Chapter 170 – MEGALOBLASTIC ANEMIAS

Megaloblastic anemias, a group of disorders characterized by a distinct morphologic pattern in hematopoietic cells, are commonly due to deficiency of vitamin B12 (cobalamin) or folates. Folates and cobalamin are both required to sustain one-carbon metabolism, which involves the transfer of one-carbon groups such as methyl-, formyl-, methylene-, forminyl-, and formimino- in reactions essential for pyrimidine and purine biosynthesis, including the synthesis of three of the four nucleotides of DNA. Thus, a deficiency in cobalamin or folate results in the common biochemical feature of a defect in DNA synthesis, with lesser alterations in RNA and protein synthesis, that leads to a state of unbalanced cell growth and impaired cell division. In contrast to a normal population of cells, the majority of megaloblastic cells are not resting but rather are vainly engaged in attempting to double their DNA, with frequent arrest in the S phase and lesser arrest in other phases of the cell cycle; as a result, an increased percentage of these cells have DNA values between 2N and 4N because of delayed cell division. This increased DNA content in megaloblastic cells is morphologically expressed as larger than normal “immature” nuclei with finely particulate chromatin, whereas the relatively unimpaired RNA and protein synthesis results in large cells with greater “mature” cytoplasm and cell volume. The microscopic appearance of this nuclear-cytoplasmic asynchrony (or dissociation) is morphologically described as megaloblastic. Megaloblastic hematopoiesis is commonly manifested as anemia, the most easily recognized manifestation of a more global defect in DNA synthesis that affects all proliferating cells. Because correct vitamin replacement is curative, precise diagnosis of the deficient vitamin is essential. In the case of cobalamin, knowledge of the causes of the deficiency ( Table 170-1 ) dictates the dose and duration of replacement therapy.

TABLE 170-1   -- ETIOPATHOPHYSIOLOGIC CLASSIFICATION OF COBALAMIN AND FOLATE DEFICIENCIES

   I.    Cobalamin deficiency
   A.    Nutritional cobalamin deficiency (insufficient cobalamin intake)—vegetarians, poverty-imposed near-vegetarians, breast-fed infants of mothers with pernicious anemia
   B.    Abnormal intragastric events (inadequate proteolysis of food cobalamin)—atrophic gastritis, partial gastritis with hypochlorhydria, proton pump inhibitors, H2 blockers
   C.    Loss/atrophy of gastric oxyntic mucosa (deficient intrinsic factor molecules)—total or partial gastrectomy, adult and juvenile pernicious anemia, caustic destruction (lye)
   D.    Abnormal events in the small bowel lumen
   1.    Inadequate pancreatic protease (R factor–cobalamin not degraded, cobalamin not transferred to intrinsic factor)
   a.    Insufficient pancreatic protease—pancreatic insufficiency
   b.    Inactivation of pancreatic protease—Zollinger-Ellison syndrome
   2.    Usurping of luminal cobalamin (inadequate binding of cobalamin to intrinsic factor)
   a.    By bacteria—stasis syndromes (blind loops, pouches of diverticulosis, strictures, fistulas, anastomosis), impaired bowel motility (scleroderma), hypogammaglobulinemia
   b.    By Diphyllobothrium latum (fish tapeworm)
   E.    Disorders of ileal mucosa/intrinsic factor-cobalamin receptors (intrinsic factor–cobalamin not bound to intrinsic factor–cobalamin receptors)
   1.    Diminished or absent intrinsic factor–cobalamin receptors—ileal bypass/resection/fistula
   2.    Abnormal mucosal architecture/function—tropical/nontropical sprue, Crohn's disease, tuberculous ileitis, infiltration by lymphomas, amyloidosis
   3.    Intrinsic factor/post–intrinsic factor–cobalamin receptor defects—Imerslund-Gräsbeck syndrome, hereditary megaloblastic anemia, TCII deficiency
   4.    Drug-effects—metformin, cholestyramine, colchicine, neomycin
   F.    Disorders of plasma cobalamin transport (TCII-cobalamin not delivered to TCII receptors)—congenital TCII deficiency, defective binding of TCII-cobalamin to TCII receptors (rare)
   G.    Metabolic disorders (cobalamin not used by cell)
   1.    Inborn enzyme errors (rare)
   2.    Acquired disorders: (cobalamin functionally inactivated by irreversible oxidation)—nitrous oxide (N2O) inhalation
   II. Folate deficiency
   A.    Nutritional causes
   1.    Decrease dietary intake—poverty and famine, institutionalized individuals (psychiatric/nursing homes)/chronic debilitating disease, prolonged feeding of infants with goat's milk, special slimming diets or food fads (folate-rich foods not consumed), cultural/ethnic cooking techniques (food folate destroyed)
   2.    Decreased diet and increased requirements
   a.    Physiologic: pregnancy and lactation, prematurity, hyperemesis gravidarum, infancy
   b.    Pathologic
   (1)  Intrinsic hematologic diseases involving hemolysis with compensatory erythropoiesis, abnormal hematopoiesis, or bone marrow infiltration with malignant disease
   (2)  Dermatologic disease—psoriasis
   B.    Folate malabsorption
   1.    With normal intestinal mucosa
   a.    Some drugs (controversial)
   b.    Congenital folate malabsorption (rare)
   2.    With mucosal abnormalities—tropical and nontropical sprue, regional enteritis
   C.    Defective cellular folate uptake—infantile cerebral folate deficiency (antifolate receptor antibodies) (rare)
   D.    Inadequate cellular utilization
   1.    Folate antagonists (methotrexate)
   2.    Hereditary enzyme deficiencies involving folate
   E.    Drugs (multiple effects on folate metabolism)—alcohol, sulfasalazine, triamterene, pyrimethamine, trimethoprim-sulfamethoxazole, diphenylhydantoin, barbiturates
   III. Miscellaneous megaloblastic anemias not caused by cobalamin or folate deficiency
   A.    Congenital disorders of DNA synthesis
   1.    Orotic aciduria
   2.    Lesch-Nyhan syndrome
   3.    Congenital dyserythropoietic anemia
   B.    Acquired disorders of DNA synthesis
   1.    Deficiency—thiamine-responsive megaloblastic anemia (DIDMOAD syndrome)
   2.    Malignancy—erythroleukemia
   a.    Refractory sideroblastic anemias (?pyridoxine responsive)
   b.    All antineoplastic drugs that inhibit DNA synthesis (including antinucleosides used against HIV and other viruses)
   3.    Toxic—alcohol

DIDMOAD = diabetes insipidus, diabetes mellitus, optic atrophy, deafness; HIV = human immunodeficiency virus; TCII = transcobalamin II.

Epidemiology

Cobalamin Nutrition

Cobalamin is a pink, water-soluble vitamin with a complex structure that generally resembles the heme molecule but with cobalt replacing iron in the center of the pyrrole ring. The recommended daily allowance of cobalamin is 2.4 μg for men and nonpregnant women, 2.6 μg for pregnant women, 2.8 μg for lactating women, and between 1.5 and 2 μg for children 9 to 18 years old. Cobalamin is produced in nature only by microorganisms, and humans receive cobalamin solely from the diet. Meat from parenchymal organs is richest in cobalamin (>10 μg per 100 g wet weight); fish and animal muscle, milk products, and egg yolk have 1 to 10 μg per 100 g wet weight. An average nonvegetarian Western diet with abundant meat, milk and dairy products, and eggs contains 5 to 7 μg/day of cobalamin, which adequately sustains normal cobalamin equilibrium. For vegetarians, consumption of eggs, milk, and dairy products provides most of the cobalamin. Other sources include contamination of plants by cobalamin-producing bacteria that grow in the roots and nodules of legumes, manure that is rich in cobalamin and used for fertilization, microscopic insects that infest plants, and nori (dried seaweed) and tempeh (fermented soybean cake) used in Asian cuisine.

Vegetarian diets that provide less than 0.5 μg of cobalamin daily cannot sustain adequate cobalamin balance. In addition to vegan diets, both lactovegetarian and lacto-ovovegetarian diets (in ascending order of cobalamin content) are generally insufficient to prevent cobalamin deficiency. These individuals have increased blood levels of homocysteine and methylmalonic acid (MMA), which is strongly suggestive biochemical evidence of cobalamin deficiency (with or without folate deficiency). Furthermore, the normalization of sensitive electroencephalographic, evoked potential, and electrophysiologic markers of cognitive ability by cobalamin treatment in otherwise apparently asymptomatic individuals with subclinical cobalamin deficiency indicates that these individuals can benefit from prophylactic cobalamin replacement. Poverty-imposed near-vegetarians who consume meat infrequently have a cobalamin status that is only marginally better than that of lacto-ovovegetarians. Because vegetarians have a lifelong low cobalamin status, superimposition of additional conditions that perturb either absorption or metabolism of cobalamin can easily tip them into frank cobalamin deficiency far earlier than nonvegetarians who have substantially greater preexistent stores.

Cobalamin is stored exceptionally well in tissues. Of the total body content of 2 to 5 mg in adults, half is in the liver. With a daily loss of 1 μg, dietary cobalamin deficiency can take up to 5 to 10 years to become apparent. However, it takes about 3 to 4 years to deplete cobalamin stores if dietary cobalamin is abruptly malabsorbed, thereby interfering with an efficient enterohepatic circulation that accounts for the turnover of 5 to 10 μg of cobalamin per day and reabsorption of 75% of the cobalamin secreted into bile. Cobalamin is stable and resists high-temperature cooking processes. A pH greater than 12 (during food processing) or high doses of ascorbic acid (vitamin C) convert cobalamin to inactive analogues.

Worldwide, the most common cause of cobalamin deficiency in all age groups is nutritional deficiency. In affluent countries, however, inadequate absorption of adequate dietary cobalamin is the more common cause.

The incidence of pernicious anemia is approximately 25 new cases per year per 100,000 persons older than 40 years, and the average age at onset is about 60 years. In one study, up to 1.9% of free-living individuals older than 60 years in southern California had undiagnosed pernicious anemia with minimal clinical manifestations of cobalamin deficiency. The prevalence was 2.7% in women and 1.4% in men, but 4.3% of African American women and 4.0% of white women had pernicious anemia. However, pernicious anemia is found in persons of all ages, race, and ethnic origin. About 30% of patients have a positive family history, and there is an association with other autoimmune diseases (i.e., polyglandular autoimmune syndrome, Graves' disease [ Chapter 244 ], Hashimoto's thyroiditis [ Chapter 244 ], vitiligo [ Chapter 467 ], Addison's disease [ Chapter 250 ], idiopathic hypoparathyroidism [ Chapter 266 ], primary ovarian failure [ Chapter 255 ], myasthenia gravis [ Chapter 448 ], type 1 diabetes [ Chapter 247 ], and adult hypogammaglobulinemia [ Chapter 271 ]).

Folates

Nutrition

Folates are synthesized by microorganisms and plants. Rich food sources include green leafy vegetables (spinach, lettuce, broccoli), beans, fruit (bananas, melons, lemons), yeast, mushrooms, and animal protein (liver, kidney). The recommended daily allowance of folate is 400 μg for adult men and nonpregnant women, 600 μg for pregnant women, 500 μg for lactating women, and 300 to 400 μg for children 9 to 18 years of age. A balanced Western diet can prevent folate deficiency, but the net dietary intake of folate in many developing countries is often insufficient to sustain folate balance. For example, in rural areas of northern India, estimates of folate intake are often less than half of optimum.

Folates are susceptible to breakdown during prolonged cooking (for >15 minutes), which is common in many cultures; for example, many ethnic cooking techniques involve prolonged boiling of lentils or beans or frying foods in an open pan—methods that destroy 50 to 95% of folate. Eating fresh raw or stir-fried vegetables or salads that are rich in folate is not common in many cultures. Finally, processing food with additives such as nitrites destroys folate, but adding ascorbic acid before processing preserves the folate.

Folate deficiency can arise from decreased supply (reduced intake, absorption, transport, or utilization) or increased requirements (from metabolic consumption, destruction, or excretion). The same individual may have multiple causes for folate deficiency, but specific tests to define each mechanism are not available clinically. Folate deficiency varies among different populations, and nutritional deficiency is the most common cause worldwide in all age groups. With food folate fortification programs, folate deficiency has been dramatically reduced in the United States; nevertheless, folate deficiency may be present in 5 to 10% of the elderly (>70 years of age).

Pathobiology

Cobalamin

Normal Physiology

 

Absorption and Transport

Cobalamin in food is usually in coenzyme form (as deoxyadenosylcobalamin and methylcobalamin) and bound to proteins ( Fig. 170-1 ). In the stomach, peptic digestion at low pH is a prerequisite for release of cobalamin from food protein. Once released by proteolysis, cobalamin preferentially binds with a high-affinity cobalamin-binding protein called R protein, which is secreted in salivary and gastric juice. These cobalamin–R protein complexes along with unbound intrinsic factor, which is produced by gastric parietal cells, pass into the second part of the duodenum, where pancreatic proteases degrade the R protein to which cobalamin is bound, thereby allowing transfer of cobalamin to intrinsic factor.

Components and mechanism of cobalamin absorption with an indication of the locus for malabsorption

FIGURE 170-1  Components and mechanism of cobalamin absorption with an indication of the locus for malabsorption. See text for details. IF = intrinsic factor; TCII = transcobalamin II.  (From Antony AC: Megaloblastic anemias. In Hoffman R, Benz EJ Jr, Shattil SJ, et al [eds]: Hematology: Basic Principles and Practice, 4th ed. Philadelphia, Churchill Livingstone, 2005, pp 519–556.)

The stable intrinsic factor–cobalamin complex then passes through the jejunum to the ileum, where it binds to membrane-associated intrinsic factor–cobalamin receptors on the microvilli of ileal mucosal cells. Within enterocytes, cobalamin is transferred to transcobalamin II, which efficiently delivers cobalamin to transcobalamin II receptors found on cell surfaces. Another protein, transcobalamin I, which binds 75% of serum cobalamin but does not participate in transport, appears to be a storage protein for cobalamin in blood. A third quantitatively minor protein, transcobalamin III, binds a wide spectrum of cobalamin analogues that are rapidly cleared by the liver into bile for fecal excretion.

Cellular Processing

More than 95% of intracellular cobalamin is bound to two intracellular enzymes, methylmalonyl–coenzyme (CoA) mutase and methionine synthase. In mitochondria, deoxyadenosylcobalamin is a coenzyme for methylmalonyl-CoA mutase, which converts the products of propionate metabolism methylmalonyl-CoA to succinyl-CoA, a form that is easily metabolized. In the cytoplasm, methylcobalamin is a coenzyme for methionine synthase, which catalyzes the transfer of methyl groups from methylcobalamin to homocysteine to form methionine. The methyl group of 5-methyltetrahydrofolate (methyl-THF) is donated to regenerate methylcobalamin, thereby forming the THF that is essential to sustain one-carbon metabolism. The methionine so formed can be adenylated to S-adenosylmethionine, which donates its methyl group in a critical series of biologic methylation reactions involving more than 80 proteins, phospholipids, neurotransmitters, RNA, and DNA.

Pathogenesis of Cobalamin Deficiency

Nutritional Cobalamin Deficiency

Vegetarianism and poverty-imposed near-vegetarianism, which is especially common in developing countries, are important causes of nutritional cobalamin deficiency.

Inadequate Dissociation of Cobalamin from Food Protein

Dietary cobalamin requires proteolytic digestion of food by gastric acid and pepsin. Thus, failure to release cobalamin from food protein can lead to cobalamin deficiency despite the presence of intrinsic factor.

Absent Secretion

Deficiency of intrinsic factor as a result of gastric parietal cell atrophy is associated with insufficient HCl secretion and can be caused by (1) total or partial gastrectomy, (2) autoimmune destruction (chronic atrophic gastritis) as found as in classic pernicious anemia, or (3) destruction of gastric mucosa by caustic (lye) ingestion.

Total gastrectomy invariably leads to cobalamin deficiency in 2 to 10 years, thus warranting prophylactic cobalamin (and iron) replacement. After partial gastrectomy, up to a third of patients may have multifactorial cobalamin deficiency from decreased secretion of intrinsic factor, hypochlorhydria, or intestinal bacterial overgrowth of cobalamin-consuming organisms. Morbidly obese patients who have undergone gastric bypass surgery have more malabsorption of cobalamin from food than do those treated by vertical banded gastroplasty. Malabsorption of cobalamin can also occur with the long-term use of H2 blockers or proton pump inhibitors.

In pernicious anemia, the primary event is autoimmune destruction and atrophy of the gastric parietal cell mucosa, thereby leading to the absence of intrinsic factor and HCl, which causes severe cobalamin malabsorption and deficiency. The autoimmune gastritis that eventually leads to chronic atrophic gastritis involves the gastric fundus and body. Intrinsic factor antibodies are found in the serum of 60% and in the gastric juice of 75% of patients with pernicious anemia. Type I anti–intrinsic factor antibodies prevent binding of cobalamin to intrinsic factor, whereas type II anti–intrinsic factor antibodies prevent binding of intrinsic factor–cobalamin complexes to ileal intrinsic factor–cobalamin receptors and can interfere with tests for cobalamin absorption. About 96% of African American women with pernicious anemia have high titers of blocking anti–intrinsic factor antibodies but lack anti–parietal cell antibodies. Juvenile pernicious anemia is manifested in the second decade, often in conjunction with endocrinopathies; recently, mutations in intrinsic factor resulting in qualitative abnormalities and cobalamin deficiency have been described.

Abnormal Events Precluding Absorption of Cobalamin

Pancreatic insufficiency ( Chapter 147 ) with a deficiency of pancreatic protease will fail to break down the R proteins to which cobalamin is preferentially bound in the stomach, thereby precluding transfer of cobalamin to intrinsic factor. However, with the widespread early use of pancreatic replacement, frank cobalamin deficiency is now uncommon. Endogenous pancreatic protease can be inactivated by massive gastric hypersecretion arising from a gastrinoma in Zollinger-Ellison syndrome ( Chapters 141 , 142 , and 205 ), where the low pH of the luminal contents in the ileum can also preclude binding of the intrinsic factor–cobalamin complex with intrinsic factor–cobalamin receptors, a process that requires a pH higher than 5.4.

Bacterial overgrowth in the small bowel ( Chapter 143 ) (arising from stasis, impaired motility, and hypogammaglobulinemia) favors colonization by bacteria, which can then usurp free cobalamin before it can bind to intrinsic factor; this problem can be reversed by a short course of antibiotic therapy. Individuals heavily infested with the fish tapeworm Diphyllobothrium latum (by consuming raw or partially cooked freshwater fish from lakes in Russia, Japan, Switzerland, Germany, and the United States) can become cobalamin deficient when these 10-m-long adult worms in the jejunum avidly usurp cobalamin. After the worms have been expelled (praziquantel, 10 to 20 mg/kg, single dose orally), cobalamin replenishment is curative.

Disorders of the Intrinsic Factor Receptors or Mucosa

Because the distal ileum has the greatest density of intrinsic factor–cobalamin receptors, removal, bypass, or dysfunction of only 1 to 2 ft of terminal ileum can result in cobalamin malabsorption. Among drugs, biguanides (i.e., metformin) decrease intrinsic factor and acid secretion and can inhibit transenterocytic transport of cobalamin in up to a third of patients, which can be avoided by intake of calcium (1.2 g/day). Other drugs (extended-release potassium chloride, cholestyramine, colchicine, and neomycin) can also impair transepithelial transport of cobalamin and interfere with the Schilling test.

Acquired Cobalamin Deficiency

Nitrous oxide (N2O) irreversibly inactivates cobalamin and results in a state of functional intracellular cobalamin deficiency, which can be bypassed by administration of 5-formyl-THF (leucovorin). Although N2O exposure during prolonged surgery can induce megaloblastosis, especially in those with marginal or low cobalamin stores, chronic intermittent (surreptitious, accidental, or occupational) exposure leads more frequently to a neuromyelopathic manifestation.

Folates

Normal Physiology

Absorption and Transport

In general, only half the folate in food, which is mainly in polyglutamylated form, is nutritionally available (bioavailable), whereas 85% of folic acid that is added to food or ingested as a supplement is bioavailable. The small intestine can absorb folic acid unchanged, but food folate polyglutamates must be hydrolyzed to monoglutamate by folate polyglutamate hydrolase at the brush border before transport into enterocytes, where it is reduced to THF and methylated before release into plasma as methyl-THF. The normal serum folate level is maintained by dietary folate intake and by an efficient enterohepatic circulation.

From plasma, there is rapid uptake of folate (methyl-THF and folic acid) into tissues by two physiologic transport processes for cellular entry. The reduced-folate carrier is a low-affinity, but high-capacity system that can mediate the uptake of physiologic methyl-THF and pharmacologic folates (methotrexate and folinic acid) into a variety of cells. In addition, membrane-associated folate receptors can also bind and take up methyl-THF and folic acid with high affinity at concentrations found in serum. The relative density of these two pathways on cells generally determines the route of entry of folates and antifolates into normal and malignant cells. Finally, physiologic transplacental folate transport involves capture of maternal folate by placental folate receptors, followed by displacement of this pool by dietary folates, a process that leads to transfer to the fetal circulation along a downhill concentration gradient. Passive diffusion also operates to transport folate across biologic membranes at supraphysiologic folate concentrations.

The acquired transport resistance in patients with relapsed acute lymphocytic leukemia is associated with decreased reduced-folate carrier expression. Antibodies to folate receptors in mothers and infants have been linked to recurrent neural tube defects and infantile cerebral folate deficiency, respectively.

Cellular Retention and Excretion

Polyglutamylation of folate is the major factor for intracellular retention. After glomerular filtration, folate receptors on the brush border membranes on proximal renal tubular cells bind luminal folate and transport it back into blood.

Intracellular Metabolism and Cobalamin-Folate Interactions

After cellular uptake, methyl-THF must first be converted to THF via methionine synthase. Only then can the THF be polyglutamated by folate polyglutamate synthase, which allows it to play a central role in one-carbon metabolism. THF can be converted to 10-formyl-THF, which can be used for de novo biosynthesis of purines, and to methylene-THF, which can be used for the synthesis of thymidylate.

The central role of methylene-THF is that it can be used either in the thymidylate cycle via thymidylate synthase for the synthesis of thymidine and DNA or in the methylation cycle via methionine synthase, but only after its conversion to methyl-THF by methylene-THF reductase. Inactivation of methionine synthase during cobalamin deficiency results in accumulation of the substrate methyl-THF, which cannot be polyglutamylated and thus leaks out of the cell, thereby resulting in an intracellular THF deficiency and reduction of one-carbon metabolism. This process explains why cobalamin deficiency responds to replacement with folic acid, which can be converted to THF via dihydrofolate reductase, or to replacement with 5-formyl-THF (folinic acid), which bypasses methionine synthase and can be converted to methylene-THF or 10-formyl-THF via intermediates.

Pathogenesis of Folate Deficiency

 

Nutritional Causes (Decreased Intake or Increased Requirements)

With an abrupt reduction in folate consumption, body stores of folate are adequate for approximately 4 months; however, these stores are depleted even faster in individuals who are chronically in negative folate balance and who often have multiple nutritional deficiencies (and diseases) that tip them into frank folate deficiency. Children and women in developing countries are at particular risk, but even the elderly in developed countries are at risk if their diet is poor because of physical disabilities or social isolation. A seasonal reduction in folate-rich food, poverty, cultural or ethnic diets that are intrinsically poor in folates, cooking techniques that destroy food folate, and the anorexia that accompanies chronic illnesses are among the myriad of reasons for folate deficiency, especially in developing countries.

Patients with hematologic diseases involving increased intrinsic cell proliferation or with compensatory erythropoiesis in response to chronic peripheral red blood cell destruction are at risk. Folate deficiency in the face of chronic hemolysis can lead to an acute reticulocytopenic aplastic crisis, an unexpected increase in transfusional requirements, or a fall in platelets. Exfoliative skin diseases ( Chapter 464 ) also cause folate deficiency when there is a combination of increased demand from excess loss of skin cells.

Pregnancy and Infancy

Pregnancy with poor folate intake is a very common cause of megaloblastic anemia in developing countries because pregnancy and lactation require additional folate for growth of the fetus and maternal tissues. Transplacental maternal to fetal folate transport relies on provision of adequate dietary folate. However, mothers with short intervals between consecutive pregnancies, with twin pregnancies, or with hyperemesis gravidarum are unable to maintain adequate folate stores, thereby leading to premature, low-birthweight infants and other predominantly midline developmental abnormalities in the fetus.

Tropical and Nontropical (Celiac) Sprue

With the development of intestinal mucosal abnormalities, patients are at increased risk for folate malabsorption. In tropical sprue ( Chapter 143 ), for example, a dramatic response to folate can cure about 60% of patients of their sprue in the first year. In the short term, malabsorption leads to folate deficiency, but later in the chronic phase of the disease (longer than 3 years), malabsorption of cobalamin also develops. In addition, iron deficiency ( Chapter 163 ), pellagra, and beriberi ( Chapter 237 ) may coexist in these patients.

Drugs

Excess alcohol consumption at the expense of a balanced diet may be the most common cause of folate deficiency in the United States. Inhibition of dihydrofolate reductase by trimethoprim and pyrimethamine or methotrexate can be acutely reversed by the administration of 5-formyl-THF (folinic acid). Sulfasalazine induces megaloblastosis in two thirds of subjects taking full doses (>2 g/day) by decreasing the breakdown of folate polyglutamates to monoglutamates before absorption or by inducing Heinz body hemolytic anemia, which leads to increased requirements. Oral contraceptives may increase folate catabolism, whereas anticonvulsants can reduce absorption and induce microsomal liver enzymes. Antineoplastics and antiretroviral antinucleosides (azidothymidine) induce megaloblastosis by perturbing DNA synthesis.

Cellular Consequences of Perturbed One-Carbon Metabolism

In either cobalamin or folate deficiency, a net decrease in methylene-THF interrupts the reaction mediated by thymidylate synthase, which converts deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). Because of decreased dTMP (and eventually deoxythymidine triphosphate [dTTP]), dUMP is converted to deoxyuridine triphosphate (dUTP), which is then misincorporated into DNA by DNA polymerase. An editorial enzyme, DNA uracil glycosylase, recognizes this misincorporation and excises dUTP; however, with persistently low dTTP, the DNA strand is not repaired. Repetition of this cycle results in repeated DNA strand breaks and, ultimately, fragmentation of DNA, which then leaks out of the cell. Defective DNA synthesis also leads to additional epigenetic changes, such as altered sensitivity of folate-sensitive fragile sites, acetylation and methylation of histones, and numerous chromosomal abnormalities, that cause progressive nuclear-cytoplasmic asynchrony as a cobalamin- or folate-deficient cell divides, thereby resulting in classic megaloblastic changes in all proliferating cells (hematopoietic cells and epithelial cells in the gastrointestinal tract, cervix, vagina, and uterus).

Clinical Manifestations

The finding of macrocytosis on a routine complete blood count may be the first clinical manifestation. In other patients, the findings may be dominated by the condition that caused the deficiency of cobalamin or folate, such as malabsorption, alcoholism, or malnutrition (see Table 170-1 ).

The clinical manifestations of folate deficiency may involve the hematologic (pancytopenia with megaloblastic marrow), cardiopulmonary (secondary to anemia), gastrointestinal (megaloblastosis with or without malabsorption), dermatologic (hyperpigmentation of the skin and premature graying), genital (megaloblastosis of the cervical epithelium), infertility (sterility), and psychiatric (with primarily a flat affect) systems. If patients have additional neurologic findings, other diseases that predispose to folate deficiency (alcoholism with thiamine deficiency) or associated cobalamin deficiency must be considered. Because megaloblastosis secondary to either folate or cobalamin deficiency results in functional folate deficiency, the hematologic manifestations of both deficiencies, including pancytopenia with megaloblastic bone marrow, are indistinguishable (see later). However, only cobalamin deficiency results in a patchy demyelination process, which is expressed clinically as cerebral abnormalities and subacute combined degeneration of the spinal cord ( Chapter 443 ). This widespread demyelination begins in the dorsal columns in the thoracic segments of the spinal cord and then spreads contiguously to involve the corticospinal tracts and later the spinothalamic and spinocerebellar tracts. Hematologic manifestations, neurologic manifestations, or both may dominate the clinical picture.

Megaloblastosis can lead to atrophy of epithelial cells lining the gastrointestinal lumen and functional defects (failure of secretion of intrinsic factor and malabsorption of cobalamin and folate). This vicious cycle, whereby megaloblastosis begets more megaloblastosis, can be interrupted only by specific therapy with cobalamin or folate.

In folate deficiency there is increased susceptibility to carcinogenesis and neural tube defects. Moreover, with chronic hyperhomocysteinemia associated with severe cobalamin or folate deficiency (or both), there may be additional clinical manifestations that stem from the fact that homocysteine is a continuous, progressive risk factor for occlusive vascular diseases such as myocardial infarction ( Chapter 72 ), stroke ( Chapter 431 ), vascular disease in end-stage renal failure ( Chapter 131 ), thromboangiitis obliterans ( Chapter 79 ), aortic atherosclerosis ( Chapter 78 ), arterial and venous thromboembolism ( Chapters 81 and 182 ), and placental abruption or infarction. For example, in western India, about 75% of ambulatory subjects have hyperhomocysteinemia consistent with nutritional deficiency of cobalamin alone or with folate and/or pyridoxine deficiency.

Diagnosis

Diagnostic Approach to the Patient

The general approach to a patient with megaloblastic anemia is first to recognize that megaloblastic anemia is present; then to distinguish whether folate, cobalamin, or combined folate and cobalamin deficiencies have led to the anemia; and finally to diagnose the underlying disease and mechanism causing the deficiency (see Table 170-1 and Fig. 170-2 ). Deficiencies of cobalamin and folate are but two of the causes of macrocytosis, although they become increasingly more likely as MCV increases ( Table 170-2 ).

Algorithm for evaluation of a patient with macrocytosis

FIGURE 170-2  Algorithm for evaluation of a patient with macrocytosis.

TABLE 170-2   -- CAUSES OF MACROCYTOSIS BY LEVEL OF MEAN CORPUSCULAR VOLUME

CATEGORY 1: MACROCYTOSIS WITH MCV >110 fL[*]
Megaloblastic anemia caused by folate or cobalamin deficiency Megaloblastic anemia caused by drug-induced disorders of DNA synthesis (antineoplastic chemotherapy, immunosuppressive agents, antiretroviral agents) Myelodysplastic syndromes (especially the 5q- syndrome) Erythroleukemia (rare) Inherited disorders affecting DNA synthesis (pediatric age group)
CATEGORY 2: MACROCYTOSIS WITH MCV <110 fL[*]
Reticulocytosis[†] secondary to the broad categories of acute blood loss or hemolysis Post-splenectomy status Hepatic disease with or without alcoholism Aplastic/hypoplastic anemia, myelophthisic anemia, multiple myeloma Myeloproliferative disease Hypothyroidism Smoking, chronic lung disease

Note: Cold agglutinins may result in an abnormal high MCV from adherent cells being counted as single cells. Severe hyperglycemia or leukocytosis can also lead to spuriously raised MCV.

MCV = mean corpuscular volume.

* This table is only a rough guide because there are several exceptions. For example, early stages of diseases in category 1 can be accompanied by smaller rises in MCV to less than 110 fL, whereas some diseases in category 2 such as hemolysis, alcoholism, hepatic disease, or myeloproliferative disease may be associated with folate deficiency and megaloblastic anemia. Furthermore, a combination of causes in category 2 such as hepatic disease or alcoholism (or both) with acute bleeding can result in an MCV greater than 110 fL.
Marked reticulocytosis could result in an MCV greater than 110.

The underlying condition that predisposed to the development of folate deficiency usually began within the previous 6 months and will often dominate the overall clinical picture. Alcoholism can be identified as the basis for folate deficiency from the history and physical examination, but associated thiamine deficiency may result in a more complex manifestation (e.g., heart failure from cardiovascular disease [“wet beriberi”] and peripheral neuropathy [“dry beriberi”] with Wernicke-Korsakoff syndrome [ Chapters 237 and 443 ]). By contrast, cobalamin deficiency takes several years to become clinically manifested. Therefore, the underlying condition is more chronic and symptoms develop more insidiously, so specific tests will often be required to define the cause.

History and Physical Examination

The dietary history may be revealing (food faddism, vegetarianism, alcohol intake), whereas the medical or family history may uncover blood diseases, gluten sensitivity, autoimmune diseases, epilepsy treated with an anticonvulsant, use of offending drugs, previous hemolytic anemia, a past surgical history (e.g., gastrectomy, fistula, or bowel resection), accidental or surreptitious inhalation of nitrous oxide, or a travel history suggestive of tropical sprue.

Physical examination of cobalamin-deficient vegetarians or those with pernicious anemia may reveal well-nourished individuals. By contrast, patients with folate deficiency are poorly nourished and may have other stigmata of multiple deficiencies from malabsorption ( Chapter 143 ). Associated deficiency of vitamins A, D, and K or protein-calorie malnutrition, or both, may give rise to angular cheilosis, bleeding mucous membranes, dermatitis, osteomalacia, and chronic infections. Varying degrees of pallor with lemon-tint icterus (a combination of pallor and icterus best observed in fair-skinned individuals) are common features of megaloblastosis. The skin may reveal either a diffuse brownish pigmentation or abnormal blotchy tanning. Premature graying is observed in both light- and dark-haired individuals.

Examination of the mouth may reveal glossitis with a smooth (depapillated), beefy red tongue and occasional ulceration of the lateral surface. Thyromegaly may be observed in the neck if there is associated autoimmune disease, but it also raises suspicion for macrocytosis related to hypothyroidism (see later). The characteristic findings of cardiovascular failure from severe anemia may be accompanied by mild splenomegaly and extramedullary hematopoiesis.

In prolonged cobalamin deficiency, neurologic examination will reveal evidence of involvement of the posterior and pyramidal spinocerebellar and spinothalamic tracts. Posterior column dysfunction results in loss of position sense in the index toes (before great toe involvement) ( Chapter 443 ) and loss of the ability to discern vibration of a high-pitched (256 cps) tuning fork. Diminished vibratory sensation and proprioception of the lower extremities are the most common early objective signs. Neuropathic involvement of the legs precedes the arms. Upper motor neuron signs may be modulated by the subsequent involvement of peripheral nerves. A positive Romberg sign and a Lhermitte sign may be elicited. Loss of sphincter and bowel control or involvement of cranial nerves, such as optic neuritis, may accompanied by other dysfunction of the cerebral cortex, including dementia, psychoses, and disturbances of mood. Folate deficiency in adults does not give rise to significant neurologic findings. Thus, the coexistence of folate deficiency with neurologic disease should prompt investigations to exclude cobalamin and other nutrient deficiencies arising from dietary insufficiency or malabsorption.

Nutritional cobalamin deficiency in developing countries can be manifested as florid pancytopenia, mild hepatosplenomegaly, fever, and thrombocytopenia, with the neuropsychiatric syndrome developing as a later manifestation. However, cobalamin-related neurologic disease has also been found in patients with only mild to moderate anemia secondary to cobalamin deficiency in both developing and developed countries. Between 25 and 50% of patients who have neuropsychiatric abnormalities attributable to cobalamin deficiency can have a normal hematocrit and MCV if they have adequate folate stores to protect them from hematologic abnormalities. In fact, in the United States there is often an inverse correlation between the hematocrit and neurologic disease in cobalamin deficiency—most subjects have mild neurologic deficits, and 25% have only moderate deficits with paresthesias or ataxia as the initial symptoms.

Laboratory Tests Megaloblastosis

To establish the diagnosis of megaloblastosis, the evaluation begins with a complete blood count, MCV (which often reveals a steady increase over a period of several months or years), peripheral smear, and reticulocyte count (see Fig. 170-2 ). Classic megaloblastosis from cobalamin or folate deficiency may be accompanied by a hemoglobin level of less than 5 g/dL. Neutropenia and thrombocytopenia occur less commonly than anemia and are not usually severe. Occasionally, however, neutrophil counts less than 1000/μL and platelet counts less than 50,000/μL can be seen. Additional abnormalities supporting intramedullary hemolysis include elevated levels of serum lactate dehydrogenase and bilirubin, as well as decreased serum haptoglobin levels.

Megaloblastic anemia can be masked when there is a coexisting condition that neutralizes the tendency to generate large cells, such as with iron deficiency ( Chapter 163 ) or thalassemia ( Chapter 166 ). In these situations, giant myelocytes and metamyelocytes in bone marrow and hypersegmented neutrophils in bone marrow and peripheral blood ( Fig. 170-3 ) are important clues to a masked megaloblastosis. This problem is clinically relevant because appropriate replacement with cobalamin or folate will elicit a maximal hematologic response only when any associated iron deficiency is corrected. Conversely, if combined iron and cobalamin deficiency (after gastrectomy) or iron and folate deficiency (pregnancy) is treated with iron alone, megaloblastosis will be unmasked. Thus, the diagnosis of megaloblastic anemia should not be excluded until bone marrow aspirates have been examined and the presence of bone marrow iron is established.

Megaloblastic anemia

FIGURE 170-3  Megaloblastic anemia. The peripheral blood has oval macrocytes (large red blood cells) and marked neutrophil hypersegmentation.

Cobalamin and Folate Levels

Laboratory evaluation of suspected cobalamin or folate deficiency begins with measurement of the serum levels of these vitamins and then progresses to confirmatory tests ( Table 170-3 ). Use of clinical information will improve the pretest probability of serum cobalamin and folate levels. Moreover, without detailed clinical information, the combined results of serum cobalamin, folate, and metabolite test results are not sufficiently unambiguous to diagnose and distinguish cobalamin deficiency from combined cobalamin plus folate deficiency.

TABLE 170-3   -- STEPWISE APPROACH TO THE DIAGNOSIS OF COBALAMIN AND FOLATE DEFICIENCY

Megaloblastic anemia or neurologic-psychiatric manifestations consistent with cobalamin deficiency
plus
Test results on serum cobalamin and serum folate
Cobalamin[*] (pg/mL) Folate[†] (ng/mL) Provisional Diagnosis Proceed With Metabolites?[‡]
> 300 > 4 Cobalamin/folate deficiency is unlikely No
< 200 > 4 Consistent with cobalamin deficiency No
200–300 > 4 Rule out cobalamin deficiency Yes
> 300 < 2 Consistent with folate deficiency No
< 200 < 2 Consistent with combined cobalamin plus folate deficiency or with isolated folate deficiency  
> 300 2–4 Consistent with folate deficiency or with an anemia unrelated to vitamin deficiency Yes
TEST RESULTS ON METABOLITES: SERUM METHYLMALONIC ACID AND TOTAL HOMOCYSTEINE
Methylmalonic Acid (Normal = 70–270 nM) Total Homocysteine (Normal = 5–14 μM) Diagnosis
Increased Inc