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Megaloblastic anemias are disorders caused by impaired DNA synthesis. The presence of megaloblastic cells is 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 fine “salt and pepper” granular chromatin that contrasts with the ground-glass texture of its normal counterpart. As the cell differentiates, the chromatin condenses more slowly than normal into darker aggregates that coalesce, but do not fuse homogeneously, giving the nucleus a characteristic fenestrated appearance. Continuing maturation of the cytoplasm as it acquires hemoglobin contrasts with the immature-looking nucleus—a feature termed nuclear-cytoplasmic asynchrony.
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Megaloblastic granulocyte precursors are also larger than normal and show nuclear-cytoplasmic asynchrony. A characteristic cell is the giant metamyelocyte, which has a large horseshoe-shaped nucleus, sometimes irregularly shaped, containing ragged open chromatin.
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Megaloblastic megakaryocytes may be abnormally large and polylobated, with deficient granulation of the cytoplasm. In severe megaloblastosis, the nucleus may show detached lobes. Further details are provided in “Laboratory Features” below and in Figs. 41–12 and 41–13.
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ETIOLOGY AND PATHOGENESIS
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Table 41–4 lists the causes of megaloblastic anemia. By far the most common causes worldwide are folate deficiency and cobalamin deficiency. There has, however, been a marked reduction in the prevalence of folate deficiency in North America and a growing number of other countries that have implemented folic acid fortification of the food supply.
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Megaloblastic cells have much more cytoplasm and RNA than do their normal counterparts, but they have a relatively normal amount of DNA,144 suggesting that cytoplasmic constituents (RNA and protein) are synthesized faster than is DNA. Evidence that maturation is retarded in megaloblastic precursors supports this conclusion.145 DNA synthesis is impaired,146 and migration of the DNA replication fork and the joining of DNA fragments synthesized from the lagging strand (Okazaki fragments) are delayed,147 and the S-phase is prolonged.146
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Slowing of DNA replication in the megaloblastic anemias of folate and cobalamin deficiency appears to arise from failure of the folate-dependent conversion of dUMP to dTMP. Because of this failure, deoxyuridine triphosphate (dUTP) levels become abundant and because DNA polymerase is promiscuous with respect to its substrate specificity, allows dUTP to become incorporated into the DNA of folate-deficient cells in place of deoxythymidine triphosphate (dTTP).148 DNA excision–repair mechanisms to repair the DNA by replacing uridine with thymidine fail for the same reason that uridine triphosphate was incorporated into the DNA in the first place. The result is a repetitive iteration of flawed DNA repair that ultimately leads to DNA strand breaks, fragmentation, and apoptotic cell death.149
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Addition of deoxyuridine (dU) to marrow cells in culture normally decreases the incorporation of tritiated thymidine into DNA, because the dU is converted via dUMP→dTMP to unlabeled dTTP, which competes with the tritiated thymidine. In megaloblastic cells, this effect of added dU is greatly diminished. This finding is consistent with impairment in the dUMP→dTMP reaction in the megaloblastic cells and was the basis for the now defunct dU suppression test.150 The failed excision–repair model following dUTP misincorporation into DNA also explains the chromosome breaks and other abnormalities that occur in megaloblastic cells.151
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All megaloblastic anemias share certain general clinical features. Because the anemia develops slowly, with opportunity for cardiopulmonary and intraerythrocytic compensatory changes,152 it produces few symptoms until the hematocrit is severely depressed. Symptoms, when they appear, are those of anemia: weakness, palpitation, fatigue, light-headedness, and shortness of breath. The blending of severe pallor and slight jaundice caused by a combination of intramedullary and extravascular hemolysis produce a characteristic lemon-yellow skin. Leukocyte and platelet counts may 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.
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All cell lines are affected. Erythrocytes vary markedly in size and shape, often are large and oval, and in severe cases can show basophilic stippling and nuclear remnants (Cabot rings and Howell-Jolly bodies). Erythroid activity in the marrow is enhanced, although the megaloblastic cells usually die before they are released, accounting for the reduced reticulocyte count. The more severe the anemia, the more pronounced the morphologic changes in the red cells. When the hematocrit is less than 20 percent, erythroblasts with megaloblastic nuclei, including an occasional promegaloblast, may appear in the blood. The anemia is macrocytic (mean corpuscular volume [MCV] is 100 to 150 fL or more), although coexisting iron deficiency, thalassemia trait,153 or inflammation can prevent macrocytosis.154 Slight macrocytosis may be the earliest sign of megaloblastic anemia. Because of the progressive nature of gradual replacement of normocytic red cells with the macrocytic progeny of a megaloblastic marrow, the earliest observable change in red cell indices is an increase in the red cell distribution width (RDW), reflecting an increase in anisocytosis.
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Neutrophil nuclei often have more than the usual three to five lobes (see Fig. 41–12).155 Typically, more than 5 percent of the neutrophils have five lobes. Cells may contain six or more lobes, a morphology rarely seen in normal neutrophils but not pathognomonic of megaloblastic hematopoiesis. In nutritional megaloblastic anemias caused by folate deficiency, hypersegmented neutrophils are an early sign of megaloblastosis5 and persist in the blood for many days after treatment.155 Neutrophil hypersegmentation was not found to be a sensitive test for mild cobalamin deficiency.156 Cytogenetic studies are nonspecific and show chromosomes that are elongated and broken. Specific therapy corrects these abnormalities, usually within 2 days, although some abnormalities do not disappear for months.151,157 Platelets are often reduced in number and slightly smaller than normal with a wider variation in size (increased platelet distribution width [PDW]).158 The morphologic features of megaloblastic anemia may be grossly exaggerated in patients who have been splenectomized or lack a functional spleen as occurs in celiac disease or sickle cell anemia. Numerous circulating megaloblasts and bizarre red cell morphology may be present.159
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Aspirated marrow is cellular and shows striking megaloblastic changes, especially in the erythroid series with well-hemoglobinized erythroblasts containing nuclei that possess less-mature, more-open nuclear chromatin than their normal counterparts. There is a preponderance of earlier basophilic erythroblasts over more mature forms which gives the overall impression of a maturation arrest (see Fig. 41–13). Sideroblasts are increased in number and contain increased numbers of iron granules. The ratio of myeloid to erythroid precursors falls to 1:1 or lower, and granulocyte reserves may be decreased. In severe cases, promegaloblasts containing an unusually large number of mitotic figures are plentiful. Macrophage iron content often is increased. Megaloblastic features in the granulocytic series is also usually present with giant forms and large horseshoe-shaped nuclei. Occasionally megakaryocytes with hyperlobated nuclei are present.
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Coexisting Microcytic Anemia
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Many features of megaloblastic anemia may be masked when megaloblastic anemia is combined with a microcytic anemia.154 The anemia can be normocytic or even microcytic, whereas the blood film may show both microcytes and macroovalocytes (a “dimorphic anemia”). The marrow may contain “intermediate” megaloblasts160 that are smaller and look less “megaloblastic” than usual. In this kind of mixed anemia, the microcytic component usually is iron-deficiency anemia,154 but it may be thalassemia minor153 or the anemia of chronic disease. Even megaloblastic anemia masked by a severe microcytic anemia usually shows hypersegmented neutrophils in the blood and giant metamyelocytes and bands in the marrow. Neutrophil myeloperoxidase levels are high.161
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Less commonly, the megaloblastic component of a mixed iron-deficiency anemia can be overlooked, and the patient may be treated only with iron. In this situation, the anemia may respond only partly to therapy, and megaloblastic features become more conspicuous as iron stores fill. The masking of macrocytosis in these situations may be responsible for delay or difficulty in diagnosis of pernicious anemia, particularly in certain geographic areas and ethnic groups where there is a high incidence of thalassemia and microcytic hemoglobinopathies.153,162,163 There are several situations that favor the coexistence of a megaloblastic state with iron deficiency. Both folate and iron deficiency occur in celiac disease,164 and cobalamin and iron deficiency both complicate gastric reduction surgery for morbid obesity.165 Furthermore, Helicobacter pylori infection is associated with gastric atrophy that can result first in iron deficiency and later lead to cobalamin malabsorption and perhaps even predispose to pernicious anemia.166,167
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Incomplete Megaloblastic Anemia
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If a patient with a full-blown megaloblastic anemia receives cobalamin or folate before marrow aspiration, the anemia persists but the megaloblastic changes may be obscured. Attenuated megaloblastic changes also are seen in patients with early megaloblastic anemia, in patients with coexisting infection,154 or in patients after transfusion.
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Megaloblastic Anemia Misdiagnosed as Acute Leukemia
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Occasionally, very severe megaloblastic anemia produces marrow morphology so bizarre as to be mistaken for acute leukemia. In some cases, the erythroid series does not mature, and the megaloblastic pronormoblast dominates the marrow with prominent mitotic figures and dysmorphic forms, raising the possibility of erythroid leukemia.
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Megaloblastic Changes in Other Cells
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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. Distinguishing these “megaloblastic” changes from the changes of malignancy may be difficult.168
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Chemical Changes in Body Fluids
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Plasma bilirubin, iron, and ferritin levels are increased.169 Serum lactate dehydrogenase-1 (LDH-1) and LDH-2, both found in red cells, are markedly elevated as a result of rapid intramedullary erythroblast turnover and increase with the severity of the anemia.170 In megaloblastic anemia LDH-1 is greater than LDH-2, whereas in other anemias LDH-2 is greater than LDH-1.171 Serum muramidase (lysozyme) levels are high,172 whereas serum glutamic oxaloacetic transaminase (aspartate transaminase [AST]) is normal.173 Erythropoietin levels rise, but less than in other anemias of similar severity.174 Surprisingly, the elevated erythropoietin levels fall sharply within 1 day of beginning treatment, an interval too short either to have been mediated by the hematocrit or to affect it.
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Megaloblastic anemia is associated with two pathophysiologic abnormalities: ineffective erythropoiesis and hemolysis. Ineffective erythropoiesis increases the red cell precursor to reticulocyte ratio, plasma iron turnover,175 LDH-1 and LDH-2 levels,171 and “early labeled” bilirubin.176 Both intramedullary and extramedullary hemolysis occurs in megaloblastic anemia, with red cell life span decreased by 30 to 50 percent.177
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Increased serum muramidase in megaloblastic anemia can be caused by increased granulocyte turnover,172 possibly induced by 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,178 perhaps reflecting ineffective thrombopoiesis. Platelets in severe cobalamin deficiency are functionally abnormal.179
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FOLIC ACID DEFICIENCY
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Etiology and Pathogenesis
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Folate deficiency is caused by (1) dietary deficiency, (2) impaired absorption, and (3) increased requirements or losses (see Table 41–4).
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Decreased Intake Caused by Poor Nutrition
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Prior to the mid-1990s, inadequate dietary intake was the major cause of folate deficiency. However, in the era of folic acid fortification, the prevalence of folate deficiency has fallen dramatically. In the United States, the prevalence of low plasma folate has dropped from 22 to 1.7 percent of the population.180 Because folate reserves are limited, deficiency develops rapidly in malnourished persons, typically the old, the poor, and the alcoholic. Folate deficiency can also occur during hyperalimentation181 and subclinical folate deficiency has been reported in subtotal gastrectomy.182 Folate deficiency can occur in premature infants, especially with infection, diarrhea, or hemolytic anemia183; in children on a synthetic diet because of inborn errors184; and in infants raised on goat’s milk, which is poor in available folate.185 Destruction of folate through excessive cooking can aggravate folate deficiency.
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In alcoholic cirrhosis, megaloblastic anemia usually is caused by folate deficiency.186 Alcohol may acutely depress serum folate, even if folate stores are replete,187 and accelerates the development of megaloblastic anemia in persons with early folate deficiency.188 Alcohol causes acute marrow suppression, decreases in reticulocyte, platelet, and granulocyte levels189; reversible vacuolation of erythroid and myeloid precursors; and dysfunction of granulocytes.190 These changes occur even if large doses of folate are given with the alcohol.191
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Decreased Intake Caused by Impaired Absorption Nontropical Sprue
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Nontropical sprue (celiac disease in children) is related to ingestion of wheat gluten.192 Pathologically, nontropical sprue shows atrophy and chronic inflammation of the small intestinal mucosa that is most severe proximally. Findings include weight loss; glossitis (typical of folate deficiency); other signs of a generalized vitamin deficiency; diarrhea; and passage of light-colored, bulky stools with a particularly foul odor caused by steatorrhea. Iron deficiency, hypocalcemia, osteoporosis, and osteomalacia may occur.
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Folate malabsorption occurs in most patients with this disorder.193 Serum folate levels are low,194 and megaloblastic anemia occurs frequently.
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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 the travelers return.195 Tropical sprue is rapidly corrected by folate therapy, even though folate deficiency does not cause the disease. The precise etiology of tropical sprue is unknown, although the response of the disease to antibiotics suggests infection.196
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Clinically and pathologically, tropical sprue is like nontropical sprue, except that tropical sprue is more severe in the distal small intestine.197 Therefore, tropical sprue eventually also leads to cobalamin deficiency198 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 occur,199 possibly because the diseased intestine fails to deconjugate folate polyglutamates.200 Consequently, megaloblastic anemia is very common in patients with this disease,201 and may result from both folate and cobalamin deficiency.
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Other Intestinal Disorders
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Malabsorption of folic acid commonly occurs in regional enteritis,201 after extensive resections of the small intestine,202 and in conditions such as lymphomatous or leukemic infiltration of the small intestine,203 Whipple disease,203 scleroderma and amyloidosis,204 and diabetes mellitus.205 Systemic bacterial infections impair folate absorption.206
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Increased Folate Requirements in Pregnancy
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During pregnancy (Chap. 8),207 folate requirements increase five- to 10-fold because of transfer of folate to the growing fetus,208 which draws down maternal folate stores even in the face of severe maternal folate deficiency.209 Further increases in requirements may result from the presence of multiple fetuses, a poor diet, infection, coexisting hemolytic anemia, or anticonvulsant medication. Lactation aggravates folate deficiency.210 Consequently, folate deficiency is very common in pregnancy and is the major cause of the megaloblastic anemia of pregnancy,211 particularly in developing countries.212
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Folate deficiency is difficult to diagnose in pregnancy because the signs of deficiency are obscured by the normal hematologic changes of pregnancy. During pregnancy, a physiologic “anemia” develops because of increased plasma volume that is only partly offset by an accompanying increase in red cell mass. Hemoglobin levels may fall to 10 g/dL. The anemia is associated with a physiologic macrocytosis; MCV may increase to 120 fL, although the average at term is 104 fL.213 Serum and red cell folate levels fall steadily during pregnancy, even in well-nourished women who are not taking a folic acid supplement.214 Conversely, hypersegmented neutrophils, usually a reliable clue to early megaloblastic anemia, are inconspicuous in early megaloblastic anemia of pregnancy.215
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Increased Cell Turnover
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Because of increased marrow cell turnover, the folate requirement rises sharply in chronic hemolytic anemia.216 During bouts of acute hemolysis that can occur in these anemias, the marrow may become megaloblastic within days.
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Folic acid deficiency may arise in chronic exfoliative dermatitis, in which folate losses of 5 to 20 mcg/day may occur.217 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.217 During hemodialysis, folate is lost in the dialysis fluid.218
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The clinical picture of folate deficiency includes all the general manifestations of megaloblastic anemia 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” below), and (3) a full response to physiologic doses of folate.
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The earliest specific indicator of folate deficiency is a low serum or plasma folate. Raised plasma levels of homocysteine may precede the lowering of plasma folate. However, elevated homocysteine has poor specificity as there are several causes of a raised plasma homocysteine.219 Plasma folate follows folate intake closely, so an isolated low serum folate (less than approximately 3 ng/mL) may simply indicate a drop in folate intake over the preceding few days.5 Similarly, a low plasma folate, except in malabsorption, rises quickly on refeeding.
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A better indicator of the tissue folate status is the red cell folate,220 which remains relatively unchanged while a red cell is circulating and thus reflects folate status over the preceding 2 to 3 months. Red cell folate usually is quite low in folate-deficient megaloblastic anemia. However, red cell folate also is low in more than 50 percent of patients with cobalamin-deficient megaloblastic anemia100 owing to the poor retention of methyltetrahydrofolate monoglutamate within the cells; consequently, red cell folate cannot be used to distinguish between folate and cobalamin 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” below).221
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The dU suppression test has been used in research on pathogenetic mechanisms in megaloblastic states. It adds little to the clinical evaluation of a megaloblastic anemia. The test is further discussed in “Deoxyuridine Suppression” below.
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Differential Diagnosis
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Macrocytosis without megaloblastic anemia occurs in alcoholism, liver disease, hypothyroidism, aplastic anemia, certain forms of myelodysplasia, pregnancy, and any condition associated with reticulocytosis (e.g., autoimmune hemolytic anemia). Macrocytosis has also been reported among smokers.222 However, MCV rarely exceeds 110 fL in these conditions, whereas in folate deficiency, uncomplicated by causes of microcytosis, the MCV is usually over 110 fL.
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A full hematologic response to physiologic doses of folate (i.e., 200 mcg daily) distinguishes folate deficiency from cobalamin deficiency, in which a response occurs only at pharmacologic doses of folate (e.g., 5 mg daily). This is not recommended as a diagnostic test because neurologic problems may develop in cobalamin-deficient patients treated with folate alone. High doses of cobalamin may produce a partial response in folate deficiency.73
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The diagnosis of nontropical sprue rests on (1) the demonstration of malabsorption, (2) noninvasive serologic testing including detection of antibodies to gliadin, endomysium, tissue transglutaminase, and deamidated gliadin,223 (3) a jejunal biopsy showing villus atrophy, and (4) the response to a gluten-free diet. In 80 percent of patients, a gluten-free diet gradually reverses the functional disorder by correcting folate malabsorption.224
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Nonhematologic Effects of Folate Deficiency
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The hematologic problems associated with folate deficiency have been recognized for decades. However, folate deficiency has been associated with a number of serious disorders not involving the hematopoietic system. Moreover, these disorders occur at folate levels usually regarded as low normal. They include developmental, neurologic, cardiovascular, and neoplasic diseases.225
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Abnormalities of Neural Tube Closure
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A close association exists 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.226 A portion of the neural tube closure defects appear to be associated with antibodies against folate receptors that may be overcome by higher folate intake.227 Mutations and polymorphisms affecting enzymes of folate metabolism, especially the common 677C→T polymorphism of the MTHFR gene (also designated as MTHFR 677C→T),228 also predispose to congenital anomalies. As noted above, this polymorphism results in diminished conversion of its substrate methylene FH4 to methyltetrahydrofolate, supporting the view that it is the role of folate in methylation through methionine synthesis (see Table 41–1) that is critical in embryonic development. Folic acid fortification programs, which were mandated in the United States and Canada in the mid-1990s, have been highly successful as a public health measure in reducing the incidence of neural tube defect births by between 20 and 50 percent.229,230
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Cobalamin also plays a significant role as a risk factor for neural tube defects. Levels of TC in normal pregnant women correlate with their likelihood of bearing an infant with a defect in neural tube closure. Patients in the lowest quintile of TC concentration are five times more likely to give birth to a defective infant as patients in the highest quintile.231 Evidence indicates that in populations exposed to folic acid fortification, there is an approximately threefold increase in the risk of neural tube defects in offspring of mothers in the lowest quartile of TC.232
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Several poorly defined neuropsychiatric abnormalities that respond to folate therapy have been reported in patients with folate deficiency. The most convincing associations are with depressive illness.225,233
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Even a mildly elevated homocysteine level is a major independent risk factor for atherosclerosis and venous thrombosis, possibly because of an effect on the vascular endothelium.234 In folate deficiency, homocysteine levels may rise considerably, resulting in varying degrees of hyperhomocysteinemia. This is true also in cobalamin and pyridoxine deficiencies; consequently, the notion of seeking to ameliorate hyperhomocysteinemia and thus diminish the risk of cardiovascular disease has seemed appealing. However, the effect of lowering homocysteine levels by the use of folate, cobalamin, and pyridoxine supplements, on the risk of recurrent vascular disease is unclear. Although there is some evidence that such supplements reduce risk,235 contradictory evidence suggests that supplement use may actually increase the risk of in-stent coronary restenosis236 or other adverse cardiovascular outcome.237 On the other hand, an accelerated rate of decrease in stroke mortality has been observed in the United States and Canada that coincided with the introduction of folic acid fortification in these countries.238 The disparate designs of these studies makes it difficult to draw firm conclusions regarding the question of whether lowering of plasma homocysteine in subjects at risk for cardiovascular disease has any ameliorative or deleterious effect on outcome. In a meta-analysis of eight randomized trials involving over 37,000 individuals, the authors concluded that supplementation with folic acid in various combinations with vitamin B12 and vitamin B6 for periods of up to 7.3 years, despite an overall reduction of plasma homocysteine of 22 percent and 25 percent in folic acid fortified and non-fortified populations, respectively, there were no significant effects on cardiovascular events, overall cancer, or mortality.239 Critical factors might relate to several considerations including the preexisting degree of vascular damage in relation to the time of the intervention and the form and dosage of administered vitamins.
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Another potential link between folate deficiency and morbidity risk that relates to hyperhomocysteinemia is the association between elevated homocysteine levels and incident dementia or cognitive impairment without dementia, independent of the vascular complications of hyperhomocysteinemia.240,241 The MTHFR polymorphism MTHFR 677C→T leads to increased homocysteine levels in subjects with low folate or cobalamin levels,242 although controversy exists as to whether MTHFR 677C→T contributes to an increased incidence of vascular disease. Like folate, cobalamin seems to be important in decreasing the risk of vascular disease.243 A 1561C→T polymorphism in the gene for glutamate carboxypeptidase-II increases serum folate and decreases serum homocysteine in the homozygote, possibly protecting against vascular disease.244
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Severe folate deficiency reportedly mimics the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome (preeclampsia with liver swelling and abnormal liver function studies in pregnant women; Chap. 8).245 In these patients, the diagnosis of severe folate deficiency can be made based on the presence of anemia and a megaloblastic blood film and marrow. Serum and red cell folate, serum cobalamin, homocysteine, and methylmalonic acid levels all should be assayed before treatment is started.246 The patient should immediately be given high doses of folate plus cobalamin, the latter in case the megaloblastic anemia actually results from cobalamin deficiency, a possibility rendered more likely in folic acid-fortified populations. A major goal of treatment is preventing preterm delivery of the fetus.
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A large study of nurses in the United States indicated that supplementation with more than 400 mcg of folic acid per day reduces the incidence of colon cancer by 31 percent.247 Furthermore, individuals who are homozygous for the 677C→T MTHFR mutation also have a decreased incidence for colon cancer compared with 677C→T heterozygotes and normal controls.248 Other evidence points to possible deleterious effect of folic acid on colon cancer incidence. Although only circumstantial, a recent epidemiologic study reported that after several successive years of a declining incidence of colorectal cancer in the United States and Canada, there was a significant increase in the rate in both countries that coincided with and followed the introduction of folic acid fortification.249 These apparently contradictory observations may be reconcilable because of the several roles of folate on cellular proliferation and repair as well as on the stage of tumorigenesis.250 Because folate is critical for de novo thymidine synthesis, it plays an important part in DNA repair, thus correcting mutations and DNA strand breaks that could potentially initiate cancer. On the other hand, the growth of established neoplastic clones might be accelerated by additional folate, allowing more rapid tumor progression. The situation is rendered even more complex if the potential role of folate in epigenetic regulation of gene expression is considered. Folate is necessary for synthesis of the universal methyl donor, SAMe, which is required for both cytosine and histone methylation. In this pathway, too, the role of folate theoretically may be cancer promoting or cancer protective, depending on whether oncogenes or tumor-suppressor genes are silenced by methylation of CpG islands in DNA or by conformational changes in chromatin resulting from histone methylation. The question of a possible effect of increased folate intake through the use of folic acid supplements on overall and site-specific cancer incidence was recently examined in a meta-analysis of 50,000 individuals. The authors concluded that there was no substantial increase or decrease in incidence over a 5-year period of folic acid supplement use.251
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Therapy, Course, and Prognosis
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Folate, usually in the form of folic acid, 1 to 5 mg/day, is given orally, although 1 mg usually is sufficient. At this dose, anemia usually is corrected even in patients with malabsorption. A parenteral preparation containing 5 mg/mL of folate also is available.
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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.
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Pregnant women must be given at least 400 mcg of folate per day.252 As to the possibility of overlooking cobalamin deficiency resulting from folate administration, although pernicious anemia (PA) in women of childbearing age is rare in whites, this is not the case among persons of African and Hispanic descent.253,254 In pregnant women at risk for cobalamin deficiency (e.g., vegans or patients with malabsorption), the risk of an associated cobalamin deficiency is easily prevented with vitamin B12, 1 mg given parenterally every 3 months during the pregnancy.
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Therapeutic doses of folate partially and temporarily correct the hematologic abnormalities in cobalamin deficiency, but the neurologic manifestations can progress, with disastrous results.255 Therefore, both folate status and cobalamin status must be evaluated 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 suitable specimens have been obtained for assay.
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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.256 Administration of folic or folinic acid can prevent or greatly diminish the major side effects without reducing the therapeutic effect of low-dose methotrexate. Coadministration of folic acid together with vitamin B12 also reduces side-effects without adversely affecting the therapeutic efficacy of the newer multitargeted antifolate drug, pemetrexed.257
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Etiology and Pathogenesis
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There are several causes and varying degrees of severity of cobalamin depletion and deficiency. From the hematologic standpoint, it is convenient to divide the causes of B12 deficiency into those that frequently lead to megaloblastic anemia and those that usually do not.64,258
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Table 41–4 lists disorders that lead to cobalamin deficiency.
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Decreased Uptake Caused by Impaired Absorption
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Cobalamin deficiency most often results from defective absorption, most commonly PA, a condition characterized by failure of gastric intrinsic factor production. Many other causes of defective cobalamin absorption involve mainly the stomach, or small intestine and to lesser extent, the pancreas.
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Gastric Disorders in Pernicious Anemia
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PA is a disease of insidious onset that generally begins in middle age or later (usually after age 40 years).259 In this condition, intrinsic factor secretion fails because of gastric mucosal atrophy. PA 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. The term pernicious anemia sometimes is 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 caused by an autoimmune process primarily directed against the parietal cells and their products.
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In patients with PA, antibodies occur that recognize the H+/K+-adenosine triphosphatase (ATPase), which resides in the secretory membrane of the parietal cell and is responsible for acidifying the stomach contents. These antiparietal cell antibodies occur in approximately 60 percent of patients with simple atrophic gastritis and in 90 percent of patients with PA, but in only 5 percent of a random 30- to 60-year-old population.260 Antiparietal cell antibodies also occur in a significant percentage of patients with thyroid disease.261 Conversely, patients with PA have a higher than expected incidence of antibodies against thyroid epithelium, lymphocytes, and renal collecting duct cells.262
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Antiparietal cell antibodies are not thought to be responsible for the pathogenesis of PA. Rather, studies in mice suggest the gastric atrophy in PA 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. CD4+ T cells from these mice produce atrophic gastritis when injected into nude mice.263
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Antibodies to intrinsic factor (“type I,” or “blocking,” antibodies) or the intrinsic factor–cobalamin (Cbl) complex (“type II,” or “binding,” antibodies) are highly specific to PA patients.264 Blocking antibodies, which prevent formation of the intrinsic factor–Cbl complex, are found in up to 70 percent of PA sera.264 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. 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.265 Second, the incidence of PA is higher than expected in patients with agammaglobulinemia, even though their sera contain none of the antibodies typical of PA.266
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Other Autoimmune Diseases
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The coexistence of several other autoimmune diseases and PA is further evidence that PA is an autoimmune disease. Antiparietal cell antibodies and PA are unexpectedly frequent in patients with other autoimmune diseases,267 including autoimmune thyroid disorders (thyrotoxicosis, hypothyroidism, and Hashimoto thyroiditis),268 type I diabetes mellitus, hypoparathyroidism,269 Addison disease, postpartum hypophysitis,270 vitiligo,271 acquired agammaglobulinemia,266 infertile female patients younger than age 40 years,272 and hypospermia and infertility in males.273,274 Infertility may, however, relate to impairment of DNA synthesis in gonadal cells rather than to an autoimmune mechanism.
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Inherited Predisposition to Pernicious Anemia
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Predisposition to PA can be inherited. The disease is associated with human leukocyte antigen types A2, A3, B7, and B12,275 and with blood group A.276 PA and antiparietal cell antibodies occur more frequently than expected in the families of PA patients.277 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 antiintrinsic factor antibodies.278 PA occurs relatively frequently in northern Europeans (especially Scandinavians)279 as well as Africans,163,280 but is uncommon in Asians. In Americans of African descent, the disease tends to begin early, occurs with high frequency in women, and often is severe.163,254
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Stomach and Intestine in Pernicious Anemia
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Gastric manifestations of PA include achlorhydria, acquired intrinsic factor deficiency previously demonstrable by the Schilling test, and an increased incidence of certain malignancies. There is an approximately 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.279 Achlorhydria may precede by many years the loss of intrinsic factor secretion and the development of PA.281 The absence of achlorhydria excludes the diagnosis of PA. H. pylori, a microorganism that infects the gastric mucosa, is a major cause of gastritis and peptic ulcers. Evidence is conflicting 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.282 One study reported that anti–H. pylori antibodies were found in only a small fraction of the sera from these patients. The other study reported that these antibodies were present in most of the PA sera, indicating that most of the patients described in the study had been infected previously. Whether H. pylori participates in the pathogenesis of PA is an open question. An intriguing hypothesis has been advanced that chronic infection with H. pylori may be responsible for triggering an autoimmune reaction directed against the host H+/K+-ATPase protein as a result of molecular mimicry.167,283
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Fasting plasma gastrin levels are high in most patients with PA, whereas somatostatin levels are low.284 In biopsies from PA stomachs, however, fundal gastrin and somatostatin levels were high, correlating with increases in argyrophilic cells in the basal crypts; antral gastrin and somatostatin were normal. Gastrin levels are high in simple achlorhydria without PA.285
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The stomach shows characteristic histologic abnormalities in PA (Fig. 41–14). 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 lymphocytes286 and plasma cells. In contrast, the antral and pyloric mucosa are normal. 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.287 Clinical response to administration of glucocorticoids or adrenocorticotropic hormone in patients with neurologic disease may reflect temporary amelioration of underlying and undiagnosed PA.288
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Megaloblastic changes reversible by cobalamin are seen in the gastrointestinal epithelium. Cells recovered by lavage are large168 and show atypical nuclei resembling early malignant change.289 Small intestinal biopsy shows decreased mitoses in crypts, shortening of villi, megaloblastic changes in epithelial cells, and infiltration in the lamina propria.290 These changes may account for the malabsorption of D-xylose and carotene observed in PA.291
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Recognizing PA may be difficult. PA combines the general features of megaloblastic anemia and features specific for cobalamin deficiency with unique clinical features related to its (probable) autoimmune etiology and gastric pathology. The disease is easily missed because of its (1) insidious onset, (2) tendency to be masked by the use of multivitamin preparations containing folic acid,292 and (3) many atypical presentations,293 including its presentation as a neurologic disease without hematologic findings,77,294 and its tendency to be overlooked in patients with another autoimmune disease.
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Antiparietal cell and antiintrinsic factor antibodies are rarely measured, even though antiintrinsic factor antibodies in particular could be of considerable diagnostic value.221 In the absence of a reliable method to assess vitamin B12 absorption, following the Schilling test becoming obsolete, measurement of antiintrinsic factor antibodies in serum represents the only available method to confirm a diagnosis of PA. Antiintrinsic factor antibody is highly specific for PA (although its sensitivity is only modest); its presence in a megaloblastic anemia makes the diagnosis of PA almost certain.
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Gastrectomy Syndromes
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Gastric surgery often leads to anemia. Iron-deficiency anemia is most common, but cobalamin deficiency with megaloblastic anemia can occur. After total gastrectomy, cobalamin deficiency develops within 5 or 6 years because the operation removes the source of intrinsic factor.295 The delay between surgery and the onset of cobalamin deficiency reflects the time needed to exhaust cobalamin stores after cobalamin absorption ceases. This may occur more rapidly because of abrogation of the enterohepatic reabsorption of biliary cobalamin.
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After partial gastrectomy, few patients show frank cobalamin deficiency, but approximately 5 percent have intermediate megaloblastosis, approximately 25 to 50 percent have low serum cobalamin levels, and many have varying degrees of decreased cobalamin absorption.296 Achlorhydria not present before surgery often develops some years after gastrectomy. Postgastrectomy patients with low serum cobalamin levels usually have low serum iron levels,297 in contrast to the high iron levels otherwise typical of cobalamin deficiency.
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Cobalamin deficiency after partial gastrectomy can be caused by mucosal atrophy in the unresected remnant of the stomach298 or, if a gastrojejunostomy was performed, by bacterial overgrowth in the afferent loop (see “Competing Intestinal Flora and Fauna: ‘Blind Loop Syndrome’” below). A surgical procedure that has gained popularity for the treatment of morbid obesity is gastric reduction surgery. This procedure results in multiple deficiencies of micronutrients including cobalamin.299
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Of the various causes of cobalamin malabsorption described, those that most often lead to megaloblastic anemia include PA, total or partial gastrectomy, intestinal blind loop syndrome, fish tapeworm, ileal resection, regional enteritis (Crohn disease) and tropical sprue.64 In addition, several of the inherited disorders affecting cobalamin absorption and metabolism, such as congenital intrinsic factor deficiency, selective cobalamin malabsorption and congenital TC deficiency can also result in megaloblastic anemia.
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Zollinger-Ellison Syndrome
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In 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 prevents transfer of Cbl from HC binder to intrinsic factor and also inactivates pancreatic proteases.300
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Because the terminal ileum is the site for physiologic cobalamin absorption, a number of intestinal disorders can lead to cobalamin deficiency, including (1) extensive resection of the ileum301; (2) inflammatory bowel disease or regional ileitis or other disease affecting the ileum (e.g., lymphoma, radiation damage302); (3) cobalamin malabsorption associated with hypothyroidism,303 or certain drugs304; (4) the effects of cobalamin deficiency itself305; and (5) sprue, either tropical or, less often, nontropical.198 In each of these disorders, administration of exogenous intrinsic factor, as was carried out in the Schilling test, would fail to correct subnormal cobalamin absorption.
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Competing Intestinal Flora and Fauna: “Blind Loop Syndrome”
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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).306 Serum cobalamin is low, but intrinsic factor secretion is normal. Cobalamin malabsorption is not corrected by exogenous intrinsic factor but may be corrected by antibiotic treatment. 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.307 Steatorrhea is also seen in the blind loop syndrome.
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Another cause of cobalamin deficiency is infestation with the fish tapeworm Diphyllobothrium latum. Prevalence is highest near the Baltic Sea, Canada, and Alaska, where raw or undercooked fish is consumed. Cobalamin deficiency results from competition between the worm and the host for ingested cobalamin.308 The clinical picture of D. latum infestation ranges from no symptoms to a full-blown megaloblastic anemia with neurologic changes. The infestation is diagnosed by finding tapeworm ova in the feces.
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Acquired Immunodeficiency Syndrome
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A substantial number of patients with AIDS have low serum cobalamin levels with associated evidence of cobalamin malabsorption.309 In addition, individuals testing seropositive for HIV infection may also have low serum cobalamin and evidence of cobalamin malabsorption.309 The cause of the malabsorption may be intestinal or gastric or a combination of both.310,311
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Some degree of cobalamin malabsorption has been demonstrated in 50 to 70 percent of patients with exocrine pancreatic insufficiency.312 Cobalamin malabsorption in pancreatic insufficiency is caused by a deficiency in pancreatic proteases, resulting in a partial failure to destroy HC–Cbl complexes whose destruction is a prerequisite for the transfer of cobalamin to intrinsic factor.313 Pancreatic insufficiency rarely causes clinically significant cobalamin deficiency.314
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Dietary Cobalamin Deficiency
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Dietary cobalamin deficiency was previously considered very unusual and restricted largely to complete vegetarians who also do not consume dairy products and eggs (vegans).315 Low serum cobalamin levels occur in 50 to 60 percent of individuals in this group. The onset of cobalamin deficiency in vegans is slower than in conditions associated with cobalamin malabsorption. Thus it may take 10 to 20 years for an individual consuming a vegan diet to manifest features of cobalamin deficiency.316 This is because the enterohepatic pathway for biliary cobalamin absorption remains intact, thus conserving body cobalamin stores.64 Breastfed infants of vegan mothers also may develop cobalamin deficiency.317 Cobalamin deficiency in vegans presents with mild megaloblastic anemia, glossitis, and neurologic disturbances. In addition to vegans, however, there is mounting evidence of cobalamin inadequacy in children and young adults in developing countries that cannot be explained on the basis of cobalamin malabsorption, and has therefore been attributed to inadequate dietary intake.318
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Cobalamin deficiency may occur in severe general malnutrition. A megaloblastic anemia not related to cobalamin deficiency may accompany kwashiorkor or marasmus.319
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Neurologic Effects of Cobalamin Deficiency
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Previously, the neurologic abnormalities of cobalamin deficiency were attributed to disordered metabolism of myelin lipids caused by an impaired methylmalonyl CoA mutase reaction.320 Similar neurologic abnormalities do not, however, occur in patients with inherited methylmalonyl CoA mutase deficiency.260,321 Authentic combined system disease has occurred in a patient with nutritional folate deficiency322 and in a patient with MTHFR deficiency.323 The latter reports suggest the neurologic lesions of cobalamin deficiency result from deranged methyl group metabolism. Animal studies support this hypothesis. Neurologic disorders closely resembling combined system disease develop in cobalamin-deficient fruit bats,324 pigs, and monkeys.325 The development of these disorders is prevented by methionine, which is produced in a cobalamin-dependent reaction and is the precursor of the biologic methylating reagent SAMe. A finding that further supports a methylation defect is that brains from cobalamin-deficient pigs contain increased levels of SAH,326 a powerful methylation inhibitor produced in SAMe-dependent methylation reactions:
+
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where RH is any unmethylated compound and RCH3 is its methylated form. Against the methylation defect hypothesis is the finding that cobalamin deficiency had no effect on SAMe, SAH, or methylation of phospholipids or myelin basic protein327 in the brains of fruit bats.
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The more typical clinical picture of cobalamin deficiency includes the nonspecific manifestations of megaloblastosis, such as anemia, thrombocytopenia, neutropenia, smooth tongue, cardiomyopathy, pale yellow skin and/or weight loss, plus specific features caused by the lack of cobalamin, chiefly neurologic abnormalities. Disturbances in either or both cellular and hormonal immune functions have been reported in cobalamin deficiency.328,329 Cobalamin deficiency may also contribute to the risk of vascular disease through elevation of homocysteine levels. Other disease associations with cobalamin deficiency have been described. These include a possible increase in breast cancer risk in premenopausal women330 and in osteoporosis.331,332 Because cobalamin reserves are large, years may pass between the cessation of cobalamin absorption and the appearance of deficiency symptoms.
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Neurologic Abnormalities
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Cobalamin deficiency causes a neurologic syndrome that is particularly dangerous because the syndrome can develop in isolation,333 with no megaloblastic anemia to suggest a lack of cobalamin,294,334 and because the syndrome cannot be reversed by treatment when it is sufficiently far advanced. The syndrome usually begins with paresthesias in feet and fingers as a result of early peripheral neuropathy and disturbances of vibratory sense and proprioception. The earliest signs, which 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.335 Left 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. 41–15).336
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The peripheral nerves, the spinal cord, and the brain are 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.337 There is recent evidence linking low cobalamin status with brain volume loss and cerebral white matter lesions.338,339 Psychological derangements, including psychotic depression and paranoid schizophrenia, can occur.340 Frank psychosis in cobalamin deficiency has been given the sobriquet megaloblastic madness.341
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The neurologic lesions of cobalamin deficiency can be detected by magnetic resonance imaging (MRI). Demyelination appears as T2-weighted hyperintensity of the white matter.342 MRI is particularly useful for confirming the diagnosis of a neurologic disorder resulting from cobalamin deficiency. MRI also has been used to follow the progress of neurologic abnormalities during treatment of cobalamin-deficient patients.342
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Subtle Cobalamin Deficiency
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Some observations suggest the existence of a large group of patients who are hematologically normal, with a normal hematocrit and MCV, but who have cobalamin-responsive neuropsychiatric disease.294 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. Most of the neuropsychiatric abnormalities appear to respond to cobalamin therapy.
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Plasma or Serum Cobalamin Levels
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Plasma or serum cobalamin is low in most but not all patients with cobalamin deficiency.219 Cobalamin levels are usually normal in cobalamin deficiency resulting from exposure to nitrous oxide, TC deficiency, and inborn errors of cobalamin metabolism. Levels also may be normal in cobalamin-deficient patients with high HC levels resulting from myeloproliferative diseases. Conversely, plasma cobalamin levels may be low in the presence of normal tissue cobalamins in vegetarians, in subjects taking megadoses of ascorbic acid,343 in pregnancy (25 percent), in the presence of HC deficiency,344,345 and in megaloblastic anemia resulting from folate deficiency (30 percent).219 Plasma folate may be high in cobalamin deficiency because of retardation in conversion of methyltetrahydrofolate, which is the predominant form in plasma. Patients deficient in both cobalamin and folate may show normal serum folate levels.
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Plasma or Serum Holotranscobalamin
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The fraction of the cobalamin in plasma that is bound to TC constitutes only 10 to 30 percent of the total plasma cobalamin. Even so, it is this fraction that is functionally important and also better reflects the integrity of the cobalamin absorptive status of an individual.142,346,347 The major fraction of plasma cobalamin bound to HC is considered functionally inert and is therefore less relevant for the consideration of cobalamin status. Consequently, and with the development of assays to measure the TC-bound fraction of the plasma cobalamin, an increasing body of evidence has accumulated to support the usefulness of TC-associated cobalamin (holoTC).64,137,140,346
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Except when caused by an inborn error, methylmalonic aciduria is a reliable indicator of cobalamin deficiency.348 Normal subjects excrete only traces of methylmalonate (0 to 3.4 mg/day). In cobalamin deficiency, urine methylmalonate usually is elevated.349 Cobalamin therapy restores excretion to normal in a few days. Another possible advantage of measurement of urine rather than plasma methylmalonic acid is that in conditions of impaired renal function, when plasma methylmalonic acid may give misleadingly elevated levels, measurement of the metabolite in urine when correlated with creatinine obviates this problem.350
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Serum or Plasma Methylmalonic Acid and Homocysteine
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Elevated plasma or 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 plasma cobalamin falls to subnormal levels.219,351 Elevated plasma methylmalonic acid and/or elevated homocysteine are both indicators of cobalamin deficiency in patients without a congenital disorder in their metabolism. Of the two, methylmalonic acid measurement is both more sensitive and more specific, and elevated methylmalonic acid will persist for several days, even after cobalamin treatment is instituted. Unlike homocysteine levels that rise in folate and pyridoxine deficiencies, as well as in hypothyroidism, methylmalonic acid elevation is seen only in cobalamin deficiency.219 In renal diseases however, both homocysteine and methylmalonate, acid levels are frequently elevated. Additionally, intestinal bacteria synthesize propionate, a precursor of methylmalonate, and in conditions of bacterial overgrowth, microbially derived methylmalonic acid may contribute to elevations in plasma methylmalonic acid.351,352 Although measurement of these metabolites may be used for population screening for evidence of cobalamin deficiency, the finding of an isolated elevation of plasma methylmalonate cannot be taken as a priori evidence of clinically attributable cobalamin deficiency, absent any demonstration of a therapeutic response to the administration of cobalamin.352,353
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Spinal fluid methylmalonic acid levels are markedly elevated in cobalamin deficiency.354
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Assays of Cobalamin Absorption and Intrinsic Factor
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Despite its numerous shortcomings the previous “gold standard” for assessment of cobalamin absorption was the Schilling test. The Schilling test assessed cobalamin absorption by measuring urinary radioactivity after an oral dose of radioactive cobalamin. The test could be performed even after cobalamin deficiency had been corrected. The test consisted of administering a physiologic dose of radiolabeled Co-CnCbl by mouth followed 2 hours later by injection of a large “flushing” dose of unlabeled CnCbl and determination and radioactivity in a 24-hour collection of urine. Subjects with normal absorption excreted 7 percent or more of the radioactivity in the urine. Subjects with subnormal urinary excretion would have the test repeated with addition of an animal-derived intrinsic factor to determine whether the malabsorption could be corrected.355 The use of the Schilling test has dropped to a point of obsolescence as a consequence of reduced availability of the test components, cost, radioactive waste disposal, and concern about the use of animal-derived tissues for human use, which were required for the intrinsic factor administered in the second part of the test.64 Replacements for the Schilling test are currently under development. One approach uses measurement of the change in holoTC following oral administration of non–radiolabeled cobalamin.346,347,356 A different approach involves the use of accelerator mass spectrometry and microbially produced 14C at attomolar concentrations.357 In this approach, 14C is measured in blood at the time of peak appearance 6 to 8 hours following the dose. Both methods show promise but have not been approved or validated for routine clinical use.
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Deoxyuridine Suppression Test
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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 through dilution of the label in the thymidine pool.358 This occurs when the thymidylate synthase reaction is functionally intact, which requires adequate quantities of both folate and cobalamin.
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The dU suppression test is chiefly a research tool. It can help diagnose certain special clinical problems,358 but these problems also can be diagnosed using other laboratory tests, therapeutic trials with vitamins or iron, or watchful waiting. Furthermore, in more than 40 years of use, the test has not moved from the research laboratory into the clinic. The dU suppression test seems unlikely to enjoy more widespread clinical use in the future.
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THERAPY, COURSE, AND PROGNOSIS
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Treatment of cobalamin deficiency consists of parenteral CnCbl (vitamin B12) or OHCbl to replace daily losses and refill storage pools, which normally contain 2 to 5 mg of cobalamin.359 Toxicity is highly unusual, and there is no defined upper limit.2 Doses exceeding 100 mcg saturate the cobalamin-binding proteins (TC and HC), and the excess is lost in the urine. A typical treatment schedule consists of 1000 mcg cobalamin intramuscular (IM) daily for 2 weeks, then weekly until the hematocrit is normal, and then monthly for life. For neurologic manifestations, 1000 mcg every 2 weeks for 6 months is recommended. Higher doses are given for certain inherited disorders (e.g., TC deficiency). Transfusion occasionally is required when the hematocrit is less than 15 percent or 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.
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Response to Treatment and Therapeutic Trial
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Following parenteral administration of cobalamin to deficient patients, elevated plasma bilirubin, iron, and LDH levels fall rapidly (Fig. 41–16).360 Decreasing plasma iron turnover and fecal urobilinogen reflect cessation of ineffective erythropoiesis. Within 12 hours, the marrow begins to change from megaloblastic to normoblastic, a process that is complete in 2 to 3 days. Consequently, morphologic diagnosis may be difficult after treatment is initiated. Reticulocytosis begins on days 3 to 5 and peaks on days 4 to 10.361 The new red cells come from new normoblasts, not from the old megaloblasts, most of which die before leaving the marrow.149 Blood hemoglobin concentration becomes normal within 1 to 2 months. If normal values are not achieved by 2 months, another cause of anemia should be sought.
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Other changes include the following: (1) prompt and dramatic improvement in the sense of well-being; (2) normalization of leukocyte and platelet counts, although neutrophil hypersegmentation may persist for 10 to 14 days; (3) rise in serum cobalamin and folate. Cobalamin deficiency does not respond to a physiologic dose of folate (100 to 400 mcg/day), although this dose produces a maximal response in folate deficiency. Larger doses of folate (5 to 15 mg/day) can produce a reticulocytosis and partially or temporarily correct the anemia in cobalamin deficiency. To avoid the risk of masking an underlying cobalamin deficiency by inducing a hematologic remission in response to folate, doses in excess of 1 mg folic acid daily should be shunned until an underlying cobalamin deficiency has been ruled out.3
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Special Circumstances
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Cobalamin should always be given after total gastrectomy. Cobalamin administration is not necessary after partial gastrectomy, but patients need to be watched for megaloblastic anemia, bearing in mind that this anemia can be masked by postgastrectomy iron deficiency.352,362
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The anemia of the blind loop syndrome can be treated by parenteral cobalamin therapy. It also responds after approximately 1 week to oral broad-spectrum antibiotics (cephalexin monohydrate [Keflex] 250 mg QID plus metronidazole 250 mg TID for 10 days),363 and cobalamin absorption is restored. Successful surgical correction of an anatomic lesion also cures the syndrome.
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Treatment consists of a single oral dose of a 50 mg/kg of niclosamide or a dose of 5 to 10 mg/kg of praziquantel.
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Rekindled Use of Oral Cobalamin
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Much interest has been kindled364 regarding the possibility of treating cobalamin deficiency with oral cobalamin as had been proposed previously.365 Oral cobalamin can be used not only for treatment of dietary cobalamin deficiency that occurs in vegans and in patients with very severe general malnutrition, but also for patients with food cobalamin malabsorption366 and for patients with PA, provided the patients are followed carefully.367 In patients lacking intrinsic factor, approximately 1 percent of an oral dose of the vitamin crosses the intestinal epithelium by mass action. Therefore, 1000 to 2000 mcg/day of oral cobalamin supplies most PA patients with their daily cobalamin requirement without the need for injections and their accompanying pain and expense. Cobalamin should be given by mouth to patients with dietary cobalamin deficiency and patients (e.g., hemophiliacs, the frail elderly) who cannot take IM injections.
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ACUTE MEGALOBLASTIC ANEMIA
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Megaloblastic anemia usually is a chronic condition that requires weeks or months to develop, but a potentially fatal megaloblastic state resulting from acute tissue folate or cobalamin deficiency can arise over the course of only a few days. Patients with acute megaloblastic anemia present with rapidly developing thrombocytopenia and/or leukopenia and counts that sometimes fall to very low levels, but little change in red cell levels unless another cause of anemia is present. The discrepancy between platelet and leukocyte counts on the one hand and red cells on the other hand is a reflection of the much longer red cell life span. 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.
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The most common cause of acute megaloblastic anemia is nitrous oxide (N2O) anesthesia.368 N2O rapidly destroys MeCbl,369 leading to a megaloblastic state. AdoCbl eventually is lost, SAMe and total folate levels decline, and the proportion of folate in the form of N5-methyltetrahydrofolate increases.370 Clinical findings develop quickly. Grossly megaloblastic changes are seen in the marrow after 12 to 24 hours.371 Hypersegmented neutrophils do not appear until 5 days after exposure but then persist for several days.372 The effects of N2O disappear spontaneously after a few days; disappearance can be hastened by folinic acid or cobalamin.373 Fatalities resulting from N2O-induced megaloblastosis have occurred in tetanus patients given N2O for weeks.368 Long-term recreational use of N2O has led to a neurologic disorder similar to combined system disease.374
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Acute megaloblastic anemia occurs in other clinical settings. A rapidly developing megaloblastic state with acute thrombocytopenia has occurred in seriously ill patients, often in intensive care units.375 Especially at risk are patients who are transfused extensively at surgery,376 those on dialysis or total parenteral nutrition, and those receiving weak folate antagonists such as trimethoprim. Morphologic clues to the diagnosis (e.g., hypersegmented neutrophils) often are absent from the blood film. Both red cell folate and serum cobalamin levels 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.
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MEGALOBLASTIC ANEMIA CAUSED BY DRUGS
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Table 41–5 lists the drugs that cause megaloblastic anemia. Aminopterin and methotrexate are almost structurally identical to folic acid. After they enter cells via the folate carrier377 and acquire a polyglutamate chain,378 they act as very powerful inhibitors of FH2 reductase.379 By blocking the FH2→FH4 reaction and perhaps inhibiting other enzymes of folate metabolism, they effect the rapid withdrawal of folates from the one-carbon fragment carrier pool, causing a fall in nucleotide (especially thymidine) biosynthesis that leads to a major derangement in DNA replication (Chaps. 10 and 22).380
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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). Folic acid itself is useless in this setting because the blocked reductase cannot convert folate to the active tetrahydro form. Folinic acid is already in the tetrahydro form, so folinic acid is effective despite reductase blockade. The usual dose of folinic acid is 3 to 6 mg/day IM. Larger doses are given in chemotherapy protocols that use folinic acid 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.381
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Zidovudine (azidothymidine [AZT]) is used for HIV infections (AIDS; Chap. 81).382 Its principal toxic effect is severe megaloblastic anemia. Anemia or neutropenia produced by zidovudine limits use of this drug.383
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HIV infection itself suppresses hematopoiesis, leading to pancytopenia with myelodysplastic features (Chaps. 81 and 87). The blood film shows vacuolated monocytes. Megaloblastosis in HIV infection may result from folate or cobalamin deficiency384 or AZT or trimethoprim toxicity.
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Hydroxyurea is used at high doses to treat chronic myelogenous leukemia, polycythemia vera, and essential thrombocythemia, and at lower doses to treat psoriasis, rheumatoid arthritis, and sickle cell disease (Chap. 22). It inhibits conversion of ribonucleotides to deoxyribonucleotides.385 Marked megaloblastic changes are routinely found in the marrow 1 to 2 days after initiating hydroxyurea therapy. These changes are rapidly reversed after the drug is withdrawn. Megaloblastosis as a result of N2O is discussed in “Acute Megaloblastic Anemia” above.
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Long-term use of omeprazole and presumably other H+/K+-ATPase inhibitors is associated with reduced serum cobalamin levels, presumably because of the ability of these drugs to inhibit parietal cell function.386 Reduced serum cobalamin levels are not a problem when these drugs are used for short intervals.387
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Pemetrexed is an antifolate approved for use in mesothelioma. It also has been used for treatment of non–small cell lung cancer. Like other antifolate agents, pemetrexed can result in a megaloblastic anemia that is treated with cobalamin and folate. Coadministration of the drug with cobalamin and folate also reduces toxicity. Trimethoprim is a FH2 reductase inhibitor that is designed to act on microbial rather than the mammalian enzyme. Still, in patients with borderline folate status, trimethoprim can precipitate a state of folate deficiency.
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MEGALOBLASTIC ANEMIA IN CHILDHOOD
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Megaloblastic anemia in childhood is usually the result of genetic disorders affecting either the cobalamin binding proteins or the enzymes concerned with intracellular trafficking of cobalamin or its conversion to coenzymatically active forms. Several recent reviews have dealt comprehensively with this topic.321,388,389
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Defects Involving Cobalamin-Binding Proteins
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Several genetic mutations and polymorphisms exist that affect the key binding proteins for cobalamin. Their effects range from being clinically benign to causing severe cobalamin deficiency with megaloblastic anemia and neurologic complications usually manifesting in infancy or early childhood, occasionally in adolescence or early adulthood. In general, the mutations and deletions affecting the encoded proteins cause serious health consequences whereas the polymorphic variants may be totally inconspicuous or result only in a modified likelihood of disease risk.
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Cobalamin malabsorption occurs in four childhood conditions associated with a genetic component: (1) cobalamin malabsorption in the presence of normal intrinsic factor secretion, (2) congenital abnormality of intrinsic factor, (3) TC deficiency, and (4) true PA of childhood. The management of cobalamin deficiency in childhood has been comprehensively reviewed.389
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Selective Malabsorption of Cobalamin, Autosomal Recessive Megaloblastic Anemia, Imerslund-Gräsbeck Disease
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Imerslund-Gräsbeck disease390 is an inherited failure of transport of the intrinsic factor–Cbl complex by the ileum, usually accompanied by proteinuria, mostly of albumin.389 It may be the most common cause of cobalamin deficiency in infancy in some populations.391 Cobalamin deficiency usually is seen before age 2 years, but may appear earlier or later. Cobalamin malabsorption is not corrected by addition of intrinsic factor. Endogenous intrinsic factor and HCl secretion, TC and HC levels, and gastric and intestinal histology are all normal. Intrinsic factor antibodies are absent. Intrinsic factor–Cbl receptors are present in some but not all patients. The molecular defect responsible for this disease has been elucidated. For the ileal phase of cobalamin absorption, two genes code distinct proteins that form part of the cobalamin–IF receptor (cobalamin-intrinsic factor receptor) complex. The first, which codes for the protein CUBN, is affected by several mutations described in Finnish patients with MGA1.95,392 The second, affecting the protein AMN results in a milder MGA1 phenotype and is found in Norwegian patients.95,393 Again, several mutations in the gene coding for the AMN protein have been described.95 Patients are treated with IM cobalamin. The anemia is corrected, but proteinuria persists.
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Congenital Intrinsic Factor Deficiency
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Congenital intrinsic factor deficiency is an autosomal recessive disease in which parietal cells fail to produce functionally normal intrinsic factor.394 Patients present with irritability and megaloblastic anemia when cobalamin stores (<25 mcg at birth) are exhausted. The disease usually presents at age 6 to 24 months. HCl secretion and gastric histology are normal, proteinuria is not present, and antiintrinsic factor antibodies are absent.395 Abnormal cobalamin absorption is corrected by oral intrinsic factor.396 Treatment consists of standard doses of IM cobalamin.
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Transcobalamin Deficiency
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TC deficiency is an autosomal recessive disorder causing a flagrant megaloblastic anemia that generally presents in early infancy.395 The disease is dangerously deceptive because it results from a very severe deficiency of tissue cobalamin, usually with serum cobalamin levels in the normal range because most of the plasma cobalamin is bound to HC resulting in a misleading test result if reliance is placed simply on serum cobalamin measurement. Undiagnosed TC deficiency causes irreversible CNS damage.397 Patients are healthy at birth but over the next few weeks develop signs and symptoms of cobalamin deficiency, such as rapidly progressive pancytopenia, mouth ulcers, vomiting, and diarrhea. Recurrent bacterial infections may occur.395 Neurologic findings are not prominent in the early stages of the disease.397
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Serum folate and cobalamin are normal (the latter because most cobalamin is carried by HC). Homocysteine and/or methylmalonic acid levels are elevated in the plasma.398 The marrow is megaloblastic and the cobalamin absorption is usually but not always abnormal and is not corrected by intrinsic factor.399 The diagnosis is made by measuring plasma TC.389 Prenatal diagnosis is possible.400 Serum should be obtained prior to treatment because TC levels in normal individuals drop sharply after cobalamin is given. TC deficiency is treated with cobalamin doses sufficiently large to force enough vitamin into the cells to allow normal function. Initial therapy can consist of oral CnCbl or OHCbl 500 to 1000 mcg twice a week, or IM OHCbl 1000 mcg/week. Blood counts and symptoms should be monitored and doses adjusted upward if necessary.
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Several single nucleotide polymorphisms in the TC gene have been described and the allele frequency of the most common form (776 C>G) is high in certain populations.401,402 HoloTC levels are lower and methylmalonate levels are higher in individuals homozygous for the G allele,401,402 suggesting that this genotype may be associated with less-favorable cobalamin status.64
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Haptocorrin Deficiency
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Congenital deficiency is not associated with clinically manifested cobalamin deficiency, although the plasma or serum cobalamin levels are well below normal,345 and this is how the condition is recognized. The absence of morbidity in these patients indicates that HCs are not essential for health.
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True Juvenile Pernicious Anemia
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True PA, with gastric atrophy and a defect in intrinsic factor secretion, is exceedingly rare in childhood.403 Patients usually present in their teens with cobalamin deficiency. Serum antiintrinsic factor antibodies usually are present.265 The diagnosis and treatment are the same as for PA in adults.
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INBORN ERRORS OF COBALAMIN METABOLISM
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Cobalamin is converted to AdoCbl and MeCbl by a complex series of transformations involving several steps.388,398,404 Eight disorders affecting this cobalamin transformation pathway have been described, one for each of the steps. Because 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 sequential capital letters preceded by a cbl prefix. The disorders can be grouped into three broad clinical syndromes based on the abnormal metabolites in the patient’s urine (Table 41–6). These disorders are usually discovered during investigation of infants with unexplained developmental delay, acidosis, anemia, or unexplained neurologic difficulties. Typically they have normal plasma cobalamin levels.
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Methylmalonic Aciduria Only (cblA, cblB, and cblH)
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In cblA and cblB, AdoCbl production is impaired but MeCbl production is normal. This may result either from an abnormal methylmalonyl CoA mutase (designated muto or mut–) or from a defect in activation or production of its cofactor, AdoCbl. The cblH variant appears to represent an interallelic variant of cblA.405 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 mcg/day of OHCbl or CnCbl, although muto and mut– patients are unresponsive.
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Homocystinuria Only (Cble and Cblg)
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In these disorders, N5-methyltetrahydrofolate-homocysteine methyltransferase is defective and lacks the capacity to produce MeCbl.406 In patients with cblG, methionine synthase is missing or defective.407 cblE results from failure to reactivate methionine synthase that was inactivated by oxidation of its bound cobalamin.408 Patients present in infancy with vomiting, mental retardation, and megaloblastic anemia. They have marked homocystinuria and hyperhomocysteinemia without methylmalonic aciduria or methylmalonic acidemia. They respond well to CnCbl 1000 mcg/day or 1000 mcg/week. Infants diagnosed prenatally and treated from birth usually show normal development. On rare occasions, this disorder may first become apparent in adult life.
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Methylmalonic Aciduria and Homocystinuria (Cblc, Cbld, and Cblf)
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In these disorders, the defect in Cbl transformation affects AdoCbl and MeCbl, probably because reduction of cobalt from Co++ to Co+ is defective. These patients have both hyperhomocysteinemia and methylmalonic acidemia. 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. Older patients present with psychological problems, progressive dementia, and motor signs and symptoms. cblC disease is the most common of the cobalamin inborn errors. In cblF the defect lies in an inability to release cobalamin from lysosomes.409 Megaloblastic anemia occurs in about half the cases. Patients respond partially to 1000 mcg/day of OHCbl or CnCbl.
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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 in “Methylmalonic Aciduria Only” or “Homocystinuria Only,” respectively. Establishing a diagnosis requires a specialized laboratory equipped to do cultured fibroblast complementation studies.388 In a patient suspected of having a cobalamin mutation, treatment should be started pending the test results because early high-dose cobalamin treatment is risk-free and may reduce the chance of damage to the CNS. Fetuses with these diseases have been successfully treated in utero with very large doses of CnCbl given parenterally to the mother.410
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INBORN ERRORS OF FOLATE METABOLISM
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Megaloblastic anemia in infancy has been described in three inherited disorders of folate metabolism.30,389,411
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Hereditary Folate Malabsorption
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Hereditary folate malabsorption is a rare inherited disorder in which patients cannot absorb folate from the gastrointestinal tract or transport it across the choroid plexus and into the cerebrospinal fluid.29,30 The molecular basis for this disorder is caused by abnormalities in the PCFT.29 Patients present with severe megaloblastic anemia, seizures, mental retardation, and other CNS findings.412 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 cerebrospinal fluid folate level. Treatment with daily folinic acid by injection maintains the spinal fluid level and can lead to normal development.389
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Dihydrofolate Reductase Deficiency
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Dihydrofolate reductase deficiency may present isolated megaloblastic anemia within days or weeks after birth. The anemia responds to folinic acid but not to folic acid.413
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N5-Methyltetrahydrofolate–Homocysteine Methyltransferase Deficiency
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Decreased methyltransferase activity was described in a liver biopsy from a child with megaloblastic anemia and mental retardation. The anemia failed to respond to folate, cobalamin, or pyridoxal phosphate.414 The phenotype of this disorder resembles the inborn errors of cobalamin metabolism affecting the methionine synthesis reaction and has not been well characterized as a distinct entity at the molecular level.
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Methylene Tetrahydrofolate Reductase Deficiency
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In this rare autosomal recessive disorder there is a severe hyperhomocysteinemia and homocystinuria with low plasma methionine. Patients have neurologic and vascular complications but no megaloblastic anemia or methylmalonic aciduria.389 The polymorphic variations in MTHFR have been discussed earlier as well as their influence on disease susceptibility and the influence of the enzyme on the distribution of major folate species toward either methylation or DNA synthetic pathways.
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Hereditary Orotic Aciduria
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Hereditary orotic aciduria is an autosomal recessive disorder of pyrimidine metabolism415 characterized by megaloblastic anemia, growth impairment, and excretion of orotic acid in the urine. Cobalamin and folate levels are normal.
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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 hypoxanthine–guanine phosphoribosyltransferase deficiency. One patient described had megaloblastic anemia.416
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Thiamine-Responsive Megaloblastic Anemia
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Seven children with severe megaloblastic anemia, sensorineural deafness, and diabetes mellitus, all beginning in infancy, have been reported. The anemia responded to thiamine (25 to 100 mg/day). The marrow was reported as myelodysplastic in two patients with the disorder.417 The gene for this puzzling disorder has been mapped to the long arm of chromosome 1, and the underlying biochemical defect is caused by reduced nucleic acid production through impairment of the thiamine dependent pentose cycle enzyme transketolase that results in cell-cycle arrest and the megaloblastic phenotype.418 This condition is also discussed in Chap. 44.
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OTHER CAUSES OF MEGALOBLASTIC ANEMIA
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Congenital Dyserythropoietic Anemia
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The congenital dyserythropoietic anemias are lifelong anemias. They often are mild, showing dysplastic changes affecting the red cell line only, most typically multinuclearity of the normoblasts. They appear to result from defects in glycosylation of polylactosaminoglycans linked to membrane proteins and ceramides.419 Of the three types, two (type I usually420 and type III occasionally421) show megaloblastic red cell precursors (Chap. 39).
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Refractory Megaloblastic Anemia
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Refractory megaloblastic anemia is regarded as a manifestation of some sideroblastic anemias (Chap. 59) and myelodysplastic disorders (Chap. 87).422 The megaloblastic changes are atypical. Dysplastic features are confined to the erythroid series. Giant metamyelocytes and bands are absent from the marrow. A few patients with refractory megaloblastic anemia respond to pharmacologic doses of pyridoxine (200 mg/day),423 perhaps because of an effect on serine transformylase, which requires both pyridoxine and folate.
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Acute Erythroid Leukemia
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In acute erythroid leukemia, a variety of acute myelogenous leukemia (Chap. 89)424 nucleated red cells appear on the blood film, there is usually marked anisocytosis and anisochromia, and macrocytes are usually present. The marrow shows pronounced erythroid hyperplasia involving very bizarre looking megaloblast-like red cell precursors, often containing multiple nuclei or nuclear fragments (see Chap. 88, Fig. 88–1) together with increased numbers of blasts. The megaloblastoid erythroid precursors frequently appear vacuolated.
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Consideration of the rarer causes of megaloblastic anemia is important when the common and correctable causes resulting from folate or cobalamin deficiencies have been excluded. This is particularly important in the pediatric age group, but also in patients who are refractory to treatment with either folate or cobalamin.