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VN: Vitamin B-12 (2011)

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

To identify the status of vitamin B12 in vegetarians (i.e., lactoovovegetarians, lactovegetarians and vegans) using total serum vitamin B12, Methylmalonic acid, total homocysteine and holotranscobalamin II concentrations and compare them with omnivorous subjects.  

Inclusion Criteria:

Report a constant dietary pattern for at least one year.

Exclusion Criteria:
  • Presence of renal disease
  • Current consumption of a diet for weight loss
  • Pregnancy
  • Medications that could affect nutritional status
  • Presence of metabolic disease.
Description of Study Protocol:


  • Vegetarian subjects were recruited from a conference sponsored by the German Federation of Vegetarians and a summer camp sponsored by the Vegan Society in the Netherlands
  • Control subjects (omnivorous participants) were recruited from the University of Saarland.


This was a cross-sectional study were vegetarians were compared to non-vegetarian subjects (i.e., controls) with regards to vitamin B12 status.

Statistical Analysis

  • ANOVA was used to compare vitamin B12 status between groups. Spearman's correlation coefficient was used to determine concordance between the various measured used to describe vitamin B12 status
  • Stepwise multiple regression was used to determine the strongest predictor of homocysteine levels among subjects taking into account their gender, creatinine, folate and other biological indicators measured.
Data Collection Summary:

Timing of Measurements

Measurements were done once after a 12-hour fast.

Dependent Variables

Vitamin B12 status:

  • Methylmalonic acid: Assayed  in serum by gas chromatography-mass spectrometry
  • Total homocysteine: Assayed  in serum by gas chromatography-mass spectrometry
  • Serum B12: Measured with chemiluminescence immunoassay
  • Serum holotranscobalamin II: Measured in serum using a radioimmunoassay-based reagent  set.

Independent Variables

  • Serum folate: Measured with chemiluminescence immunoassay
  • Serum vitamin B6: Measured by HPLC with fluorescent detection using reagents from Immundiagnostik
  • Vitamin supplement consumption: Self-reported consumption
  • Age.

Control Variables

Type of vegetarian diet:

  1. Lactovegetarian
  2. Lactovovegetarian
  3. Vegan.
Description of Actual Data Sample:

Initial N

Total: 174:

  • Omnivorous subjects: 79
  • Vegetarians: 95
    • Lactovegetarian (LV) and lactoovovegetarian (LOV): 66
    • Vegans: 29.


  • Ominivorous subjects: 51 years (median)
  • LV and LOV subjects: 48 years (median)
  • Vegans: 37 years (median).

Other Relevant Demographics

Gender: 55 % of participants were women.


 Vegan subjects were significantly younger than LV and LVO subjects.


Germany and the Netherlands.

Summary of Results:

Median Levels of B12, MMA and HoloTC by Comparison Group

Median (fifth, 95th percentiles):


Serum B12

(pmol per L)


(nmol per L)


(pmol per L)


287 (190, 471)

161 (95, 357)

54 (16, 122)

LOV/LV (all)

192 (127, 450)*

355 (138, 1,948)*

23 (3, 155)*

Use vitamins

303 (146, 771)

230 (120, 1,344)*

26 (3, 235)

No vitamins

179 (124, 330)*#

368 (141, 2,000)*#

23 (4, 84)*

Vegan (all)

148 (99, 314)%*

708 (193, 3,470)%*

10 (2, 78)%*

Use vitamins

192 (125, 299)*

708 (163, 2,651)*

14 (3, 53)*

No vitamins

126 (92, 267)*

708 (163, 2,651)*

4 (2, 3)*#

*Significantly different from omnivores.

#Significantly different than vitamin users.

%Significantly different from LOV/LV.

Proportion of Subjects with Problematic Status Relative to Cutoff


Low Vitamin B12, Less than 156pmol per L (%)

MMA More than 271nmol per L (%)

Low holoTC, Less than 35pmol per L (%)

Omnivores 1 5 11
LOV/LV (all) 26 61 73
Use vitamins 8 31 62
No vitamins 32 68 77
Vegan (all) 52 86 90
Use vitamins 29 88 88
No vitamins 83 83 92

Correlation between indicators of Vitamin B12 status among those taking vitamins:

  • MMA and Total HCY: R=0.521, P<0.001
  • MMA and HoloTC: R=-0.686, P<0.001
  • Vitamin B12 and HoloTC: R=0.769, P<0.001.
Author Conclusion:

Vegetarians in this study had different degrees of vitamin B12 deficiency, which were related to the degree of animal product restriction. Hypermocysteinemia and relative folate shortage were linked to vitamin B12 deficiency. Assessment of holoTC II accompanied by that of metabolic markers such as tHCY and MMA may offer sensitive and reliable tools of early diagnosis of vitamin B12 deficiency.

Funding Source:
University/Hospital: Saarland University Hospital
Other: Axis-Shield
In-Kind support reported by Industry: Yes
Reviewer Comments:
  • Authors did not take into account the length of time subjects had adopted their vegan lifestyle, which may influence significant differences in vitamin B12 status. Control subjects were not matched by age. Furthermore, relevant demographic characteristics among subjects were not reported.
  • Scatterplots of correlations between vitamin B12 status indicators were unclear as they included results from vitamin and non-vitamin takers. Classification of these groups resulted in small sample size increasing the probability of type II error.
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) N/A
  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) N/A
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? Yes
2. Was the selection of study subjects/patients free from bias? Yes
  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? Yes
  2.2. Were criteria applied equally to all study groups? Yes
  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? Yes
3. Were study groups comparable? No
  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? No
  3.3. Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.) Yes
  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? No
  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.) No
  3.6. If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")? N/A
4. Was method of handling withdrawals described? No
  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%.) No
  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? N/A
5. Was blinding used to prevent introduction of bias? No
  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? No
  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? N/A
6. Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described? No
  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? No
  6.3. Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? ???
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? No
  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? N/A
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? Yes
  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? ???
  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)? No
  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? Yes
  10.2. Was the study free from apparent conflict of interest? Yes