VN: Vitamin B-12 (2011)

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

To examine the correlation between serum and fasting urinary methylmalonic acid (MMA) concentrations and determine the cutoffs for detecting elevated and mildly elevated serum MMA concentrations in older persons.

Inclusion Criteria:
  • Vegetarians (vegans and lactovegetarians)
  • Older than 55 years old
  • Normal renal function
  • Written informed consent was obtained.
Exclusion Criteria:

Renal impairment (serum creatinine concentrations above 150µM per L).

Description of Study Protocol:


Vegetarians older than 55 years old from three old-age homes for Buddhist and Taoist vegtarians and community-dwelling Taoists in Hong Kong were invited to participate in the study.


  • Descriptive study. The correlation between serum MMA and urinary MMA over creatinine was examined at one point in time
  • Serum MMA levels higher than 0.4μmols were considered elevated
  • Serum MMA levels higher than 0.3μmols was considered mildly elevated.

Statistical Analysis

  • The correlation between serum MMA and urinary MMA over creatinine was examined by the use of scatter plots and Pearson's correlation analysis
  • Receiver operating characteristic curves for urinary MMA over creatinine were drawn to determine the optimal cutoffs associated with the highest sensitivitiy and specificity
  • SPSS 10 for Windows was used for statistical analysis. MedCalc for Windows was used to draw the graphs. 
  • P<0.05 (two-sided) was considered statistically significant.


Data Collection Summary:

Timing of Measurements

Serum and urinary specimens were measured at one point in time.

Dependent Variables

  • Renal function: Sodium, potassium, urea and creatinine (renal function tests were carried out using colorimetric methods on a Dimension autoanlalyzer; urinary creatinine was measured by the Jaffe reaction, without deproteinization, on a Hitachi 911 autoanalyzer)
  • Serum vitamin B12 (measured by solid-phase, no-boil, dual-count radioimmunoassay)
  • Serum folate (measured by solid-phase, no-boil, dual-count radioimmunoassay)
  • Serum MMA (measured by capillary gas chromatography-mass spectrometry method)
  • Urinary MMA (measured by stable-isotope dilution method)

Control Variables

Not reported whether subjects were taking vitamin supplements.

Description of Actual Data Sample:
  • Initial N: 118 female vegetarian subjects participated in the study. Five were exclude due to elevated serum creatinine concentrations, leaving 113 subjects to be analyzed further.
  • Age: Average age of the subjects was 77.4 years (standard deviation 7.6, age range 55 to 94)
  • Ethnicity: All subjects were Chinese.

Other Relevant Demographics

  • 109 subjects had been vegetarians longer than 10 years
  • 60 subjects (53%) were vegans
  • 62 subjects (55%) lived in old-age homes.


The dietary analysis and health profile of the subjects were not reported in the article.


Hong Kong, China.


Summary of Results:

 Key Findings




Median Value (5th Percentile, 95th Percentile)

Percentage of Subjects within Reference Ranges

Vitamin B12 (pM per L)

145 (52, 827)


     More than 150pM per L  


     More than 300pM per L  


Folate (nL per L) 50.1 (23.6, 54)  
     More than 10nL per L   0
Serum MMA (µM per L) 0.63 (0.07, 2.52)  
     Less than 0.4µM per L  


     Less than 0.3µM per L



Urinary MMA (µM per mM creatinine)

2.5 (0.6, 12.0)


     Less than 4.8µM per mM creatine




Urinary MMA (µM per mM Creatinine)

Subjects (N)

Vitamin B12 Less Than 150pM per L N (%)

Vitamin B12 Les Than 300pM per L N (%)
More than 1.5


52 (66) 

73 (92)
More than 2.0 68

45 (66)

64 (94)
More than 4.8 23

21 (91)

23 (100)


  • Fasting urinary and serum MMA levels were highly, linearly correlated (R=0.94., P<0.0001)
  • The cutoff of fasting urinary MMA of 2.0µM per mM of creatinine had a sensitivity of 79%, a specificity of 85% and a positive predictive value of 93% for elevated serum MMA (more than 0.4µM per L)
  • The cutoff of fasting urinary MMA of 1.5µM per mM of creatinine had a sensitivity of 86%, a specificity of 85% and a positive predictive value of 95% for mildly elevated serum MMA  (more than 0.3µM per L)
  • Both cutoffs had high positive predictive value for subnormal vitamin B12 concentrations.
Author Conclusion:
  • Overnight fasting urinary MMA over creatinine had a strong correlation with serum MMA in older vegetarians without significant renal impairment
  • Urinary MMA over creatinine can be used to screen for elevated and mildly elevated serum MMA with high sensitivity and specificity. It also has a high predictive value for sub-normal serum vitamin B12 concentrations.
Funding Source:
University/Hospital: Prince of Wales Hospital, The Chinese University of Hong Kong
Other: Not reported
Reviewer Comments:
  • Subjects were strict vegetarians with a high prevalence of vitamin B12 deficiency. This likely contributes to a higher positive predictive value of urinary MMA for elevated serum MMA and subnormal vitamin B12.
  • While it is unlikely that these factors would alter the relationship between urinary MMA and serum MMA, the fact that all subject were Chinese women could limit the ability to generalize findings.
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? Yes
  2.2. Were criteria applied equally to all study groups? N/A
  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? 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) N/A
  3.2. Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline? N/A
  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? N/A
  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.) N/A
  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? 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%.) N/A
  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? Yes
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? 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? No
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? 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? Yes
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? N/A
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)? No
  8.5. Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)? N/A
  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? No
  10.2. Was the study free from apparent conflict of interest? Yes