VN: Vitamin B-12 (2011)

Study Design:
- Click here for explanation of classification scheme.
Quality Rating:
Research Purpose:
  • To obtain the normal levels of MMA and Hcy in healthy infants
  • To investigate the metabolite levels and thereby the intracellular cobalamin function in macrobiotic infants
  • To evaluate the usefulness of MMA and Hcy in plasma for the individual diagnosis of cobalamin deficiency in infancy.


Inclusion Criteria:
  • Mothers of infants following macrobiotic diet for at least three years
  • Feeding of breast milk to infants from birth onwards with later complementary feedings according to macrobiotic principles
  • Provided blood samples
  • Subjects were described in detail in a previous publication on nutritional status in macrobiotic infants.
Exclusion Criteria:
  • Infants of mothers not following a macrobiotic diet for at least three years
  • Infants who were not fed breast milk from birth onward, followed by complementary feedings according to macrobiotic principles
  • Those unable to provide blood samples.
Description of Study Protocol:


Not described in this paper. Possibly detailed in previous publication on nutritional status in macrobiotic infants.


Cross-sectional study

Blinding used

Not applicable


Not applicable

Statistical Analysis

GM and GM±1.96 SD intervals were calculated for MMA, Hcy, cobalamin, methionine, and cysteine in the controls. Because of skew distribution, log-transformation of data was performed before calculation. The data were normally distributed after transformation, as judged by normal probability plots. Group GM were compared with the two-sample T test.

In the controls, the 95th percentile for MMA and total Hcy an the 5th percentile for plasma cobalamin were calculated on the basis of the log-transformed data. The 95th percentile for MCV and 5th percentile for Hb were calculated from the non-transformed data. These clues were defined as cutoff values. Logistic regression analysis was used to predict the dietary status. MMA, Hcy, cobalamin, MCV and Hb served as candidate predictor variables. Two-sided P-values less than 0.05 were considered significant. Analysis were carried out with the BMDP statistical package.



Data Collection Summary:

Timing of Measurements

Blood samples were taken. Plasma samples for measurement of both MMA and total Hcy were available from 41 macrobiotic infants and 50 controls. MMA was measured in an additional three samples from macrobiotic infants and four samples from controls. Because subjects were visited at home, care was taken to visit macrobiotic and control infants in a randomized order. The fasting period was one to four hours in about 70% of the children.

Dependent Variables

  • MMA
  • Hcy
  • Cobalamin
  • Methionine
  • Cysteine.

Independent Variables

Macrobiotic diet

Control Variables


Description of Actual Data Sample:
  • Initial N:
    • 50 macrobiotic infants
      • 24 females
      • 26 males
    • 57 group-matched controls
      • 26 females
      • 31 males
  • Attrition (final N): 41 macrobiotic infants
    • 14 females
    • 27 males
  • Age: 10-20 months
  • Ethnicity: Not applicable
  • Other relevant demographics: Not applicable
  • Location: Norway.
Summary of Results:

Key Findings

Plasma levels of metabolites and cobalamin in macrobiotic and healthy control infants

(Geometric mean±1.96 SD)


MMA (μmol/L) Hcy (μmol/L) Cobalamin (pmol/L)

Cysteine (μmol/L)

Methionine (μmol/L)

Macrobiotics (n=41)

1.44 (0.17-12.15) 13.5 (6.8-26.8) 141.4 (58.8-339.7) 163.6 (118.7-225.6) 6.0 (1.5-23.8)

Controls (n=50)

 0.18 (0.06-0.51)

7.59 (5.3-11.0)

 398.7 (93.6-820.7)

183.8 (144.7-233.4)

6.9 (1.1-41.9)







Macrobiotic Infants in reference range

  • MMA>0.43μmol/L: 85.4%
  • Cobalamin <218pmol/L: 83.0%.

Other Findings

  • Neither plasma MMA nor total plasma Hcy was significantly related to the age of the infants
  • The concentration of MMA in plasma from macrobiotic infants varied over a wide range and the GM of 1.44μmol/L was 7.5-fold higher than the mean for controls
  • The GM for total Hcy of 13.5μmol/L was increased about two-fold compared with controls
  • The plasma level of methionine was not significantly different between macrobiotic infants and controls
  • Total cysteine was slightly but significantly reduced in macrobiotic infants
  • Both MMA in plasma and total Hcy were inversely correlated with plasma cobalamin
  • There was a strong positive correlation between MMA and total Hcy in plasma, but in a few cases, only MMA or total Hcy was elevated.
Author Conclusion:

The hematologic parameters showed subtle differences between macrobiotic infants and controls, and there was considerable overlap between the groups. Plasma cobalamin was markedly reduced in macrobiotic infants, suggesting impaired cobalamin function in most macrobiotic infants. Hb was not significantly different between groups. 

Hematocrit and red blood call count were lower, and MCV, mean corpuscular Hb, and mean corpuscular Hb concentration were higher in macrobiotic infants compared with controls. Because hematologic symptoms are often subtle, and an intervention was performed in only a limited number of infants included in the present study, the data do not allow an independent discrimination between healthy and cobalamin-deficient infants.

MMA, total Hcy and cobalamin measurements identified the macrobiotic infants with high sensitivity, but regression analysis showed plasma MMA had the highest discriminative power followed closely by total plasma Hcy and plasma cobalamin. MCV and Hb were insensitive and unspecific in diagnosing cobalamin deficiency in the individual infant. 

Funding Source:
Government: Norwegian Research Council
The Nordic Insulin Foundation, and the Dutch Praeventiefonds
Other non-profit:
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) No
  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) No
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? ???
  2.2. Were criteria applied equally to all study groups? ???
  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? ???
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? ???
  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.) N/A
  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%.) ???
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? ???
  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? No
  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? 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? 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? 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? 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