VN: Vitamin B-12 (2011)

Study Design:
- Click here for explanation of classification scheme.
Quality Rating:
Research Purpose:

To determine the diganostic value of serum levels of holotranscobalamin as an early marker of vitamin B12 deficiency.

Inclusion Criteria:

Specific inclusion criteria was not disclosed by authors. However, various groups were recruited to compare their levels of vitamin B12 using several markers. The groups were characterized by:

  • Adult Germans, male and female, apparently healthy (CONTROL)
  • German and Dutch vegetarians, male and female, apparently healthy
  • Syrians, apparently healthy
  • Elderly (81±7 years old) Germans, males and females
  • Germans with renal disease (pre-dialysis).
Exclusion Criteria:

None disclosed.

Description of Study Protocol:


Authors did not provide information of how subjects were recruited to participate in the study.


This was a study of the sensitivity/specificity of different markers of B12 deficiency.

Statistical Analysis

  • Significant difference among groups with regards to demographic characteristics were determined using chi-square analysis
  • Significant differences between groups with regards to markers of vitamin B12 deficiency were done by Analysis of Variance followed by post-hoc Tukey and Tamhane tests
  • Sensitivity and specificity of holotranscobalamin to determine its diagnostic value was determined by creating ROC curves  
  • Median values and geometric means were used to account for the skewed distribution of the variables.
Data Collection Summary:

Timing of Measurements

Blood samples were collected once under fasting conditions (except renal subjects).


Biological markers of vitamin B12:

  • Serum homocysteine (Hcy): Measured by gas-chromatography
  • Serum methylmalonic acid (MMA): Measured by gas-chromatography
  • Serum folate: Using chemiluminiscence immunoassay
  • Serum vitamin B12: Using chemiluminiscence immunoassay
  • Serum holotrasncobalamin (holoTC): Using RIA-based regeant set.

Independent Variables

Group membership:

  • Vegetarian
  • Omnivorous
  • Elderly
  • Renal patient
  • Ethnic group. 
Description of Actual Data Sample:

 Initial N

Group N Age (Mean)

German controls (omnivorous)

93 (47 males, 46 females)


German and Dutch vegetarians

111 (49 males, 62 females)



122 (66 males, 56 females)


Elderly Germans (81±7 years old)

127 (40 males, 87 females)


Germans with renal disease (pre-dialysis)

92 (49 males, 43 females)



Elderly were significantly different from controls in the proportion of females.

Summary of Results:

Key Findings

  • When serum B12 was used as a marker, 25% to 52% of vegetarians had low B12 status
  • However, when holoTC was used, 73% to 90% of vegetarians were classified as B12 deficient
  • While lower holoTC alone could indicate stage I or II B12 deficiency, the combination of low holoTC (less than 35pmols per L) and elevated MMA (more than 271nmols) indicated stage III B12 deficiency. Stage IV deficiency is when clinical signs appear.

Markers of B12 Status by Comparison Group

Measure German Controls Vegetarians Syrians Elderly Renal Patients

B12, pmol per L, median (5th to 95th) percentiles

287 (195 to 473)

192 (118 to 385)*

240 (151 to 408)*

276 (146)

309 (175 to 695)

B12 less than 156 pmol per L (%)

1 30* 6 7 2

HoloTC, pmol per L median (5th to 95th) percentiles

54 (17 to 114)

23 (2 to 92)*

35 (7 to 194)*

56 (17 to 320)

74 (36 to 221)*

HoloTC less than 35pmols per L (%)

13 72* 50* 21 4

MMA, nmol per L, median (5th to 95th) percentiles

167 (95 to 409)

356 (146 to 2,099)*

265 (105 to 857)*

234 (110 to 809)*

360 (146 to 2,166)*

MMA more than 271nmol per L (%)

8 60* 48* 42* 67*

*Significantly different at P<0.05.

Status of Vitamin B12 among Groups Using Biological Markers
  • 55% of vegetarian subjects had Stage III vitamin B12 deficiency as indicated by low holoTC levels and high MMA
  • Elevated concentrations of MMA (more than 271nmol per L) were detected in 8% of German controls, 60% of vegetarians, 48% of Syrians, 42% of elderly subjects and in 67% of renal patients.
Diagnostic Value of HoloTC
  • Using German controls and vegetarians, ROC curve analysis indicated sensitivity of holoTC was 0.879 compared to 0.836 for serum vitamin B12 levels
  • This indicates holoTC is capable of detecting vitamin B12 deficiency, when present (considered when there were high MMA levels and low holoTC) more often than serum vitamin B12 levels.
Author Conclusion:

Holo TC is the most sensitive marker followed by MMA. The use of holoTC and MMA enables us to differentiate between storage depletion and functional vitamin B12 deficiency. In renal dysfunction, holoTC cannot be used as a marker of vitamin B12 status.

Funding Source:
University/Hospital: Saarland University, Germany
Reviewer Comments:
  • This study provides excellent data that supports the use of holoTC in future research studies that aim to detect and describe the levels and status of vitamin B12 in the population and populations at risk of deficiency. However, the clinical value or its practicality in the clinical setting is not discussed and it is an important factor in order for this marker to be used in this particular setting.
  • Although it was not the objective of the study to determine the degree of vitamin B12 deficiency among vegetarians according the the type of vegetarian diet, it would have helped to inform the results better as to the large proportion of vegetarians identified with abnormal B12 values. Moreover, inclusion, exclusion criteria, recruitment and demographic information for the participants was not provided.
Quality Criteria Checklist: Primary Research
Relevance Questions
  1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies) Yes
  2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? Yes
  3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice? Yes
  4. Is the intervention or procedure feasible? (NA for some epidemiological studies) Yes
Validity Questions
1. Was the research question clearly stated? Yes
  1.1. Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified? Yes
  1.2. Was (were) the outcome(s) [dependent variable(s)] clearly indicated? Yes
  1.3. Were the target population and setting specified? No
2. Was the selection of study subjects/patients free from bias? No
  2.1. Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? No
  2.2. Were criteria applied equally to all study groups? ???
  2.3. Were health, demographics, and other characteristics of subjects described? No
  2.4. Were the subjects/patients a representative sample of the relevant population? ???
3. Were study groups comparable? Yes
  3.1. Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT) N/A
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? Yes
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) No
  3.4. If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis? ???
  3.5. If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.) ???
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? Yes
4. Was method of handling withdrawals described? Yes
  4.1. Were follow-up methods described and the same for all groups? N/A
  4.2. Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.) N/A
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
  4.4. Were reasons for withdrawals similar across groups? N/A
  4.5. If diagnostic test, was decision to perform reference test not dependent on results of test under study? Yes
5. Was blinding used to prevent introduction of bias? Yes
  5.1. In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? N/A
  5.2. Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.) Yes
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? N/A
  5.4. In case control study, was case definition explicit and case ascertainment not influenced by exposure status? N/A
  5.5. In diagnostic study, were test results blinded to patient history and other test results? Yes
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? Yes
  6.1. In RCT or other intervention trial, were protocols described for all regimens studied? N/A
  6.2. In observational study, were interventions, study settings, and clinicians/provider described? N/A
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? N/A
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? N/A
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? N/A
  6.6. Were extra or unplanned treatments described? N/A
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? N/A
  6.8. In diagnostic study, were details of test administration and replication sufficient? Yes
7. Were outcomes clearly defined and the measurements valid and reliable? Yes
  7.1. Were primary and secondary endpoints described and relevant to the question? N/A
  7.2. Were nutrition measures appropriate to question and outcomes of concern? Yes
  7.3. Was the period of follow-up long enough for important outcome(s) to occur? N/A
  7.4. Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? Yes
  7.5. Was the measurement of effect at an appropriate level of precision? Yes
  7.6. Were other factors accounted for (measured) that could affect outcomes? No
  7.7. Were the measurements conducted consistently across groups? Yes
8. Was the statistical analysis appropriate for the study design and type of outcome indicators? Yes
  8.1. Were statistical analyses adequately described and the results reported appropriately? Yes
  8.2. Were correct statistical tests used and assumptions of test not violated? Yes
  8.3. Were statistics reported with levels of significance and/or confidence intervals? Yes
  8.4. Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)? N/A
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? N/A
  8.6. Was clinical significance as well as statistical significance reported? Yes
  8.7. If negative findings, was a power calculation reported to address type 2 error? N/A
9. Are conclusions supported by results with biases and limitations taken into consideration? Yes
  9.1. Is there a discussion of findings? Yes
  9.2. Are biases and study limitations identified and discussed? Yes
10. Is bias due to study's funding or sponsorship unlikely? Yes
  10.1. Were sources of funding and investigators' affiliations described? No
  10.2. Was the study free from apparent conflict of interest? Yes