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Abstract

Plant-based diets are increasingly popular worldwide. A well-planned plant-based diet lowers the risk of cardiovascular disease, type 2 diabetes and certain cancers. In contrast, a poorly planned plant-based diet increases the risk of certain micronutrient deficiencies, chiefly, vitamin B₁₂ (B₁₂). Because B₁₂ is not present in plants or in unfortified plant-based foodstuffs, the safest way to prevent its deficiency in plant-based diets is to take an oral B₁₂ supplement. Studies determining the dose and frequency of B₁₂ to be taken by healthy individuals on a plant-based diet to support an adequate B₁₂ status are scarce. Here, we summarize the natural sources, metabolic requirements, biomarker findings with and without supplementation with B₁₂, and current recommendations to help prevent vitamin B₁₂ deficiency in healthy individuals adhering or transitioning to plant-based diets. This review focuses on the prevention of vitamin B₁₂ deficiency in healthy individuals adhering to plant-based diets. The information covered in this review does not apply to individuals suffering from autoimmune-based malabsorption of vitamin B₁₂ resulting from pernicious anemia due to atrophic gastritis, other acquired causes of B₁₂ malabsorption or to those with genetic disorders that impair vitamin B₁₂ absorption, transport and utilization.

Plain language title

Vitamin B₁₂ in Plant-Based Diets

Plain language summary

Plant-based diets are increasingly popular worldwide. Because vitamin B₁₂ is not found in plants, individuals must acquire the micronutrient by consuming fortified foods or by taking an oral vitamin B₁₂ supplement. We review B₁₂ sources, required daily intake, and use of B₁₂ supplements among those on plant-based diets. The safest way to prevent B₁₂ deficiency in individuals adhering to plant-based diets is by using an oral B₁₂ supplement.

Keywords

malnutrition micronutrient deficiency plant-based diet homocysteine methylmalonic acid

Natural Sources and Chemical Forms of Vitamin B₁₂

Vitamin B₁₂ is a water-soluble vitamin synthesized exclusively by a few groups of microorganisms.^1

-6 Omnivores obtain vitamin B₁₂ by consuming animals that are environmentally exposed to vitamin B₁₂ produced by microorganisms. Because B₁₂ is not found in plants, humans adhering to unsupplemented plant-based diets have an increased risk of developing B₁₂ deficiency. Edible mushrooms⁷ and commercial sauerkraut⁸ have highly variable quantities of B₁₂, therefore they are not a reliable source of the micronutrient. A cultivated aquatic plant has been found to contain bioactive B₁₂ material.⁹ Much like humans, certain algae depend on the acquisition of B₁₂ from the environment to support function. Such algae employ specialized transport proteins to acquire the micronutrient from their natural ecosystems.¹⁰ This makes algae a potential source of B₁₂ for individuals adhering to plant-based diets. A recent study that examined 57 nutritional supplements found that certain algae such as Chlorella sp contain vitamin B₁₂ in substantial quantities.¹¹ However, the same study showed that other types of algae, such as Spirulina sp,¹¹ contain little to no B₁₂ and instead carry large quantities of the structural analogue pseudo-B₁₂, which does not support function in humans. The effects of B₁₂-analogue consumption in humans, as would possibly occur when using Spirulina sp as the sole source of B₁₂, have not been investigated. Because the presence of B₁₂ analogues in blood has, in a few cases, been correlated with severe neurological impairment,¹² the safest way to secure an adequate intake of B₁₂ in plant-based diets is the use of supplements that contain pure B₁₂. The biologically active forms of vitamin B₁₂ include cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin, and adenosylcobalamin. In healthy individuals, all of these chemical forms are absorbed and processed to support physiological needs. The enzyme MMACHC processes all 4 chemical forms of B₁₂ into a common intermediate that can be used by the 2 B₁₂-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT).¹³ There is no proven superiority of one chemical form of B₁₂ over another to supplement healthy individuals.¹⁴ Most commercial supplements contain CNCbl.

Requirement for Vitamin B₁₂ in Human Physiology

Humans require vitamin B₁₂ to support the activity of the enzymes MS and MUT. Deficiency of vitamin B₁₂ impairs the activity of the 2 vitamin B₁₂-dependent enzymes, which manifests in the elevation of their respective substrates, homocysteine (Hcy) and methylmalonic acid (MMA).¹⁵ Cells that lack vitamin B₁₂ produce larger than normal quantities of Hcy and MMA, which are exported into the extracellular milieu, and can therefore be measured in plasma and urine. The absorption of food B₁₂ and supplemental free B₁₂ is a complex process. Food B₁₂ is mostly bound to proteins (MS, MUT, haptocorrin [HC], and transcobalamin [TC]). Free B₁₂, whether in food or taken in as an oral supplement, will bind to HC in saliva and the stomach. B₁₂ is liberated from proteins by digestion and binds to gastric apo-IF (apo-intrinsic factor) to form holo-IF, which is then largely absorbed by enterocytes in the ileum.^16,17 After release from IF, B₁₂ enters portal circulation and binds to both apo-TC and apo-HC. Only the fraction of B₁₂ bound to TC, known as holo-transcobalamin (holo-TC) is the bioactive fraction of B₁₂ that undergoes uptake by all cells in the body.¹⁸ Apo-HC also binds B₁₂ analogues and is probably involved in their removal from circulation through the enterohepatic circulation of cobalamins.^19,20 Asialo-holo-HC is cleared by the liver.²¹ Thus, total plasma vitamin B₁₂ (holo-TC + holo-HC + trace free B₁₂) and specifically holo-TC are 2 biomarkers of vitamin B₁₂ status. In healthy individuals, these 2 biomarkers respond to and reflect the dietary and supplemental intake of vitamin B₁₂. Individuals taking large amounts of supplemental B₁₂ absorb small amounts by passive diffusion along the gastrointestinal (GI) tract. By this nonphysiological process, it is estimated that approximately 1% of a 1000 to 2000 µg dose of CNCbl is absorbed (10-20 µg).²² Thus, large daily doses of CNCbl have been used to successfully treat subjects with pernicious anemia.^23,24 Absorption by this nonphysiological process appears to occur across the entire length of the GI tract.²⁵

The Challenge of Acquiring Enough Vitamin B₁₂ in the Era of Sustainability

Humans and other animals are B₁₂-auxotrophs and, therefore, they must acquire B₁₂ from the environment.²⁶ Omnivores are protected from a low intake of B₁₂ by the consumption of animals or animal-derived products (dairy, eggs) that have previously acquired vitamin B₁₂ from the environment. This mode of acquisition of vitamin B₁₂ is effective in that it supplies sufficient quantities of the micronutrient; however, it is also more environmentally taxing compared to using oral supplementation while on a plant-based diet. Firstly, the consumption of foodstuffs derived from animals contributes significantly to water and land wasting and to greenhouse emissions.^27

-30 Secondly, the upscaling of meat production to satisfy increasing planetary demands has led to live-stock being husbanded under conditions wherein supplementation with either cobalt (to supply rumen microbes with the metal for B₁₂ biosynthesis) or with parenteral vitamin B₁₂ (to treat cobalt deficiency) are necessary for their own health and growth.^31

-34 This requirement appears to be linked to unnatural feeding conditions typical of intensive animal agriculture, as free grazing animals do not have a need for supplementation with vitamin B₁₂.³⁴ Thirdly, the high demand for meat on the planet does not permit the feeding of all humans with free-ranging grazing animals. Since the transition toward plant-based diets that are devoid of B₁₂ is unavoidable, every effort should be made to prevent B₁₂ deficiency in vegetarians and vegans. Vegetarians obtain vitamin B₁₂ primarily from dairy and eggs, whereas vegans rely on the use of supplements or fortified foods. Two recent studies have identified types of algae (in addition to Chlorella sp) and an aquatic duckweed plant that contain substantial amounts of vitamin B₁₂.^9,10 This presents as a potentially new source of vitamin B₁₂ for human consumption, in particular, ones that are compatible with all diets including vegan.

Intake of Vitamin B₁₂ in Plant-Based Populations

The intake of vitamin B₁₂ decreases proportionally to the greater sustainability of the diet, in rank-order omnivore > flexitarian > vegetarian > vegan.³⁵ This trend is applicable to all ages, from neonates to the elderly. The European Food Safety Agency (EFSA) recommends a daily intake between 1.5 µg/d (infants 7 to 11 months old) and 4.0 µg/d (children aged 15-17 years and adults), and between 4.5 and 5.0 µg/d for pregnant and lactating women.³⁶ The National Institutes of Health (NIH, USA) recommend 0.4 to 2.4 µg/d depending on age (adults > 19 years old: 2.4 µg/d), 2.6 µg/d for pregnant women, and 2.8 µg/d for lactating women.³⁷ The National Health Systems (NHS) in the United Kingdom recommend 1.5 µg/d for adults between 19 and 64 years old.³⁸ The DACH reference values for nutrient intake are jointly issued by the nutrition societies of Germany, Austria, and Switzerland.³⁹ The revised values are as follows: a range from 0.5 to 4.0 µg/d for children and adolescents, 4.0 µg/d for adults, 4.5 µg/d for pregnant women, and 5.5 µg/d for lactating women.³⁹ The Nordic Nutrition Recommendations (NNR) 2023 have increased the recommended intake for B₁₂, in line with the recommendations of EFSA, to 4.0 µg/d for adult males and females.⁴⁰ A systematic analysis of vitamin B₁₂ intake with respect to vitamin B₁₂ status in groups vulnerable to B₁₂ deficiency (including 4 studies on vegetarians and vegans) failed to conclude whether habitual B₁₂ intake per the NNR is sufficient to support adequate vitamin B₁₂ status in all vulnerable groups, and strongly encouraged population-based cohort studies.⁴¹ A systematic review of 13 publications determined that the intake of vitamin B₁₂ in children and adolescents following vegetarian and vegan diets does not meet the estimated average requirement.⁴² When excluding the use of B₁₂ supplements, the average B₁₂ intakes calculated from 10 independent studies were 3.49, 1.66, and 0.59 µg/d for meat-eaters, vegetarians, and vegans, respectively.⁴² This pattern changed when the analysis considered intake including B₁₂ supplements, as follows: 28.8, 5.2, and 116.6 µg/d for meat-eaters, vegetarians, and vegans, respectively.⁴² This implies that vegetarians do not consume supplemental B₁₂ as much as do vegans. A study performed in a cohort of Swiss adults who have been omnivore, vegetarian, or vegan for at least 1 year showed a similar pattern of B₁₂ intake.⁴³ Omnivores, vegetarians, and vegans had an intake of 4.1, 1.6, and 0.2 µg/d, respectively. In this study, both vegetarians and vegans had B₁₂ intakes below the estimated average requirement of 2.0 µg of B₁₂.⁴³ Two independent cross-sectional studies performed in different regions of Germany showed concurrent data. In a study conducted in northern Germany, the B₁₂ intake of flexitarians (therein defined as consumption of meat and meat products ≤50 g/d; equivalent to ≤350 g/week), vegans and omnivores was 2.12, 1.81, and 3.70 µg/d, respectively.⁴⁴ The B₁₂ intake of participants in a study in southern Germany was 0.43, 0.98, and 2.14 µg/d for vegans, lacto-ovo-vegetarians, and omnivores, respectively.⁴⁵ Beside children and adults, babies born to mothers whose pregnancy progressed with low or deficient B₁₂ can suffer from vitamin B₁₂ insufficiency or deficiency. Data from a national pilot study on expanded newborn screening (NBS) in Germany involving 176,702 children screened over 27 months detected nutritional B₁₂ deficiency in 33 neonates (incidence 1 in 5355).⁴⁶ Homocysteine was the most sensitive biomarker. However, MMA measurement was necessary to identify all 33 children with B₁₂ deficiency. Of the diagnosed children, 84% were treated with oral B₁₂ and remained without clinical symptoms.⁴⁶ Interestingly, 89% of the mothers adhered to a balanced omnivore diet. In a NBS project involving 588,793 newborns aimed at capturing propionic acidemia, methylmalonic acidemia, and vitamin B₁₂ deficiency in Spain, 89 cases were due to maternal B₁₂ deficiency (incidence 1 in 6616).⁴⁷ Of these cases of maternal B₁₂ deficiency, 5% were vegetarian mothers and 15% were mothers diagnosed with pernicious anemia.⁴⁷ These 2 large studies confirm that nutritional B₁₂ deficiency during pregnancy is common among omnivores, and its monitoring should not be exclusively restricted to individuals on plant-based diets. The early diagnosis of B₁₂ deficiency in neonates permitted the rapid initiation of treatment to prevent their health deterioration, with the added benefit of treatment to the undiagnosed mothers.

Biomarkers of Vitamin B₁₂ Status: Which One(s) to Use and Why?

Vitamin B₁₂ status is determined by the use of single or multiple biomarkers, namely, plasma vitamin B₁₂, holo-TC, Hcy, and MMA.⁴⁸ The 4 biomarkers of vitamin B₁₂ status constitute an aggregate measurement of the absorption, transport, and cellular utilization of the micronutrient. Plasma vitamin B₁₂ and holo-TC are measured by automated platforms in diagnostic laboratories. Homocysteine and MMA are often determined by liquid chromatography tandem mass spectrometry. Plasma and serum are the primary biofluids employed for the determination of biomarkers of vitamin B₁₂ status. The reference ranges for each biomarker in healthy adults are provided in Table 1.

Table 1.

Reference Ranges for Biomarkers of Vitamin B₁₂ Status in Healthy Adults.

Biomarker	Reference range or cutoff	Reference
Total vitamin B₁₂	Normal: >250 pmol/L Low: 150-249 pmol/L Acute deficiency: <149 pmol/L	^49,50
holo-TC	20-125 pmol/L	⁵¹
holo-TC	<35-40 pmol/L^a	⁵²
Hcy	5-15 µmol/L	⁵³
MMA	>260-350 nmol/L	⁵⁰

Abbreviations: Hcy, homocysteine; holo-TC, holo-transcobalamin; MMA, methylmalonic acid.

^a This reference range is based on the relationship of holo-TC with MMA.

As with all laboratory biomarkers, decisions on B₁₂ adequacy, insufficiency, and deficiency are based on reference ranges determined in a large cohort of healthy individuals, who are predominantly omnivores (>90%). Age- and sex-specific reference ranges have been reported for the assessment of vitamin B₁₂ status.^52,54
-
56 Because none of the individual biomarkers possess sufficient sensitivity and specificity to detect B₁₂ insufficiency and deficiency, a combination of at least 2 biomarkers is recommended.⁴⁸ Concurrent measurement of plasma B₁₂, holo-TC, Hcy, MMA, and the 4cB₁₂ index developed by Fedosov^57,58 in a population of 11 833 mixed patients suggested that holo-TC was the better of the 2 direct biomarkers and MMA was the better of the 2 metabolic biomarkers, with holo-TC being the preferable first-line marker for the identification of B₁₂ deficiency.⁵⁹

In a controlled trial where healthy omnivores were randomized into either a meat-rich diet or a vegan diet for 4 weeks, both plasma vitamin B₁₂ and holo-TC underwent a significant reduction at the end of the trial in the vegan group, without changes in Hcy or MMA.⁶⁰ The concentration of plasma holo-TC decreased by 28% compared to baseline concentrations after only 4-weeks on an unsupplemented vegan diet.⁶⁰ This study demonstrated that holo-TC decreases quickly upon reduction of dietary intake after transitioning from omnivore to vegan diets, and thus may serve as the preferable marker to monitor B₁₂ status in healthy individuals on plant-based diets.

Plasma Hcy is the preferred biomarker for the assessment of B₁₂ status in infants and toddlers up to 3 years old, with a cutoff of 6.5 µmol/L.^55,56,61 In older children and adults, Hcy is most responsive to folate status and thus MMA is the preferred biomarker.¹⁵ The biomarkers of B₁₂ status change distinctively during pregnancy due to increased blood volume and maternal-fetal transfer of vitamin B₁₂.^62

-65 Results from an extended NBS in Spain showed that in cases of babies born with B₁₂ deficiency due to maternal B₁₂ deficiency, the mean concentrations of plasma B₁₂ were 187.6 ± 76.9 pg/mL (139 ± 70 pmol/L) and 213.7 ± 95.0 pg/mL (158 ± 57 pmol/L) in babies and mothers, respectively.⁴⁷ Thus, pregnant women with a low concentration of plasma B₁₂ (150-249 pmol/L) are a high-risk group for neonatal vitamin B₁₂ deficiency.

In addition to the above reference range considerations that are applicable to healthy individuals irrespective of their dietary pattern, studies performed on healthy vegetarians showed that assessing B₁₂ status in individuals on plant-based diets may require adjusted reference ranges. Analysis of plasma B₁₂, holo-TC, and Hcy in healthy vegetarian Indian graduates showed that when using the reference ranges given in Table 1, 50% were B₁₂ deficient, 70% of males and 50% of females had low concentration of plasma holo-TC, and 92% of males and 50% of females had elevated Hcy.⁶⁶ Strikingly, none of the study participants exhibited clinical signs of B₁₂ deficiency.⁶⁶ As noted by Carmel, vegetarians and vegans may fall within the category of subclinical B₁₂ deficiency, wherein biomarkers of B₁₂ status are suboptimal yet individuals remain asymptomatic for long periods of time and may not always develop B₁₂ deficiency.⁶⁷ Analysis of biomarker relationships by receiver operating characteristic curves led to the suggestion of the use of a combination of biomarkers to define B₁₂ deficiency in healthy vegetarian Indians, with the following adjusted cutoffs: 100 pmol/L for plasma B₁₂, 19.6 pmol/L for holo-TC, 17.6 and 27 μmol/L for plasma Hcy in females and males, respectively.⁶⁶ While the use of adjusted reference ranges for individuals on plant-based diets merits consideration,⁶⁸ reducing the cutoff for plasma B₁₂ to 100 pmol/L to define deficiency may expose healthy vegetarian women of child-bearing age to a pregnancy that progresses with too low plasma vitamin B₁₂, which per available evidence⁴⁷ will lead to neonatal B₁₂ deficiency. A concentration of plasma vitamin B₁₂ >394 pmol/L has been recommended as a target at week 18 of pregnancy.⁶⁹ Hence, the status of vitamin B₁₂ should be monitored carefully in prospective mothers and early in pregnancy especially if they adhere to plant-based diets. Likewise, any adjustment to existing reference ranges should consider stages of life where demands for vitamin B₁₂ are high (i.e., child and adolescent development, pregnancy and lactation, elderly).

Vitamin B₁₂ Status in Plant-Based Populations

In the absence of vitamin B₁₂ supplementation or the consumption of foodstuffs fortified with B₁₂, individuals on plant-based diets exhibit a poor status of vitamin B₁₂ and are at risk of developing B₁₂ deficiency.^70,71 A randomized controlled trial with dietary intervention showed that both plasma B₁₂ and holo-TC undergo a significant decrease in individuals transitioning from omnivore to vegan diets, after only 4 weeks.⁶⁰

According to survey data and recording of B₁₂ supplement use in cross-sectional studies, the level of awareness concerning the importance of using a B₁₂ supplement while on a vegan diet has risen over the years. A survey designed to assess the level of B₁₂ supplement use among Slovak and Czech vegans showed that in a population of 1337 self-identified vegans, approximately 88% of participants supplemented with B₁₂.⁷² The mean weekly B₁₂ intake ranged from 1630.31 ± 1949.27 µg to 2938.34 ± 2566.60 µg in irregularly and regularly supplementing vegans, respectively.⁷² These weekly intakes of B₁₂ exceed the recommendations in all available guidelines. A survey examining 1565 vegans from Austria revealed that 92% took B₁₂ supplements and/or fortified foods, and 76% of the participants had their B₁₂ status checked.⁷³ A survey assessing 1530 Australian vegan women of reproductive age revealed that the use of B₁₂ supplements varied widely. In this study, when the calculated fraction of B₁₂ absorbed was taken into consideration, 39% of participants had an estimated total intake of B₁₂ below the recommended dietary intake (RDI) equivalency, versus 26% being below the RDI when estimations were based exclusively on mean daily intake.⁷⁴ Analysis of maternal B₁₂ deficiency detected by NBS in Germany revealed that in a total of 121 mother-infant dyads, 100% of mothers following a vegan diet and most mothers with a vegetarian diet took vitamin preparations during pregnancy, whereas only 55.6% of mothers with an omnivore diet took vitamin supplements.⁷⁵

Cross-sectional studies have been also valuable in examining the B₁₂ status of long-term (>1 year on the diet) vegetarians and vegans and their conduct toward the incorporation of B₁₂ supplements. In a cross-sectional study conducted in healthy Germans following flexitarian (therein defined as consumption of meat and meat products ≤50 g/d; equivalent to ≤350 g/week), vegan and omnivore diets, 32%, 82%, and 24% reported using vitamin supplements, respectively.⁴⁴ Surprisingly, analysis of B₁₂ status using the 4cB₁₂ index showed that suboptimal B₁₂ status was lowest among vegans (9%), followed by omnivores (10%) and flexitarians (13%).⁴⁴ A study conducted in Switzerland comparing omnivores, vegetarians, and vegans showed that despite low intakes of B₁₂ especially in vegans, a normal B₁₂ status was achieved by the use of oral B₁₂ supplements.⁴³ A study on healthy omnivores and vegans from the Berlin area in Germany showed comparable B₁₂ status in the 2 groups, as determined by the 4cB₁₂ index indicating adequate B₁₂ supply.⁷⁶ The adequate B₁₂ status in vegans was attributed to a high rate of supplement use in this group (97.2% of participants).⁷⁶ A study performed on healthy young adults from south Germany showed that the B₁₂ status (4cB₁₂ index) of vegans was comparable to that of omnivores, owing to the high rate of B₁₂ supplement use among vegans (90%) compared to lacto-ovo-vegetarians (51%).⁴⁵ Vegetarians exhibited the poorest B₁₂ status.⁴⁵ Examination of unsupervised supplement use over the course of 1 year showed that a median intake of 250 µg B₁₂/d was sufficient to achieve an adequate status of B₁₂ in vegans.⁴⁵ Similarly, a study performed on Czech vegans showed that those using regular supplementation with vitamin B₁₂ had similar plasma concentrations of B₁₂ and holo-TC as omnivores.⁷⁰

Clinical trials with B₁₂ interventions in individuals on plant-based diets are few and heterogeneous in design and biomarker readouts. Table 2 summarizes studies with vitamin B₁₂ intervention in groups susceptible to vitamin B₁₂ deficiency with a focus on plant-based diets. Plasma vitamin B₁₂ is the most commonly measured biomarker across different studies and was herein chosen to examine response to various supplementation regimens. The available studies show known trends in vitamin B₁₂ absorption and the relationship between B₁₂ intake and plasma vitamin B₁₂ concentration. These can be summarized as follows:

A comparatively greater fraction of B₁₂ is absorbed from low-dose supplements compared to high-dose supplements.⁸⁴

The magnitude of change in plasma vitamin B₁₂ in response to oral supplementation depends on the initial status of B₁₂ and the duration of the trial, more so than on the dose alone (see also study by Del Bo et al).⁸⁰

Neither chemical form of B₁₂ (CNCbl, HOCbl, or methylcobalamin) exhibits superiority over the others in supporting function in healthy individuals.¹⁴

Table 2.

Response of Plasma Vitamin B₁₂ to Oral Supplementation in Vulnerable Groups of Adults Including Individuals on Plant-Based Diets.

Study type	Population	Size	Duration	Intervention	Inclusion criteria	Baseline VITB₁₂ (pmol/L)	Endpoint VITB₁₂ (pmol/L)	Δ VITB₁₂ (pmol/L)	Reference
RCT, double-blind, placebo	Older adults (>75 years) free from severe illnesses	N = 81	12 months	1000 µg CNCbl once daily	107-210 pmol/L	Q1 167.2 (18.8) Q2 211.5 (8.8) Q3 251.6 (13.6) Q4 302.2 (26.4)	638.9 (221.6) 614.5 (244.5) 613.6 (99.3) 711.8 (203.8)	471.7 403.0 362.0 409.6	⁷⁷
RCT Placebo	Individuals with Diabetes	N = 271	9 months	2 × 500 µg MeCbl once daily	150-300 pmol/L	227.5 (40.0)	846 (266.9)	618.5	⁷⁸
RCT	Lactovegetarians	N = 35	8 weeks (max B₁₂ reached after 2 weeks)	2 × 2.8 µg/d CNCbl	<150 pmol/L	106	182	76	⁷⁹
RCT	Vegans and vegetarians	N = 40	12 weeks	350 µg/week given as 50 µg/d or 2000 µg/week	<220 pmol/L	146 ± 36 131 ± 56	260 325	114 194	⁸⁰
Longitudinal cohort intervention, placebo	Vegetarians and flexitarians (65% lacto-vegetarians; 35% consuming a portion of nonvegetarian food approximately twice a week)	N = 51	8 weeks	3 µg CNCbl or 3 µg HOCbl	<200 pmol/L	128 (68-191)	168 (87-302)	40	⁸¹
Double-blind, placebo controlled, randomized crossover	Vegetarians	N = 50	12 weeks	500 µg CNCbl once daily	<272 pmol/L	146 ± 126	380 ± 206	234	⁸²
RCT, 2×2 factorial design	Lactovegetarian women	N = 42	6 weeks (max B₁₂ reached after 2 weeks)	500 µg CNCbl once daily	<250 pmol/L (70% had <150 pmol/L)	125	215	90	⁸³

Abbreviations: CNCbl, cyanocobalamin; HOCbl, hydroxocobalamin; MeCbl, methylcobalamin; RCT, randomized controlled trial.

Vitamin B₁₂ Supplementation in Plant-Based Diets

Despite its importance, there is a lack of international consensus on the dose and frequency of vitamin B₁₂ supplementation required to support B₁₂ status adequacy in healthy adults on plant-based diets. Prior estimates include: (1) a daily intake of 50 to 100 µg/d B₁₂ or 2000 µg per week B₁₂ divided into 2 doses^85,86 for healthy vegetarian adults, (2) a daily dose of 50 µg sublingual B₁₂ for vegans and vegetarians with marginally low plasma B₁₂,⁸⁷ (3) a daily intake between 50 and 150 µg B₁₂ for healthy asymptomatic individuals without malabsorption (no diet specified),⁸⁸ and (4) a daily dose of 500 µg/d B₁₂ to treat individuals with vitamin B₁₂ in the low normal range (no diet specified).⁸⁹ Based on the joint consideration of intake, supplement use and plasma biomarkers of B₁₂ status, results from a cross-sectional study comparing long-term omnivores, lacto-ovo-vegetarians, and vegans suggest that a B₁₂ intake of 250 µg/d supports an adequate status of the micronutrient in individuals on plant-based diets.⁴⁵ This daily intake was calculated based on unsupervised use of B₁₂ supplements by the study participants. While supplement use by vegans in that study exceeded the daily intake of 4 to 20 µg B₁₂ recommended by an international panel of experts for individuals of all ages,³⁵ it highlights an increasing awareness and acceptance of the importance of B₁₂ supplementation in plant-based diets. Concerning pediatric and adolescent populations, intervention with B₁₂ in a study performed in Canada showed that a daily consumption of up to 10 µg B₁₂ by children older than 6 years and adolescents, and up to 10-25 µg by older adults led to an improved vitamin B₁₂ status.⁹⁰ The NNR suggest a daily intake of 3 to 4.9 µg B₁₂ in children <10 years old, and 5.3 to 10 µg/d for children >10 years old to sustain an adequate B₁₂ status.⁵⁵

Conclusions

Unfortified plant-based diets are devoid of vitamin B₁₂. Vitamin B₁₂ intakes in unsupplemented vegetarians and vegans fail to meet the estimated average requirements. The 4 biomarkers of vitamin B₁₂ status respond to changes in dietary intake, albeit differently. A combination of at least 2 biomarkers, ideally holo-TC (direct marker, rapid responder to changes in B₁₂ intake) and MMA (metabolic marker, denotes cellular deficiency) is desirable when monitoring B₁₂ status in individuals on plant-based diets. In studies performed with vegetarians and vegans, plasma biomarkers of vitamin B₁₂ changed markedly before the onset of clinical symptoms. Thus, despite their known shortcomings,^48,91 these biomarkers remain a valuable tool to monitor and to diagnose B₁₂ deficiency in individuals on plant-based diets. Certain types of algae such as Chlorella sp contain substantial quantities of vitamin B₁₂ and carry little quantities of unwanted, nonmetabolizable B₁₂ analogues. However, because the production of algae preparations is not controlled with pharmaceutical quality assurance and controlled dose–response studies with algae preparations in humans are lacking, the safest way to prevent vitamin B₁₂ deficiency in plant-based diets is to use a supplement of B₁₂. Healthy adults on plant-based diets can prevent vitamin B₁₂ deficiency by securing a daily intake of B₁₂ of 4 to 20 µg/d.³⁵ The recommendation of B₁₂ intake for children younger than 10 years is 3 to 4.9 µg/d, and for those older than 10 years 5.3 to 10 µg/d.⁵⁵

Insufficient intake of B₁₂ was documented not only in individuals on a vegan diet but also in vegetarians and flexitarians, the latter two being the fastest growing dietary regimens worldwide. Flexitarians and vegetarians did not use B₁₂ supplements as often as vegans (32%-55% vs 80%-97%), which shows an excessive reliance on food intake as the sole source of B₁₂. Survey data indicate that while the level of awareness regarding the importance of using a B₁₂ supplement when adopting a vegan diet is high, the dose and frequency of B₁₂ supplementation varied widely. The analysis of unsupervised B₁₂ supplement use by vegans in independent cross-sectional studies showed that their daily B₁₂ intake from supplements vastly exceeds the daily intakes recommended by a multitude of regulating authorities including DACH, EFSA, NIH, NHS, and NNR. While there is no toxicity associated with the use of high-doses of oral B₁₂, it is presently unknown whether long-term exposure to high-dose B₁₂ may perturb gut microbiome communities that produce B₁₂ as well as those that synthesize vitamin B₁₂ analogues from B₁₂. Dose-finding studies are urgently needed to determine the physiologically optimal upper limit of B₁₂ supplementation for individuals on plant-based diets.

Footnotes

Author Contributions

Author contributions were as follows: LH planned and wrote a first complete version of the manuscript; AKL, MAS, RH wrote and edited sections of the manuscript focused on plant-based nutrition; DWJ wrote and edited sections of the manuscript focused on vitamin B₁₂ biomarkers and B₁₂ deficiency. All authors read and approved the final version of the manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Luciana Hannibal

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Vitamin B 12 Status and Supplementation in Plant-Based Diets

Abstract

Plain language title

Plain language summary

Keywords

Natural Sources and Chemical Forms of Vitamin B12

Requirement for Vitamin B12 in Human Physiology

The Challenge of Acquiring Enough Vitamin B12 in the Era of Sustainability

Intake of Vitamin B12 in Plant-Based Populations

Biomarkers of Vitamin B12 Status: Which One(s) to Use and Why?

Vitamin B12 Status in Plant-Based Populations

Vitamin B12 Supplementation in Plant-Based Diets

Conclusions

Footnotes

Author Contributions

Declaration of Conflicting Interests

Funding

ORCID iD

References

Natural Sources and Chemical Forms of Vitamin B₁₂

Requirement for Vitamin B₁₂ in Human Physiology

The Challenge of Acquiring Enough Vitamin B₁₂ in the Era of Sustainability

Intake of Vitamin B₁₂ in Plant-Based Populations

Biomarkers of Vitamin B₁₂ Status: Which One(s) to Use and Why?

Vitamin B₁₂ Status in Plant-Based Populations

Vitamin B₁₂ Supplementation in Plant-Based Diets