The Revised D‐A‐CH‐Reference Values for the Intake of Vitamin B12: Prevention of Deficiency and Beyond

Scope The nutrition societies of Germany, Austria, and Switzerland are the joint editors of the “D‐A‐CH reference values for nutrient intake”, which are revised regularly. Methods and Results By reviewing vitamin‐B12‐related biomarker studies, the reference values for vitamin B12 were revised in 2018. For adults, the estimated intake is based on the adequate serum concentrations of holotranscobalamin and methylmalonic acid. The estimated values for children and adolescents are extrapolated from the adult reference value by considering differences in body mass, an allometric exponent, and growth factors. For infants below 4 months of age, an estimated value is set based on the vitamin B12 intake via breast milk. The reference values for pregnant and lactating women consider the requirements for the fetus and for loss via breast milk. The estimated values for vitamin B12 intake for infants, children, and adolescents range from 0.5 to 4.0 µg d−1. For adults, the estimated values are set at 4.0 µg d−1, and for pregnant and lactating women, they are set at 4.5 and 5.5 µg d−1, respectively. Conclusion Based on the data of several vitamin B12 status biomarkers studies, the reference value for vitamin B12 intake for adults is raised from 3.0 to 4.0 µg d−1.


Introduction
The D-A-CH 'reference values for nutrient intake' [1] are jointly issued by the nutrition societies of Germany, Austria, and Switzerland (the abbreviation D-A-CH arises from the initial letters of the common country identification for the countries Germany [D],

Austria [A], and Switzerland [CH]
). Reference value is a collective term for recommended intake (RI) values, estimated values, and guiding values. An RI value, according to its definition, meets the requirement of nearly any person (approximately 98%) of a defined group of metabolically healthy people. Estimated values are given when human requirements cannot be determined with desirable accuracy. Guiding values are stated in terms of aids for orientation. [1] Reference values for nutrient intake are amounts that are assumed to r protect nearly all healthy individuals in a population from deficiency-related conditions r ensure optimal physiological and psychological performance, and r create a certain body reserve. [2] Since 2012, the D-A-CH nutrition societies have published revised reference values for the intake of several nutrients [3][4][5][6][7][8][9][10][11] but not yet for vitamin B 12 . The last update of the reference values for vitamin B 12 given by the D-A-CH nutrition societies dates to a 2000 report. [12] Since that time, a number of new scientific data have become available and have investigated the relationship between vitamin B 12 intake and biomarkers of vitamin B 12 status. [13][14][15] From this background, there is a need www.advancedsciencenews.com www.mnf-journal.com to review and update the D-A-CH reference values. In 2018, the revised reference values for vitamin B 12 intake were published in German. This paper provides a summary of this work.

Nutritional Physiology of Vitamin B 12
Vitamin B 12 is the collective term for a number of substances with qualitatively equivalent biological effects. These compounds consist of a corrin ring system that is similar to porphyrin with four reduced pyrrole rings and a central cobalt ion and are therefore referred to as cobalamins. Characteristically, all cobalamins have an α-axial ligand at the cobalt ion consisting of a phosphoribosyl-5,6-dimethylbenzimidazole side chain. The β-axial position can be occupied by various substituents. Depending on the group (R), a distinction is made between cyano-(R = CN), aquo-(R = H 2 O), hydroxo-(R = OH), methyl-(R = CH 3 ), and adenosyl-(R = 5'-deoxyadenosyl) cobalamin. [16] Cobalamins are synthesized only by microorganisms, and humans and animals receive them solely through the food chain. [17] In natural foods, vitamin B 12 is present mainly as hydroxocobalamin and adenosylcobalamin and is found in milk as methylcobalamin. [18] Due to its stability, cyanocobalamin is used in fortified foods as well as in pharmaceuticals and dietary supplements. Some pharmaceuticals and dietary supplements also contain hydroxocobalamin and methylcobalamin. [19,20] Humans metabolize cyanocobalamin and hydroxocobalamin into the two physiologically active coenzymes methylcobalamin and 5'-deoxyadenosylcobalamin: [17,19] r Methylcobalamin is a coenzyme of cytosolic methionine synthase (EC 2.1.1.13) that catalyzes the remethylation of L-homocysteine to L-methionine. This reaction also involves folate as 5-methyltetrahydrofolate (5-MTHF), which is the actual methyl donor. Methylcobalamin that is bound to the methionine synthase is demethylated in this reaction and then again remethylated through 5-MTHF. Accordingly, methylcobalamin is not only involved in the regulation of L-homocysteine and L-methionine but also required to provide free tetrahydrofolate (THF), which is an important coenzyme for the transfer of one-carbon units, for example, in purine and pyrimidine synthesis. [21] r 5'-Deoxyadenosylcobalamin is a coenzyme of the mitochondrial enzyme L-methylmalonyl-CoA mutase (EC 5.4.99.2). This enzyme isomerizes L-methylmalonyl-CoA to succinyl-CoA, a central intermediate in the degradation of odd-chain fatty acids and of the amino acids methionine, threonine, and isoleucine. In a first step, propionyl-CoA is converted to D-methylmalonyl-CoA in a reaction catalyzed by the biotin-dependent propionyl-CoA carboxylase (EC 3.4.1.3). Thereafter, a racemase converts D-methylmalonyl-CoA to L-methylmalonyl-CoA. [22] Furthermore, D-Methylmalonyl-CoA, which is a product of valine and thymine degradation, is also converted after epimerization to succinyl-CoA through the L-methylmalonyl-CoA mutase reaction.
The largest vitamin B 12 pool in the human body is located in the liver (approximately 50% of the total body pool). At a mean total body pool of 3 mg, [23] this amount corresponds to a content of approximately 1.5 mg in the liver, with 61% present as adenosylcobalamin, 38% as hydroxycobalamin and only 1% as methylcobalamin. [18] Significant quantities of vitamin B 12 are also found in skeletal muscles (approximately 9%), bone marrow (approximately 4.5%) and bowel tissue (approximately 4.5%). [18] In the blood, cobalamins are transported bound to the β-globulins transcobalamin (TC)I (haptocorrin) or TC II. The most physiologically important carrier protein is TC II. The complex formed by vitamin B 12 and TC II is referred to as holo-TC II (simplified synonym holo-TC). [24] The half-life of holo-TC is 60 to 90 min [18,25] ; 10-30% of the circulating vitamin B 12 is bound to TC II. [24]

Criteria to Assess the Vitamin B 12 Status
There are different biomarkers for assessing vitamin B 12 status. These biomarkers include the two status parameters vitamin B 12 and holo-TC in serum or plasma and the functional parameters methylmalonic acid (MMA) and homocysteine in serum. [21,[26][27][28][29][30][31][32] However, none of these parameters are sufficient on their own to adequately assess vitamin B12 status. [29] Furthermore, until now, no generally accepted cut-off values exist for any of the biomarkers. Additionally, several groups have published different cut-off values. [28,33,34]

Total Circulating Vitamin B 12
A serum or plasma concentration of vitamin B 12 between 148 pmol L −1 and 221 pmol L −1 indicates marginal supply, [28] and a concentration of <148 pmol L −1 indicates vitamin B 12 deficiency. [26,28,35] A serum concentration of >221 pmol l −1 may be considered indicative of a sufficient vitamin B 12 status in adults. [28,36] However, a serum concentration of >221 pmol L −1 may not necessarily mean that the vitamin B 12 supply is sufficient since symptoms of vitamin B 12 deficiency and/or a functional metabolic vitamin B 12 deficiency may also be present. [21,31] Therefore, the total serum vitamin B 12 concentration should be determined in combination with a functional parameter such as MMA. [29,30,37]

Circulating Holo-TC
Circulating holo-TC is responsible for supplying the tissues with vitamin B 12 . [38] Thus, the serum holo-TC concentration reflects the supply of metabolically active cobalamins. [32,39] A holo-TC concentration between 40 and 100 pmol L −1 , [36] 40 and 200 pmol L −1 [39] or >37 and <190 pmol L −1 [40] is considered as the target concentration for a sufficient vitamin B 12 status in adults. A holo-TC concentration of >8.4 to <20 pmol L −1 is indicative of a possible vitamin B 12 deficiency and a concentration of <8.4 pmol L −1 is indicative of a potential vitamin B 12 deficiency. [40] For a more reliable assessment of vitamin B 12 supply, holo-TC should be determined in combination with a functional parameter such as MMA. [29,30]

Circulating MMA
Serum MMA concentration is considered as functional biomarker of vitamin B 12 status. In the case of vitamin B 12 www.advancedsciencenews.com www.mnf-journal.com deficiency, the serum concentration of MMA increases due to a reduced vitamin B 12 -dependent isomerization of L-methylmalonic acid to succinyl-CoA through the enzyme L-methylmalonyl-CoA mutase. [29,35,41] A MMA concentration of <210 [28] or <270 nmol L −1 [36] is considered the target concentration for a sufficient vitamin B 12 supply in adults. MMA concentrations of >350 to <840 nmol L −1 are indicative of an insufficient vitamin B 12 status, and MMA concentrations of >840 nmol L −1 are indicative for a vitamin B 12 deficiency. [40] However, an increased MMA serum concentration may also occur due to impaired renal function. During pregnancy and at advanced age, MMA also increases independent of the vitamin B 12 intake. Therefore, the specificity of MMA as a biomarker for vitamin B 12 is limited. [29,35] Thus, MMA should only be used in combination with a status parameter such as holo-TC. [29,30]

Circulating Homocysteine
The remethylation of homocysteine to methionine is impaired if there is an insufficient vitamin B 12 supply; consequently, plasma homocysteine concentration increases. [42] However, the homocysteine concentration is influenced not only by vitamin B 12 but also by folate, vitamin B 6 , and riboflavin (vitamin B 2 ). Insufficient supply of these vitamins can also lead to increased homocysteine concentrations. [43] The latter may also occur as a consequence of renal insufficiency, dehydration, and cystathionine β-synthase deficiency as well as certain polymorphisms of the methylenetetrahydrofolate reductase. [44] Therefore, the homocysteine concentration is a sensitive but non-specific functional marker of vitamin B 12 status. [26] Accordingly, the homocysteine concentration is suitable to assess the vitamin B 12 status only in combination with a vitamin B 12 status parameter (serum holo-TC or vitamin B 12 ). [30] In general, a plasma homocysteine concentration of <12 µmol L −1 in adults is considered to be adequate. [45]

Bioavailability of Vitamin B 12
The bioavailability of vitamin B 12 mainly depends on the intake level and decreases with an increasing dose. [46] The approximate absorption rates for the intake of 1, 5, 10, and 20 µg are 50%, 20%, 10%, and only 5%, respectively. [47] In addition to the dosage, the food matrix determines the absorption rate of vitamin B 12 . [17,47] The absorption rate when consuming lamb with a vitamin B 12 content of 2.6 µg per 100 g is 56-89%, and for fish (rainbow trout with 4.9 µg per 100 g), the absorption rate is 42%. For cow's milk (0.4 µg per 100 g), a bioavailability of 65% has been determined. [17] The absorption rate for eggs with an average vitamin B 12 content of 1.3 µg per 100 g is <9%. [17] Based on a metaregression analysis of eight bioavailability studies, absorption rates of 37% and 29% were determined for an intake of 1.2-3.1 µg vitamin B 12 , respectively, per meal complying with the usual dietary habits. [46] At an average vitamin B 12 intake of 1.2 µg per meal, the bioavailability is 37%, [46] which means that for a mixed diet, which contains vitamin B 12 also from foods with lower bioavailability, [17] an availability of 35-40% may be assumed.
Vitamin B 12 produced by colon bacteria in the human gut cannot be absorbed due to missing receptors. [48]

Adults under 65 Years of Age
As criteria to derive the reference value of vitamin B 12 intake in adults, the serum concentrations of holo-TC (ࣙ 40 pmol L −1 ) and of the functional parameter MMA in the desirable range of <270 nmol L −1 are considered as the most important ones. These criteria are based on the results of two studies, in which the adequate vitamin B 12 intake was determined using the serum concentrations of total vitamin B 12 and holo-TC as well as the functional parameters MMA and homocysteine. [13,14] In the cross-sectional study by Bor et al. [13] with subjects aged 18 to 50 years, an average serum holo-TC concentration of 65 pmol L −1 was reached with a daily median intake of 4.2 µg vitamin B 12 ; higher intake levels were not associated with higher holo-TC concentrations. The serum concentration of vitamin B 12 reached an average of 325 pmol L −1 . For the functional parameters MMA and homocysteine, average concentrations of 210 nmol L −1 and 8 µmol L −1 , respectively, were measured, [13] which indicate adequate vitamin B 12 supply. [28] The intake of more than 7 µg d −1 during this study did not result in a further increase in the plasma vitamin B 12 concentration. With an intake of 7 µg d −1 , the lowest serum concentrations of MMA and homocysteine were approximately 190 nmol L −1 and approximately 7 µmol L −1 , respectively. [13] More data on the dose-response relationship between the vitamin B 12 intake and the serum concentrations of MMA, homocysteine and vitamin B 12 are available from a randomized controlled intervention study including 231 subjects. [14] In this study, the lowest MMA and homocysteine concentrations were 190 nmol L −1 and 8.2 µmol L −1 , respectively, with an intake of approximately 7 µg vitamin B 12 d −1 (4 µg d −1 from foods plus 3.4 µg d −1 from a supplement). This dietary intake resulted in a serum concentration of vitamin B 12 of approximately 330 pmol L −1 . At a dietary intake of 4.0 µg vitamin B 12 , the serum concentrations of vitamin B 12 , MMA, and homocysteine were approximately 300 pmol L −1 , 220 nmol L −1 , and 8.3 µmol L −1 , respectively. [14] Based on the data from the studies by Bor et al. [13] and Pentieva et al. [14] as well as on the target concentrations for total serum vitamin B 12 (>221 pmol L −1 ), holo-TC (ࣙ40 pmol L −1 ), MMA (<270 nmol L −1 ), and homocysteine (<12 µmol L −1 ), the estimated value for the vitamin B 12 intake is 4.0 µg d −1 for men and women within the age range of 19 and 65 years ( Table 1).

Adults above 65 Years of Age
In a study by Bor et al. [15] on osteoporotic women and women at risk of osteoporosis at the age of 41 to 75 years, a median holo-TC serum saturation of 119 pmol L −1 was achieved at a daily intake of 6 µg vitamin B 12 . At this dose, the median serum concentration of vitamin B 12 was 380 pmol L −1 . Median concentrations of MMA and homocysteine were 120 nmol L −1 and 9.75 µmol L −1 , www.advancedsciencenews.com www.mnf-journal.com respectively, [15] which indicate adequate vitamin B 12 supply. [28] However, 36% of the subjects had a gastric pH of ࣙ3 or other indications of gastric dysfunction. [15] Those dysfunctions could cause an inhibition of gastric acid and pepsinogen secretion, which results in a reduced release of free vitamin B 12 from food proteins, hence decreasing intestinal absorption of the cobalamin protein complexes from food. Furthermore, the reduced acid secretion leads to an alkalinization of the small intestine, which may result in bacterial overgrowth and thus to a further decrease of the bioavailability of vitamin B 12 .
A cross-sectional study in German women at the age of 60 to 70 years (n = 178) showed that at an average intake of 5 µg d −1 , 43% of the women had a serum cobalamin concentration of less than 258 pmol L −1 . [49] This concentration was defined in the study as a limit for sufficient supply. To derive the reference values for vitamin B 12 intake, a concentration range of 148 to 221 pmol L −1 was defined as suboptimal, while a concentration of >221 pmol L −1 indicated sufficient supply. However, only 10% of the subjects in the study by Wolters et al. [49] had an MMA concentration >271 nmol L −1 . An MMA concentration of <270 nmol L −1 was considered the target concentration for a sufficient supply.
Currently, the available data are not sufficient to derive reference values for adults above 65 years of age for vitamin B 12 intake that are different from those for younger adults. Thus, the estimated value for vitamin B 12 intake for adults above 65 years of age does not differ from that for younger adults independent of gender and is set to 4.0 µg d −1 (Table 1).

Children and Adolescents
No data are available regarding the vitamin B 12 requirement for children and adolescents. Therefore, the reference values for children and adolescents are based on the values compiled for adults and take into account the differences in body weight, an allometric exponent and the growth factors to consider the requirements for growth ( Table 2). Growth factors at the different age groups were calculated as the proportional increase in protein requirement for growth relative to the maintenance requirement according to the WHO. [1,50] When using the age groups and reference body weights the D-A-CH reference values are based upon, [1] the resulting estimated values for vitamin B 12 intake range from 1.5 µg d −1 (for 1 to under 4 year olds) to 4.0 µg d −1 (for 15 to under 19 year olds) ( Table 2).

Infants
The reference values for the intake of vitamin B 12 for infants aged 0 to under 4 months were derived based on the vitamin B 12 content of breast milk, which is considered to be the optimal diet for infants. [51,52] The cobalamin content of breast milk varies depending on the duration of breastfeeding [53] and maternal vitamin B 12 supply. [54] It declines continuously during the lactation period. [55] In a Danish study, in which the majority of the women had taken a multivitamin supplement (1.0-4.5 µg vitamin B 12 d −1 ), the vitamin B 12 content of the breast milk was 0.1 µg per 100 mL two weeks postpartum and 0.04 µg per 100 mL four months postpartum [53] with a median vitamin B 12 content in breast milk in the first four months of 0.07 µg per 100 mL. [53] Assuming that the median vitamin B 12 content of breast milk during the first four months is 0.07 µg per 100 mL, an exclusively breastfed infant receives 0.53 µg d −1 vitamin B 12 from breast milk of mothers with adequate vitamin B 12 supply at an average breast milk intake of 750 mL d −1 . [56] Therefore, the estimated value for the adequate intake of vitamin B 12 for breastfed infants aged 0 to under 4 months is set at 0.5 µg d −1 ( Table 3).
The consumption of breast milk declines with the introduction of solid foods. Since no data are available from Germany with regard to vitamin B 12 intake via solid foods, the estimated value for infants aged 4 to under 12 months is based on the values compiled for adults and considers differences in the body weight, an allometric exponent and a growth factor to consider the requirements for growth. An estimated value of 1.4 µg d −1 vitamin B 12 for infants aged 4 to under 12 months was derived ( Table 2).

Pregnancy
The requirement of pregnant women is slightly increased to meet the vitamin B 12 requirement of the fetus. It was estimated that the fetus accumulates 0.1 to 0.2 µg vitamin B 12 per day. [57] Considering an absorption rate of 35-40%, the estimated value for vitamin B 12 intake for pregnant women is therefore set to 4.5 µg d −1 (Table 1).

Lactation
In comparison to the vitamin B 12 requirement of non-lactating women, the requirement of lactating women is increased due to vitamin B 12 secretion from breast milk. Approximately 0.5 µg d −1 www.advancedsciencenews.com www.mnf-journal.com Table 2. Estimated values for vitamin B 12 intake for infants (4 to under 12 months), children and adolescents considering differences in body weight, allometric exponent, and growth factors.

Age [years]
Gender Reference body weight [kg] a) [1] Growth factor b) [ [126] In each case, the values reflect the midpoint of the respective age range; b) Growth factors at different ages were calculated as the proportional increase in protein requirement for growth relative to the maintenance requirement according to WHO [1,50] ; c) Calculated from: estimated value adults x (reference body weight infants/children/adolescents /reference body weight adults ) 0.75 x (1 + growth factor); Estimated value adults : 4.0 µg d −1 (Table 1)

Preventive Aspects
In the following paragraphs, currently available data on vitamin B 12 are outlined in association with some health-related aspects. Dietary reference values are aimed at healthy individuals; thus, the requirements of patients are not addressed.

Vitamin B 12 and Cardiovascular Diseases
Since metabolic vitamin B 12 deficiency impairs the remethylation of homocysteine to methionine, serum homocysteine concentration increases. [42] It is well known that homocysteine is an independent risk factor for ischaemic heart disease and ischaemic stroke. [58][59][60] By increasing the homocysteine concentration by 5 µmol L −1 , the risk of coronary events increases by 18%. [60] With an increase of 3 µmol L −1 , the risk of stroke increases by 19%. [59] However, it is not clear, whether this observation reflects a causal relationship. [61] Nevertheless, genetic association studies, [62][63][64] in vitro studies and animal studies indicate a causal link. [45,65] In contrast, the results from clinical intervention studies with vitamin B 12 , vitamin B 6 and folate are contradictory. On the one hand, supplementation with B vitamins has the potential to reduce the relative risk of stroke by approximately 10%, [66,67] but vitamin B 12 alone does not seem to exert a preventive effect. [67] Additionally, vitamin B 12 supplementation was not associated with a reduced risk of coronary events and cardiovascular diseases. [66,68]

Vitamin B 12 and Cancer
Vitamin B 12 is essential for DNA methylation, genome integrity, and chromosomal stability, which suggests a preventive effect of vitamin B 12 regarding the development of cancer. [69] For breast cancer, a meta-analysis of 18 observational studies showed an inverse association with the risk of disease and vitamin B 12 intake but not for serum vitamin B 12 concentration. [70] Furthermore, no association was observed between dietary vitamin B 12 intake [71] or vitamin B 12 status [72] and the risk of endometrial cancer and colorectal carcinoma. [73][74][75][76][77] Another meta-analysis investigated the www.advancedsciencenews.com www.mnf-journal.com association between vitamin B 12 serum concentration and the risk of renal cell cancer using data from seven cohorts. Compared with the group with the lowest intake, subjects with the highest intake had a relative renal cell cancer risk of 0.72 (95% CI = 0.52-1.00). [78] On the other hand, it is discussed that increased serum vitamin B 12 concentration is associated with a higher risk of cancer. For example, in a meta-analysis of observational studies, there was a positive association between vitamin B 12 serum concentration and the risk of prostate cancer. [79] In a cohort study, an increased serum vitamin B 12 concentration (>600 pmol L −1 ) was associated with an elevated total risk of cancer incidence. The risk was highest within the first year of follow-up. [80] However, this association might be due to the increased intake of animal-based foods, which is associated with high vitamin B 12 intake ("spurious correlation"). Another cause for this association may be cancer types that are already present but not yet diagnosed, as some cancer types are associated with higher serum vitamin B 12 concentrations. [81]

Vitamin B 12 , Miscarriages, and Neural Tube Defects
In observational studies, an insufficient supply of vitamin B 12 during pregnancy is associated with an increased risk of miscarriage. [82,83] Furthermore, maternal vitamin B 12 status is associated with the occurrence of neural tube defects in the child. [84,85] However, it remains unclear whether this observation reflects a causal relationship. [86]

Vitamin B 12 and Bone-Related Diseases
Vitamin B 12 deficiency and hyperhomocysteinaemia are linked with the stimulation of osteoclast activity and accelerated degradation of the bone matrix. [87][88][89][90] The data from observational studies on the association between vitamin B 12 status or vitamin B 12 intake and bone mineral density, the risk of fracture or bone turnover markers are inconsistent. [88] In a meta-analysis of four prospective studies, for each increase of total serum vitamin B 12 concentration by 50 pmol L −1 , a 4% reduced fracture risk was calculated (95% CI = 0.92-1.00). [91] A meta-analysis of four randomized controlled intervention studies indicates that the effect of vitamin B 12 on the risk of fracture depends on the basal homocysteine concentration. A protective effect of vitamin B 12 supplementation may be expected only at homocysteine concentrations >15 µmol L −1 (75% risk reduction; 95% CI = 0.12-0.53). There is no evidence that vitamin B 12 supplementation in excess of the individual requirement for subjects with adequate supply of folate and vitamin B 12 at homocysteine concentrations <15 µmol L −1 results in a further fracture risk reduction. [92]

Vitamin B 12 , Neurodegenerative Diseases, and Cognitive Impairment
There is also a discussion about the question of whether functional vitamin B 12 deficiency, which is characterized by decreased concentrations of holo-TC and increased concentrations of MMA and/or homocysteine, is associated with an increased risk of neurodegenerative diseases and cognitive impairment. [93,94] Several epidemiological studies showed a positive correlation regarding MMA concentrations and an inverse association regarding holo-TC concentrations with the development or progression of dementia, while there is only a low or no association between total vitamin B 12 concentrations and disease risk. [72,74,75,93,94] In older people, vitamin B 12 deficiency is also associated with cerebral atrophy. [95,96] Vitamin B 12 supplementation (0.5 mg d −1 ) in combination with folate (0.8 mg d −1 ) and vitamin B 6 (20 mg d −1 ) in older people could slow cerebral atrophy over two years. This effect is, however, dependent on baseline homocysteine concentration. [97] The concentrations of holo-TC and MMA have proved to be good predictors of cognitive performance, while total vitamin B 12 concentration has not been shown to be a good predictor, as indicated by a longitudinal cohort study including 1648 senior women and men (ࣙ65 years) over a period of 10 years. An increase of the holo-TC concentration from 50 to 100 pmol L −1 is associated with a 30% lower reduction of cognitive performance; the increase in MMA and homocysteine concentrations from 0.25 to 0.5 µmol L −1 and from 10 to 20 µmol L −1 , respectively, is associated with an impairment of cognitive performance by >50%. [98] The data from intervention studies on the influence of vitamin B 12 supplementation on cognitive performance are contradictory; the effects seem to depend on the basal homocysteine concentration and duration of the intervention. A systematic review of 25 randomized controlled studies and cohort studies concluded that there is insufficient evidence regarding an association between vitamin B 12 intake or vitamin B 12 status, respectively, and cognitive performance. [94] Another systematic review of ten observational studies including subjects over 50 years had shown similar results. [99] 7. Discussion and Conclusion

Vitamin B 12 Reference Values-What has Changed?
The reference values for vitamin B 12 intake from international expert panels vary considerably. [57,[100][101][102] This variance of recommendations results from the fact that the scientific basis allows a certain variation (see Table 4). Therefore, they have been the subject of intense controversy. Since the publication of the first D-A-CH reference values for the intake of vitamin B 12 , several vitamin B 12 biomarker studies have questioned whether the current reference value of 3.0 µg vitamin B 12 d −1 is adequate for optimal vitamin B 12 status in healthy adults. [13][14][15] From this background, D-A-CH nutrition societies have reviewed and evaluated vitamin B 12 -related biomarker studies and revised the reference values for vitamin B 12 in 2018.
Developing new reference values for vitamin B 12 intake requires the definition of the best target biomarkers for a supply, corresponding to adequacy of metabolic functions. For adults, the reference value for vitamin B 12 intake is based on biomarkers of vitamin B 12 status, notably adequate serum concentrations of holo-TC and MMA. As a result, the revised vitamin B 12 Table 4. Reference values for the intake of vitamin B 12 from different nutrition societies.
World Health Organisation (WHO), Food and Agricultural Organisation of the United Nations (FAO) [57] European Food Safety Authority (EFSA) [102] Institute of Medicine (IoM) [100] Nordic Council of Ministers [101] Infants www.advancedsciencenews.com www.mnf-journal.com reference value for adults was changed from a recommended intake value of 3 µg d −1 up to an estimated value of 4 µg d −1 , which is in line with the reference value of vitamin B 12 of the European Food Safety Authority (EFSA). [102]

Vitamin B 12 Reference Values-Practical Aspects
According to the analysis of the data from the National Nutrition Survey II (NVS II, 2005-2006, the median vitamin B 12 intake in Germany in women and men between the ages of 15 and 80 years is 3.8 and 5.3 µg d −1 , respectively. [103,104] The intake of foods naturally rich in cobalamin as part of a mixed diet can ensure sufficient vitamin B 12 supply. To achieve sufficient vitamin B 12 intake, a regular intake of animal-based food such as milk and dairy products, fish, poultry and lean meat is recommended. It should be noted that during food processing, e.g., cooking or pasteurization, a vitamin B 12 loss up to 50% might occur. [105] People adopting a vegan diet that excludes animal foods should be encouraged to take a dietary vitamin B 12 supplement and should have their vitamin B 12 status regularly checked by a physician. [106] Spirulina and other cyanobacteria, which are advertised as natural vitamin B 12 sources for vegans, do not contain cobalamins that are bioavailable for humans. In addition, the cobalamin analogues that they contain can even inhibit intestinal uptake of vitamin B 12 and might suppress the formation of biologically active vitamin B 12 coenzymes. [107,108] Therefore, they are unsuitable to cover the requirements of vegans.
Partly, vitamin B 12 deficiency represents a health issue for ovolacto vegetarians. [105,109,110] Thus, it is recommended that vegetarians should have their vitamin B 12 status checked regularly by a physician, too, and should take dietary supplements to achieve sufficient vitamin B 12 supply if necessary. [105,[111][112][113] In particular, vegetarians with increased nutrient requirements, for example, due to pregnancy or lactation, should pay attention to sufficient vitamin B 12 intake.
In older people, the risk of vitamin B 12 deficiency is increased, mainly due to impaired absorption because of medication (e.g., proton pump inhibitors, H 2 blockers) or diseases (e.g., atrophic gastritis) and less due to insufficient dietary intake of vitamin B 12 . [114][115][116][117] In the case of atrophic gastritis with hypochlorhydria and related malabsorption of dietary proteinbound vitamin B 12 , [118,119] supplementation of vitamin B 12 is recommended to ensure adequate vitamin B 12 status. [114,116,120] In contrast to vitamin B 12 from natural foods, the absorption of crystalline vitamin B 12 from preparations is not impaired in atrophic gastritis type B-related hypochlorhydria. [116,120] However, investigations show that the use of dietary supplements or fortified foods reduces the prevalence of vitamin B 12 deficiency in older people, [96,[121][122][123] but the risk of insufficient supply in general persists because of the dosage, which is often too low. [124,125] Thus, a regular observation of the vitamin B 12 status is also recommended for older people. For the assessment of the vitamin B 12 status, a relevant functional parameter such as MMA should be combined with a status marker such as serum vitamin B 12 or holo-TC.