VN: Vitamin B-12 (2011)

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

The primary research purpose is to investigate whether there is an association between disturbed remethylation caused by vitamin B12 deficiency in vegetarians and DNA global methylation.

  • Using additional measurements, SAM (S-adenosylemethionine) and SAH (S-adenosylhomocysteine), the regulation of DNA methylation could be analyzed in greater detail
  • Promoter methylation of the p66Shc gene was studied.
Inclusion Criteria:
  • Healthy vegetarians
  • Informed consent signed by participant
  • Maintained a constant dietary pattern for more than one year.
Exclusion Criteria:

Not applicable.

Description of Study Protocol:


Subjects recruited at a conference of the German Federation of Vegetarians.


Cross-sectional study.

Blinding used

Not applicable.


Not applicable.

Statistical Analysis

  • Logistic regression used for homocysteine, MMA, vitamins B6 and B12, SAM, SAM/SAH ratio, HoloTC and cystathionine
  • Correlations between variables evaluated by Pearson test
  • All tests were two-tailed and significance set at P<0.05.


Data Collection Summary:

Timing of Measurements

Blood samples collected after 12-hour fast for all participants.

Dependent Variables

  • Median plasma concentrations
    • Homocysteine: assayed by gas chromatography-mass spectrometry
    • Vitamin B12: measured using chemiluminescence immunoassays
    • MMA (methylmalonic acid): assayed by gas chromatography-mass spectrometry
    • HoloTC (holotranscobalamin II): quantified by radioimmunoassay
    • Folate: measured using chemiluminescence immunoassays
    • Vitamin B6: analyzed by HPLC
    • SAM: measured using an HPLC method
    • SAH: measured using an HPLC method.
  • DNA analysis
    • Global DNA methylation status: mean methylation values for three CpG sites using Pyrosequencing method
    • Specific promotor methylation for the p66Shc gene: mean methylation values for three CpG sites in two sequencing reactions were used also using Pyrosequencing

Independent Variables

Vegetarian diet.

Control Variables

  • Age
  • Sex.
Description of Actual Data Sample:

Initial N

Not applicable.

Attrition (final N)

  • 71 vegetarians
    • 48 lacto/lacto-ovo-vegetarians
    • 23 vegans.
  • 79 omnivorous.


Median age (years)

  • Lacto/lacto-ovo-vegetarian=53 
  • Vegan=51 
  • Omnivorous=51.




Vegetarians' age was significantly different from omnivorous subjects, P<0.05.



Summary of Results:

Key Findings

  • Homocysteine levels above 12μmol/L were found in 45% of vegetarians 
  • Median and percentage of elevated homocysteine concentrations were significantly lower in vegetarians compared to age and sex matched omnivorous control group 
  • vitamin B12 deficiency (HoloTC II<35pmol/L or MMA>271nmol/L) found in 58% of the vegetarians and 30% of the deficient subjects were categorized as stage III deficient.


Omnivorous subjects, n=79

Median (5th and 95th percentiles)

Lacto-/lacto-ovo-vegetarians, n=48

Median (5th and 95th percentiles)

Vegan subjects, n=23

Median (5th and 95th percentiles)

Homocysteine, μmol/L

8.8 (5.5, 16.1)

11.1 (6.3, 21.8)*

13.0 (5.5, 38.0)*

Vitamin B12, pmol/L

 287 (189, 470)

180 (117, 356)* 

 145 (86, 596)*

HoloTC II, pmol/L

 54 (16,122)

 40 (9, 82)*

 22 (1, 160)*

MMA, nmol/L 160 (94,356) 273 (150,1198)* 695 (129,3582)*
Folate, nmol/L 21.7 (14.5, 51.4) 22.8 (11.7, 42.8) 25.4 (13.6, 66.0)
Vitamin B6, nmol/L 52.5 (24.7, 128.8) 53.1 (21.7, 166.5) 46.2 (18.3, 260.9)

 *Significantly different from omnivorous subjects, P<0.05 (Mann-Whitney test)

Other Findings

Global DNA methylation status

  • Significant correlations between global DNA methylation status and SAH (r=-0.36, P<0.01 and the SAM/SAH ratio (r=-0.27, P<0.05) were found by using the mean methylation values for three CpG sites
  • No significant association was seen between methylation and plasma homocysteine or SAM
  • A trend for a lower degree of methylation in subjects homozygous for the 677C→T mutation (12.1% in subjects) was observed.

Promoter methylation of the p66Shc gene

  • No correlation between degree of methylation (used as surrogate measurement for promotor methylation) and homocysteine or SAM
  • No correlation between promoter methylation and SAM or the SAM/SAH ratio.
Author Conclusion:
  • In vegetarians, the reverse metabolism of homocysteine to SAH is not important, due to lack of correlation in the study
  • Total global and p66Shc promoter methylation measures showed no effects of disturbed methyl group metabolism in vegetarians
  • Negative consequences for methyl group metabolism was not seen in vegetarians due to the absence of correlations found between homocysteine or SAM and the degree of methylation.
Funding Source:
University/Hospital: Department of Clinical Chemistry, Saarland Medical School, Homburg, Germany
Other: Not reported
Reviewer Comments:
  • No details given about how age- and sex- matched omnivorous control group were selected
  • No details given for funding for study; only listed primary author's contact info
  • Vegetarians can be at risk for abnormal methyl group metabolism due to low blood levels of vitamin B12, which leads to elevated serum homocysteine
    • Diets are low in methyl group donors, methionine
    • This may lead to increased risk for certain cancers, atherosclerosis, aging and other diseases
    • SAM may be an important factor in the methylation process for vegetarians because no association was seen between methylation (both global and specific gene) and diet
  • Vegetarians may not have an increased risk of disease due to dietary choices, as previously thought 
  • Good study to understand and review biochemical consequences of the vegetarian diet; difficult study to determine real-world implications of results.
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? Yes
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? Yes
  2.2. Were criteria applied equally to all study groups? No
  2.3. Were health, demographics, and other characteristics of subjects described? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? No
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) No
  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")? Yes
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? ???
  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? 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? Yes
  6.4. Was the amount of exposure and, if relevant, subject/patient compliance measured? Yes
  6.5. Were co-interventions (e.g., ancillary treatments, other therapies) described? No
  6.6. Were extra or unplanned treatments described? No
  6.7. Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? Yes
  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? Yes
  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? Yes
  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)? Yes
  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? No
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? No
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