There are approximately four million vegetarians within the UK population(1). In addition 5 % of British adults are practising semi-vegetarians, whose diet contains a greatly reduced intake of products of animal origin(2). Worldwide, there are 75 million vegetarians by choice and 1450 million by necessity(3). This agrees with the Foods Standards Agency(4) approximation of 25 % of the world's population consuming a largely vegetarian diet. The most commonly known vegetarians are vegan, lactovegetarian (LV) and lacto-ovovegetarian (LOV).
Hyperhomocysteinaemia (>15 μmol/l, as defined by Ravaglia et al. (Reference Ravaglia, Forti and Maioli5)) has been shown to be linked with chronic conditions, among which is CVD(Reference Ueland, Refsum and Beresford6, Reference Humphrey, Fu and Rogers7). Other studies have shown that CHD is linked to homocysteine concentrations, with a substantial risk occurring at >10 μmol/l plasma total homocysteine (tHcy)(Reference Malinow, Bostom and Krauss8, Reference Ubbink, Carmel and Jacobsen9). Furthermore, each 5 μmol/l increase in plasma tHcy is associated with an approximate 20 % increased risk of CHD events(Reference Ueland, Refsum and Beresford6, Reference Humphrey, Fu and Rogers7), irrespective of the diet. The present review sets out to determine the homocysteine and vitamin B12 status of vegetarians compared with omnivores, as they may be a group of the population who may have the potential to be at greater risk than omnivores to these homocysteine-related diseases. This is due to the lack of intake of animal produce, the only natural abundant source of vitamin B12, whose deficiency can raise homocysteine levels(Reference Kluijtmans, Boers and Tribels10, Reference Selhub11). Vitamin B12 is required in the important remethylation pathway, where homocysteine is remethylated to methionine in a reaction catalysed by the enzyme methionine synthase and the cofactor vitamin B12(Reference Dudman, Guo and Gordon12), but only in its methylcobalamin form(Reference Herrmann, Obeid and Schorr13). Homocysteine acquires a methyl group from 5-methyl tetrahydrofolate (THF), which is catalysed from 5,10-methyleneTHF by the enzyme methylenetetrahydrofolate reductase and folic acid/folate from the diet, which enters the remethylation pathway as THF, via dihydrofolate acid, which has been reduced to THF by the enzyme dihydrofolate reductase. The exact pathomechanisms of cobalamin deficiency that cause the typical clinical symptoms of vitamin B12 deficiency, especially the neurological symptoms, in human subjects have not been fully clarified. The methyl folate trap hypothesis(Reference Herbert and Zalusky14–Reference Scott and Weir16) has been widely accepted over decades, despite the difficulty in testing the theory in any meaningful way. The methyl trap hypothesis proposes that due to a vitamin B12 deficiency, folate can be trapped as methylfolate, which is metabolically inactive. This is due to the fact that vitamin B12 is required for the transfer of the methyl group from 5-methylTHF to form THF, so that it can return to the tetrahydrofolate pool for conversion to 5,10-methyleneTHF. As the transfer of the methyl group of 5-methylTHF to homocysteine is impaired in vitamin B12 deficiency, it results in a rise in homocysteine levels(Reference Chanarin, Deacon and Lumb17, Reference Butensky, Harmatz and Lubin18).
The RDA for vitamin B12 is 2·5 μg(19), of which the body stores considerable amounts (several mg) in the liver. The body recycles approximately 75 % of vitamin B12 it uses; serum vitamin B12 starts to decline and plasma tHcy rises when the absorption of the ingested vitamin B12 input is less than that dissipated by the body(Reference Herbert20). Thus, a delay of 5–10 years may separate the beginning of a vegetarian diet and the onset of deficiency symptoms that usually occur when serum vitamin B12 is reduced to below 150 pmol/l, which marks the onset of pernicious anaemia(Reference Herbert and Das21).
It is also noted that cell-surface receptors located in the ileum require free Ca to enable the vitamin B12 absorption – intrinsic factor complex to aid absorption of vitamin B12(Reference Carmel, Rosenberg and Lau22–Reference Herzlich, Schiano and Moussa Zimbalist24).
Lack of Ca in the vegetarian diet could, therefore, inhibit vitamin B12 absorption(Reference Carmel, Rosenberg and Lau22–Reference Herzlich, Schiano and Moussa Zimbalist24). Prolonged Fe deficiency damages the gastric mucosa and promotes atrophic gastritis and gastric atrophy, including loss of gastric acid and intrinsic factor secretion and, therefore, diminished vitamin B12 absorption. As vegetarians have reduced Fe intake(Reference Herbert, Subak-sharpe and Hammock25–Reference Simopoulos, Herbert and Jacobson27), this would cause vitamin B12 deficiency(Reference Herbert28). Furthermore, vitamin B12 is excreted in the presence of high levels of soluble fibre (such as pectin), probably via an effect on the enterohepatic cycle of vitamin B12, a common feature of vegetarian diets(29). Furthermore, vegetarian diets contain high levels of n-6 PUFA, whilst they are low in n-3 PUFA. This imbalance, together with inherent low vitamin B12 levels and consequential high concentrations of plasma tHcy, can be shown to have a thrombotic tendency that raises the risk of developing CVD(Reference Li30).
Hypothesis and objective
The hypothesis is that there is a correlation between levels of plasma tHcy and the intake of dietary animal produce, the only natural abundant source of vitamin B12.
The main objective of the present systematic review and meta-analysis is, therefore, to assess the plasma tHcy and serum vitamin B12 status of LV–LOV and vegans, as compared with omnivores, from a wide range of cohort and cross-sectional published studies that have met the set criteria.
Materials and methods
Electronic searches
The search engines selected were PubMed, as it contains entries from MEDLINE, EMBASE, JAMA, BMJ, Cochrane Databases and Lancet, together with Science Direct, ACP Journal Club, CCTR, AMED, Highwire Press and EBSCO host databases. A search for systematic reviews, meta-analyses, cohort case studies, cross-sectional studies and randomised controlled trials was carried out using the search terms ‘Hyperhomocysteinemia’; ‘Vitamin B12’; ‘Omnivores and vegetarians’; and ‘Supplementation with vitamin B12 to normalise homocysteine in vegetarians’; this revealed 443 entries for studies undertaken during the period from January 1999 to June 2011.
Participants
In the studies examined, omnivores were defined as individuals who consumed both plant and animal products. LV were defined as individuals who did not consume animal produce, but consumed dairy products. LOV had the same diet as LV, but they consumed eggs too. Vegans were defined as individuals who abstained from all types of animal products and semi-vegetarians were defined as individuals who occasionally included animal products in their diet.
Inclusion and exclusion criteria
The flow chart in Fig. 1 outlines the initial inclusion and exclusion criteria employed in the selection of six cohort case studies and eleven cross-sectional studies; this was followed by these studies being finally assessed by one author and checked by another for methodological validity employing standardised data extraction tools from JBI000308(31) with any disagreements being resolved through discussion with a third reviewer. All initially screened studies met these requirements and are included in the present systematic review and meta-analysis, and summarised in Table 1.
Flow chart. Initial inclusion and exclusion criteria for selected studies for plasma total homocysteine (tHcy) and serum vitamin B12 status of omnivores, lactovegetarians (LV) or lacto-ovovegetarians (LOV) and vegans.
Details of the selected studies of plasma total homocysteine (tHcy) and serum vitamin B12 status among omnivores, lactovegetarians or lacto-ovovegetarians and vegans (1999–2010) (Mean values and standard deviations; medians, 5th–95th percentiles and 25th–75th percentiles; geometric mean values and 95 % confidence intervals)
* Mixed population of adult volunteers.
† Male adult volunteers only.
‡ Sex of volunteers not stated.
§ Female adult volunteers only.
∥ Geometric mean.
¶ Combined measured levels of plasma tHcy and serum vitamin B12 of lactovegetarians or lacto-ovovegetarians and vegans.
Statistical analyses
Data from the selected seventeen studies in the vast majority of cases have been calculated as mean values for each diet group. In the case of the small number of cases that employed median values, it has been assumed in the calculations that these are approximately equal to the mean value, and that, in the case of two studies, the small number of vegans has been included in the LV–LOV group. As the number (n) is < 30 for the group of the studies, comparison between groups has been undertaken by Student's two-tailed unpaired test to determine the significant difference between the values of plasma tHcy and serum vitamin B12 for LV–LOV and vegans against omnivores(Reference Spiegel32). Table 2 summarises the calculated values.
Plasma total homocysteine (tHcy) and serum vitamin B12 levels of lactovegetarians or lacto-ovo vegetarians and vegans compared with omnivores from the selected seventeen studies shown in Table 1 (study by Cappuccio et al. (Reference Cappuccio, Bell, Perry and Gilg33) omitted) (Mean values and standard deviations)
Results
Of a total of 443 entries, the search revealed six cohort case studies and eleven cross-sectional studies, as summarised in Table 1.
Table 2 demonstrates that the primary outcome of the meta-analysis is that an inverse relationship exists between plasma tHcy and serum vitamin B12 for all three diets, indicating that vegans have the highest mean plasma tHcy value of 16·41 (sd 4·80) μmol/l as well as the lowest mean serum vitamin B12 value of 172 (sd 59) pmol/l.
LV–LOV exhibited a mean plasma tHcy value of 13·91 (sd 3·15) μmol/l and a mean serum vitamin B12 value of 209 (sd 47) pmol/l. Omnivores recorded a mean plasma tHcy value of 11·03 (sd 2·89) μmol/l and a mean serum vitamin B12 value of 303 (sd 72) pmol/l. Fig. 2 indicates the relationship between plasma tHcy and serum vitamin B12 for the three diets.
Correlation between plasma total homocysteine (tHcy) and serum vitamin B12 for omnivores, lactovegetarians (LV) or lacto-ovovegetarians (LOV) and vegans, with median values approximated to be equal to mean values of five studies(Reference Herrmann, Schorr and Obeid36, Reference Koebnick, Garcia and Dagnelie37, Reference Herrmann, Schorr and Purschwitz41, Reference Refsum, Yajnik and Gadkari45, Reference Waldmann, Koschizke and Leitzmann46) and combined measured levels of plasma tHcy and serum B12 of LV or LOV and vegans of two studies(Reference Yen, Yen and Cheng38, Reference Huang, Chang and Chiu43) from 1999 to 2010 (study by Cappuccio et al. (Reference Cappuccio, Bell, Perry and Gilg33) excluded) taken from Table 1. Y= 24·57 e− 0·003x (R 2 0·598).+, Omnivores; x, LV–LOV; ○, vegans.
Statistical heterogeneity
The null hypothesis states that the mean values of plasma tHcy and serum vitamin B12 for omnivores, LV–LOV and vegans are homogeneous. Table 3 reports the results of an ANOVA, which demonstrates that the null hypothesis can be rejected.
ANOVA table for differences of plasma total homocysteine and serum vitamin B12 between omnivores and vegans and omnivores and lactovegetarians or lacto-ovovegetarians*
* Mean values utilised from Table 2.
Discussion
A total of fifteen of the seventeen selected studies that met the inclusion and exclusion criteria show a good agreement that serum vitamin B12 and plasma tHcy exhibit an inverse relationship. The study by Cappuccio et al. (Reference Cappuccio, Bell, Perry and Gilg33) did not monitor serum vitamin B12 levels. The study by Haddad et al. (Reference Haddad, Berk and Kettering34) concluded that, statistically, vegans had similar plasma tHcy to omnivores (i.e. 8·0 against 7·9 μmol/l, respectively) and serum vitamin B12 levels (i.e. 313 against 312 pmol/l, respectively). In this case, it was noted that 36 % of the participating vegans were users of vitamin B12 supplements, although the type, dosage and frequency of usage were not reported. Nevertheless, this could confound the statistics, which have given a result that is incompatible with the other fifteen studies. Furthermore, a small proportion of LV–LOV and vegans were found to be consuming vitamin B12 supplements and/or consuming vitamin B12-fortified foods in the studies conducted by Bissoli et al. (Reference Bissoli, Di Francesco and Ballarin35), Herrmann et al. (Reference Herrmann, Schorr and Obeid36) and Koebnick et al. (Reference Koebnick, Garcia and Dagnelie37). None of these studies recorded details regarding type, dosage and frequency of consumption. The conclusion reached by the respective researchers was that fortification does not lower plasma tHcy or increase serum vitamin B12 levels significantly. The remaining studies stated that no vitamin B12 supplements had been used by the participants. Yen et al. (Reference Yen, Yen and Cheng38) concluded that vegetarian parents and their preschool children had lower vitamin B12 intake than omnivorous parents and their preschool children, but had similar vitamin B12 and homocysteine concentrations. As far as the adults are concerned, these results are incompatible with the observations of six studies that compared LV–LOV, vegan and omnivores’ serum vitamin B12 and plasma tHcy levels(Reference Herrmann, Schorr and Obeid36, Reference Koebnick, Garcia and Dagnelie37, Reference Mann, Li and Sinclair39–Reference Majchrzak, Singer and Manner42). Huang et al. (Reference Huang, Chang and Chiu43) concluded that vegetarians have lower vitamin B12 status than omnivores, leading to raised plasma tHcy, and that vitamin B6 and folate have little effect on plasma homocysteine concentration when individuals have adequate vitamin B6 and folate status. Kluijtmans et al. (Reference Kluijtmans, Boers and Tribels10) and Selhub(Reference Selhub11) demonstrated that hyperhomocysteinaemia can be caused by a deficiency of folate. They also demonstrated that, normally, homocysteine elevation is much less affected in cases of vitamin B6 deficiency. However, Majchrzak et al. (Reference Majchrzak, Singer and Manner42) have shown that in vegetarian diets and, particularly, in vegan diets, which contain relatively high levels of folate, folate deficiency is unlikely to occur, whereas omnivore diets are more predisposed to this. An exception to this is possibly seen in India, where traditionally folate deficiency has been linked to poverty, which may cause problems, with 33 % of the population being vegetarians by necessity(Reference Antony44). There is strong evidence, with a significance of P< 0·005 from four studies that, compared with omnivores, a large proportion of vegans develop hyperhomocysteinaemia (>15 μmol/l plasma tHcy) and serum vitamin B12 deficiency ( ≤ 150 pmol/l)(Reference Koebnick, Garcia and Dagnelie37, Reference Mann, Li and Sinclair39–Reference Herrmann, Schorr and Purschwitz41). Furthermore, a significant proportion of LV–LOV subjects in the study conducted by Koebnick et al. (Reference Koebnick, Garcia and Dagnelie37) showed a hyperhomocysteinaemia condition, with a strong significance of P< 0·001. A total of ten studies(Reference Bissoli, Di Francesco and Ballarin35, Reference Krajcovicova-Kudlackova, Blazicek and Kopvova40–Reference Huang, Chang and Chiu43, Reference Waldmann, Koschizke and Leitzmann46–Reference Hung, Huang and Lu50) reported that vegans and/or LV–LOV were found to have plasma tHcy >10 μmol/l and serum vitamin B12 of >150 pmol/l (i.e. not deficient(Reference Herbert and Das21)), although vegan and LV–LOV levels of serum vitamin B12 were substantially lower than omnivores, with mean values of 172 and 209 against 303 pmol/l, respectively (Table 2). This was generally in accordance with studies conducted by Joosten et al. (Reference Joosten, van dem Berg and Riezler51) and Herrmann et al. (Reference Herrmann, Schorr and Bodis52).
Refsum et al. (Reference Refsum, Yajnik and Gadkari45) reported that, in India, both omnivores and LV–LOV have high plasma Hcy levels (i.e. 19·4 v. 22·0 μmol/l, respectively), indicating hyperhomocysteinaemia together with low serum vitamin B12 levels (i.e. 161·0 v. 124·0 pmol/l, respectively). It is, however, noted that even the diet of non-vegetarians in India contains only low proportions of animal produce and hence relatively low amounts of vitamin B12(Reference Herrmann, Schorr and Bodis52). Also, it is noted that a high proportion of the Indian population is expected to have, in addition, folate deficiency. This could be a contributing factor for the high levels of plasma tHcy and low levels of serum vitamin B12 in Indian omnivores. Furthermore, most vegetarians and omnivores in India begin consuming essentially a vegetarian diet as infants, which leads to low vitamin B12 intake, with the only source of vitamin B12 coming from bacterial-contaminated food, for most of their lives(Reference Dong and Scott53). India has large proportions of its population who suffer from malnutrition, tropical sprue and gastrointestinal infections, which often result in malabsorption(Reference Herbert54–Reference Balaji and Dustagheer56). It would seem reasonable to deduce that the high prevalence of vitamin B12 deficiency accompanied by elevated plasma tHcy can only be expected for both omnivores and LV–LOV in India.
The examined studies took steps to eliminate possible well-known confounding factors that may distort the results and were appropriately adjusted for factors such as smoking, age and sex. However, there is a minimal risk of distortion due to inter-assay and inter-population bias and variability in the present study. It can be clearly observed from Table 2 that there is an inverse relationship between plasma tHcy and serum vitamin B12. Moreover, statistical evidence in Table 2 indicates that vegans have the highest mean values of plasma tHcy and the lowest mean levels of serum vitamin B12. LV–LOV show intermediate levels, whereas omnivores exhibit high level of serum vitamin B12 and the lowest levels of plasma tHcy. This is compatible with work done by Herbert & Das(Reference Herbert and Das21). Studies undertaken by Gilsing et al. (Reference Gilsing, Crowe and Sanders57), who researched British male omnivores, LV–LOV and vegans, found that 226 omnivores had mean serum vitamin B12 levels of 281 (95 % CI 270, 292) pmol/l, 231 LV–LOV had mean serum vitamin B12 levels of 182 (95 % CI 175, 189) pmol/l and 232 vegans had mean serum vitamin B12 levels of 122 (95 % CI 117, 127) pmol/l. Furthermore, work done by Herbert & Das(Reference Herbert and Das21), who studied vitamin B12 deficiency of LV, LOV, vegans and semi-vegetarians from the American Vegetarian Society, found that 92 % of the vegans, 64 % of the LV, 47 % of the LOV and 20 % of semi-vegetarians had serum vitamin B12 levels of ≤ 150 pmol/l, which indicates vitamin B12 deficiency(Reference Herbert and Das21).
In the research carried out by Ueland et al. (Reference Ueland, Refsum and Beresford6), Humphrey et al. (Reference Humphrey, Fu and Rogers7), Malinow et al. (Reference Malinow, Bostom and Krauss8) and Ubbink(Reference Ubbink, Carmel and Jacobsen9), it was demonstrated that a substantial risk of developing CHD exists at a plasma tHcy level of >10 μmol/l and that, furthermore, each 5 μmol/l increase in plasma tHcy is associated with an approximately 20 % increase risk of CHD events. This, together with the fact that the present study indicates that there is an inverse relationship between plasma tHcy and serum vitamin B12, is not unreasonable to deduce that these danger levels will be breached by some vegetarians well before they reach the deficiency level of serum vitamin B12 ( ≤ 150 pmol/l) and symptoms of pernicious anaemia usually occur(Reference Herbert and Das21). Levels at which this could occur apply to all vegetarian groups, with exception of Haddad et al. (Reference Haddad, Berk and Kettering34), as can be observed in Table 1. Meta-analyses conducted by the Homocysteine Studies Collaboration(58) and Wald et al. (Reference Wald, Law and Morris59) have demonstrated that lowering homocysteine concentrations by 3 μmol/l substantially reduces the risk of CVD. Moreover, Ward et al. (Reference Ward, McNulty and McPartlin60) showed that there is a benefit to health in reducing the risk of primary CVD by lowering homocysteine levels. In contrast, The Heart Outcome Prevention Evaluation (HOPE 2) Investigation(61) found that supplements combining vitamin B12 and folic acid did not reduce the risk of major secondary cardiovascular events in patients with vascular disease.
A further finding is that the mean overall homocysteine level of vegans shown in Table 2 of 16·41 (sd 4·80) μmol/l (P< 0·005) and mean serum vitamin B12 of 172 (sd 59) pmol/l (P< 0·005) indicates that most vegans can be classified as being likely to suffer from hyperhomocysteinaemia due to a deficiency of vitamin B12 that will increase their risk of developing CVD. Moreover, LV–LOV with a mean overall homocysteine level of 13·91 (sd 3·15) μmol/l (P< 0·025) and mean serum vitamin B12 of 209 (sd 47) pmol/l (P< 0·005) also have an increased risk of developing CVD. Furthermore, omnivores from the results recorded in the present review (mean plasma tHcy 11·03 (sd 2·89) μmol/l) can be considered generally to have a borderline increased risk of developing homocysteine-related CVD, probably due to inadequate status of folate(Reference Majchrzak, Singer and Manner42). Statistical tests (independent samples t tests and ANOVA) showed a significant difference in mean levels of tHcy and serum vitamin B12 between omnivores, LV–LOV and vegans. Whilst the diets of some vegetarians are aimed at the well-documented benefits of promoting health, due to the restriction or absence of food from animal origin, this as far as CVD is concerned is probably due to reduced saturated fat, lower total serum cholesterol levels, lower prevalence of obesity and slightly lower blood pressure, as compared with omnivores. However, this may not negate the risk of vegetarians with elevated plasma tHcy being susceptible to homocysteine-related CVD, as indicated by Ueland et al. (Reference Ueland, Refsum and Beresford6), Humphrey et al. (Reference Humphrey, Fu and Rogers7), Malinow et al. (Reference Malinow, Bostom and Krauss8) and Ubbink(Reference Ubbink, Carmel and Jacobsen9). The present review reveals that there is only poor evidence available of vegetarians consuming vitamin B12 supplements and/or vitamin B12-fortified food and beverages. However, supplements, fortified food and beverages normally contain the less efficient cyanocobalamin form of vitamin B12, which when it enters the bloodstream must be converted to methylcobalamin(Reference Cooper and Rosenblatt62), the only form of vitamin B12 that has a methyl donor that is required to neutralise homocysteine(Reference Pezacka, Green and Jacobsen63). It takes 4–9 weeks for this conversion to take place(Reference Kelly64), assuming there are no disruptions by genetic factors, age-related problems and metabolic obstacles that may be present. Furthermore, research suggests that vitamin B12 that is not dissolved in the mouth will not (up to 88 %) be absorbed(Reference Crane65), due to the lack of R-binder mostly obtained from saliva, which is required to start the absorption process. The aforementioned study indicates that supplementation with cyanocobalamin can be poorly absorbed, which will have little or no effect on raising vitamin B12 levels.
A well-designed study is needed to investigate supplementary methylcobalamin by, for example, a 1 mg lozenge dissolved in the mouth (that can bypass the above potential problems), and takes advantage of absorption by mediated intrinsic factor, non-intrinsic-mediated diffusion and sublingual intake and on its affects on elevated homocysteine levels of vegetarians, who may have a resultant susceptibility to hyperhomocysteinaemia-related diseases. This would fill gaps in present nutritional scientific knowledge.
Acknowledgements
The present research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. There are no conflicts of interest to declare. The contribution made by D. O. was that of lead researcher and was responsible for the compilation of the manuscript. D. C. C. and A. A. T. were responsible for checking the methodological validity of the initially selected studies employing standardised data extraction tools from JBI-QARI and JBI-MAStARI, with any disagreement being resolved through discussion with a third reviewer, together with checking the accuracy of the finalised manuscript. A. D. was responsible for providing information and advice on the treatment on the statistical aspects of the research.
