VN: Vitamin B-12 (2011)

Study Design:
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Quality Rating:
Research Purpose:

To investigate whether a moderate consumption of animal products is sufficient for achieving and maintaining normal cobalamin function in adolescents who had received a macrobiotic diet in early life.

Inclusion Criteria:
  • For the study group, adolescents who had been fed a macrobiotic diet early in life that were participating in a bone density study; a control group was also selected (similar ages) that had received an omnivorous diet since birth
  • White
  • Considered in good health
  • Not taking drugs that would affect their hematologic status.
Exclusion Criteria:

Not specified with the exception of the adolescents currently being on any medication that would affect their hematologic status.

Description of Study Protocol:


  • Dutch adolescents who had been fed a macrobiotic diet were recruited from existing groups of macrobiotic families affiliated with the Division of Human Nutrition and Epidemiology, Wagenigen Agricultural University. A control group was recruited (fed an omnivorous diet) from local schools.
  • Based on earlier studies of the macrobiotic group, families were given recommendations to increase the intake of fat, fatty fish and dairy products to children's diets. A 1993 follow-up study showed that the children had increased their intake of animal products.
  • Data for this study were gathered in 1995. At this period, the previously macrobiotic adolescents consumed lacto-, lacto-ovo-vegetarian and omnivorous diets.


Cross-sectional study using aged matched controls.

Statistical Analysis

Means, standard deviations or geometric means were calculated from the log-normal distribution for all blood indexes. Group means were compared by two-sample student T-tests. The correlation between indexes was determined by using Pearson correlation coefficients, but Spearman correlation coefficients were calculated if variables were not normally distributed. Multiple regression analysis was also used.

Data Collection Summary:

Timing of Measurements

Subjects underwent anthropometric measurements, dietary assessment and biochemical measurements within a one-month period.

Dependent Variables

  • B12
  • Folate
  • MMA
  • tHcy.

Independent Variables

Children who were fed a macrobiotic diet early in life.

Control Variables

Current moderate consumption of animal products.

Note: Intake of supplements was not recorded.

Description of Actual Data Sample:
  • Initial N: In the study group, 93 Dutch adolescents (50 male and 43 female) participated. 102 subjects were recruited from the control group (42 boys and 60 girls).
  • Attrition (final N): Complete data was only available for 167 adolescents (73 macrobiotic and 94 control) for statistical analyses after 21 adolescents refused to give blood and seven other adolescents samples were insufficient
  • Age: Age ranged from nine to 15 years. Average age of boys was 12.7 years for macrobiotic and 11.7 for control, and girls was 11.7 for macrobiotic and 11.7 for control.
  • Ethnicity: All subjects were white
  • Other relevant demographics: Socioeconomic status was determined by using Attwood scores, a five-point score based on the occupation of and highest level of education attained by both parents
  • Anthropometrics: Weight and height were measured on all subjects
  • Location: Wageningen, Netherlands.
Summary of Results:

Key Findings

The proportion of formerly macrobiotic adolescents were deficient in:
  • MMA (more than 0.41mmol per L): 21%
  • B12 (less than 218pmol per L): 37%.

Characteristics  of Formerly Macrobiotic and Control Adolescents



Formerly Macrobiotic Group

Control Group 

Statistical Significance of Group Difference

Height (m)

Boys: 1.57±0.1

Girls: 1.50±0.1

Boys: 1.53±0.1

Girls: 1.52±0.1

Weight (kg)

 Boys: 42.2±10.5

Girls: 38.4±9.2

 Boys: 41.2±9.2

Girls: 41.3±8.6


Socioeconomic status

 Boys: 1.8±0.5

Girls: 2.0±0.7

 Boys: 2.0±0.8

Girls: 2.0±0.7


Average cobalamin intake from dairy products combined (mcg per day)  

Boys: 1.13±0.99

Girls: 0.76±0.69

  Boys: 2.30±0.90

Girls: 2.34±0.92


Biochemical and Hematological Values of Formerly Macrobiotic and Control Adolescents



Formerly Macrobiotic Group

Control Group Statistical Significance (P1)
Mean corpuscular volume (fL)

Boys: 85.2±3.2

Girls: 86.1±3.8

Boys: 83.3±3.7

Girls: 84.4±3.2

Mean corpuscular hemoglobin mass (fmol)

Boys: 1.80±0.07

Girls: 1.81±0.09

Boys: 1.76±0.10

Girls: 1.78±0.08

Folate (nmol per L)

Boys: 17.9 (10.9 to 29.4)

Girls: 18.9 (11.4 to 31.2)

Boys: 14.7 (8.0 to 27.0)

Girls: 14.5 (7.8 to 26.8)

Cobalamin (pmol per L) Boys: 213 (107 to 426)

Girls: 288 (112 to 738)

Boys: 484 (238 to 985)

Girls: 458 (206 to 1,020)

Methylmaloninc acid (μmol per L) Boys: 0.29 (0.09 to 0.93)

Girls: 0.25 (0.09 to 0.70)

Boys: 0.15 (0.06 to 0.43)

Girls: 0.17 (0.07 to 0.40)

Homocysteine (μmol per L) Boys: 8.3 (5.2 to 13.4)

Girls: 7.6 (3.8 to 15.1)

Boys: 7.0 (4.2 to 11.7)

Girls: 7.2 (3.8 to 13.7)

  • In macrobiotic adolescents (treatment group) dairy products (200g milk or yogurt and 22g cheese per day) supplied on average 0.95mcg cobalamin per day
  • The treatment group also consumed fish, meat or chicken two to three times per week.
Author Conclusion:
  • There was significantly lower serum cobalamin and higher MMA concentrations in adolescents who followed a macrobiotic diet early in life than their age-matched controls
  • Because the adolescents in the study group were healthy, it was assumed that their marginal cobalamin status was a result of long-term insufficient cobalamin consumption rather than disease or malabsorption
  • Since most subjects in the treatment group switched to a lactovegetraian, lactoovovegetarian or omnivorous diet, the consumption of such moderate amount of animal products over six to seven years was not sufficient to restore and maintain adequate cobalamin function
  • In summary, the adverse effects of cobalamin deficiency in the macrobiotic community may not be restricted to just early childhood, but may cause symptoms related to impaired cobalamin status later in life.
Funding Source:
Government: Dutch Prevention Fund
University/Hospital: Division of Human Nutrition and Epidemiology, Wageningen Agricultural University.
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? Yes
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? 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? Yes
  4.1. Were follow-up methods described and the same for all groups? No
  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? Yes
  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.) 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? 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? Yes
  10.1. Were sources of funding and investigators' affiliations described? Yes
  10.2. Was the study free from apparent conflict of interest? Yes