Vegetarian Nutrition

VN: Vitamin B-12 (2011)

Citation:

Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L, Guttormsen AB, Joglekar A, Sayyad MG, Ulvik A, Ueland PM. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am J Clin Nutr. 2001 Aug; 74(2): 233-241.

PubMed ID: 11470726
 
Study Design:
Cross-Sectional Study
Class:
D - Click here for explanation of classification scheme.
Quality Rating:
Positive POSITIVE: See Quality Criteria Checklist below.
Research Purpose:

To examine indicators of cobalamin status in Asian Indians.

Inclusion Criteria:
  • No without acute or severe illnesses
  • Provided informed consent
  • Evidence of no large vessel disease, not including atherosclerosis
  • Had not received treatment with nitrous oxide anesthesia during the previous three months.
Exclusion Criteria:
  • Acute or severe illness
  • Treatment with nitrous oxide anesthesia during the previous three months
  • Evidence of large vessel disease other than atherosclerosis
  • Pregnancy
  • Excessive alcohol consumption.
Description of Study Protocol:

Recruitment

Subjects were recruited from one of the two Cardiology Clinics at King Edward Memorial (KEM) Hospital or from the Diabetic Unit at the KEM Hospital. In addition, healthy subjects recruited were staff members of KEM Hospital, their contacts or outpatients attending the KEM Hospital for treatment of minor illnesses.

Design

Case control study. Variables such as cobalamin, cobalamin deficiency, MMA, serum folate, hemoglobin, anemia, MCV, macrocytosis and thrombocytopenia are compared in vegetarians and non-vegetarians as well as among healthy subjects and subjects with diabetes, CVD and CVD + diabetes.

Statistical Analysis

Results were presented as medians, percentages, and fifth and 95th percentiles unless otherwise indicated. Student's T-test for independent samples, one-way analysis or variance and the chi-square test were used for comparisons between groups. Tukey's post-hoc test was performed to identify significantly different group means. Determinants of cobalamin status were identified by Spearman rank-order correlation coefficients, and analysis of variance was used to assess the relation between the various determinants and cobalamin concentrations. Odds ratios for tHcy concentrations were greater than 20μmol per L; for MMA concentrations higher than 0.50μmol per L; or for cobalamin deficiency, were obtained by logistic regression analyses. A logistic regression analysis of the risk of cobalamin deficiency was performed in subjects with infrequent intake for the various food groups.  P<0.05 (two-sided) was considered significant. SPSS version 10 for Macintosh was used for statistical analyses.

Data Collection Summary:

Timing of Measurements

Blood samples were collected to or more weeks after all minor illnesses were successfully treated. Questionnaires were completed by a research assistant. Information on the subjects' personal and family histories of CVD; smoking; and intakes of vitamin supplements, drugs, and alcohol were collected.  Height, weight, waist and hip circumferences; pulse rate; and blood pressure measurements were repeated twice, and the means of the two measurements were calculated. All subjects underwent a stress test on a treadmill. A fasting blood sample was collected on all subjects.

Variables

  • Cobalamin status
  • Vegetarian diet
  • Diabetes
  • CVD
  • tHcy.

A tHcy concentration of 20.0μmol per L were used as thresholds. Thrombocytopenia was defined as a platelet count less than 140 x 109L, macrocytosis as a mean corpuscular volume less than 100fL and anemia as a hemoglobin concentration less than 135g per L for men and less than 115g per L for women. Cobalamin deficiency was defined as a serum cobalamin concentration less than 150pmol per L. Low holoTC was defined as concentration less than 35pmol per L, folate deficiency was defined as a serum folate concentration less than 5nmol per L, elevated serum MMA was defined as a concentration higher than 0.26μmol per L and hyperhomocysteinemia was defined as a tHcy concentration higher than 15.0μmol per L.

Note: while intake of vitamin supplements was gathered, this information is not used in reporting results.

Description of Actual Data Sample:
  • Initial N: 204
    • 100 with CVD, 104 without CVD
    • 126 non-vegetarians, 78 vegetarians
  • Attrition (final N): 204
  • Age: 27 to 55 years
  • Ethnicity: Asian Indian
  • Other relevant demographics: 83% male, 17% female; 38% vegetarians, 41% with diabetes
  • Location: Pune, Maharashtra, India. 
Summary of Results:

 Key Findings

Markers of B12 Status by Disease Group

Variables Total Population (N=204) Healthy (N=63) Diabetes (N=41) CVD (N=58) P2
Cobalamin deficiency 47% 46% 54% 43% 0.78
Serum cobalamin 154 160 130 159 0.79
Vegetarian diet 38% 27% 44% 45% 0.17

HoloTC (pmol per L)

21 (8 to 96) 24 16 22 0.62
MMA (μmol per L) 0.49 (0.08 to 1.67) 0.40 0.4 0.50 0.33
MMA (0.26μmol per L or more) 73% 70% 71% 79% 0.65

Variables Associated with B12, holoTC and MMA

  • 52% of subjects had a low serum cobalamin concentration, 76% of subjects had hyperhomocysteinemia and 73% had an elevated MMA concentration
  • Only 6% of the subjects had a tHcy concentration lower than 10μmol per L
  • MMA was higher in men than in women (0.52 compared with 0.29μmol per L, P<0.01)
  • Total cobalamin, holoTC, tHcy and MMA concentrations were not significantly associated with age
  • The association between total cobalamin and holoTC was strong (R=0.78), and both variables showed strong, significant inverse relationships with tHcy and MMA. As serum cobalamin increased, holoTC increased.

B12 Status by Diet

Variable Non-vegetarians Vegetarians
Cobalamin deficiency 39% 60% 0.003
Low HoloTC 71% 75% 0.61
MMA higher than 0.26μmol per L 71% 76% 0.51
tHcy higher than 15μmol per L 76% 79% 0.59
Folate deficiency 6% 4% 0.60
Hemoglobin (g per L) 146 (116 to 171) 144 (108 to 164) 0.031
Anemia 15% 22% 0.21
MCV (fL) 81.4 (69.9 to 93.6) 82.1 (65.3 to 97.3) 0.85

Other Findings

About 75% of the subjects had metabolic signs of cobalamin deficiency, which was only partly explained by the vegetarian diet.

Author Conclusion:
  • Indicators of impaired cobalamin status were observed in both vegetarians and non-vegetarians. 
  • Folate deficiency  was rare and only 2.5% of the subjects were homozygous for the MTHFR 677C→T polymorphism. 
  • The mean cysteine concentration was slightly lower than the mean concentration reported in Norwegians; therefore, the high tHcy concentrations were not due to analytic errors or lyophilization of the samples. 
  • Low cobalamin concentrations were consistent with data from the Indian subcontinent of healthy subjects as well as malnourished children. Cobalamin deficiency was common, even in subjects who reported intake of eggs, poultry and mutton more than two times per week (36%). 
  • Low cobalamin concentrations were often accompanied by thrombocytopenia in the subjects. The cause of thrombocytopenia was unclear, but could be related to impaired cobalamin function or a lack of other nutrients. 
  • If these findings of approximately 75% of the adult population with evidence of cobalamin deficiency are confirmed in other parts of India, they may have important health implications.
Funding Source:
Government: EU Commission Demonstration Project, The Advanced Research Programme and the Programme for Advanced Technical Equipment of the Norwegian Research Council
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) 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? No
  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) No
  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? 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%.) No
  4.3. Were all enrolled subjects/patients (in the original sample) accounted for? Yes
  4.4. Were reasons for withdrawals similar across groups? No
  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? No
  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? 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? 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