore becomes an indication of cellular folate or cobalamin deficiency. While the foregoing theory is highly oversimplified,5,241 experience has shown that the dU suppression test can answer questions about cellular folate and cobalamin.

FIGURE 37-6 Incorporation of thymidine into DNA via the de novo and salvage pathways. (Adapted from Metz.240)

To perform the assay, cultured lymphocytes or marrow cells from a patient or control are incubated for an hour at 37°C with and without 0.1 µM dU. [3H]Thd is then added, and the cells are incubated for an additional 0.5 to 3.0 h. The incorporation of [3H]Thd is then determined, and dU suppression is calculated as 100× (3H incorporation in dU-treated cells/3H incorporation by control cells), expressed as a percentage. Normally, dU depresses the uptake of [3H]Thd into DNA to less than 10 percent of control values. Deoxyuridine suppression is relieved in megaloblastic anemias of nutritional origin and in certain inherited disorders of folate or cobalamin metabolism240,241 and 242 but not in other megaloblastic states.243 In the nutritional anemias, dU suppression can be restored with folate or cobalamin according to a pattern that depends on the nature of the deficiency (Table 37-2).241,242 Even subclinical deficiency states can be detected by the dU suppression test.244


The dU suppression test, however, is chiefly a research tool. It can help diagnose certain special clinical problems,240 but these problems can also be diagnosed using other laboratory tests, therapeutic trials with vitamins or iron, or watchful waiting. Furthermore, in over 25 years of use, the test has not moved from the research laboratory into the clinic. It seems unlikely that the dU suppression test will enjoy more widespread clinical use in the future.
Pernicious anemia combines the general features of megaloblastic anemia and features specific for cobalamin deficiency with unique clinical features related to its (probable) autoimmune etiology and its gastric pathology. The disease is easily missed, however, because of its (1) insidious onset, (2) tendency to be masked by the use of multivitamin preparations containing folic acid,245 and (3) many atypical presentations,246,247 including its presentation as a neurologic disease without hematologic findings and its tendency to be overlooked in a patient with another autoimmune disease.
Antiparietal cell and anti-intrinsic factor antibodies are rarely measured, even though the anti-intrinsic factor antibodies in particular could be of considerable diagnostic value.88 Anti-intrinsic factor antibody is highly specific for PA (although its sensitivity is only modest), and its presence in a megaloblastic anemia makes the diagnosis of PA almost certain.
Treatment consists of parenteral cyanocobalamin (vitamin B12) or hydroxocobalamin to replace daily losses and refill storage pools, which normally contain 2 to 5 mg of cobalamin.248 Toxicity is nil, but doses exceeding 100 µg saturate the transcobalamins, and the excess is lost in the urine.
A typical treatment schedule consists of 1000 µg cobalamin IM daily for 2 weeks, then weekly until the hematocrit is normal, and then monthly for life. For neurologic manifestations, 1000 µg every 2 weeks for 6 months is recommended, and higher doses are given for certain inherited disorders (e.g., TC-II deficiency). Cobalamin should be given by mouth for dietary cobalamin deficiency and to patients (e.g., hemophiliac patients) who cannot take IM injections.
Transfusion is occasionally required when the hematocrit is less than 15 percent or when the patient is debilitated, infected, or in heart failure. In such instances, packed cells should be given slowly to avoid pulmonary edema. Infections can impair the response to cobalamin and must be treated vigorously.
Following the parenteral administration of cobalamin to deficient patients, elevated plasma bilirubin, iron, and LDH levels fall rapidly (Fig. 37-7).249 Decreasing plasma iron turnover and fecal urobilinogen reflect cessation of ineffective erythropoiesis. Within 12 h the marrow begins to change from megaloblastic to normoblastic, a process that is complete in 2 to 3 days. Reticulocytosis begins on day 3 to 5 and peaks on day 4 to 10.250 The new red cells come from new normoblasts, not from the old megaloblasts, most of which die before leaving the marrow. The blood hemoglobin concentration becomes normal within 1 to 2 months; if normal values are not achieved by then, another cause of anemia should be sought.

FIGURE 37-7 Effect of CNCbl on reticulocyte count, serum iron, serum bilirubin, stool urobilinogen, and plasma iron turnover. (Adapted from Finch et al.249)

Other changes include the following: (1) a prompt improvement in the sense of well-being; (2) normalization of the leukocyte and platelet counts, although neutrophil hypersegmentation persists for 10 to 14 days; (3) a rise in serum cobalamin and folate; and (4) a drop in serum potassium.251 Cobalamin deficiency will not respond to a physiologic dose of folate (100 to 400 µg/day), although this dose will produce a maximal response in folate deficiency. Larger doses of folate (5 to 15 mg/day) can produce a reticulocytosis and partially correct the anemia in cobalamin deficiency.
After Gastrectomy Cobalamin should always be given after total gastrectomy. Cobalamin administration is not necessary after partial gastrectomy, but these patients need to be watched for megaloblastic anemia, bearing in mind that this anemia could be masked by postgastrectomy iron deficiency.
Blind Loop Syndrome The anemia of the blind loop syndrome can be treated by parenteral cobalamin therapy. It also responds after a week or so to oral broad-spectrum antibiotics [cephalexin monohydrate (Keflex) 250 mg qid plus metronidazole 250 mg tid for 10 days],167 and the Schilling test becomes normal. Successful surgical correction of an anatomic lesion will cure the syndrome.
Fish Tapeworm Treatment consists of a single 2-g dose of niclosamide.
Much interest has recently been kindled in the possibility of treating cobalamin deficiency with oral cobalamin.253,254 and 255 Oral cobalamin can be used not only for the treatment of the dietary cobalamin deficiency that occurs in vegans and in patients with very severe general malnutrition but also for the treatment of patients with PA, provided these patients are followed carefully.256 In patients lacking intrinsic factor, about 1 percent of an oral dose of the vitamin is forced across the intestinal epithelium by mass action. Therefore, 1000 to 2000 µg/day of oral cobalamin will supply most PA patients with their daily cobalamin requirement without the need for injections and their accompanying pain and expense.
Although megaloblastic anemia is usually a chronic condition that requires weeks or months to develop, a potentially fatal megaloblastic state due to acute tissue folate or cobalamin deficiency can sometimes arise over the course of only a few days. Patients with acute megaloblastic anemia present with rapidly developing thrombocytopenia and/or leukopenia, counts sometimes falling to very low levels, but with little change in red cell levels unless another cause of anemia is present. The clinical picture can suggest an immune cytopenia. The diagnosis is made from the marrow aspirate, which is floridly megaloblastic, and confirmed by the rapid response to appropriate replacement therapy.
The most common cause of acute megaloblastic anemia is nitrous oxide (N2O) anesthesia. N2O rapidly destroys MeCbl,257 leading quickly to a megaloblastic state. AdoCbl (adenosylcobalamin) is eventually lost, and S-adenosylmethionine and total folate decline as well, with an increase in the proportion of folate in the form of N5-methyl FH4.257,258 and 259
Clinical findings develop quickly. An impairment in dU suppression with a cobalamin-deficiency pattern (see Table 37-2) appears after 6 h of exposure, and grossly megaloblastic changes are seen in the marrow after 12 to 24 h.260,261 Hypersegmented neutrophils do not appear until 5 days after exposure but then persist for several days.262 Some say that the hematologic effects of N2O can be prevented by folinic acid (30 mg at surgery and 12 h later).261,263,264 The effects of N2O disappear spontaneously after a few days; disappearance can be hastened by folinic acid or cobalamin.260
Fatalities due to N2O-induced megaloblastosis have occurred in tetanus patients given N2O for weeks.265,266 Long-term recreational use of N2O has led to psychosis267 and to a neurologic disorder similar to combined system disease.268,269 Operating room personnel, however, are not at risk for N2O-induced megaloblastic anemia.270
Acute megaloblastic anemia also occurs in other clinical settings. A rapidly developing megaloblastic state with acute thrombocytopenia has occurred in seriously ill patients, often in intensive care units.271,272 and 273 Especially at risk are patients transfused extensively at surgery,274 those on dialysis or total parenteral nutrition, and those receiving weak folate antagonists such as trimethoprim.275,276 Morphologic clues to the diagnosis (e.g., hypersegmented neutrophils) are often absent from the blood film, and both the red cell folate and the serum cobalamin may be normal, but the marrow is always megaloblastic. A rapid response to therapeutic doses of parenteral folate (5 mg/day) and cobalamin (1 mg) is the rule.
Drugs that cause megaloblastic anemia are listed in Table 37-3. Aminopterin and methotrexate are almost identical in structure to folic acid. After entering cells via the folate carrier314 and acquiring a polyglutamate chain,315 they act as very powerful inhibitors of dihydrofolate reductase.277 By blocking the FH2®FH4 reaction and perhaps inhibiting other enzymes of folate metabolism, they effect the rapid withdrawal of folates from the 1-carbon fragment carrier pool, causing a fall in nucleotide (especially thymidine) biosynthesis that leads to a major derangement in DNA replication278,279 (see Chap. 25).


Toxic effects include necrotic mouth lesions; ulcerations of the esophagus, small intestine, and colon, with abdominal pain, vomiting, and diarrhea; megaloblastic anemia; alopecia; and hyperpigmentation. The drug is excreted by the kidney, so effects and toxicity are prolonged and enhanced if renal function is impaired.
Toxicity caused by these folate antagonists is treated with folinic acid (N5-formyl FH4). Folate itself is useless because the blocked reductase cannot convert it to the active tetrahydro form. Folinic acid, however, is already in the tetrahydro form and is therefore effective despite the reductase blockade. The usual dose of folinic acid is 3 to 6 mg/day IM. Larger doses are given in chemotherapy protocols in which folinic acid is used to rescue patients deliberately treated with otherwise fatal doses of methotrexate. Folinic acid was used intrathecally in a patient in whom a large overdose of methotrexate was accidentally delivered into the subarachnoid space.316
Zidovudine (azidothymidine, AZT) is used for HIV infections (AIDS) (see Chap. 89).317 Its principal toxic effect is severe megaloblastic anemia. Anemia or neutropenia produced by zidovudine may limit the use of this drug.290
HIV infection itself suppresses hematopoiesis, leading to pancytopenia with myelodysplastic features (see Chap. 89). The blood film shows vacuolated monocytes. Megaloblastosis in HIV infection may be due to folate or cobalamin deficiency318,319 or to AZT or trimethoprim toxicity.
Hydroxyurea is used at high doses to treat chronic myelogenous leukemia, polycythemia vera, and essential thrombocythemia, and at lower doses to treat psoriasis (see Chap 14). It inhibits the conversion of ribonucleotides to deoxyribonucleotides.320 Marked megaloblastic changes are routinely found in the marrow within 1 to 2 days of initiating hydroxyurea therapy.291,321 These changes are rapidly reversed after withdrawing the drug.
Megaloblastosis due to nitrous oxide (N2O) is discussed under “Acute Megaloblastic Anemia.”
Long-term use of omeprazole and presumably other H+/K+-ATP ase inhibitors is associated with reduced serum cobalamin levels, presumably because of the ability of these drugs to inhibit parietal cell function.309 This is not a problem when these drugs are used for short intervals.322,323,324 and 325
Cobalamin malabsorption occurs in five childhood conditions: (1) cobalamin malabsorption in the presence of normal intrinsic factor secretion, (2) congenital abnormality of intrinsic factor, (3) transcobalamin II deficiency, (4) congenital R-binder deficiency, and (5) true pernicious anemia of childhood. The management of cobalamin deficiency in childhood has been thoughtfully reviewed.326
This disorder is an inherited failure of transport of the intrinsic factor-Cbl complex by the ileum, usually accompanied by proteinuria (mostly albumin).74,328,329 and 330 It may be the most common cause of cobalamin deficiency in infancy.115,328,331 Cobalamin deficiency is usually seen before age 2 but may appear later.74,330,332 Both parts of the Schilling test are abnormal, but intrinsic factor and HCl secretion, TC-I and -II levels, and gastric and intestinal histology are all normal, and intrinsic factor antibodies are absent.119,332,333 Intrinsic factor-Cbl receptors are present in some but not all patients.334,335 The molecular defect responsible for this disease is unknown.
Patients are treated with IM cobalamin. The anemia is corrected, but proteinuria persists.
This is an autosomal recessive disease in which parietal cells fail to produce functionally normal intrinsic factor.336,337 and 338 Patients present with irritability and megaloblastic anemia when their cobalamin stores (<25 µg at birth) are exhausted. The disease usually presents at 6 to 24 months of age. HCl secretion and gastric histology are normal, there is no proteinuria, and anti-intrinsic factor antibodies are absent.74,115,326,332 The abnormal Schilling test is corrected by oral intrinsic factor.339 Treatment is with standard doses of IM cobalamin.
This is an autosomal recessive disorder causing a flagrant megaloblastic anemia that generally presents in early infancy.341 The disease is dangerously deceptive, because it results from a very severe deficiency of tissue cobalamin, usually with normal serum cobalamin levels, and if not diagnosed will cause irreversible CNS damage.342 Patients are healthy at birth but develop signs and symptoms of cobalamin deficiency over the first few weeks of life: a rapidly progressive pancytopenia, mouth ulcers, vomiting, and diarrhea. Recurrent bacterial infections may occur.340,343,344 and 345 Neurologic findings are not prominent in the early stages of the disease.342
Serum folate and cobalamin are normal (the latter because most cobalamin is carried by TC-I, not TC-II) and little homocysteine or methylmalonic acid is found in the urine,346,347 and 348 but the marrow is megaloblastic (a few patients showed severe erythroid hypoplasia349). The Schilling test is usually340 but not always350 abnormal and is never corrected by intrinsic factor. The diagnosis is made by measuring serum TC-II.351 Prenatal diagnosis may be possible.352 Serum should be obtained prior to treatment, because TC-II levels in normals drop sharply after cobalamin is given.340 TC-II deficiency is treated with doses of cobalamin large enough to force enough vitamin into the cells to allow normal function. Initial therapy can be with oral vitamin B12 or hydroxocobalamin, 500 to 1000 µg twice a week, or IM hydroxocobalamin, 1000 µg/week, following blood counts, symptoms, and immune function, and adjusting doses upward if necessary.
Congenital R-binder deficiency has been reported in six patients,215,227,353,354 none of whom had a clinical manifestation of cobalamin deficiency, although the patients’ serum cobalamin levels were well below normal. R binders were deficient in leukocytes and saliva as well as in plasma. These patients show that the R binders are not essential for health.
True PA, with gastric atrophy and a defect in intrinsic factor secretion, is exceedingly rare in childhood.355,356 Patients usually present in their teens with cobalamin deficiency. Serum anti-intrinsic factor antibodies are usually present.123 The diagnosis and treatment are the same as for PA in adults.
Cobalamin is converted to AdoCbl and MeCbl by a complex series of transformations involving several steps (see Chap. 34). Seven disorders affecting this cobalamin transformation pathway have been described, one for each of the steps. Since the molecular causes of these disorders have not yet been fully characterized, the disorders themselves are not named for a defective protein but instead are designated by letter, as in “cobalamin mutant class ‘cobalamin A,’” or “cblA.” Based on the abnormal metabolites in the patients’ urine, these disorders can be grouped into three clinical syndromes (Table 37-4).


METHYLMALONIC ACIDURIA ONLY (cblA, cblB, AND cblF359,360 361 and 362)
In cblA and cblB, AdoCbl production is impaired,363,364 but MeCbl production is normal; in cblF, cobalamin export from lysosomes to cytosol is defective. Patients present in infancy with acidosis because they cannot catabolize methylmalonic acid. Symptoms include lethargy and failure to thrive, vomiting, and neurologic problems. Mental retardation is not prominent, and megaloblastic anemia is absent. Most patients respond to 1000 µg/day of OHCbl or CNCbl.364
HOMOCYSTINURIA ONLY (cblE AND cblG365,366 and 367)
In these disorders, N5-methyltetrahydrofolate-homocysteine methyltransferase is able to produce methionine but has difficulty making MeCbl. In patients with cblG, methionine synthase is missing or defective368; cblE is due to a failure to reactivate methionine synthase that has been inactivated by oxidation of its bound cobalamin369 (see Chap. 34). Patients present in infancy with vomiting, mental retardation, and megaloblastic anemia. They respond well to CNCbl at 1000 µg per day or per week. Infants diagnosed prenatally and treated from birth show normal development.
In these disorders, the defect in Cbl transformation affects both AdoCbl and MeCbl, probably because the reduction of the cobalt from Co3+ to Co1+ is defective. The age at initial presentation ranges from early infancy to adolescence. In addition to lethargy and failure to thrive, affected infants present with serious neurologic difficulties, while older patients present with psychological problems and progressive dementia along with motor signs and symptoms. In one fetus at risk for cblC, the diagnosis was excluded prenatally by chorionic villus sampling.372 Megaloblastic anemia occurs in about half the cases. Patients respond partially to 1000 µg/day of OHCbl or CNCbl.
A tentative diagnosis of a cobalamin mutation can be made by demonstrating methylmalonic aciduria and/or homocystinuria in a patient with the clinical findings described above. Establishing a diagnosis requires a specialized laboratory. In a patient suspected of having a cobalamin mutation, treatment should be started while awaiting test results, because early high-dose cobalamin treatment is risk-free and may reduce the chance of damage to the central nervous system. Fetuses with these diseases have been successfully treated in utero with very large doses of cyanocobalamin given parenterally to the mother.373,374
Megaloblastic anemia in infancy has been described in three inherited disorders of folate metabolism.
Patients cannot absorb folate from the gastrointestinal tract or transport it into the cerebrospinal fluid. They present with severe megaloblastic anemia, seizures, mental retardation, and other central nervous system findings. Folate levels are low in the serum and nil in the cerebrospinal fluid. Folate given parenterally has corrected the anemia and seizures in some patients but has had no effect on other CNS symptoms or on the CSF folate level.
A patient postulated to have dihydrofolate reductase deficiency presented with isolated megaloblastic anemia at 6 weeks of age. His anemia responded to folinic acid but not to folic acid.
Decreased methyltransferase activity was found in a liver biopsy from a child with megaloblastic anemia and mental retardation. The anemia failed to respond to folate, cobalamin, or pyridoxal phosphate.
Hereditary orotic aciduria is an autosomal recessive disorder of pyrimidine metabolism380,381 and 382 characterized by megaloblastic anemia, growth impairment, and excretion of orotic acid in the urine. Cobalamin and folate levels are normal.
The Lesch-Nyhan syndrome is an X-linked disorder of purine metabolism characterized by hyperuricemia, hyperuricosuria, and a neurologic disease with self-mutilation. It is caused by a deficiency of hypoxanthine-guanine phosphoribosyltransferase. One patient had megaloblastic anemia.383
Seven children have been reported with severe megaloblastic anemia, sensorineural deafness, and diabetes mellitus, all beginning in infancy.384,385 and 386 The anemia responded to thiamine (25 to 100 mg/day). In two patients with this disorder, the marrow was reported to be myelodysplastic.387 The gene for this puzzling disorder was recently mapped to the long arm of chromosome 1,388 but the biochemical defect is completely unknown.
The congenital dyserythropoietic anemias are lifelong anemias, often mild, showing dysplastic changes affecting the red cell line only, most typically multinuclearity of the normoblasts. They present as iron storage disorders. Of the three types, two (type I389 usually and type III390 occasionally) show megaloblastic red cell precursors (see Chap. 43).
Refractory Megaloblastic Anemia
Refractory megaloblastic anemia is now regarded as a manifestation of myelodysplastic and sideroblastic syndromes (see Chap. 63 and Chap. 92). The megaloblastic changes are atypical, with dysplastic features confined to the erythroid series; giant metamyelocytes and bands are absent from the marrow. The combination of a myeloproliferative disorder and true cobalamin deficiency can give rise to a confusing picture.391,392 A few patients with refractory megaloblastic anemia respond to pharmacologic doses of pyridoxine (200 mg/day),393 perhaps because of an effect on serine transformylase, which requires both pyridoxine and folate.
Erythroleukemia is the earliest stage of M6 acute myelogenous leukemia (see Chap. 93). Nucleated red cells appear on the blood film, and the marrow shows hyperplasia involving very bizarre-looking megaloblastic red cell precursors, often containing multiple nuclei or nuclear fragments. The disease usually evolves fairly quickly into classical acute myelogenous leukemia.
*Poorly defined neuropsychiatric abnormalities that respond to folate therapy have been reported in patients with folate deficiency.89,90 and 91
*The term pernicious anemia is sometimes used as a synonym for cobalamin deficiency, but it should be reserved for the condition resulting from defective secretion of intrinsic factor by an atrophic gastric mucosa.

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Zammarchi E, Lippi A, Falorni S, et al: Case report and monitoring of a pregnancy at risk by chorionic villus sampling. Clin Invest Med 13:139, 1980.

Ampola MG, Mahoney MJ, Nakamura E, Tanaka K: Prenatal therapy of a patient with vitamin B12-responsive methylmalonic acidemia. N Engl J Med 293:313, 1975.

Van der Meer SB, Spaapen LJM, Fowler B, et al: Prenatal treatment of a patient with vitamin B12-responsive methylmalonic acidemia. J Pediatr 117:923, 1990.

Erbe RW: Inborn errors of folate metabolism: II. N Engl J Med 293:807, 1975.

Luhby AL, Eagle FJ, Roth E, et al: Relapsing megaloblastic anemia in an infant due to a aspecific defect in gastrointestinal absorption of folic acid. Am J Dis Child 102:482, 1961.

Lanzkowsky P: Congenital malabsorption of folate. Am J Med 48:580, 1970.

Walters T: Congenital megaloblastic anemia responsive to N5-formyl tetrahydrofolic acid administration. J Pediatr 70:686, 1967.

Arakawa T, Narisawa K, Tanno K, et al: Megaloblastic anemia and mental retardation associated with hyperfolic-acidemia: probably due to N5-methyltetrahydrofolate transferase deficiency. Tohoku J Exp Med 93:1, 1967.

Huguley CM Jr, Bain JA, Rivers SL, Scoggins RB: Refractory megaloblastic anemia associated with excretion of orotic acid. Blood 14:615, 1959.

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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology


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