Detection and Diagnosis
“It is particularly important that the diagnosis of cobalamin deficiency be established with a high degree of certainty because cobalamin therapy almost always must be given for the life-time of the patient.” Stabler and Allen, 2004
Keeping vitamin B12 deficiency on your radar screen means staying vigilant during your review of the patient’s history and during the physical exam. Watch for even subtle signs of neurologic or cognitive impairment. Also, note any elements of the patient’s history that might suggest potential malabsorption or malnutrition, such as previously diagnosed pernicious anemia, previous gastrointestinal surgery, vegan diet, and advanced age. Maintain an especially high index of suspicion of vitamin B12 deficiency in new patients who report they were treated with vitamin B12 injections or high doses of oral vitamin B12 supplements by a former provider, but have since discontinued their use. Elderly patients often fail to understand that a true vitamin B12 deficiency due to malabsorption requires life-long treatment.
Early detection and prompt treatment of a vitamin B12 deficiency are essential to prevent development of irreversible neurologic damage, but making an accurate and timely diagnosis can be challenging. The list of related signs and symptoms is long, varied, and non-specific. Many risk factors have been identified, but there are no known necessary or sufficient causes. Complicating things further is the fact that because the liver is a very efficient storage organ for vitamin B12, even completely deficient diets in healthy adults might not result in low serum vitamin B12 levels for several years. Conversely, apparently healthy adults, especially the elderly, consuming diets rich in naturally occurring vitamin B12 can still develop a significant deficiency because of undetected malabsorption. It is possible for vitamin B12 deficiency to develop in a much shorter period of time (months) in some people.
The vitamin B12 literature contains many articles on the relative merits and limitations of the various laboratory testing options. Some tests are more commonly used for the initial assessment, while others, because of their cost, inconvenience, or difficulty of interpretation, are reserved for confirmatory testing in ambivalent situations or are used only in the research setting.
The CBC, smear, and the serum cobalamin (B12) test should be included in the initial laboratory assessment of vitamin B12 deficiency.
After conducting a thorough history and physical examination, if you suspect vitamin B12 deficiency, you should include a complete blood count (CBC), peripheral blood smear, and serum cobalamin (B12) as part of the initial laboratory assessment.58 The serum cobalamin test is readily available and generally affordable, and can detect low serum vitamin B12 levels even among patients who are not anemic.59,60 However, not all patients with a vitamin B12 deficiency will have hematologic manifestations. As Carmel succinctly noted, “the proscription that cobalamin deficiency should not be diagnosed unless megaloblastic changes are found is akin to requiring jaundice to diagnose liver disease.”11
While serum vitamin B12 concentrations are generally accurate,61 many conditions can complicate the interpretation of vitamin B12 laboratory values. Falsely low values have been associated with multiple myeloma, oral contraceptives,62-64 folate deficiency,58-59 and pregnancy.10 Additionally, a low serum B12 level does not automatically mean a deficiency. From 20% - 40% of elderly people with low serum B12 levels have normal metabolite (homocysteine [Hcy] and methylmalonic acid [MMA]) levels and should not be considered deficient. 11
“Accepted lower limits of serum B12 levels in adults range between 170 and 250 pg/ml; however, higher levels (but less than 350 pg/ml) have been recorded in 15% of ostensibly healthy elderly patients with other findings suggestive of a deficiency state, most notably increased levels of serum methylmalonic acid. The true lower limits of normal serum B12 would therefore appear to be somewhat poorly defined.”
Sometimes, a true cobalamin deficiency will not be detected by the serum vitamin B12 test. Some examples of falsely normal serum cobalamin results might be seen with (but not limited to) liver disease,58 myeloproliferative disorders,58 and renal insufficiency.4,65 If a patient has clinical evidence of vitamin B12 deficiency and a normal serum B12 level, it is important to evaluate further.
Opinions differ as to the optimal laboratory cutpoint for the serum vitamin B12 test, due in part to the insidious onset and slow progression of the disorder and limitations of current assays. Research studies and clinical laboratories have tended to dichotomize low values at 200 picograms per milliliter(pg/mL).18,66-67 Stabler and Allen note the following range of serum cobalamin levels among patients with a clinically confirmed B12 deficiency (defined as those who “have objective clinical responses to appropriate therapy”): less than 100 pg/mL, approximately 50%; 100 to 200 pg/mL, approximately 40%; 200 to 350 pg/mL, approximately 10%; and more than 350 pg/mL, approximately 0.1% to 1%.7
Adequate follow up for suspect normal or low normal results is needed through either additional confirmatory testing or a prolonged therapeutic trial followed by metabolic and clinical reassessment.
When the serum vitamin B12 results are suspect, it is helpful to obtain more information.1 Several tests can be used to rule out a vitamin B12 deficiency in either patients with borderline serum cobalamin levels or symptomatic patients with normal serum cobalamin levels.
Homocysteine and Methylmalonic Acid
By far, the most common, accurate, and widely used confirmatory tests for identifying vitamin B12 deficiency are tests for homocysteine (Hcy) and methylmalonic acid (MMA).1 Because cobalamin is necessary for the synthesis of methionine from Hcy, low levels of vitamin B12 lead to increases in total serum Hcy. The total serum Hcy test is a sensitive indicator for a vitamin B12 deficiency; however, its utility is limited as a sole confirmatory test because Hcy levels are also elevated among patients by familial hyperhomocysteinemia, levodopa therapy,68 renal insufficiency and folate deficiency.1,7,69,70
The serum MMA test is more specific for vitamin B12 deficiency than the Hcy test.1,2,7,69-70 MMA levels also increase in the presence of low vitamin B12 levels because cobalamin is required to convert methylmalonyl coenzyme A to succinyl coenzyme A.2 In one study, 98.4% of people with a vitamin B12 level less than 200 pg/mL also had elevated MMA levels (defined as values more than 376 nanomoles per liter [nmol/L]).70 Note that false positive increases in serum MMA have been identified in patients with impaired renal function. It is necessary to rule out whether a patient has either marked intravascular volume depletion or renal insufficiency when interpreting the MMA level, especially in the absence of a low cobalamin level.70 Elevated MMA levels in most patients indicate tissue depletion of vitamin B12. Data from the National Health and Nutrition Examination Survey (NHANES) 2001–2004 in the Table 4 shows the prevalence of vitamin B12 deficiency using combinations of serum B12 levels and MMA levels.
1,000 nmol/L* = 1 µmol/L†
376 nmol/L = 0.376 µmol/L
*nanomols per liter
†micromols per liter
Two popular methods for interpreting diagnostic thresholds for MMA and Hcy elevations are the use of cutpoints determined by laboratory norms (e.g., 3 standard deviations above the mean) and specific values (e.g., MMA >0.26 micromole per liter [µmol/L]69 or >0.4 µmol/L;59 Hcy >15 µmol/L71,72). Many clinicians rely on ranges specified by the clinical laboratories they use.
The cost of testing for MMA or Hcy might be a concern. Quotes from a national laboratory (Atlanta, Georgia, May 2006) state that the direct patient (no insurance) cost for a serum MMA is $212 and for a serum Hcy is $191.
Other metabolites, serum propionate and serum 2-methylcitrate, are also present in vitamin B12 deficiency. However, measuring either of these metabolites has no advantage over measuring MMA for the diagnosis of vitamin B12 deficiency,2 and they are not available routinely in many clinical laboratories.
Again, it is important to remember that abnormal metabolite levels might be due to conditions other than vitamin B12 deficiency, such as renal insufficiency. In one study of the elderly, renal insufficiency was associated with 20% or greater of all abnormal metabolite levels. 11
If the root cause of vitamin B12 deficiency is not obvious, you should consider ordering additional tests to determine it. Antibodies to intrinsic factor (IF) and gastrin or pentagastrin I levels are often used to diagnose pernicious anemia. 10,36,60
Serum holotranscobalamin II measures one of the blood binding proteins used to transport vitamin B12.60 Some investigators recommend it;73 others are concerned about lack of convincing evidence of its value.2,9,10,60 Theoretically, it is attractive, but early claims of its value have been poorly documented. While immunoassays have replaced the older crude methods, it is too early to determine whether measurement of holotranscobalamin II is better than measurement of serum cobalamin. 1
The deoxyuridine suppression test (“DUST” test) has been described as a sensitive indicator of impaired thymidine synthesis due to either deficiency or metabolic inactivation of vitamin B12 or folate.58 However, DUST test is used rarely in the clinical setting because it is not necessary in the evaluation of a vitamin B12 deficiency. DUST is a complicated, expensive, and time-consuming test. 58
The Schilling test is included in most lists of possible vitamin B12 deficiency confirmatory tests, but it is not available in U.S. clinical practices at this time. The Shilling test is the classic test for determining whether a person can absorb vitamin B12. However, a person’s ability to absorb crystalline vitamin B12 can differ from his or her ability to absorb the naturally occurring vitamin B12.59 While it is not an accurate test for identifying cobalamin deficiency, it can be a helpful tool in determining the root of an identified deficiency. It reveals cobalamin malabsorption such as that found in pernicious anemia and ileal disease. A normal Schilling test cannot rule out vitamin B12 deficiency.29,36
There is no gold standard for determining cobalamin deficiency. Part of the problem is related not to the tests used, but to “an uncertain boundary between cobalamin depletion and disease.”1,20
A diagnostic approach, to tailor testing to the nature of the patient’s clinical problem, is suggested by Carmel and summarized in Table 5.1
Flagging the patient’s chart will help you remember to follow up if choosing to “watch and wait” with an asymptomatic patient.
Experienced clinicians differ on the importance of tracking down the root cause of a vitamin B12 deficiency before initiating treatment; however, determining the cause of the deficiency is important ultimately in individualizing the treatment approach.1
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