VN: Vitamin B-12 (2011)

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

To investigate vitamin B12-dependent metabolism and oxidative stress in groups with various or no intake of meat or animal products.

Inclusion Criteria:
  • Healthy individuals
  • Following a constant dietary pattern for one year
  • Values for basic hematologic variables within the appropriate reference intervals
  • No renal disease
  • No use of vitamin supplementation
  • No use of lipid-lowering drugs
  • No weight loss diets
  • No medications or metabolic diseases that affect nutritional status
  • No pregnancy.
Exclusion Criteria:
  • Inconsistent dietary pattern during previous year
  • Values for basic hematologic variables outside of appropriate reference intervals
  • Renal disease
  • Vitamin supplementation
  • On lipid-lowering drugs
  • Weight loss diets
  • Medications or metabolic diseases that affect nutritional status
  • Pregnancy.
Description of Study Protocol:


All vegetarians were volunteers recruited at a conference of the German Federation of Vegetarians. High meat eaters and low meat eaters were selected from students and staff members.


Cross-sectional study

Statistical Analysis

Median values and 5th and 95th percentiles were calculated, and the Mann-Whitney test, correlation analysis by the Spearman p, and backward regression analysis were performed with the software package SPSS (Ver 9.0 for Windows; SPSS).

Data Collection Summary:

Timing of Measurements

The average of three 24-hour dietary recalls from the previous three days was used to calculate qualitative daily consumption of different nutrients.

Dependent Variables

  • Homocysteine
  • Folate
  • Vitamin B12
  • Methylmalonic acid.

Independent Variables

Dietary pattern defined in terms of the following groups:

  • HME: High meat intake,
  • LME: Low meat intake
  • LOV/LV: Lacto-ovo- and lacto-vegetarians
  • Vegans



Description of Actual Data Sample:
  • Initial N: 104
    • 44 high meat eaters
    • 19 low meat eaters
    • 34 lacto-ovo/lacto-vegetarians
    • Seven vegan vegetarians
  • Attrition (final N): 104
  • Anthropotmetrics: Average BMI of subjects was in normal range. BMI of LOV/LV and all vegetarian groups combined were significantly lower than HME group.
  • Age (median):
    • HME=23
    • All vegetarians=22
    • LME=24
    • LOV/LV=22
    • Vegan=22
  • Location: Germany.


Summary of Results:

Frequency of Increased Metabolites and Decreased Vitamins in Vegetarians

Variables HME (N=44) All vegetarians (n=60) LME (n=19) LOV/LV (n=34) Vegans (n=7)
Increased metabolites, percentage      
HCY mmol/l, median (5th, 95th percentiles)  9.8 (5.9, 16.7)  11.6 (6.3, 19.3)*  11.8 (6.1, 17.0)*  11.0 (5.7, 20.8)  15.2 (9.3, 18.5)* 
HCY>15μmol/L 20  21  12  57
MMA nmol/l median (5th, 95th percentiles) 169 (111, 280)  209 (111, 522)*  210 (95, 257)  205 (110, 572)  246 (153, 292)* 
MMA>271 nmol/L 25  32  43 
HCY and/or MMA (at least one increased)  37  21  38  71 
Decreased vitamins, percentage      
Folate nmol/l median (5th, 95th percentiles)  17.3 (7, 36.5) 17.7 (9.1, 33.8)  19.1 (10, 26.1)     
Folate <7 nmol/L 
B12 pmol/l median (5th, 95th percentiles) 276 (172, 406)  243 (148, 386)  240 (118, 331)*  253 (153, 376)  217 (153, 438) 
B12<156 pmol/L 11  14 
Folate and/or B12  10  11  14 

*Significantly different than high meat eaters (HME)

Other Findings

  • Compared with age- and sex-matched members of the HME group, the serum HCY concentrations of vegetarians were significantly increased
  • The serum concentrations of vitamin B12 and folate did not differ significantly between both groups
  • Only vegans showed a median HCY>15µmol/L
  • Compared with the HME group, the HCY median value was increased in the other subgroups, but did not reach the pathologic range of >15μmol/L
  • The serum MMA concentrations of the vegans was significantly increased compared with the HME group, while in the other subgroups the MMA concentration was only slightly increased
  • Compared with the HME group, all subgroups of vegetarians showed lower vitamin B12 serum concentrations, but the median vitamin B12 was significantly lower only in the LME group
  • Folate concentrations were uniform in all subgroups
  • Correlation analysis showed a highly significant correlation between MMA and HCY
  • Vitamin B12 correlated with the other investigated variables (MMA, HCY, folate, TAS) at a 5% significance level
  • HCY concentration was significantly and independently influenced MMA, age and sex. MMA was independently modulated by age, vitamin B12 and HCY. The TAS was influenced by vitamin B12 and sex only.


Author Conclusion:

This study demonstrates that vegetarians have higher serum concentrations of HCY than omnivorous controls. The HCY concentration increased as the vegetarian diet became more restrictive and peaked in the group of vegans.

The correlations analysis indicated a significant correlation between MMA and HCY and inversely between vitamin B12 and HCY, which is in agreement with results obtained by other investigators.

This analysis also indicated that MMA is significantly influenced by HCY and serum vitamin B12. Smoking, alcohol consumption and physical exercise were not included in the multiple regression model. 

It can be assumed that the general healthier lifestyle of vegetarians could be partly reversed by increases in HCY as a consequence of vitamin B12 deficiency. Vitamin B12 supplementation for persons on restrictive vegetarian diets is very important. 

Additional studies confirming these results that focus on determining the diagnostic value of vitamin B12 markers are needed. It can be concluded that MMA is a sensitive and specific predictor of dietary group and, therefore, it may be expected that MMA is an early, sensitive and specific marker of impaired cobalamin status. 

Funding Source:
Other: Not reported
Reviewer Comments:
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? 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? Yes
  2.4. Were the subjects/patients a representative sample of the relevant population? No
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) No
  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.) 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? Yes
  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.) Yes
  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.) No
  5.3. In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? Yes
  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? Yes
  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? 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? 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? 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? N/A
  10.1. Were sources of funding and investigators' affiliations described? No
  10.2. Was the study free from apparent conflict of interest? Yes