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Chapter 39 Megaloblastic Anemias 527

Modified Therapeutic Trials Serum Homocysteine and Methylmalonic Acid Levels in Cobalamin and
Folate Deficiencies
The traditional therapeutic trial using physiologic doses of vitamins
(100 µg of folate or 1 µg of cobalamin given daily while monitoring The combined use of homocysteine and methylmalonic acid (MMA)
the reticulocyte response) has given way to a modified therapeutic levels can differentiate cobalamin from folate deficiency, because
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trial. Rather than making the diagnosis of a deficiency, the intention most patients with folate deficiency have normal MMA levels, and
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is often to confirm the clinical suspicion that the patient does not the remainder have only mild elevations. These two tests are useful
have deficiency. This can be demonstrated by lack of response to full diagnostically. The abnormally high levels of metabolites return to
replacement doses of both vitamins (1 mg of folic acid orally for 10 normal only when the patient receives replacement with the appropri-
days and 1 mg of cobalamin intramuscularly or subcutaneously daily ate (deficient) vitamin. A positive response to cobalamin, documented
for 10 days). Clinical scenarios in which such trials may be applicable by falling levels of homocysteine and MMA, is evidence of cobalamin
(after drawing blood for serum cobalamin and folate levels) are as deficiency. Conversely, therapy with folate results in a decrease in the
follows: isolated homocysteine level if folate deficiency is present. Indeed,
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1. There is a clinical suspicion that the underlying disease is not because several variables that are not related to vitamin deficiency
caused by a vitamin deficiency, but this idea is not supported (such as age, mild renal dysfunction) can falsely elevate serum
by results of clinical, morphologic, and biochemical evaluations. homocysteine and MMA levels, if there is ambiguity, proof of vitamin
Such conditions include anemia with a megaloblastic bone deficiency would require clear-cut demonstration of a reduction in
marrow that may be secondary to chemotherapy, myelodysplastic metabolite levels after specific vitamin supplementation. 22,114
syndromes, or acute leukemia; when time is of the essence in
making the diagnosis; when the levels of cobalamin are likely to
be falsely abnormal because of these diseases; or when there is buildup and elevated serum levels of homocysteine, which can be
underlying dehydration or renal dysfunction that predictably gives 22
falsely high levels of metabolites. measured by a sensitive assay. In addition, cobalamin deficiency
2. In other situations, (i.e., pregnancy, acquired immunodeficiency perturbs the activity of methylmalonyl-CoA mutase, which leads to
syndrome [AIDS], or alcoholism) with a multifactorial basis for elevated serum MMA levels. Thus homocysteine and MMA are sensi-
anemia, the response or lack thereof to full replacement doses tive tests for cobalamin deficiency (see Table 39.2).
can eliminate cobalamin or folate deficiency and thereby narrow Folate deficiency also results in elevated levels of homocysteine
the (often extensive) differential diagnosis. because of reduced activity of the methionine synthase–catalyzed
3. In instances when severe anemia with megaloblastosis is clinically reaction. Total homocysteine concentration, which comprises the
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obvious and so serious that the physician cannot wait for the sum of all homocysteine species in plasma/serum, including free and
results of specific tests for deficiency. Full doses of both vitamins protein-bound forms, can be measured in plasma or serum. 22,113 In
are administered, and if there is a response manifested by brisk
reticulocytosis by days 5 to 7, retrospective assignment of the general, plasma levels are slightly lower. Thus an elevation of both
deficiency is based on the results of blood samples drawn before homocysteine and MMA, while consistent with cobalamin deficiency,
beginning the trial. cannot rule out a combined cobalamin and folate deficiency (see
In all therapeutic trials, if there is no evidence of response within 10 Table 39.2); see box on Serum Homocysteine and Methylmalonic
days, bone marrow aspiration is indicated to identify another primary Acid Levels in Cobalamin and Folate Deficiencies.
hematologic disease. Both homocysteine and MMA levels are elevated in patients
with dehydration and renal failure; propionic acid derived from
anaerobic fecal bacterial metabolism can also substantially contribute
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to methylmalonate production. In this setting, the fraction of
Laboratory tests are more likely to be accurate when there is a high gut flora contribution to MMA can be reduced by treatment with
pretest probability of a particular disease. This can, however, be more metronidazole.
vexing in the case of diagnosis of early cobalamin deficiency when The normal value for serum homocysteine is 5.1 to 13.9 µM and
symptoms are subtle, nonspecific, or not yet fully manifest. If there serum MMA is 70 to 270 nM, and in general the higher the values,
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is macrocytosis and cobalamin levels are borderline or just below the the more severe the clinical abnormalities. However, there is a
normal, unequivocal elevation of MMA will support the diagnosis. fairly wide range of “normalcy” in homocysteine values because of
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However, in elderly patients with anemia, there may be several other age-, creatinine-, gender-, diet-, and race-dependent variables.
causes for anemia, and depending on the population studied, cobala- Basal levels of MMA are usually less than 500 nM, and in renal
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min deficiency may be only one among several possible causes in the failure, it rarely increases by more than 1000 nM. If unseparated
differential diagnosis. This is when a modified therapeutic trial—in blood stands at room temperature, homocysteine levels will increase
which a patient is treated with full doses of both cobalamin and folate over 4 to 24 hours. Frozen serum (from measurements of serum
for 10 days—and the lack of objective response to cobalamin would folate or cobalamin) can be used for serum MMA and homocysteine
effectively rule out cobalamin deficiency as a cause (see box on Modi- determinations.
fied Therapeutic Trials). Alternatively, attribution of the cause of Serum MMA levels are elevated in more than 95% of patients
anemia to cobalamin deficiency would be reasonably confirmed ret- with clinically confirmed cobalamin deficiency (with median values
rospectively if there was evidence for resolution of anemia following of 3500 nM). Serum homocysteine concentrations are elevated in
such therapy with cobalamin. Although reductions from high serum both cobalamin deficiency (median values of 70 µM) and folate
MMA and homocysteine values to baseline following therapy would deficiency (median values of 50 µM).
also be confirmatory, this is impractical because of the expense of
multiple testing.
Serum Cobalamin Levels

BIOCHEMICAL EVALUATION OF COBALAMIN AND For the most part, a low serum cobalamin level is an established
FOLATE DEFICIENCIES biochemical indicator of cobalamin deficiency. In general, in patients
with clinical cobalamin deficiency and megaloblastic anemia or
Total Serum Homocysteine and Methylmalonic neurologic disease consistent with cobalamin deficiency, the sensitiv-
ity of cobalamin concentration less than 200 pg/mL (or less than
Acid Levels 148 pmol/L) exceeds 95% when the pretest probability is high.
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However, up to 10% of adults with true cobalamin deficiency have
Cellular nutrient deficiency of cobalamin or folate is reflected by cobalamin values in the low-normal (200 to 300 pg/mL) range and
decreased intracellular concentrations. Cobalamin deficiency perturbs only metabolite testing with homocysteine and MMA will reveal the
methionine synthase activity; this results in substrate (homocysteine) deficiency (see Table 39.2).