ANNUAL REVIEWS
1991. 42:311-22 1991 by Annual Reviews Inc.
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DIAGNOSIS OF MEGALOBLASTIC ANEMIA William S. Beck, M.D.
Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114 KEY
WORDS:
cobalamin, folate, homocysteine, methylmalonate
ABSTRACT Megaloblastic anemia can be due to cobalamin deficiency, folate deficiency, or refractory forms of bone marrow disease. This essay reviews current thinking on the diagnostic procedures available to a physician considering these disorders. The questions to be answered are as follows: Is a megaloblastic anemia present? Is there a deficiency of cobalamin or folate? If a deficiency is present, what is its cause? Various diagnostic tests are discussed with regard to the differences in their sensitivity and metabolic implications. In particular, we consider the newest diagnostic tests for cobalamin deficiency, serum homocysteine, and methylmalonate, which appear to be highly sensitive predictors of clinical morbidity. Appli cation of these tests suggests that many more patients are cobalamin deficient than had been supposed.
INTRODUCTION Throughout its long and colorful hist ory, megaloblastic anemia research has yielded surprising new concepts, just when everyone felt safe with the old ones. The most recent to emerge concerns new diagnostic tests with interesting implications. After summarizing current views on the nature of megaloblastic anemia, this brief essay reviews old and new tests and their roles in diagnosis. 311 0066-4219/91/0401-0311 $02.00
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WHAT IS MEGALOBLASTIC ANEMIA? Megaloblastic anemia, a common disorder, is a manifestation of impaired DNA synthesis that has various underlying causes. Although anemia is usually more prominent than thrombocytopenia and neutropenia, there is often a pancytopenia associated with a familiar morphologic pattern in blood and bone marrow cells-and indeed in all proliferating cells-that includes gigantism of these cells and various signs of impaired cell division (1,2). Description
The anemia is typically macrocytic, though not all macrocytic anemias are megaloblastic and not all megaloblastic anemias are macrocytic. Mega loblastic blood cell precursors (megaloblasts) contain a normal or increased amount of DNA and an increased amount of RNA per cell, which accounts for cytoplasmic basophilia in Wright's-stained smears. Defective DNA replication generally reflects impaired conversion of deoxy uridylate (dUMP) to thymidylate (dTMP), as a result of which there is decreased intracellular dTMP and dTTP and increased dUMP and dUTP.
Thus the dUTP/dTTP ratio rises (3-5) and dUTP is misincorporated into DNA. DNA uracil is removed by uracil-DNA-glycosylase (6), but dTTP is unavailable for repair and DNA becomes increasingly fragmented. This irreversibly impairs cell division and causes eventual cell death. Causes
The many disorders leading to megaloblastic anemia occur in three broad etiological categories: (a ) those due to cobalamin (vitamin B12) deficiency that respond to cobalamin therapy, (b) those due to folate deficiency that respond to folic acid therapy, and (c) refractory marrow disorders not due to cobalamin or folate deficiency and not reversed by their administration. It is rarely possible to infer the underlying cause from clinical features of the anemia alone. Deficiencies of cobalamin and folate themselves have many specific causes and are the most common categories. Pernicious anemia, for exam ple, is but one cause of cobalamin deficiency. Both of these vitamin deficiencies lead to tissue coenzyme deficiencies that are usually correctable by vitamin repletion. Hematopoiesis then reverts from megaloblastic to normoblastic. Diagnostic study of megaloblastic anemia is imperative because it guides the choice of therapy and often discloses a significant underlying disorder. Other mechanisms obviously underlie megaloblastic anemias that are unresponsive to cobalamin and folic acid. The main underlying factors
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in this category are (a) cytotoxic drugs such as cytosine arabinoside or methotrexate, and (b) a variety of refractory anemias that probably rep resent clones of cells with defective enzymes of DNA biosynthesis. These disorders, which are usually associated with normal serum vitamin levels and lack of response to vitamin therapy, are not discussed further in this essay. Awareness of the major disorders leading to cobalamin and folate deficiency is important in diagnosis-and the history should probe for circumstances such as those mentioned below. Although exceptions abound, it is useful to keep in mind certain familiar prototypes-the elderly widower subsisting on tea and toast,the chronic alcoholic,and so on. In addition,the history can provide other clues useful in the differential diagnosis of cobalamin and folate deficiency, e.g. myelopathy suggests cobalamin deficiency and coexisting disease such as cancer or chronic hemolytic anemia suggests folate deficiency. COBALAMIN DEFICIENCY Deficiency of cobalamin, as of all vitamins, may result from inadequate intake, increased requirements or impaired acti vation or utilization in tissues. Poor diet, a rare cause of cobalamin deficiency, occurs mainly in vegetarians who abstain from dairy products and eggs. Most deficiencies result from diminished intestinal absorption of various etiologies. In pernicious anemia,a gastric mucosal defect diminishes intrin sic factor synthesis. Other causes include total (occasionally subtotal) gastrectomy; pancreatic disease; overgrowth of intestinal bacteria in the "blind loop" syndrome, anastomoses, diverticula, and other conditions producing intestinal stasis; infestation with the cobalamin-utilizing fish tapeworm Diphyllobothrium tatum; and organic disease of the ileum that interferes with cobalamin absorption despite the presence of adequate intrinsic factor. Protein-bound or food-bound cobalamin may be malabsorbed without impaired adsorption of free cobalamin. Hence, Schilling test results are normal. This situation may give rise to cobalamin deficiency (7, 8) and, as noted below,has stimulated wider use of the "food Schilling test." Cobalamin deficiency resulting from increased requirements occurs mainly in pregnancy, especially when fetal demands supervene in a setting of poor nutrition. Impaired utilization of cobalamin occurs in various genetic defects, involving deletions or defects of methylmalonyl CoA mutase, transcobalamin II, and enzymes in the pathway of cobalamin adenosylation (9,10). FOLATE DEFICIENCY Unlike cobalamin deficiency, folate deficiency is often nutritional, mainly because the amount of dietary folate is not greatly
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in excess of nutritional requirements and body folate reserves are relatively meager. The frequency of nutritional folic acid deficiency was unap preciated until a serum folate assay became available in the 1960s. Among the factors that lead to inadequate folate intake is excessive cooking of vegetables, a common practice among many peoples who live on finely divided food such as rice. Intestinal malabsorption, another common cause of folate deficiency, is associated with subtotal gastrectomy, nontropical and tropical sprue, regional enteritis, and use of anticonvulsants and other drugs (11). Besides pregnancy, a variety of diseases increase folate requirements, among them hyperactive hematopoiesis of hemolytic anemia, exfoliative skin diseases, malignant tumors (especially when metastatic), and the leukemias and lymphomas. Activation of folic acid is impaired by methotrexate and other folate antagonists.
DIAGNOSTIC APPROACH General Considerations In a clinical setting, diagnosis requires (a) recognition of the presence of
megaloblastic anemia (from evidence of marrow failure and morphologic changes); (b) serum vitamin assays and other tests (described below) to elucidate the broad etiologic category; (c) appropriate studies aimed at elucidating the underlying cause; and Cd) observation of response to specific treatment. Too often patients with megaloblastic anemia are given coba lamin and folic acid and dismissed without further investigation. The Meaning of "Vitamin Deficiency"
Leaving aside the well-known difficulties of establishing meaningful ranges of normal, which are discussed elsewhere (12, 13), and the problem of precision and comparability of various assay kits (14, 15), it is important to recognize that in differing settings the term "vitamin dcficiency" may imply total body vitamin deficiency, low serum vitamin level, or decreased levels of the intracellular coenzymes derived from the vitamin in question. There are no practical methods for measuring total body cobalamin or folate. Certainly, serum levels are not measures of total body levels. A corollary of defining "deficiency" by serum levels is the practical need of physicians to designate a given level as a "cutoff," below which they will initiate further diagnostic studies. The problem with such a definition is that low serum levels (assuming the validity of the "range of normal") may occur without clinical morbidity (16, 17). When there is a decrease in coenzyme levels within body cells sufficient to inhibit relevant coenzyme dependent enzymes, then it will lead to clinical signs and symptoms. It
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appears that diagnostic tests are now available to assess cobalamin and folate coenzyme levels in a clinical setting, providing a more useful defi nition of deficiency. Such considerations are relevant in considering the following diagnostic tests, old and new, which are useful in the evaluation of megaloblastic anemia.
DIAGNOSTIC QUESTIONS TO BE RESOLVED Is There a Deficiency of Cobalamin or Folate?
The first question to be answered requires assays of the serum levels of cobalamin and folate. These may be considered screening tests. The assay of serum cobalamin has long been a diag nostic mainstay (18), despite the discovery in 1978 that normal human serum contains cobalamin analogues, which were assayed as cobalamin by commercial radioisotope dilution assay (RIDA) kits containing R pro tein as binder (19). These analogues do not support the growth of Lacto bacillus leichmannii or other cobalamin-dependent microorganisms. Hence, laboratories, which performed routine assays by microbiological methods, were obtaining lower, but more accurate, results. After 1978, kit-makers replaced R-protein binders with intrinsic factor. Thereafter, the two methods yielded comparable results (12). The nature and possible pathophysiologic significance of serum coba lamin analogues remain unknown. When analogues were quantified by subtracting the results of RIDA assays with intrinsic factor as binder from results with R protein as binder, some observations suggested that analogue levels may be higher in patients with the neuropathy of cobalamin deficiency (20). Cited ranges of normal serum cobalamin levels vary with the assay method and the laboratory. Typical published ranges (in pgjmll) are 175725 (12), 115-1150 (21), 130-750 (22), and 245-822 (23). The following points merit emphasis: (a) Serum cobalamin levels generally fall before megaloblastosis or myelopathy appear. (b) These manifestations are very probably (but not always) present when the serum level has fallen below 100 pgjml. (c) Since a level of less than 150 pgjml probably indicates a deficiency state, many physicians regard 200 pgjml as the level below which further studies are initiated. (d) Several conditions (e.g. pregnancy, transcobalamin deficiency) can lower serum cobalamin without inducing intracellular cobalamin deficiency (24). (e) Several conditions (e.g. coex-
SERUM COBALAMIN
I
Cobalamin levels are expressed here in picograms per milliliter, though some prefer
picomoles per liter in accordance with the SI system (29). In this system, a level of 200 pg/ml would be equivalent to 147 pmol/liter.
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isting myelocytic leukemia, other myeloproliferative disease) can elevate serum cobalamin levels enough to invalidate them as indicators of intra cellular cobalamin levels. Studies of the relation between serum and tissue cobalamin levels in normal subjects (25), postgastrectomy patients (26), and patients with pernicious anemia (27, 28) indicate that although the correlation is fairly good in normal subjects, serum levels are often poor indicators of tissue levels. One reason is the poorly understood influence of serum cobalamin binding proteins. Also, kinetic studies do confirm that as deficiency develops serum levels are maintained at the expense of the tissue level. Because cobalamin reserves are normally large, a low serum cobalamin level implies a long-term abnormality. It is, therefore, a compelling reason for further investigation. Although serum cobalamin levels may be normal in the presence of intracellular cobalamin deficiency (and the clinical signs it produces), the opposite pattern also occurs-low serum cobalamin levels in the absence of megaloblastic anemia. Some of the patients with this pattern display neurological disturbances (16). SERUM FOLATE When folate intake is abruptly decreased, serum folate levels fall weeks before megaloblastic anemia appears (30). Hence, low serum folate (below about 3 ng/ml) may signify an actual deficiency of total body folate but only an imminent deficiency of intracellular folate coenzymes. Folic acid deficiency is often diagnosed and treated without a serum folate assay or other diagnostic tests. Such tests, however, are necessary when the diagnosis is in question. As with the assay for serum cobalamin, serum should be taken early in the course of hospitalization for exam ination and saved in case later assay is necessary. This procedure is more important for serum folate than for serum cobalamin assays because improved diet is mOre likely to begin the early repletion of a folate-deficient patient. Serum folate may be elevated in cobalamin deficiency, although fre quently it is not (2). The rise in serum folate is most striking in cobalamin deficient individuals who are replete in folic acid. Cobalamin-deficient individuals who are also folate deficient have serum folate levels in the normal Or low range, although their levels would be lower were it not for cobalamin deficiency.
Other Tests for Cobalamin Deficiency
Methylmalonic aciduria indicates coba lamin deficiency (31, 32) except in rare cases in which it is due to an URINARY METHYLMALONIC ACID
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inborn metabolic error (10). Urinary methylmalonate may be assayed colorimetrically (33), by paper (34), thin-layer (35), or gas (36) chro matography,or by mass spectrometry (37). None of these methods,unfor tunately, is ideally suited for many diagnostic laboratories. Normal subjects excrete only trace amounts of methylmalonate: 0-3.5 mg (0-38 /lmoles) per 24 hr. Excretion increases in cobalamin deficiency, sometimes to 300 mg (3260 /lmoles) or more per 24 hr. In many studies, urinary methylmalonic acid [measured with and without an oral loading dose of D,L-valine (38)] has proved to be normal in folate deficiency and elevated in cobalamin deficiency,often rising before cobalamin levels have fallen below 200 pgjml. Hence,elevated urinary methylmalonic acid may be considered an earlier indication of cobalamin deficiency than depressed serum cobalamin (39), and thus a better indicator of intracellular coba lamin deficiency. SERUM METHYLMALONIC ACID AND HOMOCYSTEINE For years, the closest approach to a test for cobalamin coenzyme levels was the urinary methyl malonic acid. Lacking that measure, which was hard to obtain, it was necessary to guess whether intracellular cobalamin deficiency existed on the basis of serum cobalamin levels and clinical signs. A pitfall of this reasoning is that the clinical signs of intracellular cobalamin deficiency (megaloblastosis, myelopathy) have other possible sources. For example, megaloblastic anemia can be caused by folate deficiency, various drugs, etc,and the myelopathy is mimicked by other disorders. Two new tests-serum methylmalonic acid and serum homocysteine appear to go far toward permitting a direct assessment of intracellular cobalamin levels, though these tests are still not readily available. Proceeding on the logical premise that intracellular cobalamin deficiency should be reflected most sensitively by elevated concentrations of the unmetabolized substrates of the two cobalamin-dependent enzymes-and on the basis of earlier work showing elevations of urinary methylmalonic acid and intracellular homocysteine (40) in cobalamin deficiency-Stabler and coworkers in the laboratories of Allen and Lindenbaum demonstrated the diagnostic value of serum methylmalonic acid and total homocysteine (free plus bound) assays performed by innovative methods employing capillary gas chromatography and mass spectrometry (41-44). Although these costly techniques are still beyond the scope of routine laboratories, the assays are increasingly available in commercial and reference labor atories. Significantly, their precision and careful exploration has redefined the inquiry and in consequence opened a new chapter in the study of an old problem. These studies revealed the following normal ranges (45):
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Serum methylmalonic acid (nmoles/liter) Serum total homocysteine ({lmoles/liter)
Mean+2 SD Mean+3 SD 73-271 53-376 4.1-21.3 5.4-16.2
Data do suggest that these tests are more sensitive than the serum coba lamin level in detecting intracellular cobalamin deficiency. Early studies of normal and deficient subjects showed that about 95% of cobalamin deficient patients had elevated serum methylmalonic acid and total homo cysteine levels (43, 44). 0[86 consecutive cobalamin-deficient patients with serum cobalamin levels below 200 pg/ml, 77% had marked elevations (> 3 standard deviations above the normal mean) of both metabolites, while 9% had elevated methylmalonic acid alone and 8% had a marked elevation of homocysteine alone. Only 6% had normal levels of both metabolites. In a study of 40 nonanemic cobalamin-deficient patients, only 22 had serum cobalamin levels under 100 pg/ml, yet all but one had elevated serum methyl malonic acid levels and all but two had elevated homocysteine levels (46). More recent work (45) indicates that 95% of patients who relapse because of suboptimal therapy display early elevations of serum methyl malonic acid, total homocysteine, or both metabolites, compared to 69% with low serum cobalamin levels. Of 419 consecutive patients with rec ognized Significant cobalamin deficiency, 12 had serum cobalamin levels greater than 200 pg/ml, mild or absent anemia, and (in 5) prominent neurological signs that responded to cobalamin. In all 12 cases, both serum methylmalonic acid and total homocysteine were increased. Observations by others (47) confirm the value of total homocysteine assays in cobalamin deficiency. Such data apparently establish that serum cobalamin is normal in a significant minority of cobalamin-deficient patients and that assay of these serum metabolites is diagnostically essential. What is the Cause of Cobalamin Deficiency? The Schilling Test
Schilling's demonstration that a large parenteral dose (1 mg) of non radioactive cobalamin increases excretion of radioactive cobalamin, pre sumably by blocking cobalamin-binding sites in plasma and liver (48), led to a standard procedure for assessing cobalamin absorption by studies of urinary excretion following oral administration. Though a bedrock of diagnosis, the Schilling test was long more popular in the US than in Europe, where assays of intrinsic factor in gastric juice were widely preferred. Today, it is Universally used despite such sources of error as incomplete urine collections, renal disease, etc. An uncertain number of patients display low serum cobalamin levels with normal Schilling test results. Some of these have been shown to have various gastric disorders that impair the absorption of food-bound
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cobalamin but not the pure cobalamin tracer used in the Schilling test. Among the factors underlying this situation are gastrectomy (49), vago tomy (50), and cimetidine therapy (51). In many, there is no evidence of gastric dysfunction. Recent awareness of this pattern-unexplained low serum cobalamin levels with normal Schilling test results-has stimulated widespread use of a "food Schilling test" (or egg-yolk cobalamin absorption test), in which the ingested labeled cobalamin derives from eggs produced by hens injected with labeled cobalamin (8, 52, 53). One study (54), for example, reported on 47 patients with low serum cobalamin levels and normal Schilling test results. Egg test results were significantly lower than normal, while routine Schilling test results were normal. Twenty subjects had egg test excretions below 1.5%. No other clinical features distinguished them from the 27 who excreted more than 1.5% other than the presence of lower ratios of pepsinogen I: II. Interestingly, 60% of tested patients had neurologic, cerebral, or psychiatric abnormalities. If food cobalamin
malabsorption is often associated with otherwise unexplained low coba lamin levels, then low cobalamin levels in the presence of normal Schilling test results should not be dismissed without first testing for food cobalamin malabsorption, whether or not thcrc is known gastric dysfunction. It is emphasized that the Schilling test is not a test for cobalamin deficiency but for the basis of deficiency, clearly revealing the typical absorptive patterns of such disorders as pernicious anemia and ileal disease. One should not rule out cobalamin deficiency on the basis of a normal Schilling test.
Other Tests for Cobalamin and Folate Deficiency Although the assay of red cell folate is said to provide a better assessment of the level of coenzymes in tissue than serum folate (55), this assay is still not widely used. The test is based on the observation that serum folate· decreases before red cell folate decreases and megaloblastic anemia appears. The test for formiminoglutamic acid in the urine after a loading dose of histidine, the so-called FIGlu test, although a useful and simple test for folate deficiency, is less specific than the serum folate assay (56). It becomes abnormal later than the serum folate and thus gives a better measure of tissue coenzyme levels. Its greatest usefulness is in subjects taking antifolate drugs or drugs simulating their actions in whom serum folate levels may be normal and tissue coenzyme levels drastically reduced. The so-called deoxyuridine suppression test is an isotopic procedure that assays the ability of nonradioactive deoxyuridine to suppress the . incorporation of labeled thymidine into DNA via a pathway that is
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impaired in folate and cobalamin deficiency. The test is useful in inves tigative settings, particularly in detecting the effect of various added metab olities or temporal changes. Though it is said to become abnormal prior to the emergence of clinical signs (57), the complexity of its metabolic basis has been a source of concern (7,58). NOTE ADDED IN PROOF
Since this manuscript was completed, a useful paper has reported studies on 300 unselected consecutive patients with serum cobalamin levels below 200 pg/ml (59). Although the ranges of normal for serum methylmalonate and homocysteine differ from those reported earlier (45) because an older method was employed, there is evidence that many of the patients were not anemic. Of the patients given cobalamin therapy, 59% showed some response and 41% did not. Serum levels of methylmalonate and/or total homocysteine were elevated by more than three standard deviations above the mean for normal subjects in 94% of the responsive patients.
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8. Dawson, D. W., Sawers, A. H., Sharma, R. K. 1984. Malabsorption of protein bound vitamin B12. Br. Med. J. 288: 67578 9. Kano, Y., Sakamoto, S., Miura, Y., Takaku, F. 1985. Disorders of coba lamin metabolism. CRe. Crit. Rev. Oncol. Hematol. 3: 1-34 10. Rosenberg, L. E., Fenton, W. A. 1989. Disorders of propionate and methyl malonate metabolism. In The Metabolic Basis of Inherited Disease, ed. C. R. Scriver, A. L. Beaudet, W. S. Sly, D.
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43. Stabler, S. P., Marcell, P. D., Podell, E. R., Allen, R. H., Lindenbaum, J. 1986. Assay of methylmalonic acid in the serum of patients with cobalamin deficiency using capillary gas chro matography-mass spectrometry. J. C li n. Invest. 77; 1606-12 44. Stabler, S. P., Marcell, P. D., Podell, E. R., Allen, R. H., Savage, D. G., Lin denbaum, J. 1988. Elevation of total homocysteine in the serum of patients with cobalamin or folate deficiency detected by capillary gas chromato graphy-mass spectrometry. J. Clin. Invest. 8!; 466-74 45. Li ndenb aum, J., Savage, D. G., Stabler, S. P., Allen, R. H. 1990. Diagnosis of cobalamin deficiency II. Relative Sen sitivities of serum cobalamin, methyl malonic acid, and total homocysteine concentrations. Am. J. Hematol. 34: 99107 46. Lindenbaum, J., Healton, E. B., Savage, D. G., Brust, J. C. M., Garrett, T. J. et al. 1988. N europ sy chiatri c disorders caused by cobalamin de ficiency in the absence of anemia or macrocytosis. N. Engl. J. Med. 318; 1720-28 47. Chu, R. C, Hall, C A. 1988. The total serum homocysteine as in indicator of vitamin BI2 and folate status. Am. J. Clin. Pathol. 90; 446-49 48. Schilling, R. 1953. Intrinsic factor stud ies. II. The effect of gastric juice on the urinary excretion of radioactivity after the oral administration of radioactive vitamin B12• J. Lab. Clin. Med. 42; 86065 49. Mahmud, K., Kaplan, M. E., R ipley, D., Swaim, W. R., Doscherholmen, A. 1974. The importance of red cell B12 and folate levels after parti al gastrectomy. Am. J. Clin. Nutr. 27; 51-54 50. Streeter, A. M., Duraiappah, B., Boyle, R., O'Neill, B. J., Pheils, M. T. 1974.
51.
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Malabsorption of vitamin B12 after vagotomy. Am. J. Surg. 128: 340-43 Streeter, A. M., Goulston, K. J., Bathur, F. A., Hilmer, R. S., Crane, G. G., Pheils, M. T. 1982. Cimetidine and malabsorption of cobalamin. Dig. Dis. Sci. 27; 13-16 Doscherholmen, A., Swaim, W. R. 1973. Impaired assimilation of egg Co57 vit amin B12 in patients with hypochlor hydria and achlorhydria and after gastric resection. Gastroenterology 64; 913-19 Doscherholmen, A., Silvis, S., McMa hon, J. 1983. Dual isotope Schilling test for measuring absorption of food-bound and free vitamin H\2 simult aneous ly . Am. J. Clin. Pathol. 80: 490-95 Carmel, R., Sinow, R. M., Siegel, M. E., Samloff, L M. 1988. Food cobalamin malabsorption occurs frequently in pati ents with unexplained low serum coba lamin levels. Arch. Intern. Med. 148: 1715-19 Hoffbrand, A. V., Newcombe, F. A., Mallin, D. L. 1966. Method of assay of red cell folate activity and the value of the assay as a test for folate deficiency. J. Clin. Pathol. 19; 17-28 Beck, W. S. 1983. Metabolic aspects of vitamin B12 and folic acid. See Ref. I, pp. 311-31 Carmel, R., Karnaze, D. S. 1985. The deoxyuridine suppression test identifies subtle cobalamin deficiency in patients without typical me galobl astic anemia. J. Am. Med. Assoc. 253: 1284-87 Pelliniemi T. -T. , Beck, W. S. 1980. Bio chemical mechanisms in the Killmann experi ment. Critique of the deoxyuridine suppression test. J. Clin. Invest. 65: 44960 Stabler, S. P., Allen, R. H., Savage, D. G., Lindenbaum, J. 1990. Clinical spec trum and diagnosis of cobalamin defici ency. Blood 76 : 871-81
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DIAGNOSIS OF MEGALOBLASTIC ANEMIA William S. Beck, M.D.
Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114 KEY
WORDS:
cobalamin, folate, homocysteine, methylmalonate
ABSTRACT Megaloblastic anemia can be due to cobalamin deficiency, folate deficiency, or refractory forms of bone marrow disease. This essay reviews current thinking on the diagnostic procedures available to a physician considering these disorders. The questions to be answered are as follows: Is a megaloblastic anemia present? Is there a deficiency of cobalamin or folate? If a deficiency is present, what is its cause? Various diagnostic tests are discussed with regard to the differences in their sensitivity and metabolic implications. In particular, we consider the newest diagnostic tests for cobalamin deficiency, serum homocysteine, and methylmalonate, which appear to be highly sensitive predictors of clinical morbidity. Appli cation of these tests suggests that many more patients are cobalamin deficient than had been supposed.
INTRODUCTION Throughout its long and colorful hist ory, megaloblastic anemia research has yielded surprising new concepts, just when everyone felt safe with the old ones. The most recent to emerge concerns new diagnostic tests with interesting implications. After summarizing current views on the nature of megaloblastic anemia, this brief essay reviews old and new tests and their roles in diagnosis. 311 0066-4219/91/0401-0311 $02.00
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WHAT IS MEGALOBLASTIC ANEMIA? Megaloblastic anemia, a common disorder, is a manifestation of impaired DNA synthesis that has various underlying causes. Although anemia is usually more prominent than thrombocytopenia and neutropenia, there is often a pancytopenia associated with a familiar morphologic pattern in blood and bone marrow cells-and indeed in all proliferating cells-that includes gigantism of these cells and various signs of impaired cell division (1,2). Description
The anemia is typically macrocytic, though not all macrocytic anemias are megaloblastic and not all megaloblastic anemias are macrocytic. Mega loblastic blood cell precursors (megaloblasts) contain a normal or increased amount of DNA and an increased amount of RNA per cell, which accounts for cytoplasmic basophilia in Wright's-stained smears. Defective DNA replication generally reflects impaired conversion of deoxy uridylate (dUMP) to thymidylate (dTMP), as a result of which there is decreased intracellular dTMP and dTTP and increased dUMP and dUTP.
Thus the dUTP/dTTP ratio rises (3-5) and dUTP is misincorporated into DNA. DNA uracil is removed by uracil-DNA-glycosylase (6), but dTTP is unavailable for repair and DNA becomes increasingly fragmented. This irreversibly impairs cell division and causes eventual cell death. Causes
The many disorders leading to megaloblastic anemia occur in three broad etiological categories: (a ) those due to cobalamin (vitamin B12) deficiency that respond to cobalamin therapy, (b) those due to folate deficiency that respond to folic acid therapy, and (c) refractory marrow disorders not due to cobalamin or folate deficiency and not reversed by their administration. It is rarely possible to infer the underlying cause from clinical features of the anemia alone. Deficiencies of cobalamin and folate themselves have many specific causes and are the most common categories. Pernicious anemia, for exam ple, is but one cause of cobalamin deficiency. Both of these vitamin deficiencies lead to tissue coenzyme deficiencies that are usually correctable by vitamin repletion. Hematopoiesis then reverts from megaloblastic to normoblastic. Diagnostic study of megaloblastic anemia is imperative because it guides the choice of therapy and often discloses a significant underlying disorder. Other mechanisms obviously underlie megaloblastic anemias that are unresponsive to cobalamin and folic acid. The main underlying factors
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in this category are (a) cytotoxic drugs such as cytosine arabinoside or methotrexate, and (b) a variety of refractory anemias that probably rep resent clones of cells with defective enzymes of DNA biosynthesis. These disorders, which are usually associated with normal serum vitamin levels and lack of response to vitamin therapy, are not discussed further in this essay. Awareness of the major disorders leading to cobalamin and folate deficiency is important in diagnosis-and the history should probe for circumstances such as those mentioned below. Although exceptions abound, it is useful to keep in mind certain familiar prototypes-the elderly widower subsisting on tea and toast,the chronic alcoholic,and so on. In addition,the history can provide other clues useful in the differential diagnosis of cobalamin and folate deficiency, e.g. myelopathy suggests cobalamin deficiency and coexisting disease such as cancer or chronic hemolytic anemia suggests folate deficiency. COBALAMIN DEFICIENCY Deficiency of cobalamin, as of all vitamins, may result from inadequate intake, increased requirements or impaired acti vation or utilization in tissues. Poor diet, a rare cause of cobalamin deficiency, occurs mainly in vegetarians who abstain from dairy products and eggs. Most deficiencies result from diminished intestinal absorption of various etiologies. In pernicious anemia,a gastric mucosal defect diminishes intrin sic factor synthesis. Other causes include total (occasionally subtotal) gastrectomy; pancreatic disease; overgrowth of intestinal bacteria in the "blind loop" syndrome, anastomoses, diverticula, and other conditions producing intestinal stasis; infestation with the cobalamin-utilizing fish tapeworm Diphyllobothrium tatum; and organic disease of the ileum that interferes with cobalamin absorption despite the presence of adequate intrinsic factor. Protein-bound or food-bound cobalamin may be malabsorbed without impaired adsorption of free cobalamin. Hence, Schilling test results are normal. This situation may give rise to cobalamin deficiency (7, 8) and, as noted below,has stimulated wider use of the "food Schilling test." Cobalamin deficiency resulting from increased requirements occurs mainly in pregnancy, especially when fetal demands supervene in a setting of poor nutrition. Impaired utilization of cobalamin occurs in various genetic defects, involving deletions or defects of methylmalonyl CoA mutase, transcobalamin II, and enzymes in the pathway of cobalamin adenosylation (9,10). FOLATE DEFICIENCY Unlike cobalamin deficiency, folate deficiency is often nutritional, mainly because the amount of dietary folate is not greatly
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in excess of nutritional requirements and body folate reserves are relatively meager. The frequency of nutritional folic acid deficiency was unap preciated until a serum folate assay became available in the 1960s. Among the factors that lead to inadequate folate intake is excessive cooking of vegetables, a common practice among many peoples who live on finely divided food such as rice. Intestinal malabsorption, another common cause of folate deficiency, is associated with subtotal gastrectomy, nontropical and tropical sprue, regional enteritis, and use of anticonvulsants and other drugs (11). Besides pregnancy, a variety of diseases increase folate requirements, among them hyperactive hematopoiesis of hemolytic anemia, exfoliative skin diseases, malignant tumors (especially when metastatic), and the leukemias and lymphomas. Activation of folic acid is impaired by methotrexate and other folate antagonists.
DIAGNOSTIC APPROACH General Considerations In a clinical setting, diagnosis requires (a) recognition of the presence of
megaloblastic anemia (from evidence of marrow failure and morphologic changes); (b) serum vitamin assays and other tests (described below) to elucidate the broad etiologic category; (c) appropriate studies aimed at elucidating the underlying cause; and Cd) observation of response to specific treatment. Too often patients with megaloblastic anemia are given coba lamin and folic acid and dismissed without further investigation. The Meaning of "Vitamin Deficiency"
Leaving aside the well-known difficulties of establishing meaningful ranges of normal, which are discussed elsewhere (12, 13), and the problem of precision and comparability of various assay kits (14, 15), it is important to recognize that in differing settings the term "vitamin dcficiency" may imply total body vitamin deficiency, low serum vitamin level, or decreased levels of the intracellular coenzymes derived from the vitamin in question. There are no practical methods for measuring total body cobalamin or folate. Certainly, serum levels are not measures of total body levels. A corollary of defining "deficiency" by serum levels is the practical need of physicians to designate a given level as a "cutoff," below which they will initiate further diagnostic studies. The problem with such a definition is that low serum levels (assuming the validity of the "range of normal") may occur without clinical morbidity (16, 17). When there is a decrease in coenzyme levels within body cells sufficient to inhibit relevant coenzyme dependent enzymes, then it will lead to clinical signs and symptoms. It
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appears that diagnostic tests are now available to assess cobalamin and folate coenzyme levels in a clinical setting, providing a more useful defi nition of deficiency. Such considerations are relevant in considering the following diagnostic tests, old and new, which are useful in the evaluation of megaloblastic anemia.
DIAGNOSTIC QUESTIONS TO BE RESOLVED Is There a Deficiency of Cobalamin or Folate?
The first question to be answered requires assays of the serum levels of cobalamin and folate. These may be considered screening tests. The assay of serum cobalamin has long been a diag nostic mainstay (18), despite the discovery in 1978 that normal human serum contains cobalamin analogues, which were assayed as cobalamin by commercial radioisotope dilution assay (RIDA) kits containing R pro tein as binder (19). These analogues do not support the growth of Lacto bacillus leichmannii or other cobalamin-dependent microorganisms. Hence, laboratories, which performed routine assays by microbiological methods, were obtaining lower, but more accurate, results. After 1978, kit-makers replaced R-protein binders with intrinsic factor. Thereafter, the two methods yielded comparable results (12). The nature and possible pathophysiologic significance of serum coba lamin analogues remain unknown. When analogues were quantified by subtracting the results of RIDA assays with intrinsic factor as binder from results with R protein as binder, some observations suggested that analogue levels may be higher in patients with the neuropathy of cobalamin deficiency (20). Cited ranges of normal serum cobalamin levels vary with the assay method and the laboratory. Typical published ranges (in pgjmll) are 175725 (12), 115-1150 (21), 130-750 (22), and 245-822 (23). The following points merit emphasis: (a) Serum cobalamin levels generally fall before megaloblastosis or myelopathy appear. (b) These manifestations are very probably (but not always) present when the serum level has fallen below 100 pgjml. (c) Since a level of less than 150 pgjml probably indicates a deficiency state, many physicians regard 200 pgjml as the level below which further studies are initiated. (d) Several conditions (e.g. pregnancy, transcobalamin deficiency) can lower serum cobalamin without inducing intracellular cobalamin deficiency (24). (e) Several conditions (e.g. coex-
SERUM COBALAMIN
I
Cobalamin levels are expressed here in picograms per milliliter, though some prefer
picomoles per liter in accordance with the SI system (29). In this system, a level of 200 pg/ml would be equivalent to 147 pmol/liter.
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isting myelocytic leukemia, other myeloproliferative disease) can elevate serum cobalamin levels enough to invalidate them as indicators of intra cellular cobalamin levels. Studies of the relation between serum and tissue cobalamin levels in normal subjects (25), postgastrectomy patients (26), and patients with pernicious anemia (27, 28) indicate that although the correlation is fairly good in normal subjects, serum levels are often poor indicators of tissue levels. One reason is the poorly understood influence of serum cobalamin binding proteins. Also, kinetic studies do confirm that as deficiency develops serum levels are maintained at the expense of the tissue level. Because cobalamin reserves are normally large, a low serum cobalamin level implies a long-term abnormality. It is, therefore, a compelling reason for further investigation. Although serum cobalamin levels may be normal in the presence of intracellular cobalamin deficiency (and the clinical signs it produces), the opposite pattern also occurs-low serum cobalamin levels in the absence of megaloblastic anemia. Some of the patients with this pattern display neurological disturbances (16). SERUM FOLATE When folate intake is abruptly decreased, serum folate levels fall weeks before megaloblastic anemia appears (30). Hence, low serum folate (below about 3 ng/ml) may signify an actual deficiency of total body folate but only an imminent deficiency of intracellular folate coenzymes. Folic acid deficiency is often diagnosed and treated without a serum folate assay or other diagnostic tests. Such tests, however, are necessary when the diagnosis is in question. As with the assay for serum cobalamin, serum should be taken early in the course of hospitalization for exam ination and saved in case later assay is necessary. This procedure is more important for serum folate than for serum cobalamin assays because improved diet is mOre likely to begin the early repletion of a folate-deficient patient. Serum folate may be elevated in cobalamin deficiency, although fre quently it is not (2). The rise in serum folate is most striking in cobalamin deficient individuals who are replete in folic acid. Cobalamin-deficient individuals who are also folate deficient have serum folate levels in the normal Or low range, although their levels would be lower were it not for cobalamin deficiency.
Other Tests for Cobalamin Deficiency
Methylmalonic aciduria indicates coba lamin deficiency (31, 32) except in rare cases in which it is due to an URINARY METHYLMALONIC ACID
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inborn metabolic error (10). Urinary methylmalonate may be assayed colorimetrically (33), by paper (34), thin-layer (35), or gas (36) chro matography,or by mass spectrometry (37). None of these methods,unfor tunately, is ideally suited for many diagnostic laboratories. Normal subjects excrete only trace amounts of methylmalonate: 0-3.5 mg (0-38 /lmoles) per 24 hr. Excretion increases in cobalamin deficiency, sometimes to 300 mg (3260 /lmoles) or more per 24 hr. In many studies, urinary methylmalonic acid [measured with and without an oral loading dose of D,L-valine (38)] has proved to be normal in folate deficiency and elevated in cobalamin deficiency,often rising before cobalamin levels have fallen below 200 pgjml. Hence,elevated urinary methylmalonic acid may be considered an earlier indication of cobalamin deficiency than depressed serum cobalamin (39), and thus a better indicator of intracellular coba lamin deficiency. SERUM METHYLMALONIC ACID AND HOMOCYSTEINE For years, the closest approach to a test for cobalamin coenzyme levels was the urinary methyl malonic acid. Lacking that measure, which was hard to obtain, it was necessary to guess whether intracellular cobalamin deficiency existed on the basis of serum cobalamin levels and clinical signs. A pitfall of this reasoning is that the clinical signs of intracellular cobalamin deficiency (megaloblastosis, myelopathy) have other possible sources. For example, megaloblastic anemia can be caused by folate deficiency, various drugs, etc,and the myelopathy is mimicked by other disorders. Two new tests-serum methylmalonic acid and serum homocysteine appear to go far toward permitting a direct assessment of intracellular cobalamin levels, though these tests are still not readily available. Proceeding on the logical premise that intracellular cobalamin deficiency should be reflected most sensitively by elevated concentrations of the unmetabolized substrates of the two cobalamin-dependent enzymes-and on the basis of earlier work showing elevations of urinary methylmalonic acid and intracellular homocysteine (40) in cobalamin deficiency-Stabler and coworkers in the laboratories of Allen and Lindenbaum demonstrated the diagnostic value of serum methylmalonic acid and total homocysteine (free plus bound) assays performed by innovative methods employing capillary gas chromatography and mass spectrometry (41-44). Although these costly techniques are still beyond the scope of routine laboratories, the assays are increasingly available in commercial and reference labor atories. Significantly, their precision and careful exploration has redefined the inquiry and in consequence opened a new chapter in the study of an old problem. These studies revealed the following normal ranges (45):
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Serum methylmalonic acid (nmoles/liter) Serum total homocysteine ({lmoles/liter)
Mean+2 SD Mean+3 SD 73-271 53-376 4.1-21.3 5.4-16.2
Data do suggest that these tests are more sensitive than the serum coba lamin level in detecting intracellular cobalamin deficiency. Early studies of normal and deficient subjects showed that about 95% of cobalamin deficient patients had elevated serum methylmalonic acid and total homo cysteine levels (43, 44). 0[86 consecutive cobalamin-deficient patients with serum cobalamin levels below 200 pg/ml, 77% had marked elevations (> 3 standard deviations above the normal mean) of both metabolites, while 9% had elevated methylmalonic acid alone and 8% had a marked elevation of homocysteine alone. Only 6% had normal levels of both metabolites. In a study of 40 nonanemic cobalamin-deficient patients, only 22 had serum cobalamin levels under 100 pg/ml, yet all but one had elevated serum methyl malonic acid levels and all but two had elevated homocysteine levels (46). More recent work (45) indicates that 95% of patients who relapse because of suboptimal therapy display early elevations of serum methyl malonic acid, total homocysteine, or both metabolites, compared to 69% with low serum cobalamin levels. Of 419 consecutive patients with rec ognized Significant cobalamin deficiency, 12 had serum cobalamin levels greater than 200 pg/ml, mild or absent anemia, and (in 5) prominent neurological signs that responded to cobalamin. In all 12 cases, both serum methylmalonic acid and total homocysteine were increased. Observations by others (47) confirm the value of total homocysteine assays in cobalamin deficiency. Such data apparently establish that serum cobalamin is normal in a significant minority of cobalamin-deficient patients and that assay of these serum metabolites is diagnostically essential. What is the Cause of Cobalamin Deficiency? The Schilling Test
Schilling's demonstration that a large parenteral dose (1 mg) of non radioactive cobalamin increases excretion of radioactive cobalamin, pre sumably by blocking cobalamin-binding sites in plasma and liver (48), led to a standard procedure for assessing cobalamin absorption by studies of urinary excretion following oral administration. Though a bedrock of diagnosis, the Schilling test was long more popular in the US than in Europe, where assays of intrinsic factor in gastric juice were widely preferred. Today, it is Universally used despite such sources of error as incomplete urine collections, renal disease, etc. An uncertain number of patients display low serum cobalamin levels with normal Schilling test results. Some of these have been shown to have various gastric disorders that impair the absorption of food-bound
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cobalamin but not the pure cobalamin tracer used in the Schilling test. Among the factors underlying this situation are gastrectomy (49), vago tomy (50), and cimetidine therapy (51). In many, there is no evidence of gastric dysfunction. Recent awareness of this pattern-unexplained low serum cobalamin levels with normal Schilling test results-has stimulated widespread use of a "food Schilling test" (or egg-yolk cobalamin absorption test), in which the ingested labeled cobalamin derives from eggs produced by hens injected with labeled cobalamin (8, 52, 53). One study (54), for example, reported on 47 patients with low serum cobalamin levels and normal Schilling test results. Egg test results were significantly lower than normal, while routine Schilling test results were normal. Twenty subjects had egg test excretions below 1.5%. No other clinical features distinguished them from the 27 who excreted more than 1.5% other than the presence of lower ratios of pepsinogen I: II. Interestingly, 60% of tested patients had neurologic, cerebral, or psychiatric abnormalities. If food cobalamin
malabsorption is often associated with otherwise unexplained low coba lamin levels, then low cobalamin levels in the presence of normal Schilling test results should not be dismissed without first testing for food cobalamin malabsorption, whether or not thcrc is known gastric dysfunction. It is emphasized that the Schilling test is not a test for cobalamin deficiency but for the basis of deficiency, clearly revealing the typical absorptive patterns of such disorders as pernicious anemia and ileal disease. One should not rule out cobalamin deficiency on the basis of a normal Schilling test.
Other Tests for Cobalamin and Folate Deficiency Although the assay of red cell folate is said to provide a better assessment of the level of coenzymes in tissue than serum folate (55), this assay is still not widely used. The test is based on the observation that serum folate· decreases before red cell folate decreases and megaloblastic anemia appears. The test for formiminoglutamic acid in the urine after a loading dose of histidine, the so-called FIGlu test, although a useful and simple test for folate deficiency, is less specific than the serum folate assay (56). It becomes abnormal later than the serum folate and thus gives a better measure of tissue coenzyme levels. Its greatest usefulness is in subjects taking antifolate drugs or drugs simulating their actions in whom serum folate levels may be normal and tissue coenzyme levels drastically reduced. The so-called deoxyuridine suppression test is an isotopic procedure that assays the ability of nonradioactive deoxyuridine to suppress the . incorporation of labeled thymidine into DNA via a pathway that is
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impaired in folate and cobalamin deficiency. The test is useful in inves tigative settings, particularly in detecting the effect of various added metab olities or temporal changes. Though it is said to become abnormal prior to the emergence of clinical signs (57), the complexity of its metabolic basis has been a source of concern (7,58). NOTE ADDED IN PROOF
Since this manuscript was completed, a useful paper has reported studies on 300 unselected consecutive patients with serum cobalamin levels below 200 pg/ml (59). Although the ranges of normal for serum methylmalonate and homocysteine differ from those reported earlier (45) because an older method was employed, there is evidence that many of the patients were not anemic. Of the patients given cobalamin therapy, 59% showed some response and 41% did not. Serum levels of methylmalonate and/or total homocysteine were elevated by more than three standard deviations above the mean for normal subjects in 94% of the responsive patients.
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liams, E. Beutler, A. J. Erslev, M. A. Lichtman, pp. 434-65. New York: McGraw Hill (Blakiston Div.). 3rd ed 2. Beck, W. S. 1988. Megaloblastic an emias. In Cecil's Textbook of Medicine, ed. J. B. Wyngaarden, L. H. Smith, Jr., pp. 900--7. Philadelphia: Saunders. 18th ed 3. Grafstrom, R. H., Tseng, B. Y., Goulian, M. 1978. The incorporation of uracil into animal cell DNA in vitro. Cell
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4. Goulian, M., Bleile, B., Tseng, B. Y. 19!10. Methotrexate-induced misincor poration of uracil into DNA. Proc. Natl. Acad. Sci. USA 77: 1956-60 5. Goulian, M., Bleile, B., Tseng, B. Y. 1980. The effect of methotrexate on levels of dUTP in cells. J. Bioi. Chem. 255: 10630--37 6. Lindahl, T. 1976. New class of enzymes acting on damaged DNA. Nature 259: 64-66 7. Carmel, R., Sinow, W. M., Karnaze, D. S. 1987. Atypical cobalamin deficiency. Subtle biochemical evidence of deficiency is commonly demonstrable in patients without megaloblastic anemia and is often associated with protein hound cobalamin malabsorption. J. Lab. CUn. Med. 109: 454-63
8. Dawson, D. W., Sawers, A. H., Sharma, R. K. 1984. Malabsorption of protein bound vitamin B12. Br. Med. J. 288: 67578 9. Kano, Y., Sakamoto, S., Miura, Y., Takaku, F. 1985. Disorders of coba lamin metabolism. CRe. Crit. Rev. Oncol. Hematol. 3: 1-34 10. Rosenberg, L. E., Fenton, W. A. 1989. Disorders of propionate and methyl malonate metabolism. In The Metabolic Basis of Inherited Disease, ed. C. R. Scriver, A. L. Beaudet, W. S. Sly, D.
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