Journal of Undergraduate Research
Volume 8, Issue 2
2 - November/December 2006

Effects of Dietary Restriction of Animal-Based Products on Vitamin B12 Intake and Status

Arash Harzand, David R. Maneval, Amanda R. Brown, & Lynn B. Bailey

ABSTRACT

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.

INTRODUCTION

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 neurological abnormalities2-4.

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 diet10. 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 tube defects4.

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 B12 status12.

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

Subjects

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 min to obtain plasma, and serum tubes were centrifuged at 650 x g at room temperature for 15 min to obtain serum. Samples were immediately processed and stored at -20?C until analyzed. Plasma vitamin B-12 concentration was determined by competitive protein binding radioassay (SimulTRAC; MP Biomedicals, Orangeburg, NY). Serum homocysteine 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 homocysteine concentrations below 12 mmol/L were considered normal (19).

RESULTS

Dietary Vitamin B12 Intake

Total vitamin B12 intake (mean ± SD) among the two dietary groups is presented in Table 1. Values are expressed as total B12 intake (μg/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 (µg) among the two dietary groups was at or above the Recommended Dietary Allowance (RDA) of 2.4 μg/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.

Table 1.
Daily Total Dietary Vitamin B12 Intake1
Vitamin Omnivore
(n = 181)		 Vegetarian
(n = 121)
Vitamin B12 (µg) 6.8 ± 4.0 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).
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)
Figure 2. Methylmalonic Acid concentration of vegetarians and omnivores (P = 0.001)

There was no significant difference (P = 0.078) in serum homocysteine concentration between the dietary groups. The mean homocysteine concentration for both groups was below the normal limit of 12 μmol/L. The percentage of individuals with elevated homocysteine 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 ± SD) are presented in Table 2.

Table 2.
Indices of vitamin B12 status1
Vitamin Status  Omnivore
(n = 181)		 Vegetarian
(n = 121)
Plasma B12 (pmol/L)  313 ± 124  280 ± 1462
Methylmalonic acid (nmol/L) 195 ± 116 260 ± 2293
Homocysteine (µmol/L)  7.3 ± 2.5 7.7 ± 2.7
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)

DISCUSSION

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 homocysteine 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 homocysteine 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 homocysteine and the isomerization of succinyl-CoA (Figure 3)20.

Figure 3. Metabolic interconversions involving B12

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.

REFERENCES

  1. Stabler SP. Vitamin B12. In: Bowman BA, Russell RM, eds. Present Knowledge in Nutrition. Washington, D.C.: ILSI Press, 2001:230-240.
  2. Mezzano D, Kosiel K, Martinez C, et al. Cardiovascular Risk Factors in Vegetarians:  Normalization of Hyperhomocysteinemia with Vitamin B12 and Reduction of Platelet Aggregation with n-3 Fatty Acids. Thrombosis Research 2000;100:153-160.
  3. Pittock SJ, Payne TA, Harper CM. Reversible myelopathy in a 34-year-old man with vitamin B12 deficiency. Mayo Clin Proc 2002;77:291-4.
  4. Groenen PMW, van Rooij IALM, Peer PGM, Gooskens RH, Zielhuis GA, Steegers-Theunissen RPM. Marginal maternal vitamin B12 status increases the risk of offspring with spina bifida. American Journal of Obstetrics and Gynecology 2004;191:11-17.
  5. Seetharam B AD. Absorption and transport of cobalamin (vitamin B12). Annu Rev Nutr 1982;2:343-69.
  6. Herrmann W GJ. Vegetarian lifestyle and monitoring of vitamin B-12 status. Clin Chim Acta 2002;326:47-59.
  7. Miller DR, Specker BL, Ho ML, Norman EJ. Vitamin B-12 status in a macrobiotic community. Am J Clin Nutr 1991;53:524-9.
  8. Rauma AL, Torronen R, Hanninen O, Mykkanen H. Vitamin B-12 status of long-term adherents of a strict uncooked vegan diet ("living food diet") is compromised. J Nutr 1995;125:2511-5.
  9. Leblanc JC YH, Kombadjian A, Verger P. Nutritional intakes of vegetarian populations in France. Eur J Clin Nutr 2000;54.
  10. Haddad EH, Tanzman JS. What do vegetarians in the United States eat? Am J Clin Nutr 2003;78:626S-632.
  11. Barr SI, Broughton TM. Relative Weight, Weight Loss Efforts and Nutrient Intakes among Health-Conscious Vegetarian, Past Vegetarian and Nonvegetarian Women Ages 18 to 50. J Am Coll Nutr 2000;19:781-788.
  12. Vitamin B12. Dietary reference intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, D.C.: National Academy Press, 1998:306-356.
  13. Beck W. Cobalamin (Vitamin B12). In: Ruckber RB SJ, McCormick DB, Machlin LJ, ed. Handbook of Vitamins. 3rd ed. New York: Marcel Dekker, 2001:463-512.
  14. Marcell PD, Stabler SP, Podell ER, Allen RH. Quantitation of methylmalonic acid and other dicarboxylic acids in normal serum and urine using capillary gas chromatography-mass spectrometry. Anal Biochem 1985;150:58-66.
  15. Stabler SP, Allen RH. Quantification of Serum and Urinary S-Adenosylmethionine and S-Adenosylhomocysteine by Stable-Isotope-Dilution Liquid Chromatography-Mass Spectrometry. Clin Chem 2004;50:365-372.
  16. Stabler S, Lindenbaum J, Savage D, Allen R. Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency. Blood 1993;81:3404-3413.
  17. Allen RH, Stabler SP, Savage DG, Lindenbaum J. Elevation of 2-methylcitric acid I and II levels in serum, urine, and cerebrospinal fluid of patients with cobalamin deficiency. Metabolism 1993;42:978-88.
  18. Lindenbaum J, Allen R. Clinical spectrum and diagnosis of folate deficiency. In: Bailey LB, ed. Folate in Health and Disease. New York, NY: Marcel Decker, 1995:43-73.
  19. Selhub J, Jacques PF, Rosenberg IH, et al. Serum total homocysteine concentrations in the third National Health and Nutrition Examination Survey (1991-1994): population reference ranges and contribution of vitamin status to high serum concentrations. Ann Intern Med 1999;131:331-9.
  20. Herbert V. Staging vitamin B-12 (cobalamin) status in vegetarians. The American Journal Of Clinical Nutrition 1994;59:1213S-1222S.
  21. Afman LA, Van Der Put NMJ, Thomas CMG, Trijbels JMF, Blom HJ. Reduced vitamin B12 binding by transcobalamin II increases the risk of neural tube defects. QJM 2001;94:159-166.
  22. Krajcovicova-Kudlackova M, Blazicek P, Kopcova J, Bederova A, Babinska K. Homocysteine levels in vegetarians versus omnivores. Ann Nutr Metab 2000;44:135-8.

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