CHAPTER 6: Megaloblastic Anemias

Hematopoiesis depends upon orderly cell division for the logarithmic expansion of progenitor cells into large numbers of circulating blood cells. In the megaloblastic anemias, DNA synthesis is impaired, leading to slowing or arrest of cellular division during the DNA synthesis phase of the cell cycle (S phase). A high fraction of cells suffering from such defects undergo programmed cell death (apoptosis). In the bone marrow, the decreased survival of hematopoietic progenitors leads to reduced production of circulating cells (ineffective hematopoiesis). Because RNA synthesis and cytoplasmic differentiation are relatively unaffected, progenitors and progeny that survive are enlarged (macrocytic). The main cause of megaloblastic anemias is deficiency of either cobalamin (vitamin B₁₂) or folic acid, vitamins that are essential for DNA replication and repair. In addition, chemotherapeutic drugs that inhibit DNA synthesis can result in findings similar to those seen in cobalamin or folate deficiency. It is not surprising that the clinical phenotype extends to other tissues that rely on continuous and robust cellular proliferation and differentiation, particularly the gastrointestinal tract.

Understanding the pathophysiology of the megaloblastic anemias requires knowledge about the absorption, transport, and utilization of folate and cobalamin as well as familiarity with the key chemical reactions in which these vitamins are essential cofactors.

Cobalamin is a complex organic molecule consisting of a tetrapyrole corrin ring, similar in structure to heme except that the divalent metal atom in the center of the ring is cobalt rather than iron. Like heme iron, the cobalt atom in the corrin ring binds to two axial ligands. One is a benzimidazole nucleotide, whereas the other can be either a methyl group (methylcobalamin) or an adenosyl group (adenosylcobalamin). Cobalamin is found in all foods of animal origin including meat, fish, and dairy products. Food cobalamin is tightly bound to proteins. Following ingestion, some cobalamin in food is transferred to human haptocorrin in saliva. As depicted in Figure 6-1, the acidic environment of the stomach enables efficient release and transfer of the remaining food cobalamin to haptocorrin in gastric juice. After transit to the duodenum, the increase in pH enables the transfer of cobalamin from haptocorrin to intrinsic factor, a transport protein secreted by gastric parietal cells. The cobalamin-intrinsic factor complex resists digestion and travels down the gut until it encounters epithelial cells in the distal ileum that express cubilin, a receptor with specificity for this bimolecular complex. The cobalamin that is absorbed in the ileum exits the basolateral side of the mucosal epithelial cell into the plasma where it traverses the portal circulation into the liver. Here cobalamin binds transcobalamin, a plasma transport protein that is functionally analogous to transferrin, the transport protein for iron. As in iron homeostasis, the liver is the principal storage site for cobalamin. The uptake of the circulating transcobalamin-cobalamin complex by receptors on plasma membranes is the principal and probably the only way that the vitamin is taken into cells other than liver cells. In keeping with the broad importance of cobalamin for the biochemical reactions described below, transcobalamin receptors are expressed on a wide variety of cells.

The transcobalamin-cobalamin complex makes up only a minority of the cobalamin in plasma. About 70% to 90% is bound to haptocorrin, derived primarily from leukocytes of the myeloid lineage. Haptocorrin is secreted into a number of bodily fluids including saliva and gastric juice, as mentioned earlier. Its biologic function is unclear.

Folic acid, commonly designated folate, refers to a group of compounds consisting of a tricyclic pteroyl group linked to one or more glutamic acid residues. Food sources include fruits, vegetables, liver, and meat, in all of which folate exists primarily as polyglutamate conjugates. The bioactivity of folate in food may be attenuated or destroyed by prolonged cooking. As shown in Figure 6-2, during transit in the gut, folate polyglutamates are hydrolyzed to the monoglutamate derivative, which is readily absorbed by a specific transmembrane channel into the enterocytes of the duodenum and jejunum. It is further metabolized in enterocytes into N5-methyl tetrahydrofolate (N5-methyl THF), which exits these cells and circulates freely in the plasma. Several types of folate receptors have been identified on cell surfaces, but their role in mediating uptake has not been explained. Like cobalamin and iron, folate is stored mainly in the liver.

These cofactors participate in a series of reactions involved in the biosynthesis of a variety of products including amino acids, metabolic intermediates, purines, and nucleotides. As summarized in Figure 6-3, the two forms of cobalamin mentioned earlier participate in different reactions.

Adenosylcobalamin is required for the conversion of methylmalonyl-coenzyme A (CoA) to succinyl-CoA. As discussed later in this chapter, measurement of methylmalonate in the serum is a useful test for identifying individuals with cobalamin deficiency. As shown in Figures 6-3 and 6-4, methylcobalamin is required for the conversion of homocysteine to methionine.

After N5-methyl THF enters the cell from the plasma, it is conjugated to polyglutamate, which prevents egress back into the plasma. Transfer of the methyl group from N5-methyl THF to methionine results in the formation and maintenance of adequate intracellular levels of THF. This reaction exemplifies the primary function of folate as a conduit for the addition of one-carbon moieties such as methyl and formyl groups to organic compounds. The conversion of the amino acid serine to glycine enables the transfer of a methyl group to THF to form N5,10-methylene THF, which is then used for both de novo purine biosynthesis and for the conversion of deoxyuridylate to deoxythymidylate. Thus cobalamin and folate play critical roles in production of the building blocks for DNA synthesis. As discussed in detail in the next section, deficiency in either of these two vitamins results in impairment of cell division, with adverse consequences particularly in proliferating cells such as in the bone marrow and gastrointestinal epithelium.

The impairment in DNA synthesis imposed by cobalamin or folate deficiency results in striking morphologic abnormalities in the bone marrow and peripheral blood, often referred to as megaloblastic changes. The bone marrow reveals increased cellularity and hyperplasia of erythroid precursors. The model diagram at the bottom of Figure 6-5 compares the maturation of normal and megaloblastic erythroid cells. Throughout the stages of erythroid development, the megaloblastic cells are larger. Although cytoplasmic differentiation, as assessed by increasing hemoglobin production, is normal, nuclear maturation is retarded, a phenomenon called nuclear-cytoplasmic dyssynchrony. Megaloblastic cells may acquire non-clonal chromosomal abnormalities, owing to incomplete DNA repair. These erythroid precursor cells are defective and vulnerable to destruction within the bone marrow prior to their maturation and release into the peripheral circulation as reticulocytes. Thus, erythropoiesis is ineffective.¹ Myeloid precursors reveal similar abnormalities: increased size and retarded nuclear maturation. The increased rate of apoptosis of erythroid precursor cells results in a slight increase in the level of non-conjugated bilirubin in the serum and a marked increase in serum lactate dehydrogenase (LDH).

The peripheral blood smear reveals striking variation in red cell size (anisocytosis) and frequent large oval red cells (macro-ovalocytes), placing megaloblastic anemias in the differential diagnosis of macrocytic anemias (see Chapter 3). The mean cell volume (MCV) increases progressively as the hemoglobin falls. An MCV of >110 fL generally indicates megaloblastic maturation. Less marked degrees of "non-megaloblastic" macrocytosis can be seen in patients with hemolysis, marrow aplasia or myelodysplasia, liver disease, and alcoholism. The reticulocyte count is low in megaloblastic anemia, in keeping with ineffective erythropoiesis. In severe megaloblastic anemia, the white count and platelet count also often are decreased but rarely to levels that put the patient at risk of infection or bleeding. Of greater diagnostic value is the presence of neutrophils that have an increased number of lobes, often called hypersegmented polys, shown in Figure 6-6. The mechanism underlying this finding is unclear. Abnormalities in bone marrow and blood cell morphology and in cell numbers are the same in cobalamin deficiency and folic acid deficiency. Morphologic abnormalities, including increased cell size and megaloblastic nuclei also are seen in epithelial cells that line the gastrointestinal tract.

Similar morphologic changes in the bone marrow and gastrointestinal tract are commonly seen following administration of chemotherapy drugs that inhibit DNA replication and cell division. A good example is the drug methotrexate (shown in Fig. 6-4 as MTX), which inhibits the enzyme dihydrofolate reductase.

ETIOLOGY

The causes of cobalamin deficiency are listed in Table 6-1. The vast majority of affected individuals have some type of gastrointestinal malabsorption. In the United States and in other areas with temperate climate, most patients have either pernicious anemia or food cobalamin malabsorption.

Pernicious anemia is an autoimmune disorder that destroys the parietal cells in the gastric mucosa. These cells are the site of both intrinsic factor production and the pumping of protons into the gastric lumen. A gastric biopsy may reveal an infiltrate of lymphocytes and plasma cells in the lamina propria. With time, the mucosa atrophies, and the gastric wall becomes thin. Some studies have suggested that the autoimmune attack on gastric parietal cells is a consequence of inflammation induced by Helicobacter pylori infection. As shown in Figure 6-1, the absence of intrinsic factor prevents cobalamin from being absorbed in the distal ileum. Normally, 2 to 3 mg of cobalamin is stored in the liver. With loss of intrinsic factor, cobalamin can no longer be absorbed; after 2 to 5 years, the stores in the liver are exhausted, and the patient is at risk of developing severe deficiency.

An even more common cause of cobalamin deficiency is malabsorption due to the absence of gastric acid that is necessary for the transfer of cobalamin in food to haptocorrin (Fig. 6-1). The prevalence of gastric achlorhydria increases sharply with age, affecting 40% of individuals over age 80. Cobalamin deficiency is also encountered among younger individuals following long-term administration of proton pump inhibitors for treatment of various disorders of the upper gastrointestinal tract. Because intrinsic factor is present, the impairment in cobalamin absorption is less marked than in pernicious anemia. Accordingly, the deficiency takes longer to develop and is usually less severe.

Because intrinsic factor is produced only in the stomach, it follows that total gastrectomy will lead to malabsorption of cobalamin. By the same token, surgical resection of the terminal ileum, where cobalamin is absorbed, has the same effect. However, these surgeries are seldom performed and are therefore uncommon causes of cobalamin deficiency. In contrast, deficiency of both cobalamin and folate is often encountered in certain parts of the world near the equator including Puerto Rico and Haiti, owing to a chronic malabsorption condition known as tropical sprue. The cause of this disorder is unclear, but the fact that patients respond to antibiotic therapy offers a clue to pathogenesis. Other chronic inflammatory diseases of the ileum such as regional enteritis (e.g., Crohn disease) also can lead to cobalamin malabsorption. Deficiency of cobalamin also may arise because of competition from organisms within the gastrointestinal tract. Anatomical stasis due to diverticula, stricture, or surgically created "blind loops" can lead to overgrowth of bacteria that usurp cobalamin-intrinsic factor complexes that are en route to the ileum. In Scandinavia, infestation with the fish tapeworm results in a similar parasitic confiscation of cobalamin.

Two other rare causes of cobalamin deficiency deserve mention. Individuals who are strict vegans and ingest no animal products at all (including dairy products) may, over a protracted time period, become deficient. In contrast, acute cobalamin deficiency is occasionally encountered in individuals following prolonged exposure to the anesthetic agent nitrous oxide, which can combine chemically with cobalamin and abolish its biological activity.

CLINICAL PRESENTATION

Patients with cobalamin deficiency come to medical attention because of anemia and/or neurological symptoms. They tend to be elderly. Those with pernicious anemia may have other autoimmune manifestations such as vitiligo or antibody-mediated endocrinopathies. The onset of the anemia is insidious, with symptoms and signs no different than those seen in other causes of chronic anemia. The anemia is often severe. It is not unusual for a patient with cobalamin deficiency to present with a hemoglobin level as low as 5 g/dL. In addition to pallor, a patient's skin may have a lemon tinge, and there may be slight scleral icterus, owing to increased levels of bilirubin in the plasma arising from destruction of erythroid cells in the bone marrow (ineffective erythropoiesis).

Although cobalamin deficiency can impair proliferation of mucosal cells in the gastrointestinal tract, this seldom causes symptoms. Some patients develop a sore, beefy red, smooth tongue (glossitis) due to atrophy of the papillae (Fig. 6-7). Cobalamin deficiency can also cause blunting of the mucosal villi in the small intestine and mild malabsorption. These findings are normalized by treatment.

Neurological manifestations are an important and sometimes overlooked consequence of cobalamin deficiency. They are generally associated with severe anemia. However, patients may present with significant neurological findings without anemia or even macrocytosis. Most patients complain of numbness or tingling, generally in the legs. Less often patients develop ataxia and loss of proprioception as a result of damage to the posterior columns of the spinal cord. They also may have muscle weakness and spasticity as a result of damage to the lateral columns (Fig. 6-8). Dementia may be observed in patients with severe cobalamin deficiency. As a rule of thumb, any patient who presents with neurological manifestations of peripheral neuropathy, ataxia, or dementia should be evaluated for cobalamin deficiency, even in the absence of concurrent anemia or macrocytosis.

LABORATORY EVALUATION

The hematologic features of the megaloblastic anemias are described earlier and do not distinguish in any way between cobalamin and folate deficiency. Demonstration of a low serum cobalamin level is a useful initial step in establishing a diagnosis. However, if the result is at the lower end of the normal range, cobalamin deficiency can be verified by an elevation in the serum levels of methylmalonate and homocysteine, which are the substrates for the respective adenosylcobalamin and methylcobalamin reactions (Fig. 6-3). Once cobalamin deficiency is documented, its cause should be investigated. About half of patients with pernicious anemia will have antibodies in the serum against intrinsic factor. Gastric achlorhydria will be found in all those with either pernicious anemia or food cobalamin malabsorption. If stomach acid is present, it is likely that the patient has either bacterial overgrowth or malabsorption in the terminal ileum.

TREATMENT

In all patients with cobalamin deficiency, replacement therapy results in complete cure of the hematologic abnormalities, unless there is a second process that independently suppresses erythropoiesis. Unfortunately, therapy is much less effective in reversing the neurological complications. It is important to establish a diagnosis prior to instituting a treatment plan because the administration of folate to a cobalamin-deficient patient will partially correct the anemia but (for unknown reasons) may exacerbate the neurological manifestations.

Patients with severe anemia are at risk of becoming hypokalemic immediately following initiation of therapy, owing to the anabolic burst of orderly cell division. Moreover, these patients, particularly the elderly, may be at the brink of cardiac decompensation. Therefore, sudden expansion of blood volume should be carefully avoided. Typically, these patients mount a brisk hematologic recovery, and red cell transfusions should be avoided unless the patient evinces symptoms or signs of ischemic damage to vital organs such as the brain or heart.

Because the vast majority of cobalamin deficiency is due to malabsorption, replacement therapy is given parenterally. Regular injections of cobalamin (initially weekly, then monthly) are effective in keeping patients in hematologic remission. A typical response following the initiation of treatment is shown in Figure 6-9. A rapid rise in the reticulocyte count, peaking at about day 7, is followed by a slower but steady rise in hemoglobin and hematocrit concentrations, accompanied by a decline in the MCV to normal. Oral administration of cobalamin can be equally effective when given in doses high enough to penetrate the intestinal mucosal barrier by mass action. However, because many patients (particularly the elderly) fail to take their medications regularly, most physicians favor parenteral administration.

ETIOLOGY

In contrast to cobalamin deficiency, which is generally isolated, folate deficiency is usually accompanied by deficits in other nutrients. As indicated in Table 6-2, there are three major causes of folate deficiency: malnutrition, malabsorption, and increased requirements.

As mentioned earlier, folate is present primarily in fruits, vegetables, liver, and certain meat products and can be inactivated by prolonged cooking. Unlike cobalamin deficiency, which is rarely due to an inadequate diet, decreased intake of folate is quite common, particularly among certain population groups. Infants who are fed goat milk exclusively become markedly folate deficient. Also at risk are adolescents who subsist on "junk" food. In these two instances, the deficiency is the result of a combination of inadequate intake and the increased demand imposed by rapid growth. Indigent and house-bound people are also prone to folate deficiency because of the expense and difficulty in obtaining fresh food. Alcoholics commonly become folate deficient, owing to poor diet along with ethanol-induced suppression of folate absorption and impaired enterohepatic circulation.

Folate malabsorption can be caused by a variety of gastrointestinal disorders, particularly those that affect the mucosa of the duodenum and jejunum. Of particular importance are celiac disease and tropical sprue. As mentioned earlier, the latter also can cause cobalamin deficiency. Finally, folate deficiency often is encountered in conditions in which growth or pathologic cell proliferation leads to an increased demand for folate. Infancy and adolescence already have been mentioned. In the past, folate deficiency was commonly encountered in pregnant women, particularly those who were indigent and malnourished. Now folate is administered as part of an iron-vitamin supplement given routinely to all expectant mothers. Occasionally folate deficiency is seen in patients with disorders associated with enhanced cell proliferation. Those with chronic hemolytic anemias such as sickle cell disease are commonly given prophylactic folate to satisfy the enhanced demand of the hyperplastic erythroid progenitors. In like manner, patients with rapidly growing tumors are prone to folate deficiency, particularly if they are not maintaining a good diet.

CLINICAL PRESENTATION

The hematologic and gastrointestinal manifestations of folate deficiency are identical to those of cobalamin deficiency. Both have megaloblastic maturation and identical morphologic features. However, the conditions differ in one important respect: patients with folate deficiency rarely, if ever, develop neurologic manifestations.

If a pregnant woman is folate deficient during the first trimester, there is increased risk that the baby will be born with a neural tube defect such as spina bifida. Why folate deficiency leads to these particular defects is not understood; it may be related to a requirement for folate in the biochemical reactions that lead to DNA methylation, a modification that controls gene expression. Whatever the mechanism, the risk of neural tube defects is obviated by administration of folic acid early in pregnancy. Since 1998, fortification of flour and other grains with folate has been compulsory in the United States and has resulted in a marked decrease in the incidence of these birth defects.

Because the turnover of folate in the body is much more rapid than that of cobalamin, the 10 to 12 mg stores of folate in the liver can be exhausted in a few months under circumstances in which intake or absorption is abruptly curtailed.

LABORATORY EVALUATION

In folate deficiency, blood cell counts as well as marrow and peripheral blood morphology are indistinguishable from those of cobalamin deficiency. Both conditions cause megaloblastic maturation. The standard diagnostic test is measurement of serum folate, which is usually low in patients who are deficient. However, serum levels can change quite rapidly with alterations in diet. In contrast, red cell folate is stable over a period of several days and therefore can provide a more accurate index of deficiency. As predicted from the chemical reactions shown in Figure 6-3, in folate deficiency, the serum homocysteine level is elevated (as in cobalamin deficiency), and the serum methylmalonate level is normal (in contradistinction to cobalamin deficiency).

TREATMENT

Oral folic acid, 1 mg/day, is remarkably safe, inexpensive and effective therapy. The hematologic response is identical to that with cobalamin deficiency. Because folate deficiency is often accompanied by deficits in other vitamins and nutrients, a full nutritional evaluation should be done.