The proportion of humans adopting a plant-based lifestyle is on the rise worldwide. Benefits on economics, climate and health have been documented and projected to continue over the next decades(
Reference Appleby and Key
1
–
Reference Springmann, Godfray and Rayner
3
). Apart from its sustainability(
Reference Sabate and Soret
4
), well-planned plant-based diets are nutritionally complete for human beings of all ages(
Reference Melina, Craig and Levin
5
), with vitamin B12 being the only micronutrient not found in plants(
Reference Rizzo, Laganà and Rapisarda
6
–
Reference Rizzo, Jaceldo-Siegl and Sabate
8
). Vitamin B12 is only synthesised by a few bacteria and archaea(
Reference Raux, Schubert and Warren
9
–
Reference Eschenmoser
11
); hence, humans adhering to a plant-based lifestyle can obtain the micronutrient from foods fermented with bacteria that naturally produce vitamin B12, foods fortified with vitamin B12 and over-the-counter supplements. However, the high prevalence of vitamin B12 insufficiency in vegetarians across nations(
Reference Pawlak, Lester and Babatunde
12
) suggests that food literacy programmes(
Reference Azevedo Perry, Thomas and Samra
13
) are not yet meeting the needs of the current dietary shift. Vitamin B12 deficiency, whether acquired or inherited, leads to the inactivation of the two vitamin B12-dependent enzymes, cytosolic methionine synthase and mitochondrial methylmalonyl-CoA mutase(
Reference Green, Allen and Bjorke-Monsen
14
,
Reference Green
15
). This manifests with elevated homocysteine (Hcy) and methymalonic acid (MMA), and in severe cases with reduction of total serum vitamin B12 and its bioactive fraction holo-transcobalamin (holo-TC).
The study by Naik et al.
(
Reference Naik, Mahalle and Bhide
16
) examined vitamin B12 status in a cohort of 119 young, healthy unsupplemented vegetarian Indian graduates. Using standard cut-off values for the assessment of vitamin B12 status Naik et al. determined that 50 % were vitamin B12 deficient, 50–70 % exhibited low plasma holo-TC and 70–90 % presented elevated plasma total Hcy (tHcy). All participants in this study were asymptomatic of clinical vitamin B12 deficiency(
Reference Naik, Mahalle and Bhide
16
). These findings are in good agreement with a previous study by Refsum et al. in a different Indian cohort(
Reference Refsum, Yajnik and Gadkari
17
). Interestingly, nineteen participants with plasma vitamin B12 concentrations between 113 and 122 pmol/l had normal values of holo-TC (34–52 pmol/l), while exhibiting elevated tHcy according to standard cut-off reference values (discussed in Hannibal et al.
(
Reference Hannibal, Lysne and Bjorke-Monsen
18
) and references therein). Naik et al.
(
Reference Naik, Mahalle and Bhide
16
) propose a new set of cut-off values to improve the diagnosis of vitamin B12 deficiency in young vegetarian Indians. The authors propose the use of a combination of biomarkers and cut-off values of 100 and 19·6 pmol/l for plasma vitamin B12 and holo-TC, respectively, and values of tHcy of 17·6 and 27 µmol/l for females and males, respectively(
Reference Naik, Mahalle and Bhide
16
). This proposal appears in line with previously documented data in a study performed on a cohort of American vegetarians by the late Dr Victor Herbert(
Reference Herbert
19
). Herbert divided vitamin B12 status in vegetarians in four distinct stages, namely I, II, III and IV(
Reference Herbert
19
). Herbert and independent colleagues established that vegetarians can withstand long-term insufficient intake of vitamin B12 due to up-regulation of enterohepatic circulation and intestinal reabsorption of traces of vitamin B12
(
Reference Herbert
19
–
Reference Kanazawa, Herbert and Herzlich
22
). In healthy vegetarians, this represents a mechanism to optimise the recycling of the scarce micronutrient.
One strength of the study by Naik et al. is that the level of accuracy for identifying vitamin B12 deficiency using Hcy as a metabolic biomarker did not depend on the concentration of vitamin B12 chosen (see fig. 2, ROC, tHcy in Naik et al.
(
Reference Naik, Mahalle and Bhide
16
)). Using the current standard cut-off of 150 pmol/l for plasma vitamin B12, the sensitivity was 91·8 % and the specificity 79·31 %. Applying the new proposed cut-off value of 100 pmol/l for plasma vitamin B12, the sensitivity was 82·72 % and the specificity 89·47 %(
Reference Naik, Mahalle and Bhide
16
).
An important consideration that emerges from this analysis concerns the origin of reference intervals used worldwide to diagnose vitamin B12 deficiency. Reference ranges have been established by examining plasma vitamin B12, Hcy, methylmalonic acid and holo-TC, in healthy individuals residing in Western industrialised nations of whom the vast majority (>95 %) pursue an omnivorous lifestyle. An omnivorous diet provides sufficient vitamin B12 to keep tHcy and MMA at a minimum, and holo-TC above the established cut-off for deficiency (<35 pg/ml). Are these reference ranges established in omnivores appropriate cut-offs to assess vitamin B12 status in populations that adhere to vegetarian diets? More generally, what are the optimal intracellular concentrations of Cbl, Hcy and MMA required to support function? And how well do serum levels of Cbl, Hcy and MMA reflect cellular cobalamin status?
What follows is the question of whether slightly elevated tHcy and MMA as seen in asymptomatic vegetarians represent a prelude to clinical deficiency of vitamin B12 or if instead, these are metabolically satisfactory levels of metabolites that will cause no harm in the long term, that is subclinical cobalamin deficiency. According to Carmel, subclinical cobalamin deficiency is a condition where mild biochemical changes are documented (elevated tHcy and MMA, low vitamin B12 and holo-TC) but the patient is asymptomatic(
Reference Carmel
23
). According to this definition, asymptomatic individuals presenting with elevated tHcy and low vitamin B12 and holo-TC in the study by Naik et al.
(
Reference Naik, Mahalle and Bhide
16
) would classify as having subclinical cobalamin deficiency(
Reference Carmel
23
). Carmel described that subclinical deficiency of cobalamin rarely progresses into clinical deficiency(
Reference Carmel
23
), which brings us to the next issue: should vegetarian individuals with subclinical cobalamin deficiency receive treatment with cobalamin? If so, what should be the dose and form of administration? A possibility exists that what classifies as subclinical cobalamin deficiency in omnivores may not represent a status of abnormal vitamin B12 homoeostasis in plant-based individuals. Conceivably, applying reference values and cut-offs established from studying omnivorous populations may lead to the over-diagnosis of vitamin B12 deficiency in vegetarians and vegans. More broadly, the meaning of elevated tHcy and the impact of hyperhomocystinaemia on health remain a debate(
Reference Hannibal and Blom
24
). The finding that patients with classical homocystinuria (deficiency of the enzyme cystathionine β-synthase) sustain good health by staying at a target level of tHcy under 120 µm, that is well beyond the accepted normal range(
Reference Morris, Kozich and Santra
25
), questions the suitability of the tHcy reference values in plasma whereby 15 µm is defined as the upper limit. Vegetarian populations who naturally consume lower amounts of vitamin B12 may exhibit tHcy concentrations higher than 15 µm, without it representing a health threat.
Should a new reference interval for assessing vitamin B12 status be considered when examining human populations that pursue a predominantly plant-based lifestyle as proposed by Naik et al.? In light of current knowledge this proposal is a reasonable one, yet the definite answer to this question demands further study in larger cohorts of humans who have adopted vegetarianism both short and long term. Further, a mathematical expression that combines two, three or four biomarkers of vitamin B12 status, namely the cB12 index, has been shown to be a more reliable indicator of vitamin B12 status(
Reference Fedosov, Brito and Miller
26
–
Reference Brito, Verdugo and Hertrampf
28
). It would be valuable to examine the performance of the cB12 index with and without the introduction of new cut-off points for the accurate and timely diagnosis of vitamin B12 deficiency in vegetarians.
Another consideration that emerges from this analysis is what supplemental dose of cobalamin should be recommended to support good health in vegetarians? The most recent recommendation for strict vegetarians is a small oral dose of 2–6 µg daily(
Reference Carmel
29
). Higher doses were only recommended if absorption problems are confirmed in individual cases. Further, patients who recovered from a clinical cobalamin deficiency and have no absorption problems can be maintained safely with low-dose daily supplements in the range of 5–10 µg of cobalamin(
Reference Carmel
29
). Therapeutic doses of cobalamin (1 mg daily doses, 4 weeks(
Reference Carmel
29
)) are recommended to individuals with subclinical cobalamin deficiency only when the patient exhibits ‘sufficiently suspicious clinical findings’(
Reference Carmel
29
).
Special populations, whether omnivore or vegetarian, where vitamin B12 demands may not be met satisfactorily should be considered with caution. This includes the elderly and women in reproductive age attempting to conceive or who are pregnant. Elders with inefficient absorption of vitamin B12, a natural condition that together with lower dietary intake may increase the risk of acquiring a deficiency in vegetarians more so than in omnivores. In the case of women attempting to conceive or already pregnant, it must be remembered that depletion of serum vitamin B12 occurs much later than cellular depletion and elevation of metabolites in serum, making serum vitamin B12 a poor stand-alone marker of cobalamin status(
Reference Herbert
19
,
Reference Nexo, Christensen and Hvas
30
). Therefore, a conceiving woman with depleted cellular vitamin B12 may seriously compromise embryogenesis and early development, as the fetus relies entirely on the maternal supply of vitamin B12 via the placenta. It is highly recommended that vegetarian and vegan women in reproductive age take a vitamin B12 supplement before conception as well as during pregnancy and breast-feeding to ensure sufficient supplies of the micronutrient to the baby.
With the worldwide increase of humans invoking a vegetarian lifestyle, a demand exists to address questions concerning the diagnosis, interpretation and management of vitamin B12 insufficiency in these populations. The study by Naik et al.
(
Reference Naik, Mahalle and Bhide
16
) sets the stage for further large-population studies both at the epidemiological and basic research fronts to reassess the criteria for diagnosing vitamin B12 deficiency in vegetarian populations. Careful examination of these criteria will allow clinicians to define the appropriate chemical form, mode of administration and dose of cobalamin supplementation required to prevent the onset of acute vitamin B12 deficiency, as well as the timely implementation of therapy.
The proportion of humans adopting a plant-based lifestyle is on the rise worldwide. Benefits on economics, climate and health have been documented and projected to continue over the next decades( Reference Appleby and Key 1 – Reference Springmann, Godfray and Rayner 3 ). Apart from its sustainability( Reference Sabate and Soret 4 ), well-planned plant-based diets are nutritionally complete for human beings of all ages( Reference Melina, Craig and Levin 5 ), with vitamin B12 being the only micronutrient not found in plants( Reference Rizzo, Laganà and Rapisarda 6 – Reference Rizzo, Jaceldo-Siegl and Sabate 8 ). Vitamin B12 is only synthesised by a few bacteria and archaea( Reference Raux, Schubert and Warren 9 – Reference Eschenmoser 11 ); hence, humans adhering to a plant-based lifestyle can obtain the micronutrient from foods fermented with bacteria that naturally produce vitamin B12, foods fortified with vitamin B12 and over-the-counter supplements. However, the high prevalence of vitamin B12 insufficiency in vegetarians across nations( Reference Pawlak, Lester and Babatunde 12 ) suggests that food literacy programmes( Reference Azevedo Perry, Thomas and Samra 13 ) are not yet meeting the needs of the current dietary shift. Vitamin B12 deficiency, whether acquired or inherited, leads to the inactivation of the two vitamin B12-dependent enzymes, cytosolic methionine synthase and mitochondrial methylmalonyl-CoA mutase( Reference Green, Allen and Bjorke-Monsen 14 , Reference Green 15 ). This manifests with elevated homocysteine (Hcy) and methymalonic acid (MMA), and in severe cases with reduction of total serum vitamin B12 and its bioactive fraction holo-transcobalamin (holo-TC).
The study by Naik et al. ( Reference Naik, Mahalle and Bhide 16 ) examined vitamin B12 status in a cohort of 119 young, healthy unsupplemented vegetarian Indian graduates. Using standard cut-off values for the assessment of vitamin B12 status Naik et al. determined that 50 % were vitamin B12 deficient, 50–70 % exhibited low plasma holo-TC and 70–90 % presented elevated plasma total Hcy (tHcy). All participants in this study were asymptomatic of clinical vitamin B12 deficiency( Reference Naik, Mahalle and Bhide 16 ). These findings are in good agreement with a previous study by Refsum et al. in a different Indian cohort( Reference Refsum, Yajnik and Gadkari 17 ). Interestingly, nineteen participants with plasma vitamin B12 concentrations between 113 and 122 pmol/l had normal values of holo-TC (34–52 pmol/l), while exhibiting elevated tHcy according to standard cut-off reference values (discussed in Hannibal et al. ( Reference Hannibal, Lysne and Bjorke-Monsen 18 ) and references therein). Naik et al. ( Reference Naik, Mahalle and Bhide 16 ) propose a new set of cut-off values to improve the diagnosis of vitamin B12 deficiency in young vegetarian Indians. The authors propose the use of a combination of biomarkers and cut-off values of 100 and 19·6 pmol/l for plasma vitamin B12 and holo-TC, respectively, and values of tHcy of 17·6 and 27 µmol/l for females and males, respectively( Reference Naik, Mahalle and Bhide 16 ). This proposal appears in line with previously documented data in a study performed on a cohort of American vegetarians by the late Dr Victor Herbert( Reference Herbert 19 ). Herbert divided vitamin B12 status in vegetarians in four distinct stages, namely I, II, III and IV( Reference Herbert 19 ). Herbert and independent colleagues established that vegetarians can withstand long-term insufficient intake of vitamin B12 due to up-regulation of enterohepatic circulation and intestinal reabsorption of traces of vitamin B12 ( Reference Herbert 19 – Reference Kanazawa, Herbert and Herzlich 22 ). In healthy vegetarians, this represents a mechanism to optimise the recycling of the scarce micronutrient.
One strength of the study by Naik et al. is that the level of accuracy for identifying vitamin B12 deficiency using Hcy as a metabolic biomarker did not depend on the concentration of vitamin B12 chosen (see fig. 2, ROC, tHcy in Naik et al. ( Reference Naik, Mahalle and Bhide 16 )). Using the current standard cut-off of 150 pmol/l for plasma vitamin B12, the sensitivity was 91·8 % and the specificity 79·31 %. Applying the new proposed cut-off value of 100 pmol/l for plasma vitamin B12, the sensitivity was 82·72 % and the specificity 89·47 %( Reference Naik, Mahalle and Bhide 16 ).
An important consideration that emerges from this analysis concerns the origin of reference intervals used worldwide to diagnose vitamin B12 deficiency. Reference ranges have been established by examining plasma vitamin B12, Hcy, methylmalonic acid and holo-TC, in healthy individuals residing in Western industrialised nations of whom the vast majority (>95 %) pursue an omnivorous lifestyle. An omnivorous diet provides sufficient vitamin B12 to keep tHcy and MMA at a minimum, and holo-TC above the established cut-off for deficiency (<35 pg/ml). Are these reference ranges established in omnivores appropriate cut-offs to assess vitamin B12 status in populations that adhere to vegetarian diets? More generally, what are the optimal intracellular concentrations of Cbl, Hcy and MMA required to support function? And how well do serum levels of Cbl, Hcy and MMA reflect cellular cobalamin status?
What follows is the question of whether slightly elevated tHcy and MMA as seen in asymptomatic vegetarians represent a prelude to clinical deficiency of vitamin B12 or if instead, these are metabolically satisfactory levels of metabolites that will cause no harm in the long term, that is subclinical cobalamin deficiency. According to Carmel, subclinical cobalamin deficiency is a condition where mild biochemical changes are documented (elevated tHcy and MMA, low vitamin B12 and holo-TC) but the patient is asymptomatic( Reference Carmel 23 ). According to this definition, asymptomatic individuals presenting with elevated tHcy and low vitamin B12 and holo-TC in the study by Naik et al. ( Reference Naik, Mahalle and Bhide 16 ) would classify as having subclinical cobalamin deficiency( Reference Carmel 23 ). Carmel described that subclinical deficiency of cobalamin rarely progresses into clinical deficiency( Reference Carmel 23 ), which brings us to the next issue: should vegetarian individuals with subclinical cobalamin deficiency receive treatment with cobalamin? If so, what should be the dose and form of administration? A possibility exists that what classifies as subclinical cobalamin deficiency in omnivores may not represent a status of abnormal vitamin B12 homoeostasis in plant-based individuals. Conceivably, applying reference values and cut-offs established from studying omnivorous populations may lead to the over-diagnosis of vitamin B12 deficiency in vegetarians and vegans. More broadly, the meaning of elevated tHcy and the impact of hyperhomocystinaemia on health remain a debate( Reference Hannibal and Blom 24 ). The finding that patients with classical homocystinuria (deficiency of the enzyme cystathionine β-synthase) sustain good health by staying at a target level of tHcy under 120 µm, that is well beyond the accepted normal range( Reference Morris, Kozich and Santra 25 ), questions the suitability of the tHcy reference values in plasma whereby 15 µm is defined as the upper limit. Vegetarian populations who naturally consume lower amounts of vitamin B12 may exhibit tHcy concentrations higher than 15 µm, without it representing a health threat.
Should a new reference interval for assessing vitamin B12 status be considered when examining human populations that pursue a predominantly plant-based lifestyle as proposed by Naik et al.? In light of current knowledge this proposal is a reasonable one, yet the definite answer to this question demands further study in larger cohorts of humans who have adopted vegetarianism both short and long term. Further, a mathematical expression that combines two, three or four biomarkers of vitamin B12 status, namely the cB12 index, has been shown to be a more reliable indicator of vitamin B12 status( Reference Fedosov, Brito and Miller 26 – Reference Brito, Verdugo and Hertrampf 28 ). It would be valuable to examine the performance of the cB12 index with and without the introduction of new cut-off points for the accurate and timely diagnosis of vitamin B12 deficiency in vegetarians.
Another consideration that emerges from this analysis is what supplemental dose of cobalamin should be recommended to support good health in vegetarians? The most recent recommendation for strict vegetarians is a small oral dose of 2–6 µg daily( Reference Carmel 29 ). Higher doses were only recommended if absorption problems are confirmed in individual cases. Further, patients who recovered from a clinical cobalamin deficiency and have no absorption problems can be maintained safely with low-dose daily supplements in the range of 5–10 µg of cobalamin( Reference Carmel 29 ). Therapeutic doses of cobalamin (1 mg daily doses, 4 weeks( Reference Carmel 29 )) are recommended to individuals with subclinical cobalamin deficiency only when the patient exhibits ‘sufficiently suspicious clinical findings’( Reference Carmel 29 ).
Special populations, whether omnivore or vegetarian, where vitamin B12 demands may not be met satisfactorily should be considered with caution. This includes the elderly and women in reproductive age attempting to conceive or who are pregnant. Elders with inefficient absorption of vitamin B12, a natural condition that together with lower dietary intake may increase the risk of acquiring a deficiency in vegetarians more so than in omnivores. In the case of women attempting to conceive or already pregnant, it must be remembered that depletion of serum vitamin B12 occurs much later than cellular depletion and elevation of metabolites in serum, making serum vitamin B12 a poor stand-alone marker of cobalamin status( Reference Herbert 19 , Reference Nexo, Christensen and Hvas 30 ). Therefore, a conceiving woman with depleted cellular vitamin B12 may seriously compromise embryogenesis and early development, as the fetus relies entirely on the maternal supply of vitamin B12 via the placenta. It is highly recommended that vegetarian and vegan women in reproductive age take a vitamin B12 supplement before conception as well as during pregnancy and breast-feeding to ensure sufficient supplies of the micronutrient to the baby.
With the worldwide increase of humans invoking a vegetarian lifestyle, a demand exists to address questions concerning the diagnosis, interpretation and management of vitamin B12 insufficiency in these populations. The study by Naik et al. ( Reference Naik, Mahalle and Bhide 16 ) sets the stage for further large-population studies both at the epidemiological and basic research fronts to reassess the criteria for diagnosing vitamin B12 deficiency in vegetarian populations. Careful examination of these criteria will allow clinicians to define the appropriate chemical form, mode of administration and dose of cobalamin supplementation required to prevent the onset of acute vitamin B12 deficiency, as well as the timely implementation of therapy.
Acknowledgements
The author is extremely grateful to Dr Donald W. Jacobsen for critical reading of this manuscript.
The author declares that there are no conflicts of interest.