" I I , I - _
Effects of Dietary Restnction of Animal-Based Products on Vitamin Bl2
Intake and Status
Arash Harzand, David R. Maneval, Amanda R. Brown, Et Lynn B. Bailey
Consumption of a vegetarian diet can adversely affect vitamin B12 (B12) intake and functional status, which
may lead to B12 deficiency. B12 deficiency can contribute to clinical conditions including cardiovascular
disease, impaired fetal development and neurological degeneration. The objectives of this study were to
determine the differences in dietary B12 intake between vegetarians and omnivores and examine the effect
of vegetarian diets on several indicators of B12 status, including plasma B12, serum methylmalonic acid and
serum homocysteine concentrations. Healthy, non-smoking men and women (n = 302; aged 19-49 years) not
taking prescriptions or B12 supplements were surveyed to determine dietary B12 intake and to assess their
plasma B12, serum methylmalonic acid and serum homocysteine concentrations. Consumption of meat resulted
in increased total B12 intake (P < 0.0001). Increased B12 intake was associated with increased plasma B12 (P
= 0.0068) and lower serum methylmalonic acid concentrations (P = 0.001). The proportion of individuals
considered deficient based on these biochemical indicators was found to be greater among vegetarians
than omnivores. These data suggest that omnivores have a reduced risk for developing a B12 deficiency
as determined by plasma B12 concentration and a functional indicator of B12 status.
Vitamin B12 is an essential water-soluble nutrient that functions as a key participant in several metabolic
processes vital to the maintenance of vascular and reproductive health. In humans it functions as a coenzyme for
two metabolic reactions, the isomerization of L-methylmalonyl-CoA to succinyl-CoA in the form of
adenosylcobalamin and the remethylation of homocysteine to methionine as methylcobalamin 1. Vitamin
B12 deficiency can impair this latter process, leading to elevated levels of homocysteine, a known risk factor
for several clinical abnormalities including cardiovascular disease, impaired fetal development and
Vitamin B12 is synthesized by intestinal bacterial in animals and thus is present only in dietary sources of
animal origin5. Consumption of an animal-restricted diet, as in vegetarianism, may limit dietary intake of vitamin
B12 and negatively affect vitamin B12 status6. Previous studies have reported a decrease in dietary intake of
vitamin B12 in vegetarians in comparison to omnivores when the vegetarian group does not consume vitamin
B12 supplements or vitamin B12-fortified products 7 9. Approximately 4.8 million individuals (2.5%) among the U.
S. adult population report consuming vegetarian diets, and approximately 1% report consuming a vegan diet1o.
The younger segment of the population has shown an increasing trend towards consuming vegetarian
diets, especially among females of reproductive age of which 20-25% are known to be vegetarians.11 The role
of vitamin B12 in proper cell division places these individuals at high risk for producing offspring with neural
Several biochemical indicators are commonly employed to assess vitamin B12 status in humans. Although
plasma vitamin B12 concentration is the default method used in clinical settings, reliance on this technique as
the sole diagnostic tool may lead to erroneous interpretations and it has been recommended that additional
status indicators be evaluated 1. Methylmalonic acid (MMA) concentration is considered to be a more
functional indicator of intracellular, metabolically active vitamin B1212,13. Studies have suggested that
serum homocysteine concentration additionally may provide an accurate indication of intracellular vitamin
The objectives of the present study were to determine the differences in mean dietary B12 intake
between vegetarians and omnivores, and to examine the effect of vegetarian diets on several indicators of
vitamin B12 status including plasma vitamin B12, serum MMA and serum homocysteine concentrations
when compared to omnivores.
SUBJECTS AND METHODS
I 1W I Journal of UndcrgriAdUatC IkCIC University of Florida
Subjects were healthy males and females aged 19-49 years (n = 302) recruited and screened to participate in
this study. Prospective subjects were interviewed by phone to determine eligibility on the basis of the
following exclusion criteria: (a) chronic use of tobacco or alcohol products, (b) use of any prescription
medications (excluding oral-contraceptives), (c) history of chronic disease or major surgery, (d) pregnant/
lactating, (e) use of vitamin B12 supplements within the last 6 months, and (i) major dietary changes within the
last 3 years. The University of Florida Institutional Review Board approved this study, and written informed
consent was obtained from each subject.
Dietary Intake Analysis
The National Institutes of Health (NIH) dietary history questionnaire (DHQ) was modified to include questions
for meat-containing foods and vitamin B12-fortified foods. This modified DHQ was used to assess the contribution
of meat and meat-containing foods to total vitamin B12 intake. Analysis of the questionnaire was performed
using software provided by the NIH (Diet*Calc, NIH, Bethesda, MD), employing nutrient databases that
are commonly used for these purposes (Survey Nutrient Database, USDA & NDS-R, University of Minnesota).
Modifications to the DHQ were made in a manner that allowed for the isolation of vitamin B12-containing foods
for data analysis. Questions were modified or added that specifically inquired about vitamin B12-containing
foods such as meat, eggs and dairy products.
Specimen Collection and Analytical Methods
Fasting blood samples were drawn into ethylenediaminetraacetic acid (EDTA)-coated tubes and serum separator
clot activator tubes. EDTA-coated tubes were centrifuged at 2000 x g at 4?C for 30 mm to obtain plasma, and
serum tubes were centrifuged at 650 x g at room temperature for 15 mm to obtain serum. Samples
were immediately processed and stored at -202C until analyzed. Plasma vitamin B-12 concentration was
determined by competitive protein binding radioassay (SimulTRAC; MP Biomedicals, Orangeburg, NY).
Serum homocystelne and MMA concentrations were measured by an adaptation of the capillary gas
chromatography-mass spectrometry method of Marcell et al. (14-17).
Normal values for indexes of vitamin B-12 status assessment. Plasma vitamin B12 concentrations
between 148 and 221 pmol/L were considered marginally deficient (low-normal) and values < 148 pmol/L
were considered deficient (18). Serum MMA concentrations between 73 and 270 nmol/L (13) and
homocystelne concentrations below 12 mmol/L were considered normal (19).
Dietary Vitamin B12 Intake
Total vitamin B12 intake (mean ï¿½ SD) among the two dietary groups is presented in Table 1. Values are
expressed as total B12 intake (pg/day) consumed. Unadjusted for calories, total vitamin B12 intake in the
vegetarian group was significantly lower (P < 0.0001) than that of the omnivore group.
The total vitamin B12 intake (pg) among the two dietary groups was at or above the Recommended
Dietary Allowance (RDA) of 2.4 pg/day. The omnivore group consumed 2.8 times more B12 than the RDA, in
contrast to the vegetarian group, which consumed 1.4 times more than the RDA.
Daily Total Dietary Vitamin B12 IntakeI
Vitamin (n = 181) (n = 121)
Vitamin B12 (pg) 6 8 40 3 4 +3 02
1 Values expressed as mean + SD
2 Significantly lower than omnivores (P< 0 0001) One-way ANOVA (adjusted for age/gender)
Vitamin B12 Status
Plasma vitamin B12 concentration was significantly higher in the omnivore group (P = 0.0068) than the
vegetarian group (Figure 1). The mean plasma vitamin B12 concentration for both groups was above the normal
limit of 221 pmol/L. The percent of individuals that were deficient in the vegetarian group (17%) was higher (P
= 0.01) than the percent of individuals that were deficient in the omnivore group (8%).
Figure 1. Plasma vitamin B12 concentration of vegetarians and omnivores (P = 0.0068).
Serum MMA concentration was significantly lower (P = 0.001) in the omnivore group than the vegetarian
group (Figure 2). The mean serum MMA concentration for both groups was within the normal range of 73 to
270 nmol/L. The percent of individuals with elevated MMA concentrations in the vegetarian group (27%) was
higher (P= 0.0004) than the percent of individuals that were elevated in the omnivore group (11%).
Figure 2. Methylmalonic Acid concentration of vegetarians and omnivores (P = 0.001)
There was no significant difference (P = 0.078) in serum homocystelne concentration between the dietary
groups. The mean homocystelne concentration for both groups was below the normal limit of 12 pmol/L.
The percentage of individuals with elevated homocystelne concentrations in the vegetarian group (6%) was higher
(P = 0.0461) than the percentage of individuals that were elevated in the omnivore group (2%). All
measured biochemical indicators of vitamin B12 status (mean ï¿½ SD) are presented in Table 2.
Indices of mtamin B12 status
Vitamin Status (n = 181) (n = 121)
Plasma B12 (pmolL) 313 + 124 280 + 1462
Methylmalonic acid (nmolL) 195 + 116 260 + 2293
Homocysteine (pmolL) 7 3 +25 7 7 +27
1 Values express as mean + SD
2 Significantly lower than omnivores (P = 0 0068)
One-way ANOVA (controlled for BMI)
3 Significantly higher than omnivores (P = 0 001)
The focus of the present study was to determine the differences in mean dietary B12 intake between vegetarians
and omnivores, and to examine the effect of vegetarian diets on various indicators of vitamin B12 status,
including plasma vitamin B12, serum MMA and serum homocystelne concentrations when compared to
omnivores. Since vitamin B12 is only present in animal-derived products (i.e., meat, eggs, dairy), vegetarians are
at an increased risk for developing a vitamin B12 deficiency 6 that may result in severe clinical aberrations
including impaired fetal development, irreversible neurological damage and cardiovascular disease. Vitamin
B12 depletion may take years and individuals may be in the pre-clinical state of vitamin B12 deficiency prior
to developing such severe symptoms. During the pre-clinical stage, plasma vitamin B12 is depleted followed by
an intracellular B12 deficiency resulting in metabolic irregularities such as elevated homocystelne and
MMA concentrations. These biochemical changes are due to the role of vitamin B12 as a cofactor for two
important reactions in humans, the remethylation of homocystelne and the isomerization of succinyl-CoA (Figure 3)20.
2 Methionine Synhese1 Methylmalonyl-CoA Mutase
Figure 3. Metabolic interconversions involving B12. A. Remethylation of homocysteine to methionine.
B. Isomerization of succinyl-CoA to methylmalonic acid.
Based upon the DHQ, the intake of vitamin B12 was significantly lower in individuals that excluded dietary
meat compared to omnivores. Individuals excluding dietary meat ingested significantly lower amounts of vitamin
B12 compared to omnivores. This underscores the importance of meat as a contributor to vitamin B12
intake. Although both dietary groups consumed vitamin B12 at a level above the RDA, omnivores consumed
2.8 times more B12 than the RDA compared to vegetarians, who consumed only 1.4 times more than the RDA.
These data indicate that intake of vitamin B12 increased along with increased consumption of meat in the diet.
Mean plasma vitamin B12 concentration was significantly higher for omnivores than vegetarians. Although
mean plasma vitamin B12 concentration was above the normal level for both groups, the vegetarian group
contained a significantly larger proportion of individuals (17%) that were deficient (< 148 pmol/L) than the
omnivore group (8%), a nearly two-fold difference. Although plasma vitamin B12 concentration is widely used as
the primary vitamin B12 status indicator in clinical settings, it has been recommended that additional
status indicators be evaluated in order to obtain a more accurate indication of vitamin B12 status (1). In one
previous study, it was observed that a small percentage of patients exhibiting clinical symptoms of vitamin
B12 deficiency that also responded positively to vitamin B12 therapy had plasma vitamin B12 concentrations in
the range of low-normal (148-200 pmol/L)21.
There was a significant elevation in serum MMA concentration in the vegetarian group when compared to
omnivores. Serum MMA concentration is a highly specific indicator of vitamin B12 status. Unlike the
methionine synthase reaction that requires both vitamin B12 and folate, the synthesis of succinyl-CoA from
MMA requires only vitamin B12. Elevated MMA is therefore a specific indication of an impaired B12-
dependent process. The significant elevation of this functional indicator of vitamin B12 status in the vegetarian
group indicates the importance of dietary or supplemental B12 in order to prevent a vitamin B12 deficiency.
There was a notably higher fraction of individuals with elevated levels of MMA in the vegetarian group (27%) than
in the omnivore group (11%).
There was no significant difference in serum homocysteine concentration between the dietary groups.
Homocysteine concentration has been shown to be inversely associated with vitamin B12 status and is elevated in
the majority of individuals with low vitamin B12 status2, 22. Since homocysteine remethylation is dependent on
both vitamin B12 and folate, it is possible that individuals with an impaired vitamin B12 status may exhibit
no difference in homocysteine concentration if they are folate replete. Folic acid fortification has also been
widely effective in lowering plasma homocysteine in the U.S. population and makes it unlikely that any individuals
in our study population were folate deficient. Homocysteine is also remethylated in the liver during a vitamin
B12-independent reaction, further confounding the utility of homocysteine concentration as an indicator of B12 status.
Users of vitamin B12 supplements were excluded from participating in this study. The DHQ was also modified
to ensure the inclusion of specific questions regarding vitamin B12-containing foods and vitamin B12-fortified
foods. Both of these attributes contributed to the effectiveness of this study in accurately assessing vitamin
B12 intake from dietary sources. A possible weakness of this study is the lack of separation of the vegetarian
groups into more specific groupings (e.g., lacto-ovo vegetarian, lacto-vegetarian and vegan). The results may
have been different when comparing the contribution of vitamin B12 intake from eggs, for example, since vegans
and lacto-vegetarians do not consume them.
This study used a modified DHQ to compare the difference in vitamin B12 intake between omnivores and
vegetarians and determine the effects of meat-restriction on several indicators of vitamin B12 status. It
was determined that an increased frequency of dietary meat intake is associated with a greater intake of total
dietary vitamin B12. Increased vitamin B12 intake was associated with increased plasma vitamin B12
concentration and lower serum MMA concentrations. Vegetarians were also found to have a larger proportion
of individuals considered deficient based on these biochemical values than omnivores. These data suggest
that omnivores have a reduced likelihood of developing a vitamin B12 deficiency. Dieticians, educators and
physicians can use these data to promote consumption of more vitamin B12-containing foods and/or
B12 supplements in order to optimize vitamin B12 status.
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