Plasma total homocysteine status of vegetarians compared with omnivores: a systematic review and meta-analysis

Abstract

There is strong evidence indicating that elevated plasma total homocysteine (tHcy) levels are a major independent biomarker and/or a contributor to chronic conditions, such as CVD. A deficiency of vitamin B₁₂ can elevate homocysteine. Vegetarians are a group of the population who are potentially at greater risk of vitamin B₁₂ deficiency than omnivores. This is the first systematic review and meta-analysis to appraise a range of studies that compared the homocysteine and vitamin B₁₂ levels of vegetarians and omnivores. The search methods employed identified 443 entries, from which, by screening using set inclusion and exclusion criteria, six eligible cohort case studies and eleven cross-sectional studies from 1999 to 2010 were revealed, which compared concentrations of plasma tHcy and serum vitamin B₁₂ of omnivores, lactovegetarians or lacto-ovovegetarians and vegans. Of the identified seventeen studies (3230 participants), only two studies reported that vegan concentrations of plasma tHcy and serum vitamin B₁₂ did not differ from omnivores. The present study confirmed that an inverse relationship exists between plasma tHcy and serum vitamin B₁₂, from which it can be concluded that the usual dietary source of vitamin B₁₂ is animal products and those who choose to omit or restrict these products are destined to become vitamin B₁₂ deficient. At present, the available supplement, which is usually used for fortification of food, is the unreliable cyanocobalamin. A well-designed study is needed to investigate a reliable and suitable supplement to normalise the elevated plasma tHcy of a high majority of vegetarians. This would fill the gaps in the present nutritional scientific knowledge.

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. ⁽⁵⁾) has been shown to be linked with chronic conditions, among which is CVD^{(6, 7)}. Other studies have shown that CHD is linked to homocysteine concentrations, with a substantial risk occurring at >10 μmol/l plasma total homocysteine (tHcy)^{(8, 9)}. Furthermore, each 5 μmol/l increase in plasma tHcy is associated with an approximate 20 % increased risk of CHD events^{(6, 7)}, 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^{(10, 11)}. 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⁽¹²⁾, but only in its methylcobalamin form⁽¹³⁾. 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^(14–16) 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^{(17, 18)}.

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⁽²⁰⁾. 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⁽²¹⁾.

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^(22–24).

Lack of Ca in the vegetarian diet could, therefore, inhibit vitamin B₁₂ absorption^(22–24). 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^(25–27), this would cause vitamin B₁₂ deficiency⁽²⁸⁾. 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⁽³⁰⁾.

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.

Fig. 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.

Table 1 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⁽³²⁾. Table 2 summarises the calculated values.

Table 2 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. ⁽³³⁾ 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.

Fig. 2 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^{(36, 37, 41, 45, 46)} and combined measured levels of plasma tHcy and serum B₁₂ of LV or LOV and vegans of two studies^{(38, 43)} from 1999 to 2010 (study by Cappuccio et al. ⁽³³⁾ 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.

Table 3 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. ⁽³³⁾ did not monitor serum vitamin B₁₂ levels. The study by Haddad et al. ⁽³⁴⁾ 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. ⁽³⁵⁾, Herrmann et al. ⁽³⁶⁾ and Koebnick et al. ⁽³⁷⁾. 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. ⁽³⁸⁾ 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^{(36, 37, 39–42)}. Huang et al. ⁽⁴³⁾ 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. ⁽¹⁰⁾ and Selhub⁽¹¹⁾ 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. ⁽⁴²⁾ 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⁽⁴⁴⁾. 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)^{(37, 39–41)}. Furthermore, a significant proportion of LV–LOV subjects in the study conducted by Koebnick et al. ⁽³⁷⁾ showed a hyperhomocysteinaemia condition, with a strong significance of P< 0·001. A total of ten studies^{(35, 40–43, 46–50)} 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⁽²¹⁾), 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. ⁽⁵¹⁾ and Herrmann et al. ⁽⁵²⁾.

Refsum et al. ⁽⁴⁵⁾ 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⁽⁵²⁾. 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⁽⁵³⁾. India has large proportions of its population who suffer from malnutrition, tropical sprue and gastrointestinal infections, which often result in malabsorption^(54–56). 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⁽²¹⁾. Studies undertaken by Gilsing et al. ⁽⁵⁷⁾, 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⁽²¹⁾, 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⁽²¹⁾.

In the research carried out by Ueland et al. ⁽⁶⁾, Humphrey et al. ⁽⁷⁾, Malinow et al. ⁽⁸⁾ and Ubbink⁽⁹⁾, 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⁽²¹⁾. Levels at which this could occur apply to all vegetarian groups, with exception of Haddad et al. ⁽³⁴⁾, as can be observed in Table 1. Meta-analyses conducted by the Homocysteine Studies Collaboration⁽⁵⁸⁾ and Wald et al. ⁽⁵⁹⁾ have demonstrated that lowering homocysteine concentrations by 3 μmol/l substantially reduces the risk of CVD. Moreover, Ward et al. ⁽⁶⁰⁾ 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⁽⁴²⁾. 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. ⁽⁶⁾, Humphrey et al. ⁽⁷⁾, Malinow et al. ⁽⁸⁾ and Ubbink⁽⁹⁾. 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⁽⁶²⁾, the only form of vitamin B₁₂ that has a methyl donor that is required to neutralise homocysteine⁽⁶³⁾. It takes 4–9 weeks for this conversion to take place⁽⁶⁴⁾, 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⁽⁶⁵⁾, 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.