VN: Vitamin B-12 (2011)

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

To investigate the effect of varying degrees of vitamin B12 deficiency in the hematological status of older Chinese vegetarian women.

Inclusion Criteria:
  • Female ambulatory vegetarians older than 55 years
  • Vegetarian for more than three years
  • Consent for blood sampling was obtained.
Exclusion Criteria:
  • Iron deficiency (iron saturation less than 15%)
  • Serum creatinine more than 150mmol per L.
Description of Study Protocol:


  • Cross-sectional study design. Subjects were classified into three groups based on their vitamin B12 status: 
    • No vitamin B12 deficiency
    • Possible vitamin B12 deficiency
    • Definite vitamin B12 deficiency
  • Possible vitamin B12 deficiency defined as low serum vitamin B12 levels (less than 150pmol per L) or raised serum methylmalonic acid (MMA) (more than 0.4mmol per L)
  • Definite vitamin B12 deficiency defined as low serum vitamin B12 levels AND raised MMA.

Statistical Analysis

  • ANOVA was used to compare the hematological profile and baseline characteristics of the three vitamin B12 status groups. (Hematological profile included serum iron saturation, serum vitamin B12, MMA, serum folate, hemoglobin, mean corpuscular volume, white cell count and platelet count. Baseline characteristics consisted of age, old-age home residence, dependency level, milk intake, egg intake and vegetarian duration.)
  • A correlation analysis (chi-square test) was performed to examine the potential correlation between serum vitamin B12 or MMA and hematological indices of anemia, macrocytosis and thrombocytopenia
  • A univariate analysis of variance was performed to examine the association between serum vitamin B12 or MMA and hematological indices, adjusting for old-age home residence, dependency level, age, iron saturation and folate levels.
Data Collection Summary:

Timing of Measurements

Cross-sectional study, therefore hematological profile was measured at one point in time.

Dependent Variables

  • Serum iron saturation
  • Serum vitamin B12
  • Serum MMA 
  • Serum folate
  • Hemoglobin (HB)
  • Mean corpuscular volume (MCV)
  • White cell count (WBC)
  • Platelet count (PLT).

Note: Authors did not report whether subjects took vitamin supplements.

Description of Actual Data Sample:

Initial N

119 female vegetarian subjects participated in the study.

Attrition (Final N)

11 had iron deficiency anemia, nine were missing serum iron levels and three had raised serum creatinine levels, leaving 96 to be analyzed further.

  • 23 had no vitamin B12 deficiency
  • 28 had possible vitamin B12 deficiency
  • 45 had definite vitamin B12 deficiency.


  • No vitamin B12 deficiency: 78.1±9.1
  • Possible vitamin B12 deficiency: 80.2±7.2
  • Definite vitamin B12 deficiency: 78.0±6.8.


All subjects were Chinese.

Other Relevant Demographics

  Old-age Home Residence Partially Dependent Milk Intake and Nutritional Supplements Egg Intake Vegetarian Duration (Years)
No Vitamin B12 Deficiency 13 21 19 7 35.3±15.4
Possible Vitamin B12 Deficiency 18 5 20 9 42.5±22.1
Definite Vitamin B12 Deficiency 23 2 18 6 33.4±17.6



Hong Kong, China. 

Summary of Results:

Key Findings

Hematological Profile of Older Chinese Women (by Vitamin B12 Status)

Means ± SD:


No Vitamin B12 Deficiency

Possible Vitamin B12 Deficiency

Definite Vitamin B12 Deficiency

Serum Iron Saturation (%)




Serum Vitamin B12 (pmol per L)*




Serum MMA (µmol per L)*




Serum Folate (µmol per L) 49.0+9.2 46.3+9.0  45.4+10.5 
HB (g per dL)* 13.0+1.3 12.1+1.0   12.4+1.2 
MCV (fL) 90.6+9.6 88.8+8.2  90.2+9.0 
WBC (x109 per L) 6.3+1.7 7.1+2.3 6.6+1.6 
PLT (x109 per L) 221+57 226+66  206+62 

*Statistically significant differences across groups (post-hoc test not reported).

Percentages of Abnormal Hematological Indices in Different Degrees of Metabolic Vitamin B12 Deficiency


Normal MMA (Less than 0.4µmol per L)

Borderline Raised MMA (0.4 to 1.0µmol per L)

Raised MMA (more than 1.0µmol per L)

Anemia (HB less than 12g per dL)

3 (9.4%)

9 (22.5%)

12 (48.0%)

Macrocytosis (MCV more than 97fL)

2 (6.5%)

3 (7.5%)

4 (16.0%)

Thrombocytopenia (PLT less than 140 x109 per L)

1 (3.1%)

1 (2.5%)

4 (16.0%)

Total 31 (100%) 40 (100%) 25 (100%)

Author Conclusion:
  • Vitamin B12 deficiency was associated with a significant decrease in hemoglobin concentration
  • Anemia associated with vitamin B12 deficiency was seldom macrocytic; therefore, MCV should not be relied upon to distinguish vitamin B12-related anemia from other causes
  • Due to the high prevalence of vitamin B12 deficiency and the limitations of serum vitamin B12 in detecting deficiency states, older vegetarians should be given vitamin B12 supplements routinely.
Funding Source:
University/Hospital: Prince of Wales Hospital, The Chinese University of Hong Kong
Other: Not reported
Reviewer Comments:

Ability to generalize findings is limited:

  • Women only
  • Chinese only
  • Advanced age
  • Many subjects were vegans. 
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? 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? 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) Yes
  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.) N/A
  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.) ???
  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? 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%.) Yes
  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? 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? 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? No
  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? 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? 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)? Yes
  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? Yes
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