1. J Neurol (2011) 258:393–401
DOI 10.1007/s00415-010-5764-4
ORIGINAL COMMUNICATION
Utility of measuring vitamin B12 and its active fraction,
holotranscobalamin, in neurological vitamin B12 deficiency
syndromes
Wiebke Schrempf • Marco Eulitz • Volker Neumeister
• Gabriele Siegert • Rainer Koch • Heinz Reichmann •
Alexander Storch
Received: 9 May 2010 / Revised: 30 August 2010 / Accepted: 16 September 2010 / Published online: 2 October 2010
Ó Springer-Verlag 2010
Abstract Vitamin B12 (VitB12, cobalamin) deficiency has renal function patients, 13.2% had VitB12 deficiency. In
been associated with various neuropsychiatric conditions, receiver operating characteristic curve (ROC) analysis,
such as peripheral neuropathy, subacute combined degen- correlation of VitB12 and holoTC with vitamin B12 defi-
eration, affective disorders, and cognitive impairment. ciency was generally weak, and the areas under the curve
Current assays analyze vitamin B12, of which only a small (AUC) were not significantly different for holoTC com-
percentage is metabolically active. Measurement of its pared to vitamin B12 in all subjects (AUC: 0.66 [95%CI:
active fraction, holotranscobalamin, might be of greater 0.51–0.82]; p = 0.04 vs. 0.72 [0.65–0.78], p 0.0001) and
relevance, but data in populations with neuropsychiatric in subcohorts of patients with classical VitB12 deficiency
populations are lacking. In this study, in order to validate syndromes. The positive predictive values for holoTC and
VitB12 and holotranscobalamin (holoTC) serum levels for vitamin B12 were low (14.7 vs. 21.0%) and both were
the detection of VitB12 deficiency in neuropsychiatric associated with more false-positive than true-positive test
conditions, we compared the validity of VitB12 and holoTC results. holoTC does not show superior diagnostic accuracy
in a patient cohort with neuropsychiatric conditions suspi- compared to VitB12 for the detection of VitB12 deficiency in
cious for VitB12 deficiency. The cohort included all patients subjects with neuropsychiatric conditions. Neither test can
admitted to the Department of Neurology at our university be recommended to diagnose VitB12 deficiency in subjects
between 2005 and 2009 with at least two parameters of the with neuropsychiatric disorders.
VitB12 metabolism available (n = 1,279). We used ele-
vated methylmalonic acid as the external validation crite- Keywords Vitamin B12 deficiency Á Cobalamin Á
rion for VitB12 deficiency and restricted our analyses to Holotranscobalamin (holoTC) Á Methylmalonic acid
subjects with normal renal function. Among all normal (MMA) Á Neuropathy Á Dementia
Electronic supplementary material The online version of this
article (doi:10.1007/s00415-010-5764-4) contains supplementary Introduction
material, which is available to authorized users.
Vitamin B12 (cobalamin derivatives) is a water-soluble
W. Schrempf Á M. Eulitz Á H. Reichmann Á A. Storch (&)
Department of Neurology, Dresden University of Technology, vitamin produced in animals and microorganisms. As
Fetscherstrasse 74, 01307 Dresden, Germany cobalamin stores in human liver are high, clinical signs
e-mail: alexander.storch@neuro.med.tu-dresden.de normally appear after years of insufficient intake. Vitamin
B12 (VitB12) deficiency is mainly due to malabsorption
V. Neumeister Á G. Siegert
Institute of Clinical Chemistry, caused by chronic gastritis and gastric atrophy [3], diseases
Dresden University of Technology, of the terminal ileum, alcoholism, long-term use of proton-
01307 Dresden, Germany pump inhibitors, or histamine H2 receptor blockers. Less
common causes are decreased oral intake, for example in
R. Koch
Department of Biometrics and Medical Informatics, vegetarians or vegans, increased requirements in preg-
Dresden University of Technology, 01307 Dresden, Germany nancy, or accelerated loss of the vitamin.
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2. 394 J Neurol (2011) 258:393–401
VitB12 deficiency is not only associated with its classical data in well-characterized large populations with neuro-
symptoms such as pernicious anemia, peripheral neuropa- psychiatric conditions regarding the diagnostic value of
thy, and subacute combined degeneration but also with VitB12 are rather weak [15, 24, 45], and data on holoTC are
various other neurological and/or psychiatric (called herein completely lacking. We therefore investigated the diag-
neuropsychiatric) conditions including affective disorders nostic utility of VitB12 and holoTC in patients with neu-
and cognitive impairment [15, 24, 36, 39, 43]. The prev- ropsychiatric conditions suspicious for VitB12 deficiency.
alence of VitB12 deficiency depends largely on the detec- Since generally accepted diagnostic standards for diagnosis
tion method, and is reported to be around 1–2% in the of VitB12 deficiency are lacking, we used elevated meth-
northern European population [4] and has been estimated ylmalonic acid (MMA) serum levels as the external vali-
to be as high as 5–20% in the elderly [2, 13]. In different dation criterion. MMA levels seem to be the most suitable
trials, the portion of patients presenting with neuropsy- marker for VitB12 deficiency in clinical chemistry studies
chiatric symptoms without any gastrointestinal or hema- using large cohorts [10, 16, 40], since even the gold stan-
tologic symptoms varies widely, ranging between 20 and dard—meaning resolution of symptoms or abnormalities
75%, as macrocytosis and anemia are late indicators of under cobalamin supplementation—is limited by its poor
VitB12 deficiency [24]. Moreover, neuropsychiatric syn- clinical definition and false-negative cases, which are most
dromes might already occur when VitB12 levels are still likely due to irreversible neuropsychiatric deficits [15, 24,
within the normal range [2]. 30, 40].
Pitfalls in detecting VitB12 deficiency are lack of sen-
sitivity and specificity of the established laboratory tests
especially measurement of serum and plasma cobalamin [6, Materials and methods
42]. This is partly due to the fact that only a small per-
centage of total cobalamin is metabolically active, whereas Study population
about 80% is biologically unavailable. Cobalamin bound to
transcobalamin II (holoTC) is the active fraction available The study population included subjects admitted to the
for tissues and cells. Cobalamin is needed as a co-factor in Department of Neurology at the Dresden University of
various enzymatic reactions catalyzing the transformation Technology with neuropsychiatric conditions suspicious for
of L-methlymalonyl-CoA to succinyl-CoA and methionine VitB12 deficiency between March 2005 and January 2009.
to homocysteine as well as ribonucleotides to deoxyribo- We retrospectively analyzed the data of 1,279 patients with
nucleotides. Lack of cobalamin leads to the accumulation at least two parameters of the VitB12 metabolism available
of methylmalonyl acid (MMA) and homocysteine. Early (VitB12, holoTC and/or MMA). Reasons for analysis were
detection of VitB12 deficiency is, however, essential to either suspected peripheral neuropathy (n = 925), cogni-
prevent complications and even irreversible conditions. As tive disorder (n = 107), subacute combined degeneration
strategies of folate supplementation are broadly discussed, (n = 76), or various other causes (n = 171), e.g., suspected
particularly in the older population, and as high folate multiple sclerosis (Table 1 and Supplementary Table S1).
intake may adversely affect patients with cobalamin defi- All patients gave informed consent for laboratory diag-
ciency leading to worsening of neurological dysfunction nostics. Data regarding VitB12 supplementation or intake
though correcting macrocytic anemia, the interest in ade- were not available.
quate diagnostic tools is rising [37].
HoloTC is a relatively new laboratory marker used for Laboratory methods
detecting cobalamin deficiency, as commercially available
tests were implemented just recently. Data regarding its Blood samples were collected by standard venipuncture into
usefulness are, however, conflicting [5, 21, 46]. Trials in serum tubes, allowed to clot at room temperature, centri-
unselected patient populations reported a superior diag- fuged, and serum was collected. Creatinine and VitB12 were
nostic utility of holoTC compared to VitB12 measurements directly analyzed. Samples for analysis of holoTC were
[34]. Clarke and coworkers detected only a modestly frozen at -20°C and thawed before measurement. Samples
higher diagnostic accuracy of holoTC compared to VitB12 for measurement of MMA were sent to a reference labora-
in a population-based study in the elderly [10]. Others tory (Synlab; Weiden, Germany). All parameters were
showed that holoTC and VitB12 have equal diagnostic measured in standard routine diagnostic clinical laboratory
accuracy as screening tools for VitB12 deficiency [29]. with routine intra- and inter-laboratory quality assurance
Although the measurement of VitB12 and/or holoTC (laboratory is certified according to Guidelines of the
serum levels is recommended in various international ¨
Bundesarztekammer, Germany). VitB12 was measured by
management guidelines for dementia, multiple sclerosis, electrochemiluminescence immunoassay (ECLIA; Modular
and/or peripheral neuropathy [8, 11, 14, 18, 23, 31, 41], PPEÒ, Roche Diagnostics, Grenzach-Wyhlen, Germany,
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3. J Neurol (2011) 258:393–401 395
Table 1 Characteristics of study populations by renal function and vitamin B12 deficiency
Study MMA Number Gender (female Age (mean ± SD) Neurological diagnosis/indication
population of subjects [%]/male [%]) [range] in years
(lg/l) (N [%])b Peripheral Subacute Cognitive Various
neuropathy combined impairment (%)c
(%) degeneration (%)
(%)
All All 1,279 (100) 626 (48.9)/653 (51.1) 67.7 ± 15.2 [18–98] 925 (72.3) 76 (5.9) 107 (8.4) 171 (13.4)
B47 638 (85.2) 279 (43.7)/359 (56.3) 62.6 ± 15.1 [21–96] 435 (68.2) 51 (8.0) 34 (5.3) 118 (18.6)
[47 111 (14.8) 39 (35.2)/72 (64.8) 70.4 ± 10.8 [30–93] 85 (76.) 12 (10.8) 5 (4.5) 9 (8.1)
Normal All 851 (66.5) 475 (72.7%)/376 (60.1%) 65.7 ± 15.2 [18–98] 591 (69.4) 58 (6.8) 82 (9.6) 120 (14.1)
renal
functiona
B47 468 (86.8) 185 (39.5)/283 (60.5) 61.8 ± 14.7 [21–96] 304 (65.0) 41 (8.8) 30 (6.4) 93 (19.9)
[47 71 (13.2) 27 (38.0)/44 (62.0) 69.4 ± 11.1 [30–93] 54 (76.1) 7 (9.9) 5 (7.0) 5 (7.0)
Abnormal All 288 (22.5) 97 (14.9)/191(30.5) 77.3 ± 9.9 [34–96] 228 (79.2) 12 (4.2) 25 (8.7) 23 (8.0)
renal
functiona
B47 82 (73.2) 50 (61.0)/32 (39.0) 72.1 ± 11.0 [34–95] 65 (79.3) 7 (8.5) 5 (6.1) 5 (6.1)
[47 30 (26.8) 10 (33.3)/20 (66.7) 73.9 ± 9.6 [48–92] 23 (76.7) 3 (10) 0 (0.0) 4 (13.3)
a
Normal renal function was defined as creatinine B106 lg/l (males), B80 lmol/l (females)
b
Since MMA levels were not available for all participants, the addition of numbers is smaller than the total numbers of participants. Percentages
are of the total participants with available MMA values
c
Refer to Supplementary Table S1 for details
reference from manufacturer: 211–911 pg/ml). HoloTC was Spearman rank coefficient q [ j0.5j. Results are presented
measured by microparticle enzyme immunoassay [MEIA, as mean values ± standard deviations (SD) and range,
Turbidimetrie (AxsymÒ, Fa. Abbot, Chicago, USA, refer- mean values ± 95 confidence intervals (95%CIs) or as
ence from manufacturer: 19–119 pmol/l). Methylmalonic numbers (%). Variables were compared with Student’s
acid was measured by liquid chromatography tandem mass t test or v2 test as appropriate. Statistical significance was
spectrometry [27]; reference: 47 lg/l]. Creatinine was defined for all analyses as p 0.05. Statistical tests were
´
measured by enzymatic color reaction (Jaffe-method; stan- performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA).
Ò
dard spectrometry, Modular PPE , Roche Diagnostics;
reference: 40–80 lmol/l for females/62–106 lmol/l for
males; [28]). Results
Statistical analyses Study population
We defined metabolic VitB12 deficiency if MMA was The whole study population with at least two parameters of
higher than 47 lg/l. We defined renal function as normal if the VitB12 status (VitB12, holoTC and/or MMA) consists of
creatinine was 80 lmol/l in female and 106 lmol/l in 1,279 subjects (Table 1). One of the VitB12 parameters or
male patients. We assessed the performance of specific cut- creatinine was missing for some individuals (see Table 1).
off points for either test to detect VitB12 deficiency using No significant differences of VitB12, holoTC or MMA
specificity and sensitivity, positive and negative predictive between the various cohorts with all or with incomplete
value (PPV, NPV) according to the Bayes’ theorem, and VitB12 parameters available were detected (p values
the likelihood ratio. We used receiver operating curve (F values) of 0.553 (0.757), 0.089 (1.211) and 0.781
(ROC) plots to display the sensitivity and specificity of (0.438) for VitB12, holoTC and MMA, respectively,
holoTC and VitB12 for detection of VitB12 deficiency. The [ANOVA]). The cohort of patients with normal renal
AUC for either test was estimated in the overall population function included 851 subjects (66.5%), while 288 subjects
and in subsets with normal and abnormal renal function. (22.5%) showed abnormal renal function. Mean ± SD age
Correlations of values were estimated using the Spearman of all patients was 67.7 ± 15.2 [range: 18–98] years; and
rank correlation test defining a relevant correlation with a 48.9% were female (for demographic details refer to
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4. 396 J Neurol (2011) 258:393–401
Table 1). Indications for screening for VitB12 deficiency MMA was increased above the normal range ([47 lg/l),
were peripheral neuropathy (72.3%), subacute combined indicating VitB12 deficiency in 14.8% of all individuals.
degeneration (5.9%), cognitive impairment (8.4%), and Increased MMA was significantly more common in renal
other various differential diagnoses for VitB12 deficiency, insufficiency compared to subjects with normal renal
i.e., multiple sclerosis (13.4%, see Supplementary Table S1 function (26.8 vs. 13.2%, p = 0.0008). This is consistent
for details). There were no significant differences of with a mild direct correlation of MMA with creatinine in
diagnoses between all groups (Table 1; p = 0.652; v2 test). the overall cohort (q = 0.342, p 0.0001). However, no
correlations of VitB12 and holoTC with creatinine were
Laboratory measures observed [q = 0.016, p = 0.581 (VitB12); q = 0.069,
p = 0.084 (holoTC)]. The correlations between MMA and
Table 2 shows the distribution of mean values ± SD VitB12 or holoTC were not significant in the overall and
[range] for VitB12, holoTC, MMA, and creatinine for the both renal function cohorts, while we found a relevant
overall cohort and with respect to renal status. Concen- correlation between VitB12 and holoTC in all cohorts (q
trations of MMA were similar in females and males in the values between 0.577 and 0.637; p 0.0001). These
overall cohort (32.8 ± 44.3 vs. 36.9 ± 38.3 lg/l; p = results prompted us to restrict all further calculations to the
0.182), and also after adjusting for creatinine. Concentra- cohort with normal renal function (see Supplementary
tions of VitB12 and holoTC did not differ signifi- Table S2 for data on the overall patient cohort).
cantly between females and males in all cohorts (398 vs.
388 pg/ml; p = 0.564, for VitB12; 51 vs. 47 pmol/l; Comparison of VitB12 with holoTC
p = 0.062, for holoTC in the overall cohort). MMA,
VitB12, and holoTC showed no correlations with age ROC analysis revealed that the sensitivity and specificity of
(Spearman rank coefficients j0.5j for all correlations), any particular cut-off value of both VitB12 and holoTC
and no significant differences with respect to diagnoses were inversely related in subjects with normal renal func-
(Table 1). tion (see Table 3). The AUC values did not show any
Table 2 Distribution of vitamin status and other covariates by renal function and vitamin B12 deficiency
Study MMA Vitamin B12 (pg/ml) n holoTC (pmol/l) n MMA (lg/l) n Creatinine (lg/l) n
population (lg/l) MW ± SD [range] MW ± SD [range] MW ± SD [range] MW ± SD [range]
All All 392.2 ± 337.1 [52–4,000] 1,272 49.5 ± 26.1 [4–128] 687 35.2 ± 41.0 [3.5–704.0] 751 87.5 ± 47.7 [21–901] 1,139
B47 410.5 ± 364.7 [117–4,000] 632 48.7 ± 23.1 [4–128] 143 24.7 ± 8.6 [3.5–47.0] 636 79.2 ± 25.6 [31–312] 550
[47 327.1 ± 331.7 [52–2,100] 111 40.3 ± 17.4 [18–77] 24 95.8 ± 81.7 [47.3–704.0] 111 108.4 ± 106.9 [21–901] 101
Normal renal All 375.3 ± 292.4 [52–4,000] 844 46.6 ± 24.3 [4–128] 429 34.9 ± 46.0 [3.5–704.0] 541 72.8 ± 15.2 [21–106] 851
functiona
B47 408.9 ± 361.9 [117–4,000] 462 47.7 ± 23.5 [4–128] 109 24.1 ± 8.5 [3.5–47] 466 72.9 ± 15.5 [32–106] 468
[47 253.0 ± 107.6 [52–560] 71 35.1 ± 15.5 [18–67] 16 107.3 ± 98.8 [47.3–704.0] 71 75.1 ± 17.3 [21–106] 71
Abnormal All 436.9 ± 423.4 [98–4,000] 288 54.0 ± 29.6 [8–128] 201 41.7 ± 22.9 [12.4–136] 112 130.9 ± 76.2 [81–901] 288
renal
functiona
B47 453.4 ± 478.5 [118–4,000] 82 53.8 ± 26.0 [22–128] 20 30.2 ± 8.7 [12.4–46.4] 82 115.6 ± 38.5 [81–312] 82
[47 492.3 ± 556.5 [177–2,100] 30 54.7 ± 13.8 [44–77] 7 73.0 ± 20.2 [48.9–136.0] 30 187.0 ± 172.0 [875–901] 30
a
Normal renal function was defined as B106 lg/l (males), B80 lmol/l (females)
Table 3 Statistical parameters of ROC analysis
Study populationa Vitamin B12 holoTC
a
AUC [95%CI] p value n AUC [95%CI]a p value n
All 0.72 [0.65–0.78] 0.0001 533 0.66 [0.51–0.82] 0.04 125
Classical VitB12 deficiency syndrome subcohortb 0.71 [0.64–0.78] 0.0001 399 0.59 [0.46–0.72] 0.16 116
Neuropathy subcohort 0.73 [0.66–0.80] 0.0001 355 0.62 [0.45–0.79] 0.14 103
a
Only subjects with normal renal function as defined as B106 lg/l (males), B80 lmol/l (female) were included in the analyses
b
Subcohort of subjects with peripheral neuropathy and/or subacute combined degeneration
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5. J Neurol (2011) 258:393–401 397
differences between VitB12 and holoTC. Figure 1a shows a (85.4–86.8% for VitB12; 96.6–98.9% for holoTC; refer to
comparison of the ROC plots for VitB12 and holoTC to Supplementary Table S3). These data clearly show that the
detect VitB12 deficiency in the normal renal function lower cut-off values provided by the manufacturer are too
cohort, while Fig. 1b, c shows the same for its subcohort low for our cohort.
with clinical syndromes that can clearly result from VitB12 Table 4 compares the utility of VitB12 with holoTC in
deficiency (peripheral neuropathy and/or subacute com- the total normal renal function cohort, the subcohort with
bined degeneration; n = 649) and the peripheral neuropa- classical VitB12 deficiency syndromes (neuropathy and/or
thy subcohort as the most important indications for VitB12 subacute combined degeneration) as well the peripheral
testing (n = 591 [15, 36, 43]). Among the normal renal neuropathy subcohort using cut-off scores from ROC
function cohort, the intervals of cut-off points for VitB12 analyses that provided most approximate sensitivity and
were 200 pg/ml (90th percentile specificity) and 394 pg/ml specificity. Using these cut-off points, both tests performed
(90th percentile sensitivity). The corresponding intervals of very similar with PPVs for VitB12 and holoTC in the low
cut-off points for holoTC were 26 pmol/l (90th percentile range (21.0 and 14.7%, normal renal function cohort) and
specificity) and 55 pmol/l (90th percentile sensitivity). A more false-positive than true-positive test results. Consis-
sensitivity that mostly approximated specificity was tently, sensitivities and specificities were low, while NPVs
observed for VitB12 at 280 pg/ml and for holoTC at showed acceptable values ([85% for all tests/cohorts; see
42 pmol/l. The values obtained when the sensitivity Table 4). As shown in Table 4, similar results were
equaled the specificity were greater for VitB12 with 63 and obtained for the subcohorts.
56% for holoTC. Similar results from the ROC analyses To check whether both tests are suitable screening tools
were obtained for the subcohorts (Table 3). Hence, cut-off for VitB12 deficiency, we calculated the utility of both
points of 42 pmol/l for holoTC and 280 pg/ml for tests for cut-off values generated from ROC analyses with
VitB12 were initially used to compare the utility of both a sufficient sensitivity of 95% (395 pg/ml [VitB12];
tests for diagnosis of VitB12 deficiency. 67 pmol/l [holoTC]). Although NPVs were in suitable
ranges for screening purposes, specificities (35.5 and
Utility of total VitB12 and holoTC for detection 13.8% VitB12 and holoTC, respectively) and PPVs (17.7
of VitB12 deficiency and 13.1%) were rather low (see Supplementary Table S4
for results in all cohorts).
We first analyzed the utility parameters for measuring
VitB12 and holoTC using the cut-off values provided by the
manufacturers (see ‘‘Material and methods’’ section) to Discussion
detect VitB12 deficiency (MMA [ 47 lg/l). In general,
sensitivities of both parameters were low in all analyzed This is the first systematic observation on the utility of
cohorts (36.1–38.9% for VitB12; 6.3–6.7% for holoTC), VitB12 and holoTC to detect VitB12 deficiency in a large
while specificities were in an acceptable range patient cohort with various neuropsychiatric diseases. Our
Fig. 1 Receiver operating characteristics (ROC) curves displaying neuropathy). c Subcohort of patients with peripheral neuropathy
the sensitivity and specificity of serum vitamin B12 and holoTC alone. ROC curves indicated that holoTC had a slightly lower AUC
concentrations to detect patients with metabolic vitamin B12 defi- for the diagnosis of metabolic vitamin B12 deficiency in neuropsy-
ciency in subjects with normal renal function. a Analysis of the total chiatric patients compared to vitamin B12 concentrations in all cohorts
patient cohort. b Subcohort of patients with classical VitB12 (refer to Table 3 for AUC values, 95% confidence intervals and
syndromes (subacute combined degeneration and/or peripheral statistics)
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6. 398 J Neurol (2011) 258:393–401
Table 4 Comparison of the utility of vitamin B12 and holoTC for detection of vitamin B12 deficiency (MMA [ 47 lg/l)
Total vitamin B12 holoTC
All Classical VitB12 Peripheral All Classical VitB12 Peripheral
deficiency neuropathy deficiency neuropathy
syndromesb syndromesb
Sensitivity (a/(a ? b)), n 47/71 (66.2%) 40/61 (65.6%) 37/54 (68.5%) 9/16 (56.3%) 8/16 (50.0%) 8/15 (53.3%)
(%) [95%CI] [55.2–77.2] [54.3–78.1] [56.2–80.8] [32.0–80.6] [25.5–74.5] [28.1–78.5]
Specificity (d/(c ? d)), n 287/462 (62.1%) 212/342 (62.0%) 180/301 (59.8%) 55/109 (50.5%) 45/90 (50.0%) 43/88 (48.9%)
(%) [95%CI] [57.7–66.5] [56.9–67.1] [54.3–65.3] [41.1–59.9] [39.7–60.3] [38.5–59.3]
Positive predictive valuea 21.0 23.5 23.3 14.7 15.1 15.6
(%)
Negative predictive 92.4 91.0 91.4 88.4 84.9 85.5
valuea (%)
Likelihood ratio ([a/ 1.7 1.7 1.7 1.1 1.0 0.9
(a ? b)]/[c/(c ? d])
Only subjects with normal renal function as defined as B106 lg/l (males), B80 lmol/l (female) were included in the analyses
a
Calculated using Bayes’ theorem
b
Subcohort of subjects with peripheral neuropathy and/or subacute combined degeneration
data show rather poor external validity for both tests when of VitB12 deficiency is highly variable between the trials
estimated by ROC analyses using MMA of 47 lg/l or [22, 25, 32, 35] largely precluding direct comparisons
higher as the external validation criterion. The relation- of frequencies of MMA elevations in different studies/
ships between sensitivity and specificity were generally populations.
weak for both tests with similar AUCs from the ROC There is a long-lasting discussion of the most valid
analyses (0.59–0.73, Table 3). This weak relationship led measure for clinically relevant VitB12 deficiency [1, 12, 19,
to insufficient utility parameters for both tests for all cut- 26]. In the present study, we used elevated MMA as the
off values with low sensitivity (NPV) at high specificity external validation criterion because it seems the most
(PPV) or—even more important for screening tests—low suitable marker for VitB12 deficiency in clinical chemistry
specificity (PPV) at high sensitivity (NPV) (Table 4 and studies using large cohorts [10, 16, 40]. In the former lit-
Supplementary Table S3 and S4). Together, our data do not erature, there is a relevant amount of patients with normal
support the use of both VitB12 and holoTC to screen or VitB12 levels, but with significant neurological symptoms
detect VitB12 deficiency in neuropsychiatric patients. It is associated with VitB12 deficiency which consequently
again noted that we only discuss the results in the nor- respond to cobalamin substitution [24]. Most of these
mal renal function cohort because MMA as the external patients showed elevated MMA [24]. Moreover, in patients
validation marker is known to correlate with creatinine with normal renal function, almost all MMA elevations
[16, 17, 20]. respond to cobalamin substitution, and also show patho-
Metabolic VitB12 deficiency was detected in 13.2% of logical results in the deoxyuridine suppression test in a
the subjects with normal renal function (Table 1). There small study, indicating a high specificity of elevated MMA
are—to our knowledge—no similar data to compare these for detecting VitB12 deficiency [7, 24]. Consistently, we
values with those from the literature. A total of 9.4% of learned from studies using elevated MMA and/or respon-
patients with classical VitB12 deficiency syndromes (neu- siveness to cobalamin substitution as markers for VitB12
ropathy and/or subacute combined degeneration) and 9.1% deficiency that its neuropsychiatric spectrum is much wider
of patients with peripheral neuropathy showed VitB12 than formerly suspected using VitB12 concentrations as the
deficiency. These values are comparable to those reported standard measure and thus missing many patients with mild
in previous studies that also used elevated MMA as a VitB12 deficiency (see below). These data have raised the
marker for VitB12 deficiency [30, 38]. Interestingly, our question of whether all elevations of MMA really predict
frequencies are similar to those found in elderly popula- clinically relevant VitB12 deficiency [19]. However, most
tions (difference of mean age of population of approx. studies suggesting low PPV of elevated MMA did not
13 years compared to present population) using a compa- characterize the patients with respect to neuropsychiatric
rable cut-off value of MMA ([52 lg/l ([0.45 lmol/l) vs. symptoms in detail [34] or strongly focus on the classical
47 lg/l ([0.40 lmol/l) in the present study [10]). It is neurological symptoms, such as peripheral neuropathy or
noted that normal ranges of MMA and thus the frequencies subacute combined degeneration, and might thus oversee
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7. J Neurol (2011) 258:393–401 399
patients [19]. There is no final answer to this question modestly superior diagnostic accuracy compared to
because we have neither a clear-cut clinical syndrome nor VitB12, but this test also did not show sufficient diag-
the chance to confirm VitB12 deficiency by another gold nostic validity to screen or diagnose VitB12 deficiency
standard test. The deoxyuridine suppression test with [10, 34]. These findings are consistent with those in our
presumable high validity [7] is not available for larger neuropsychiatric cohort.
studies and thus its validity and its specificity for VitB12 Our study has some methodological limitations: First,
deficiency remains enigmatic. Even the clinical gold not all VitB12 parameters were available in all subjects,
standard—meaning resolution of symptoms or abnormal- since we performed a retrospective analysis using data
ities under cobalamin supplementation—is limited by its from our laboratory information system (see Table 2).
poor clinical definition and false-negative cases, which are However, we calculated the 95%CIs using the actual
most likely due to irreversible neuropsychiatric deficits numbers of subjects in our subcohorts under the worst-case
[15, 24, 30, 40]. Together, even though generally accepted assumption (meaning that sensitivity/specificity are 50%)
diagnostic standards for diagnosis of VitB12 deficiency are using standard statistical procedures [44], demonstrating
not available, elevated MMA serum levels as the external that the size of all analyzed subcohorts is potentially suf-
validation criterion seem the most suitable marker for ficient to provide 95%CIs below 9% (refer to Table 4). We
VitB12 deficiency in large cohort clinical chemistry studies did not detect significant differences of VitB12, holoTC, or
[10, 16, 40]. MMA between the groups of subjects with complete and
Recent studies on VitB12 deficiency have shown a wider incomplete VitB12 parameters available, which largely
spectrum of neuropsychiatric symptoms than previously excludes any biases by this condition. Second, the test–
assumed, including other syndromes like cognitive retest reliabilities of the various tests were not assessed in
impairment, affective disorders, and personality changes our neuropsychiatric population. This is of particular
[9, 15, 24, 36, 39, 43, 48]. Moreover, these studies con- interest because the variation of MMA over time is high
firmed the frequent dissociation of nervous system and [19]. Data on the other VitB12 parameters are not available.
hematological signs noted in the early research with about Third, we did not have any information on cobalamin
a third of patients with VitB12 deficiency severe enough to intake/supplementation therapy, and—due to the retro-
produce megaloblastic anemia but no neuropsychiatric spective nature of our trial—no information on the
symptoms [43] and, conversely, about a fifth of patients response of symptoms to supplementation.
presenting with nervous system disorders but no anemia or
macrocytosis [24]. Most of these investigations used MMA
and/or response to VitB12 supplementation to confirm Conclusions
VitB12 deficiency as the major cause for the symptoms.
The utility of using the response to supplementation as a In conclusion, neither cobalamin nor holoTC showed
marker for VitB12 deficiency is, however, still under suitable diagnostic accuracy for detecting VitB12 defi-
debate, because unresponsive neuropsychiatric symptoms ciency in neuropsychiatric patients when compared to
as a sign for an irreversible neuropsychiatric damage in this elevated MMA levels as the external validation criterion,
disease is well accepted [15, 24, 39]. independent of their renal function state. Therefore, neither
In contrast to most of these previous studies, we used a test can be recommended to investigate patients with sus-
neuropsychiatric population including a wider spectrum of pected VitB12 deficiency. Our data are of interest for
neuropsychiatric syndromes based on the diagnostic work- national guideline committees for dementia, multiple
up of the university-based Department of Neurology to sclerosis, and/or peripheral neuropathy still using VitB12 or
estimate the validity of VitB12 and holoTC to detect holoTC as a screening measure [8, 11, 14, 18, 23, 31, 41].
VitB12 deficiency. Previous studies used data from labo- Data in well-characterized large neuropsychiatric popula-
ratory information systems without clinical data [34] or tions regarding the choice of MMA as the biomarker with
with subsequent clinical examinations of the classical the presumably highest sensitivity is of high relevance
VitB12 deficiency symptoms [19], unselected elderly from a practical point of view: Cobalamin supplementation
patient populations [35, 47] or healthy (elderly) people therapy is cheap and safe and has thus a reasonable risk-to-
[10, 33]. These studies are thus of limited significance for benefit ratio without the problem of overuse in patients
neuropsychiatric populations, particularly concerning the with isolated serum MMA elevations, particularly con-
wider range of symptoms mentioned above. However, cerning the irreversibility of neuropsychiatric symptoms in
similar to our results, all studies showed insufficient a relevant portion of patients [15, 24, 39].
utility of VitB12 to detect VitB12 deficiency as defined by
elevated MMA and/or response to treatment [10, 24, 34]. Conflict of interest The authors declare that they have no conflicts
The few recent studies investigating holoTC revealed a of interest.
123

8. 400 J Neurol (2011) 258:393–401
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