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Articles |
1
Laboratory of Clinical Biochemistry, Haukeland University Hospital, N-5021 Bergen, Norway.
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Medical Department,
3
Department of Clinical Chemistry, and
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Department of Pathology, N-4604 Kristiansand, Norway.
5
LOCUS for Homocysteine and Related Vitamins, Armauer Hansens hus, University of Bergen, N-5020 Bergen, Norway.
a Author for correspondence. Fax 47-5-5973115; e-mail bjorn.bolann{at}ikb.uib.no
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Abstract |
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Methods: We investigated the ability of hematological variables, serum cobalamin, plasma total homocysteine (tHcy), serum and erythrocyte folate, gastroscopy, age, and gender to predict cobalamin deficiency. Patients (n = 196; age range, 17–87 years) who had been referred from general practice for determination of serum cobalamin were studied. Cobalamin deficiency was defined as serum methylmalonic acid (MMA) >0.26 µmol/L with at least 50% reduction after cobalamin supplementation. ROC and logistic regression analyses were used.
Results: Serum cobalamin and tHcy were the best predictors, with areas under the ROC curve (SE) of 0.810 (0.034) and 0.768 (0.037), respectively, but age, intrinsic factor antibodies, and gastroscopy gave additional information.
Conclusions: When cobalamin deficiency is suspected in general practice, serum cobalamin should be the first diagnostic test, and the result should be interpreted in relation to the age of the patient. When a definite diagnosis cannot be reached, MMA and tHcy determination will provide additional discriminative information, but MMA, being more specific, is preferable for assessment of cobalamin status.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence is accumulating that cobalamin deficiency occurs more frequently than previously anticipated, and prevalences of 5–15% in elderly populations have been reported (5)(6)(7). Thus, early detection of impaired cobalamin status is of clinical importance. However, determination of serum cobalamin as a test of cobalamin deficiency has certain limitations. Nearly one-half of patients with subnormal serum cobalamin concentrations do not have evidence of intracellular deficiency, whereas many patients with clinically overt cobalamin deficiency have serum concentrations within the reference interval (8). Therefore, various auxiliary diagnostic strategies have been developed.
Patients with classical pernicious anemia show gastroscopic and biochemical signs of chronic atrophic gastritis, which support the diagnosis of cobalamin deficiency (9)(10). However, in the majority of cases, chronic atrophic gastritis is not the result of an autoimmune disease, but is caused by a Helicobacter pylori infection, which has a more uncertain relation to cobalamin deficiency (11)(12)(13).
Hematologists may base a diagnosis on characteristic changes in the whole blood smear and bone marrow (9), but the diagnostic precision of these morphological variables is limited, and patients without hematological manifestations will be missed by this approach (1)(14).
The imperfection of the classical diagnostic strategies has motivated the development of more reliable tests of functional cobalamin status, including plasma total homocysteine (tHcy)1 and serum methylmalonic acid (MMA). Cobalamin serves as a cofactor in the enzymatic conversion of methylmalonyl CoA to succinyl CoA, and the synthesis of methionine from homocysteine. Impaired intracellular cobalamin status leads to the accumulation of MMA and tHcy in the blood (15)(16)(17)(18).
There are few known causes of increased serum MMA other than cobalamin deficiency. Apart from impaired kidney function (19), states of dehydration, and inherited methylmalonic aciduria (20), the only other cause is small-bowel overgrowth with bacteria producing high amounts of propionic acid, the precursor of MMA (21)(22). tHcy, on the other hand, is increased in folate deficiency, vitamin B6 deficiency, renal failure, proliferative disorders, in response to certain drugs, and in some inborn errors of metabolism (23)(24).
In this study, we evaluated traditional clinical and laboratory tests for diagnosing cobalamin deficiency, using reduction of increased serum MMA after cobalamin supplementation as the gold standard of deficiency. Our aim was to establish a better decision strategy for the diagnosis of this common clinical condition in general practice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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300 pmol/L were eligible for the study. Patients with known
hematological or malignant disease, or heart failure (New York Heart
Association classes III and IV), were excluded. A total of 196
subjects, 63 males and 133 females, fulfilled the inclusion criteria.
study protocol
In all participants we determined hemoglobin, hematocrit, mean
corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), platelet
count, serum cobalamin, serum and erythrocyte folate, tHcy, MMA, and
serum creatinine. In a subgroup consisting of subjects with serum
cobalamin 120 pmol/L, MMA >0.26 µmol/L, or tHcy above the 95
percentile (age- and sex-adjusted) of the values found in The Hordaland
Homocysteine Study (25), additional investigations were
carried out, which included a peripheral blood smear, bone marrow
aspiration, gastroscopy with biopsies from the corpus and antrum
ventriculi, and determination of intrinsic factor antibodies.
Cobalamin deficiency was defined by initial MMA values >0.26 µmol/L
(the upper reference limit), which subsequently decreased by at least
50% 4 weeks after the start of cobalamin injections (1 mg of
cyanocobalamin intramuscularly twice weekly for 2.5 weeks). According
to this definition, 51 persons had cobalamin deficiency. Subjects with
MMA 0.26 µmol/L (n = 129) or subjects with MMA >0.26 µmol/L
who did not respond to cobalamin treatment by at least a 50% reduction
(n = 7) were considered nondeficient. Six subjects with MMA >0.26
µmol/L who for various reasons did not receive cobalamin injections,
and three subjects with missing data on MMA were excluded from the
statistical analyses.
The study protocol was in accordance with the Helsinki II declaration, and was approved by the regional ethics committee.
laboratory methods
Serum cobalamin and serum and erythrocyte folate were determined
by Bio-Rad Quantaphase II Radioassay (June 1994 to March 1995) or
Abbott IMx System (from March 1995). When we compared the two methods
as described by Hollis (26), we found no significant bias
for serum cobalamin values 300 pmol/L [n = 17; 95% confidence
interval for the mean difference (Abbott - Bio-Rad) was 0–15
pmol/L]. For folate, the Abbott method gave, on average, results 6%
higher than the Bio-Rad method.
Hematological variables were determined by a Cobas Argos analyzer, whereas creatinine was measured by the Vitros System 950. Intrinsic factor antibodies were determined by a 57Co radioassay. Serum MMA (27) and plasma tHcy (28) were determined by published methods.
Bone marrow aspirate was obtained by sternal puncture and stained with May-Grünwald/Giemsa. Peripheral blood smears were stained with polychrome methylene blue and eosin. Blood and bone marrow smears were investigated by two experienced clinical hematologists blinded to each other, and without knowledge of laboratory data from the patients. The results of blood and bone marrow smears were categorized as normal (0), possibly megaloblastic (1), and megaloblastic (2).
Gastroscopy was performed with an Olympic Videoscope after acid
stimulation with 6 µg/kg pentagastrin. On the basis of visual
inspection, the results were divided into four categories: normal (0),
superficial gastritis (1), atrophic gastritis localized to fundus (2),
and generalized atrophic gastritis (3). Gastric acidity after
pentagastrin stimulation was used either as a continuous variable (pH)
or divided into three categories: achlorhydria (0), pH 
3 (1), and pH
<3 (2). Biopsies were taken from the corpus and antrum ventriculi and
were stained with hematoxylin-eosin and investigated at the Department
of Pathology, Vest-Agder Central Hospital, Kristiansand, Norway. In
addition, the biopsies were stained with combined Alcian-Blue-PAS
stain. The results were grouped into three categories: normal (0),
superficial (not atrophic) gastritis (1), or atrophic gastritis (2).
statistical analyses
The ability of continuous and categorical variables to predict
cobalamin deficiency was evaluated by ROC analysis, using the Astute
Statistics Add-in for Microsoft Excel (29)(30)(31)(32). To
investigate the ability of combinations of variables to predict
cobalamin deficiency, logistic regression analysis was performed
(32)(33), using SPSS for Windows, Ver. 9.0
(SPSS).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution of blood test results is shown in Table 1
. In nondeficient subjects, serum cobalamin was 51–300 pmol/L,
with a median value of 187 pmol/L. In comparison, deficient subjects
had overlapping serum cobalamin (range, 24–260 pmol/L) but with a
lower median value of 105 pmol/L (Table 1
).
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None of the cobalamin-deficient subjects had serum creatinine >114
µmol/L. Five nondeficient subjects had serum creatinine
concentrations of 115–132 µmol/L and MMA values of 0.07–0.25
µmol/L. The mean creatinine value in the deficient patients was not
different from that in the nondeficient group (Table 1
). We found no
correlation between serum creatinine and MMA values in any of the
groups.
roc analyses
For tests that were performed in all participants, the results of
ROC analysis are summarized in Table 2
. The ROC curve for the sensitivity-specificity pairs of serum
cobalamin is shown in Fig. 2
. A large area under the ROC curve suggests good discriminatory
power of the variable. If the area under the ROC curve is not
significantly >0.5, the variable in question does not distinguish
between deficient and nondeficient subjects. Only the first five
variables listed in Table 2
had areas under the ROC curves
significantly >0.5, and among these, serum cobalamin and tHcy were the
best discriminators. Age, MCV, and MCH afforded less discrimination. No
significant discriminatory power was provided by serum or erythrocyte
folate, hematocrit, or hemoglobin.
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When the decision threshold for tHcy was set to 15.0 µmol/L, the sensitivity was 0.73 and the specificity was 0.68. In comparison, the same sensitivity was obtained by serum cobalamin at a threshold of 116 pmol/L, and the corresponding specificity was slightly higher, at 0.74. Thus, these two tests provided essentially the same discrimination at these values. At the commonly used cutoff value for serum cobalamin of 150 pmol/L, the sensitivity was 0.90 and the specificity was 0.60. The same sensitivity was obtained for tHcy at a threshold of 11.3 µmol/L, and the corresponding specificity was only 0.38.
Increased tHcy can also be caused by folate deficiency (23). Of 81 patients with tHcy >15.0 µmol/L, 37 had cobalamin deficiency and the remaining 44 had erythrocyte folate concentrations of 127–829 nmol/L (median, 373 nmol/L; mean, 413 nmol/L; SD, 152 nmol/L). Of these, only seven patients had erythrocyte folate <300 nmol/L, of which two were <150 nmol/L. Thus, factors other than cobalamin or folate deficiency appeared to be responsible for increased tHcy in some subjects.
logistic regression analyses
To study the diagnostic accuracy provided by combinations of
tests, logistic regression analysis (32)(33) was
performed. We included cobalamin, tHcy, age, hemoglobin, MCV, MCH,
hematocrit, erythrocyte and serum folate, and gender in a forward
likelihood ratio stepwise procedure. In this model, serum cobalamin was
the best predictor of cobalamin deficiency. With serum cobalamin in the
model, only age and tHcy gave significant additional information (i.e.,
regression coefficients were significantly different from zero,
P <0.05; Table 3
).
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The correlation between serum cobalamin and probability of cobalamin
deficiency at ages 20–80 years (in a model not including tHcy) is
shown in Fig. 3
A. At the age of 20 years, a serum cobalamin value <47 pmol/L
was associated with a >50% probability of cobalamin deficiency.
At 40, 60, and 80 years, the cobalamin concentrations associated with a
>50% probability of cobalamin deficiency were <72, <97, and <120
pmol/L, respectively.
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The correlation between serum cobalamin and probability of cobalamin
deficiency at various tHcy concentrations at age 60 years is
illustrated in Fig. 3B
. Notably, the probability of cobalamin
deficiency was positively related to tHcy over a wide range of
cobalamin concentrations.
ROC analyses were performed using the logistic result as the variable, with and without tHcy in the model. The areas under the ROC curves were as follows (SE in parentheses): with cobalamin, age, and tHcy in the model, 0.836 (0.033); with only cobalamin and age in the model, 0.825 (0.034).
evaluation of diagnostic procedures carried out in subjects with
low serum cobalamin, high mma, or high tHcy
In a subgroup (n = 108) of the included patients, selected on
the basis of serum cobalamin 120 pmol/L, MMA >0.26 µmol/L, or tHcy
above the 95 percentile of a reference population (25),
invasive procedures such as bone marrow aspiration and gastroscopy with
biopsies were performed. In addition, peripheral blood smears were
performed in these patients. The results of ROC analysis of these data
are shown in Table 4
.
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Low gastric acidity and atrophic gastritis seen at gastroscopy or in
gastric biopsies were significant predictors of cobalamin deficiency
(Table 4
). However, logistic regression analysis showed that when any
one of these three variables was included in the model, the other two
did not give significant additional information. A close correlation
between the results of these investigations may account for this
observation.
Among patients with achlorhydria (n = 16), 69% had cobalamin deficiency. For the remaining patients, there was a positive relationship between cobalamin deficiency and gastric pH over the pH range 0.6–8.2. The logistic regression coefficients (SE in parentheses) were as follows: pH, 0.2451 (0.0935); constant, -1.039 (0.3529).
Peripheral blood smears or bone marrow aspirates had no significant
discriminatory power (Table 4
). The kappa values for interobserver
agreement between the two hematologists were 0.37 for peripheral blood
smear and 0.12 for bone marrow aspirate.
Intrinsic factor antibodies were determined in patients who had invasive procedures. The sensitivity and specificity to detect cobalamin deficiency were 0.17 (95% confidence interval, 0.06–0.28) and 0.96 (95% confidence interval, 0.92–0.999), respectively. When patients were subgrouped according to high and low hemoglobin values, the sensitivity and specificity were about the same in both groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When we used a reduction in increased MMA after cobalamin treatment as
the gold standard, ROC analyses demonstrated that serum cobalamin and
plasma tHcy were the best noninvasive predictors of cobalamin
deficiency (Table 2
). These two variables provided essentially the same
discrimination. Because impaired folate status causes increased tHcy
(35), tHcy may have low diagnostic specificity in an
unselected population. In addition, there are other causes of increased
tHcy (23)(24).
Cobalamin deficiency is associated with increased serum folate,
possibly because of failure to take up or retain methyltetrahydrofolate
in cells ("folate trapping") (36)(37).
However, the serum folate increase that we observed was not sufficient
to be used as a diagnostic tool for detection of cobalamin deficiency
(Table 2
).
ROC analysis gives information about only one test at a time. If results from two tests are correlated, the discriminatory information provided by combining both tests is less than the sum of information from each separate test. In such cases, multivariate logistic regression analysis can be used to determine the additional discriminatory information given by each test in successive diagnostic procedures (32)(33).
Beyond the serum cobalamin concentration, the age of the patient
provided additional predictive information (Fig. 3A
). In addition,
inclusion of tHcy increased the predictive power (Table 3
and Fig. 3B
).
However, when ROC analysis was done with the logistic result as the
variable, the area under the curve was only slightly greater for the
combinations of tests than for cobalamin alone (see
Results).
Intrinsic factor antibodies (determined only in the group selected on the basis of low serum cobalamin, high tHcy, or high MMA) achieved very low sensitivity (0.17) but high specificity (0.96). Obviously, many of our patients were cobalamin-deficient from causes other than classic pernicious anemia.
No other blood test provided significant additional information; the
only exception was serum MMA, which we used as the gold standard to
define cobalamin deficiency. In other words, our data suggest that the
only blood tests that add significant diagnostic information beyond
that provided by serum cobalamin are tHcy, MMA, and if positive,
intrinsic factor antibodies. The erythrocyte indices MCV and MCH may be
of value when used alone (Table 2
), but they add no predictive power
beyond serum cobalamin (Table 3
).
Evaluation of blood and bone marrow smears had no predictive value in
our study (Table 4
). This finding was corroborated by the poor
interobserver agreement between the two hematologists. Changes in
peripheral blood and bone marrow probably do not become evident before
late stages of cobalamin deficiency, and the majority of our patients
were probably in an early phase of the disease. One may object that
these diagnostic procedures were performed in selected patients.
However, a similar selection is done in clinical practice because these
tests are usually preceded by less time- and resource-demanding tests.
The presence of atrophic gastritis, a high pH in the stomach aspirate,
and achlorhydria were highly correlated phenomena. Any one of these
observations significantly increased the probability of cobalamin
deficiency even after serum cobalamin and/or plasma tHcy had been
determined (Table 4
).
Seven patients with MMA above our cutoff of 0.26 µmol/L had a <50% reduction in MMA after cobalamin supplementation and were therefore classified as nondeficient. That means that using MMA >0.26 µmol/L (without cobalamin supplementation) as a diagnostic test for cobalamin deficiency would have a sensitivity of 1.00 and a specificity of 0.95 according to our criteria. Because 0.26 µmol/L is our reference limit for MMA, 2.5% of healthy individuals can be expected to have higher values, and the expected specificity of this approach is thus 0.975.
It cannot be ruled out that some cobalamin-deficient patients have MMA
0.26 µmol/L or fail to show a 50% reduction in MMA after
cobalamin supplementation. These patients are not recognized by our
approach. The number of such false negatives is not known. On the basis
of other criteria, Lindenbaum et al. (15) suggested a
sensitivity for MMA of 0.95, but they used a higher cutoff value.
In a recent report, Hølleland et al. (38) defined cobalamin
deficiency by serum MMA >0.376 µmol/L, which is 3 SD above the mean
of healthy controls. In our study, 13 patients who were
cobalamin-deficient according to our criteria had serum MMA between
0.26 and 0.376 µmol/L, whereas all patients with MMA >0.376 µmol/L
had a serum MMA reduction 50%. Thus, in our study population, use of
a cutoff for serum MMA of >0.376 µmol/L (without cobalamin
supplementation) affords diagnostic sensitivity and specificity of 0.75
and 1.00, respectively.
On the basis of a cost-benefit analysis, Hølleland et al. (38) recommended measurement of MMA in patients when serum cobalamin is >60–90 pmol/L and <200–220 pmol/L. A similar recommendation was made in a recent review (39). Other algorithms also emphasize that cobalamin deficiency should not be ruled out on the basis of normal serum cobalamin values alone (40).
In this study, we emphasized the importance of taking a patient’s age
into consideration. From the calculations presented here, we conclude
that in the individual patient a definite diagnosis of cobalamin
deficiency can be based with reasonable certainty on serum cobalamin
only when a very low cobalamin value is found in a patient of advanced
age (Fig. 3A
). On the other hand, exclusion of cobalamin deficiency
seems justified in subjects with high cobalamin values, especially in
young people, although the results shown in Fig. 3
should be
interpreted with caution. In other cases, the definite diagnosis or
exclusion of cobalamin deficiency should not be based on serum
cobalamin alone, but auxiliary tests such as measurement of MMA or tHcy
should be performed.
The patients in this study were referred from their primary physicians for determination of serum cobalamin. Thus, the above decision strategy is relevant for the diagnosis of cobalamin deficiency in general practice. It may be prudent to determine folate status as well, because concomitant folate deficiency may be present.
The main limitation to a more widespread use of MMA as the
primary test is its high cost. The costs of MMA analysis are 
10-fold
higher than that of serum cobalamin. Until more inexpensive methods for
MMA determination are developed, serum cobalamin may keep its position
as the first-line diagnostic test of cobalamin deficiency.
In conclusion, the results of the present study of subjects with suspected cobalamin deficiency in general practice support the use of serum cobalamin as a first-line diagnostic procedure. Low serum cobalamin values should be interpreted in relation to the age of the patient because the probability of a deficiency state increases with increasing age. In those cases where a likely diagnosis cannot be reached, determination of serum MMA and tHcy may provide additional discriminative information, but MMA is preferable because it combines high sensitivity and specificity. Blood and bone marrow morphologies have poor discriminative power, whereas invasive procedures such as gastroscopy, gastric biopsy, and determination of gastric acidity provide both diagnostic and causal information.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: tHcy, total homocysteine; MMA, methylmalonic acid; MCH, mean corpuscular hemoglobin; and MCV, mean corpuscular volume. 
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References |
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The following articles in journals at HighWire Press have cited this article:
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  A.-L. Bjorke-Monsen, I. Torsvik, H. Saetran, T. Markestad, and P. M. Ueland Common Metabolic Profile in Infants Indicating Impaired Cobalamin Status Responds to Cobalamin Supplementation Pediatrics, July 1, 2008; 122(1): 83 - 91. [Abstract] [Full Text] [PDF]
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 A. Schmedes and I. Brandslund Analysis of methylmalonic Acid in plasma by liquid chromatography-tandem mass spectrometry. Clin. Chem., April 1, 2006; 52(4): 754 - 757. [Abstract] [Full Text] [PDF]
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 R. A. Dhonukshe-Rutten, M. van Zutphen, L. C. de Groot, S. J. Eussen, H. J Blom, and W. A van Staveren Effect of supplementation with cobalamin carried either by a milk product or a capsule in mildly cobalamin-deficient elderly Dutch persons Am. J. Clinical Nutrition, September 1, 2005; 82(3): 568 - 574. [Abstract] [Full Text] [PDF]
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 L. R. Solomon Cobalamin-responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid, and homocysteine testing Blood, February 1, 2005; 105(3): 978 - 985. [Abstract] [Full Text] [PDF]
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H. Refsum, A. D. Smith, P. M. Ueland, E. Nexo, R. Clarke, J. McPartlin, C. Johnston, F. Engbaek, J. Schneede, C. McPartlin, et al. Facts and Recommendations about Total Homocysteine Determinations: An Expert Opinion Clin. Chem., January 1, 2004; 50(1): 3 - 32. [Abstract] [Full Text] [PDF]
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A.-L. B. Monsen, H. Refsum, T. Markestad, and P. M. Ueland Cobalamin Status and Its Biochemical Markers Methylmalonic Acid and Homocysteine in Different Age Groups from 4 Days to 19 Years Clin. Chem., December 1, 2003; 49(12): 2067 - 2075. [Abstract] [Full Text] [PDF]
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 M Vrethem, E Mattsson, H Hebelka, K Leerbeck, A Osterberg, A-M Landtblom, B Balla, H Nilsson, M Hultgren, L Brattstrom, et al. Increased plasma homocysteine levels without signs of vitamin B12 deficiency in patients with multiple sclerosis assessed by blood and cerebrospinal fluid homocysteine and methylmalonic acid Multiple Sclerosis, June 1, 2003; 9(3): 239 - 245. [Abstract] [PDF]
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R. M. Kisabeth Laboratory Adaptations--Changing Expectations Clin. Chem., August 1, 2001; 47(8): 1509 - 1515. [Full Text] [PDF]
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, females; 
, males.
