VN: Vitamin B-12 (2011)
Geisel J, Schorr H, Bodis M, Isber S, Hübner U, Knapp JP, Obeid R, Herrmann W. The vegetarian lifestyle and DNA methylation. Clin Chem Lab Med. 2005; 43(10): 1164-1169.
PubMed ID: 16197315
The primary research purpose is to investigate whether there is an association between disturbed remethylation caused by vitamin B12 deficiency in vegetarians and DNA global methylation.
- Using additional measurements, SAM (S-adenosylemethionine) and SAH (S-adenosylhomocysteine), the regulation of DNA methylation could be analyzed in greater detail
- Promoter methylation of the p66Shc gene was studied.
- Healthy vegetarians
- Informed consent signed by participant
- Maintained a constant dietary pattern for more than one year.
Not applicable.
Recruitment
Subjects recruited at a conference of the German Federation of Vegetarians.
Design
Cross-sectional study.
Blinding used
Not applicable.
Intervention
Not applicable.
Statistical Analysis
- Logistic regression used for homocysteine, MMA, vitamins B6 and B12, SAM, SAM/SAH ratio, HoloTC and cystathionine
- Correlations between variables evaluated by Pearson test
- All tests were two-tailed and significance set at P<0.05.
Timing of Measurements
Blood samples collected after 12-hour fast for all participants.
Dependent Variables
- Median plasma concentrations
- Homocysteine: assayed by gas chromatography-mass spectrometry
- Vitamin B12: measured using chemiluminescence immunoassays
- MMA (methylmalonic acid): assayed by gas chromatography-mass spectrometry
- HoloTC (holotranscobalamin II): quantified by radioimmunoassay
- Folate: measured using chemiluminescence immunoassays
- Vitamin B6: analyzed by HPLC
- SAM: measured using an HPLC method
- SAH: measured using an HPLC method.
- DNA analysis
- Global DNA methylation status: mean methylation values for three CpG sites using Pyrosequencing method
- Specific promotor methylation for the p66Shc gene: mean methylation values for three CpG sites in two sequencing reactions were used also using Pyrosequencing
Independent Variables
Vegetarian diet.
Control Variables
- Age
- Sex.
Initial N
Not applicable.
Attrition (final N)
- 71 vegetarians
- 48 lacto/lacto-ovo-vegetarians
- 23 vegans.
- 79 omnivorous.
Age
Median age (years)
- Lacto/lacto-ovo-vegetarian=53
- Vegan=51
- Omnivorous=51.
Ethnicity
German
Anthropometrics
Vegetarians' age was significantly different from omnivorous subjects, P<0.05.
Location
Germany.
Key Findings
- Homocysteine levels above 12μmol/L were found in 45% of vegetarians
- Median and percentage of elevated homocysteine concentrations were significantly lower in vegetarians compared to age and sex matched omnivorous control group
- vitamin B12 deficiency (HoloTC II<35pmol/L or MMA>271nmol/L) found in 58% of the vegetarians and 30% of the deficient subjects were categorized as stage III deficient.
Variables |
Omnivorous subjects, n=79 Median (5th and 95th percentiles) |
Lacto-/lacto-ovo-vegetarians, n=48 Median (5th and 95th percentiles) |
Vegan subjects, n=23 Median (5th and 95th percentiles) |
Homocysteine, μmol/L |
8.8 (5.5, 16.1) |
11.1 (6.3, 21.8)* |
13.0 (5.5, 38.0)* |
Vitamin B12, pmol/L |
287 (189, 470) |
180 (117, 356)* |
145 (86, 596)* |
HoloTC II, pmol/L |
54 (16,122) |
40 (9, 82)* |
22 (1, 160)* |
MMA, nmol/L | 160 (94,356) | 273 (150,1198)* | 695 (129,3582)* |
Folate, nmol/L | 21.7 (14.5, 51.4) | 22.8 (11.7, 42.8) | 25.4 (13.6, 66.0) |
Vitamin B6, nmol/L | 52.5 (24.7, 128.8) | 53.1 (21.7, 166.5) | 46.2 (18.3, 260.9) |
*Significantly different from omnivorous subjects, P<0.05 (Mann-Whitney test)
Other Findings
Global DNA methylation status
- Significant correlations between global DNA methylation status and SAH (r=-0.36, P<0.01 and the SAM/SAH ratio (r=-0.27, P<0.05) were found by using the mean methylation values for three CpG sites
- No significant association was seen between methylation and plasma homocysteine or SAM
- A trend for a lower degree of methylation in subjects homozygous for the 677C→T mutation (12.1% in subjects) was observed.
Promoter methylation of the p66Shc gene
- No correlation between degree of methylation (used as surrogate measurement for promotor methylation) and homocysteine or SAM
- No correlation between promoter methylation and SAM or the SAM/SAH ratio.
- In vegetarians, the reverse metabolism of homocysteine to SAH is not important, due to lack of correlation in the study
- Total global and p66Shc promoter methylation measures showed no effects of disturbed methyl group metabolism in vegetarians
- Negative consequences for methyl group metabolism was not seen in vegetarians due to the absence of correlations found between homocysteine or SAM and the degree of methylation.
University/Hospital: | Department of Clinical Chemistry, Saarland Medical School, Homburg, Germany |
Other: | Not reported |
- No details given about how age- and sex- matched omnivorous control group were selected
- No details given for funding for study; only listed primary author's contact info
- Vegetarians can be at risk for abnormal methyl group metabolism due to low blood levels of vitamin B12, which leads to elevated serum homocysteine
- Diets are low in methyl group donors, methionine
- This may lead to increased risk for certain cancers, atherosclerosis, aging and other diseases
- SAM may be an important factor in the methylation process for vegetarians because no association was seen between methylation (both global and specific gene) and diet
- Vegetarians may not have an increased risk of disease due to dietary choices, as previously thought
- Good study to understand and review biochemical consequences of the vegetarian diet; difficult study to determine real-world implications of results.
Quality Criteria Checklist: Primary Research
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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? | 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? | No | |
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? | No | |
3. | Were study groups comparable? | No | |
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? | No | |
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? | No | |
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.) | No | |
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? | 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? | ??? | |
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? | N/A | |
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? | No | |
6.6. | Were extra or unplanned treatments described? | No | |
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? | 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? | No | |
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 | |