VN: Vitamin B-12 (2011)

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

The purpose of the study was to describe the association between vitamin B12 status biochemical markers and bone metabolism, measured by bone turnover markers, among German and Asian Indian vegetarians.

Inclusion Criteria:
  • Self-reported vegans, lacto-ovovegetarians or lactovegetarians
  • Have stable dietary habits for the last two years
  • Apparently healthy without history of coronary disease, thrombosis, stroke or diabetes.  
Exclusion Criteria:
  • Subjects taking vitamin B, D and calcium supplements
  • They were diagnosed with a condition that may interfere with homocysteine metabolism which included the following:
    • Depression
    • Dementia
    • Infection
    • Thyroid dysfunction.
Description of Study Protocol:


Recruited from three different centers from the Sultanate Oman. Invited to participate through several health centers in the Oman area.


Cross-sectional design

Blinding used

Not applicable


Not applicable

Statistical Analysis

  • Variables were log transformed to achieve normality
  • Statistical differences among groups (i.e., German vegetarians and omnivorous, Asian Indian vegetarians and omnivorous) were evaluated using ANOVA and chi-square depending on nature of variable measurement
  • A stepwise multiple regression analysis was used to identify predictors/determinants of PINP, BAP and OC levels among all participants.
Data Collection Summary:

Timing of Measurements

Blood samples were collected during fasting conditions. Venous samples were preserved with EDTA and serum without anticoagulants. Serum and venous samples were centrifuged following standard procedures. Serum and plasma were separated from cellular blood components and stores are -80 centigrades until measurement.


  • Bone turnover markers (BTM)
    • C-terminal telopeptides of collagen 1 (CTx): Measured using electro chemiluminescence immunoassays with an Elecsys 2010 automated analyzer
    • Osteocalcin (OC): Measured using electro chemiluminescence immunoassays with an Elecsys 2010 automated analyzer
    • Bone alkaline phosphatase (BAP): Measured using ELISA
    • Pro-collagen type N-terminal peptide (PINP): Measured using electro chemiluminescence immunoassays with an Elecsys 2010 automated analyzer
  • Vitamin B12 status markers
    • Total vitamin B12: Measured with commercial chemiluminescence immunoassays on an ACS Centaur automated analyzer
    • Holo transcobalamin (holoTC): Measured using an enzymatic fluorescence polarization-immunoassay from Abbott with an Axsym automated analyzer
    • Methylmalonic acid (MMA): Measured using gas chromatography mass spectrometry
    • Total homocysteine (tHCY): Measured using gas chromatography mass spectrometry
    • Cystathione (Cys): Measured using gas chromatography mass spectrometry
    • 25(OH)-vitamin D: Was determined using competitive chemiluminescence immunoassays
    • Folate: Measured with a commercial, competitive chemiluminescence immunoassay using an ACS Centaur automated analyzer.


Description of Actual Data Sample:
  • Initial N:
    • 122 Germans (all male: 35 omnivores, 54 vegetarians)
    • 73 Asian-Indians (all male: 54 omnivores, 19 vegetarians)
  • Attrition (final N): Not applicable
  • Age:
    • German vegetarians; mean age 50*
    • German omnivorous; mean age 41
    • Asian Indian vegetarians; mean age 45
    • Asian Indian omnivorous; mean age 43

*Statistically significant difference when compared with German vegetarians at P<0.001

  • Ethnicity: Not available
  • Other relevant demographics: Not provided
  • Anthropometrics: Not provided
  • Location: Sultanate Oman.


Summary of Results:

66% of German vegetarians and 69% of Asian Indians vegetarians were deficient in vitamin B12 defined as levels of holoTC ≤35pmol/l and MMA>271nmol/l. 

Vitamin B12 status marker*
Germans P-value
Asian Indian P-value
Vegetarians Omnivorous
All Lacto and lacto-octo-veg Vegans
holoTC (pmol/l)
47 (28-72)  <0.001  57 (30-106)  18 (7-49)  <0.001 
MMA (nmol/l) 553 (189-1,836)  424 (171-1,706)  727 (243-2,428)  194 (146-244)  <0.001  155 (93-313)  723 (285-2,098)  <0.001 
Cobalamin (pmol/l) 156 (120-232)  163 (112-239)  138 (117-223)  279 (182-50)  <0.001  261 (179-430)  107 (57-177)  <0.001 

*Data are median and 10th-90th percentiles.

  • Serum folate was significantly higher in vegetarians than omnivores (23.1 vs. 19, P=0.034) in both ethnic groups although Asian Indians had higher serum folate than German vegetarians. It is unclear if the higher serum folate among Asian Indian vegetarians was statistically significant.
  •  There were no significant differences in bone turnover markers (e.g., OC, BAP) according to the type of vegetarian diet among Germans
  • Bone turnover markers were significantly higher among vegetarians than omnivorous.
    Bone turnover markers Germans
    Vegetarians Omnivorous P-value
    CTx  0.47  0.43  .047 
    OC 27.7  21.8  <0.001 
    BAP  13.3  11.3  0.047 
    PINP 55.3 49.5 0.099
  • After stratification of participants according to their vitamin B12 status:
    • Deficient=holoTC≤35pmol/l and MMA>271nmol/l
    • Non-deficient=holoTC>35pmol/l and MMA<271nmol/l
    • Those who were classified as deficient also had elevated tHcy (9.5 vs. 17.1, P<0.001), bone turnover markers and low 25(OH)D concentrations (18.4 vs. 13.0, P=0.001)
  • Stepwise regression analysis indicated the significant predictors of several bone turnover markers were MMA, holoTC and tHcy. These are vitamin B12 status markers.


Author Conclusion:

In conclusion, the results of the present study show that metabolic signs of vitamin B12 deficiency are related to accelerated bone turnover in vegetarians, which may potentially have adverse effects on bone health in these individuals. Concentrations of BTMs were not related to the type of vegetarian diet.

Funding Source:
University/Hospital: University Hospital, Saarland University
Other: Not reported
Reviewer Comments:

The study was thorough in all the measures collected to establish vitamin B12 deficiency. It provides valuable data on confirming vitamin B12 status among participants and supports the use of some of these measures in future studies and possibly as a clinical tool.

It was a small sample, which the authors acknowledge, and it is unclear as to why they decided to compare Asian Indians with Germans. Furthermore, significant differences in age among German vegetarians and omnivorous may have influenced results or could have been accounted for in statistical analysis.

Dietary intake was not measured and would have helped inform results better with regards to Asian Indians difference in 25(OH) D levels besides sun exposure. Inclusion criteria indicated participants must have had stable dietary habits for at least two years. However, the authors noted that deficiencies may take years to develop. Perhaps they should have informed as to the number for years each participant had adopted their vegetarian pattern. 

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? 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.) 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? 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.) ???
  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? N/A
  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? N/A
  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? N/A
  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.) N/A
  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? 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? 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? 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? 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? N/A
  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)? 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