VN: Vitamin B-12 (2011)

Study Design:
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Quality Rating:
Research Purpose:
  • To investigate the vitamin B12 and folate status of subjects using a combination of static and functional and biochemical indices. The study also examined the subjects' vitamin B12 and folate status according to whether the women received more of their dietary energy from unrefined maize or from fermented enset products.
  • To investigate the role of vitamin B12 and folate status as well as protein, other micronutrients, infection and gravida on hemoglobin concentrations.
Inclusion Criteria:
  • Subsistence farmers from three rural communities in the Sidama Zone of southern Ethiopia. A
  • Third trimester of pregnancy
  • Afebrile
  • Aged 27.8±4.6 years.
Exclusion Criteria:

Not noted.

Description of Study Protocol:


Subjects were self-selected. 


Cross-sectional study. 

Statistical Analysis

  • Mann-Whitney U statistic: Used to assess between-group differences between plasma vitamin B12, cystathionine and tHcys
  • Spearman correlation coefficients: Used to explore the relation between the variables
  • Student's T-test: Used for independent samples or Fisher's exact test
  • Multiple linear regression analysis: Used to examine the predictors of hemoglobin concentrations in all the subjects.


Data Collection Summary:

Timing of Measurements

  • After recruitment, trained research assistants interviewed the women using a pre-tested questionnaire and observations to obtain sociodemographic and health data. Dietary intakes were calculated from one-day weighed food records using an Ethiopian food composition table based on literature and analyzed values for calcium, iron, zinc and phytate.
  • Observed intakes of energy and protein were adjusted for digestibility and protein intakes for amino acid score. The women were then classified into two groups according to whether maize (N=68) or enset (N=31) was the predominant energy source. Samples of each of the two fermented enset products, kocho and bulla, were purchased from local markets and analyzed for folate and vitamin B12 content. Vitamin B12 was assayed using a newly developed liquid chromatography-MS method in the laboratory of S. Stabler. Weight and height were obtained and BMI calculated.
  • Anticoagulated whole blood and plasma were collected from non-fasting morning venipuncture samples to obtain complete blood count, plasma albumin, ferritin, zinc and C-reactive protein. Plasma vitamin B12 was analyzed using the Solid Phase No Boil Dual count Radioassay B12/Folic acid kit, MMA, tHcys and cystathionine were assayed in plasma by isotope dilution GC-MS.  

Dependent Variables

  • Vitamin B12 and folate levels
  • Serum MMA
  • Hemoglobin concentrations.

Independent Variables

Diet (unrefined maize vs. fermented enset products). 

Control Variables

  • Gender
  • Age
  • Pregnancy.

Note: Supplementation was not reported, though given the population (women in Southern Ethiopia), supplementation is unlikely.

Description of Actual Data Sample:
  • Initial N: N=99 women
  • Attrition (final N): N=99 women
  • Age: 27.8±4.6 years of age
  • Ethnicity: Ethiopian
  • Other relevant demographics:
    • Maize-based group (N=68): 77.9% had no schooling, 39.7% rarely had enough food
    • Enset-based group (N=31): 74.2% had no schooling, 32.3% rarely had enough food
  • Anthropometrics:
    • Maize-based group (N=68): 27.6±4.3 years of age, 7.1±1.0cm fundal height, 52.0±6.3kg weight, 154.8±6.4cm height, -1.48±1.07 height for age Z score, 21.7±2.0kg/m2 BMI
    • Enset-based group (N=31): 28.4±5.4 years of age, 7.0±1.1cm fundal height, 52.4±5.7kg weight, 154.9±6.7cm height, -1.46±1.12 height for age Z score, 21.8±2.0kg/m2 BMI
  • Location: Sidama Zone of southern Ethiopia.


Summary of Results:

Key Findings

Descriptive Findings and Group Differences

  • Both groups had a very monotonous diet, low in energy, with more than 85% provided by foods from only two food sources (unrefined maize and enset products)
  • Less than 1% of energy in both diet groups was provided by dairy products and no cellular animal foods were eaten
  • The enset diet group had lower intakes (P<0.001) of utilizable protein, fat, zinc, dietary fiber, Phy and lower Phy:Zn and Phy:Fe molar ratios but higher intakes (P<0.001) of calcium than the maize diet group
  • Kocho contributed 58.1μg per day folate and 6.8μg per day vitamin B12 for the enset diet group compared with 18.0μg per day folate and 2.1μg per day vitamin B12 for the maize diet group
  • Mean plasma vitamin B12 level tended to be higher (P=0.052), whereas MCV and plasma cystathionine were lower in the enset diet group compared to the maize diet group (P<0.05). There were no other significant differences between the two diet groups for hematological or biochemical indices or for the prevalence of abnormal values.
  • Of the women, 29% (N=27) had anemia from all causes; none had severe anemia (hemoglobin less than 70g per L)
  • A total of 13% (N=12) of the women had IDA with a mean cell hemoglobin concentration of 343±11.7g per L and a MCV of 88.3±5.6fL.
  • For all subjects:
    • Serum B12 (pmol per L) (median, 25th, 75th percentile): 268 (152, 372)
    • Plasma MMA (nmol per L) (mean, SD): 407±206

Relationships among Measures

  • Spearman rank correlation coefficients were significant between plasma folate and plasma cobalamin (R=0.265; P=0.015), plasma folate and MMA (R=0.298; P=0.006) and plasma zinc and hemoglobin (R=0.328; P=0.001)
  • Negative correlations were noted between hemoglobin and plasma cystathionine (R=-0.281; P=0.007) and plasma folate and plasma cystathionine (R=-0.357; P=0.001)
  • No significant correlations existed between plasma zinc and ferritin or between plasma tHcys and any of the other folate or vitamin B12 indices
  • Plasma zinc had the largest standardized β coefficient, followed by CRP, gravida and plasma ferritin
  • None of the biochemical indices of folate or vitamin B12 status, plasma retinol or plasma albumin were significant predictors of hemoglobin concentrations.
Author Conclusion:
  • The most notable finding of this study was the low prevalence of IDA and the absence of any hematopoietic defects associated with folate or vitamin B12 deficiency during pregnancy
  • Consumption of fermented enset products may have increased vitamin B12 levels in the diet and plasma and thus reduced the risk of vitamin B12 deficiency anemia, although there was some evidence of an early stage of functional vitamin B12 deficiency based on elevated MMA values.

Interrelationship Between Diet and Vitamin B12 and Folate Status

  • The tendency for women in the enset diet group to have higher plasma vitamin B12 concentrations than those in the maize diet group might be associated with the microbial contribution of vitamin B12 from fermented kocho
  • Vitamin B12 intakes in the enset-diet group were more than twice the level set by WHO/FAO for the estimated average requirement during pregnancy. Even in the maize diet group, the contribution of vitamin B12 from fermented enset was considerable
  • Mean MMA levels did not differ between the two diet groups. The prevalence of elevated MMA concentrations, considered to be the most sensitive indicator of vitamin B12 deficiency was very high in both groups. This apparent discrepancy suggest that the fermented enset might have contained some non-functional analogs of vitamin B12 as well as the cobalamins that were measured in the rodioassay. This may have lead to the apparently normal plasma vitamin B12 concentrations in some of the women whose plasma MMA values were unexpectedly high
  • L. planatarum is also a folate-producing microorganism; the fermented enset also contributed some folate, albeit a much smaller amount in relation to the WHO/FAO estimated average
  • There were several additional sources of folate in the diets of both groups, most notably kale, kidney beans and haricot beans, which probably  accounted for the absence of folate deficiency.

Predictors of Hemoglobin

  • Plasma zinc concentrations were the strongest predictor of hemoglobin, followed by the indicator variables for infection, gravida and plasma ferritin
  • The strongest positive association between plasma zinc and hemoglobin was unexpected
  • An elevated CRP was negatively associated with hemoglobin, notwithstanding the relatively small number of subjects (N=8) with elevated values indicative of underlying acute infection or inflammation
  • Gravida also had an independent and significant inverse association with hemoglobin
  • Plasma ferritin was also a significant predictor of hemoglobin, although its importance based on the absolute value of its standardized coefficient was less than that for plasma zinc
  • Dietary iron intake in Ethiopia is characteristically high
  • Unlike ferritin, neither plasma vitamin B12 nor MMA was a significant predictor of hemoglobin, despite the high prevalence of elevated concentrations of MMA. This discrepancy is attributed to non-functional vitamin B12 analogs present in the plasma of women consuming fermented enset as well as the absence of any hematopoietic defects associated with vitamin B12 deficiency anemia
  • Plasma folate was also not a significant predictor of hemoglobin, although levels were negatively correlated with plasma cystathionine
  • Plasma retinol and plasma albumin concentrations were not positively associated with hemoglobin
  • There was some evidence of early functional deficiency of vitamin B12 but not folate deficiency amount pregnant women and not vitamin B12 deficiency anemia despite very low intakes of animal products
  • The prevalence of IDA was also low compared with elsewhere in Africa
  • Consumption of enset fermented by vitamin B12 producing microorganisms may have increased vitamin B12 levels in the diets and in plasma while at the same time enhancing non-heme iron absorption
  • Two micronutrients (zinc and iron) and two non-dietary factors, infection and gravida, were the major predictors of hemoglobin.
Funding Source:
Government: NIH grants R21 TW06729, M01 RR00069 and MRDDR Center grant P30 HD004024
University/Hospital: University of Otago Research Grant
Reviewer Comments:
  • Causal inferences cannot be made from the associations reported in this study and the results cannot be extrapolated to all women in the third trimester of pregnancy living in the Sidama Zone of Ethiopia
  • The sample size was small and this may have contributed to the null findings of differences in micronutrient status between two groups.
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? ???
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? 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? 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? 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? Yes
  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? Yes
  6.6. Were extra or unplanned treatments described? Yes
  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)? 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