Total Serum Homocysteine and Methylmalonic Acid Levels

Cellular nutrient deficiency of cobalamin or folate is reflected by decreased intracellular concentrations. Cobalamin deficiency per turbs methionine synthase activity; this results in substrate (homocysteine) buildup and elevated serum levels of homocysteine, which can be measured by a sensitive assay. In addition, cobalamin deficiency perturbs the activity of methylmalonyl-CoA mutase, which leads to elevated serum MMA levels. Thus homocysteine and MMA are sensitive tests for cobalamin deficiency (see Table 1).

Table1. Stepwise Approach to the Diagnosis of Cobalamin and Folate Deficiency

Folate deficiency also results in elevated levels of homocysteine because of reduced activity of the methionine synthase–catalyzed reaction. Total homocysteine concentration, which comprises the sum of all homocysteine species in plasma/serum, including free and protein bound forms, can be measured in plasma or serum. In general, plasma levels are slightly lower. Thus an elevation of both homocysteine and MMA, while consistent with cobalamin deficiency, cannot rule out a combined cobalamin and folate deficiency (see Table 1).

Both homocysteine and MMA levels are elevated in patients with dehydration and renal failure. In addition, propionic acid derived from anaerobic fecal bacterial metabolism can also substantially con tribute to methylmalonate production; in this setting, the fraction of gut flora contribution to MMA can be reduced by treatment with metronidazole.

The normal value for serum homocysteine is 5.1 to 13.9 μM and serum MMA is 70 to 270 nM, and in general the higher the values, the more severe the clinical abnormalities. However, there is a fairly wide range of “normalcy” in homocysteine values because of age-, creatinine-, gender-, diet-, and race-dependent variables. Basal levels of MMA are usually less than 500 nM, and in renal failure, it rarely increases by more than 1000 nM. If unseparated blood stands at room temperature, homocysteine levels will increase over 4 to 24 hours. Frozen serum (remaining from measurements of serum folate or cobalamin) can be used for serum MMA and homocysteine determinations.

Serum MMA levels are elevated in more than 95% of patients with clinically confirmed cobalamin deficiency (with median values of 3500 nM). Serum homocysteine concentrations are elevated in both cobalamin deficiency (median values of 70 μM) and folate deficiency (median values of 50 μM).

Serum Cobalamin Levels

 For the most part, a low serum cobalamin level is an established bio chemical indicator of cobalamin deficiency. In general, in patients with clinical cobalamin deficiency and megaloblastic anemia or neurologic disease consistent with cobalamin deficiency, the sensitivity of cobalamin concentration less than 200 pg/mL (or less than 148 pmol/L) exceeds 95% when the pretest probability is high. However, up to 10% of adults with true cobalamin deficiency have cobalamin values in the low-normal (200 to 300 pg/mL) range and only metabolite testing with homocysteine and MMA will reveal the deficiency (see Table 1).

Thus a serum cobalamin concentration is less than 300 pg/mL in 99% of patients with clinical hematologic or neurologic manifestations of cobalamin deficiency, and a cobalamin level of more than 300 pg/mL predicts folate deficiency or another hematologic or neurologic disease (see Table 1). However, a low serum cobalamin concentration is not synonymous with cobalamin deficiency, and several associated diseases and conditions can falsely raise or lower cobalamin levels (Table 2). Studies have also identified patients with true cobalamin deficiency who have cobalamin levels in the low-normal range. Among 173 unambiguously cobalamin-deficient patients about 5% had normal cobalamin levels.

Table2. Serum Cobalamin: False-Positive and False-Negative Test Results

If the serum cobalamin test is broadly used as a screening test without clinical context, by virtue of the way normalcy is defined, 2.5% of nondeficient individuals will have low levels, which reflects our definition of the lower limit of normal for this test. However, the finding in 2011 that the same blood sample can give different cobalamin results (one below normal versus one above normal) using different commercial assays has been of significant concern. The more recent assays have periodically had such problems, apparently arising from a lack of transparency related to these tests, poor vali dation using low-cobalamin sera, and poor track record of continuous proficiency testing and tracking of assay performance. It is a particularly serious issue when chemiluminescent tests for serum cobalamin give spuriously elevated levels and fail to detect clinically significant severe pernicious anemia. Such a false-negative test result has been attributed to the in vitro binding of anti-IF antibodies (present in the serum of patients with pernicious anemia) to the IF found in the manufacturer’s reagent. However, a reevaluation in 2014 of five different (currently used) automated cobalamin assays found that they are accurate and do not suffer from earlier problems. Nevertheless, the principle that “a clinical presentation which strongly suggests cobalamin deficiency should always lead to a therapeutic trial (with cobalamin replacement) even if the laboratory assay is nonconcordant” must be upheld against future vagaries that can lead to dangerous false-negative errors in laboratory tests.

Therefore in the absence of availability of metabolite tests, if there are hematologic or neurologic findings that are consistent with clinical cobalamin deficiency, and the serum cobalamin level is normal or borderline low, it is entirely appropriate to empirically treat as for a cobalamin deficiency. If there is no improvement in hematologic parameters within a couple of months, provided there are no other conditions that limit a full response to cobalamin (e.g., iron deficiency or underlying thalassemia trait, hypothyroidism, renal disease, infection, alcoholism, or intrinsic hematologic disease in the bone marrow), cobalamin deficiency would be unlikely.

Cobalamin deficiency can falsely raise serum folate by 20% to 30% via methyl-folate trapping. This will seriously underestimate the prevalence of an associated folate deficiency among populations (particularly in resource-limited countries) where the dietary intake of both vitamins is consistently low. Folate deficiency can also reduce serum cobalamin, but the mechanism is unclear. (See box on Summary of the Clinical Usefulness of Tests for Cobalamin and Folate Deficiencies.)

Serum Folate Levels

 The serum folate level is clinically relevant and widely used. Indeed, when combined with a clinical picture of megaloblastic anemia and additional results of cobalamin levels, the serum folate concentration is the cheapest and most useful initial biochemical test to diagnose folate deficiency (see Table 1). Microbiologic assays for folate, which measure all biologically active forms equally, have been replaced in the West by competitive folate-binding protein assays (from various commercial sources) that are indirect immunoassays, which rely on chemiluminescence methods. These tests are notorious for considerable lack of agreement with one another. Alignment with a new higher-order precision isotope-dilution liquid chromatography–tandem mass spectrometry assay, which demonstrates excel lent agreement with the traditional Lactobacillus casei method, will allow better standardization of the current competitive folate-binding protein assays.

The serum folate level is highly sensitive to folate intake, and a single nutritious hospital meal may normalize it in a patient with true folate deficiency. Rapidly developing nutritional folate deficiency first leads to a decline in the serum folate level below normal (less than 2 ng/mL) in about 3 weeks because it is a sensitive indicator of negative folate balance. However, in 25% to 50% of cases (predominantly alcoholics) with folate-deficient megaloblastosis, the serum folate levels may be below normal or borderline (2 to 4 ng/mL). The serum folate level alone should never dictate therapy.

Based on the lower costs of serum cobalamin and folate compared with serum MMA and homocysteine levels, it is recommended (see Table 1) to first use the cheaper tests that can assist in the diagnosis of cobalamin and folate deficiency. Clinicians should also restrict use of serum MMA and homocysteine (a) to patients with borderline cobalamin and folate levels; (b) to patients with existing clinical conditions known to be associated with difficulties in the interpretation of test results; (c) to situations in which cobalamin and folate levels are low, when a high MMA level is useful in confirming cobalamin deficiency (rather than attributing the condition to folate deficiency alone); and (d) to patients with clearly low serum levels but for whom there is an alternative explanation for the findings that caused an unusual serum cobalamin level to be obtained (e.g., a diabetic or alcoholic with peripheral neuropathy, an alcoholic with a high mean corpuscular volume and a low serum cobalamin without anemia). In these cases, serum levels of metabolites can assist in the diagnosis of vitamin deficiency. Diagnostic algorithms consistently stress the value of clinical data to improve the pretest probability of serum cobalamin and serum folate tests. Without detailed clinical information, the combined test results for serum cobalamin, folate, and metabolite (homocysteine and MMA) are not sufficiently unambiguous to diagnose and distinguish cobalamin deficiency from combined cobalamin plus folate deficiency. In combined cobalamin plus folate deficiency, both vitamins would be needed to restore baseline values, particularly of homocysteine.

When negative folate balance continues, hepatic folate stores are depleted in about 4 months. This leads to tissue folate deficiency, which clinically correlates with a decrease in RBC folate (less than 150 ng/mL) by the microbiologic assay. However, current RBC folate tests using different commercial kits have major limitations in sensitivity and specificity and are notoriously unreliable in alcoholics and in pregnancy; furthermore, a reduction of RBC folate also occurs in about 60% of patients with cobalamin deficiency.

The use of red-cell folates as a measure of long-term folate status is valid during clinical trials in which a single kit is used for a cohort of patients; however, it is not valuable for routine clinical diagnosis because of the significant variability of performance between different commercial kits and lack of clinical validation. For these reasons, the serum folate level, although labile, is the preferred initial choice.

However, there are important caveats to measuring serum folate levels in certain clinical settings. First, the serum folate level can be artificially raised in a patient with either pure cobalamin deficiency or combined cobalamin and folate deficiency (Table 3). This is because cessation of the cobalamin-dependent methionine synthase reaction leads to a failure in utilization of intra cellular folate for one-carbon metabolism. As a result, folate leaks out of cells into the plasma, thereby raising the patient’s serum folate level; indeed, replacement of cobalamin alone will return the serum folate level to baseline. Thus associated nutritional cobalamin deficiency has the potential to consistently mask the coexistence of mild-to-moderate folate deficiency if the unwary clinician uses the serum folate level as a gold standard for diagnosing folate deficiency in this clinical setting. Second, such patients (with combined nutritional folate and cobalamin deficiency) often reside in malarious regions where there may be ongoing hemolysis from malaria perse as well as hemolysis from associated hemoglobinopathies that are common in these regions (e.g., thalassemia, sickle cell disease, glucose 6-phosphate dehydrogenase deficiency). In a patient with malaria during hemolysis of Plasmodium falciparum infected erythroid pre cursors, reticulocytes, and mature erythrocytes, there will be substantial release of the 30-fold more folate-rich intraerythrocyte contents into serum, thereby artificially raising the baseline serum folate level. Moreover, red cells normally contain substantial amounts of various forms of folate, that is, 5-methyltetrahydrofolate (monoglutamates) and folate-polyglutamates of different glutamate chain lengths; whereas clearance of such released folate monoglutamates would be hindered with associated cobalamin deficiency, we also know that the released folate polyglutamates are also inefficiently transported back into cells relative to monoglutamates, thereby also resulting in poor clearance. In this clinical context, the current assays for serum folate may not consistently discriminate among these forms of folates. The net result is that a high serum folate could be reported in all such individuals with malaria, even when the patient’s tissue folates are significantly depleted. This predictable masking of tissue folate depletion argues against the use of serum tests for folate deficiency in this clinical setting, where a dietary history of the intake of folate-rich foods in the diet is a better clinical method to assess folate status.

Table3.Serum Folates Are Misleadingly Elevated in Cobalamin (Vitamin B12) Deficiency and/or Malaria Which Are Both Common in Resource-Limited Settings^a,b

Other Tests

The clinical use of low holo-TCII (holo-TCII) levels, to provide information on the extent of saturation of serum TCII as an early marker of cobalamin homeostasis or to diagnose cobalamin deficiency in lieu of serum cobalamin values, is still unclear. This test has not yet been sufficiently clinically validated to define sensitivity, specificity, and other clinical confounders that can alter the results; it has neither supplanted the use of the serum cobalamin test for diagnosis of deficiency, nor is it widely available or used in United States.

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