Efficacy and Safety of Vitamin B12 Fortification

Chapter 26

Efficacy and Safety of Vitamin B12 Fortification Lindsay H. Allen USDA, ARS Western Human Nutrition Research Center, Davis, CA, United States

Chapter Outline 26.1 26.2 26.3 26.4 26.5

Prevalence of Vitamin B12 Deficiency Why Vitamin B12 Status Is Important Cofortification of Vitamin B12 and Folic Acid Diagnosis of Deficiency and Depletion Requirements, Bioavailability, and Safety

255 255 256 257 257

26.6 Expert Consensus on Recommended Vitamin B12 Fortification 26.7 Experience With Vitamin B12 Fortification 26.8 Conclusions References

258 258 259 260

26.1 PREVALENCE OF VITAMIN B12 DEFICIENCY

them to be repleted. Fortification is an opportunity to prevent depletion and deficiency in populations over time.

While recognition and documentation of the high global prevalence of vitamin B12 deficiency is increasing, it is not generally appreciated that this may be the most common nutrient deficiency in the world (Fig. 26.1). The explanation for the widespread deficiency is that the vitamin is only found naturally in animal source foods, and usual daily consumption of these foods is low in many population groups due to economic and storage constraints, and cultural, religious, and other beliefs. In general vitamin B12 status is directly correlated with B12 intake (Tucker et al., 2000; Allen, 2008; Allen et al., 2018). Omnivores have better status than lacto-ovo vegetarians (nonmeat eaters), who in turn have better status than lactovegetarians, while strict vegetarians often have signs of deficiency (Herrmann et al., 2003) unless they consume supplements or fortified foods. Unlike iron deficiency, vitamin B12 deficiency occurs at all stages of the lifespan, from birth to old age, and both genders are probably equally at risk (Allen, 2009). Moreover in wealthier countries, apart from those who avoid animal source foods, the highest prevalence may occur in the elderly, who have difficulty absorbing the vitamin from food due to lack of gastric acid or the intrinsic factor needed for active absorption of B12. Once vitamin B12 stores in the liver are low or depleted, it can take months or years for

26.2 WHY VITAMIN B12 STATUS IS IMPORTANT The symptoms of severe vitamin B12 deficiency are well established—and exemplified in the autoimmune disease Pernicious Anemia where the intrinsic factor required for the vitamin to be actively and efficiently absorbed is lacking. Without regular injections or high oral doses of the vitamin, the resulting symptoms will include megaloblastic anemia, and neurological, cognitive, and developmental disorders among other problems (Allen et al., 2018; Green et al., 2017). More commonly at the populationwide level where B12 deficiency is predominantly caused by low intakes, the deficiency is less severe, and results in biochemical abnormalities such as low plasma B12 or elevated methylmalonic acid rather than anemia or neurological problems. This has been termed “subclinical cobalamin deficiency” due to the lack of obvious clinical symptoms (Carmel, 2013). However evidence is accumulating to show that even marginal deficiency has adverse effects on human function. For example, especially in regions where flour is now fortified with folic acid, poor maternal vitamin B12 status has been identified as a probable risk factor for neural tube defects (Wang et al., 2012). If pregnant women have low stores of the vitamin, their infant will be born with low stores at birth and this

Food Fortification in a Globalized World. DOI: https://doi.org/10.1016/B978-0-12-802861-2.00026-2 2018 Published by Elsevier Inc.

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Canada and United States

Low B12 <148 pmol/L Marginal B12 148–221 pmol/L

23

Canada 2011 (6–79 year) Canada 2015 (adult women) NHANES III (<4 year) NHANES III (12–19 year) NHANES III (20–29 year) NHANES III (30–39 year) NHANES III (40–49 year) NHANES III (50–59 year) NHANES III (60–69 year) NHANES III (>70 year) SALSA 2003 (elderly Latino)

34

17

8

15

20 21 21

26

29

23

Latin America Brazil (first trimester pregnancy) Brazil (second trimester pregnancy) Brazil (third trimester pregnancy) Chile 2003 (adult women) Chile 2010 (elderly) Colombia 2010 (<18 years) Colombia 2010 (pregnant women) Colombia 2010 (adult women) Costa Rica 2007 (adults) Ecuador 2009 (elderly) Guatemala 1997 (lactating women) Guatemala 2003 (school children) Guatemala 2007 (infants) Guatemala 2009–2010 (adult women) Mexico 1999 (preschoolers) Mexico 1999 (school children) Mexico 1999 (adolescent girls) Mexico 1999 (adult women) Venezuela 2007 (elderly)

17

43 48

23 25 21 37 31

60 42 47

33

49 49

39

47 47

35

58

Europe and oceania 5

Denmark 2009 (adults) Italy 2012 (elderly) United Kingdom OHAP (elderly) United Kingdom MRC (elderly) United Kingdom NDNC (elderly) Australia 2013 (refugees) New Zealand 1996–1997 (elderly)

28

8

16 20 17

40

Africa and Asia 49

Botswana (school children) Cameroon 2015 (children) Cameroon 2015 (adult women) Ghana (pregnant women) Kenya 2003 (school children) Malawi (pregnant women) Niger (pregnant women) The Gambia (first trimester pregnancy) The Gambia (third trimester pregnancy) The Gambia (12 weeks postpartum) The Gambia (infants 12 weeks) The Gambia (infants 24 weeks) Jordan 2014 (>19 years) Turkey 2015 (adult women) Bangladesh 2011 (adult women) Bangladesh 2016 (pregnant women) India (preschoolers) India 2001 (adults) South India 2016 (adults) China 2003 (adult women)

29

33

11 69 10 78 23 34

22

43 37 44

30 27

66 72

35 37

0

20

40

60

80

80

100

Prevalence of low and marginal serum B12 (%) FIGURE 26.1 Prevalence of low (,148 pmol/L) and marginal (148 221 pmol/L) plasma or serum B12 concentrations in selected representative national surveys and large nonrepresentative studies. From reference Allen, L.H., Miller, J.W., de Groot, L., Rosenberg, I.H., Smith, D.A., Refsum, H., et al., 2018. Biomarkers of Nutrition for Development (BOND): vitamin B-12 review. J. Nutr. (in review).

situation is difficult to fully overcome by maternal supplementation in pregnancy (Duggan et al., 2014; Siddiqua et al., 2014); in contrast, fortification provides the opportunity for women to enter pregnancy with adequate B12 stores. Moreover, breast milk B12 concentrations are often very low in B12 depleted women (Deegan et al., 2012; Allen, 2012). Infants and children born to mothers with poor B12 status have impaired motor and mental development (Dror and Allen, 2008; Strand et al., 2013). In the elderly poor vitamin B12 status and accompanying high plasma homocysteine concentrations are associated

with brain atrophy and white matter damage (Smith and Refsum, 2009), a situation improved by supplementation (Smith et al., 2010). Other observed consequences of poor B12 status are described in more detail elsewhere (Allen et al., 2018; Green et al., 2017).

26.3 COFORTIFICATION OF VITAMIN B12 AND FOLIC ACID There is substantial evidence that consuming high amounts of folic acid—either from high levels used in

Efficacy and Safety of Vitamin B12 Fortification Chapter | 26

fortification (Brito et al., 2016) or when folic acid supplements are taken in addition to folic acid-fortified food— can exacerbate vitamin B12 deficiency. The evidence is both biochemical (Miller et al., 2009; Selhub et al., 2007) and clinical (Morris et al., 2007). Discussion of this issue is beyond the scope of this chapter but is available in published documents (Selhub and Paul, 2011; Smith, 2007) (see Chapter 24: Assessing all the Evidence for Risks and Benefits With Folic Acid Fortification and Supplementation). The benefits of folic acid fortification for prevention of neural tube defects likely outweigh any potential adverse effects on vitamin B12 status. However, it has been argued that given the high global prevalence of B12 deficiency and of folic acid fortification programs, concern about adverse effects of high folic acid intakes on B12 status would be alleviated by cofortification with vitamin B12 (Selhub and Paul, 2011; Allen, 2012).

26.4 DIAGNOSIS OF DEFICIENCY AND DEPLETION The recent Biomarkers of Nutrition and Development (BOND) report on vitamin B12, supported by the National Institutes of Health in the United States, provides values for diagnosis of vitamin B12 deficiency and depletion—which are not much different from those generally used (Allen et al., 2018). There are four potential biomarkers of B12 status, two of which (plasma B12 and holotranscobalamin) are lower, and two of which (plasma methylmalonic acid and total homocysteine) increase indicating inadequate amounts of the vitamin for its role as a cofactor for important enzyme reactions. The BOND report provides detailed information on the pros and cons of each indicator and factors that can cause erroneous values during sample collection, preparation, and analysis. The most practical marker of B12 status at the population level is plasma (or serum) vitamin B12. Concentrations .221 pmol/L (300 pg/mL) indicate adequacy, 150 221 pmol/L depletion and low stores, 75 150 deficiency, and ,75 severe deficiency. Levels in infants are normally higher than in adults. For serum methylmalonic acid, ,271 nmol/L indicates adequate status, 271 376 nmol/L depletion, and .376 nmol/L deficiency. Methylmalonic acid is more expensive to measure than plasma B12 and requires a mass spectrometer. Holotranscobalamin, the form in which B12 is transported in the body, has been used more recently and ,35 40 pmol/L indicates B12 deficiency. However holotranscobalamin is more expensive to analyze, requires serum, and in population groups estimates a similar prevalence of deficiency to that defined using plasma B12 (Shahab-Ferdows et al., 2012), except in pregnant women

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in whom plasma B12 is normally lower but holotranscobalamin is not. Elevated plasma homocysteine is not a specific indicator of B12 status as it is also elevated by deficiencies of folate, riboflavin, or vitamin B6. However, it will be lowered by B12 fortification or supplementation in population groups who are B12-depleted. Commonly, estimates of the prevalence of depletion or deficiency differ with each indicator, making interpretation of the actual status situation difficult. A recentlyproposed solution to this challenge is to combine these indicators into a new indicator called “combined B12” or cB12 (Fedosov et al., 2015; Fedosov, 2013). This indicator can be calculated from a combination of two, three, or four of the common biomarkers and can also correct for the effect of folate deficiency on homocysteine. Cutpoints are , 2.5 for deficiency, 2.5 to 1.5 for possible deficiency, 1.5 to 0.5 for low B12, 0.5 to 1.5 for adequacy, and .1.5 for elevated. This model has the capacity to diagnose B12 depletion and deficiency more accurately and may be very useful in studies of the relationship of status to function (Brito et al., 2016). Due to the higher cost of using more than one marker of status, plasma B12 is likely to be the indicator of choice in most surveys, and is correlated with usual intakes of the vitamin.

26.5 REQUIREMENTS, BIOAVAILABILITY, AND SAFETY Estimated Average Requirements (EARs) for vitamin B12 range from 0.7 µg/day in early childhood to 2.0 µg/day for adults; EARs meet the requirements of 50% of population groups, and the percent of these groups failing to meet their EAR is used as an indicator of the adequacy of intake. The goal should be for about 95% of each lifestage group to consume their EAR, and fortification programs should be designed to fill the gap between usual intake and the EAR. Recommended Dietary Intakes meet the needs of 97.5% of the population and for B12 range from 0.9 to 2.4 µg/day during the life span. It is generally accepted that there is no health risk from high doses of vitamin B12. The Institute of Medicine concluded that there appear to be essentially no risks of adverse effects to the general population even at high intakes (Institute of Medicine, 2000). Moreover, an important fact is that while about 50% of a low oral dose (1 µg) will be absorbed, the efficiency of absorption falls off rapidly and strongly above this intake so that efficiency is 20% from 5 µg, 5% from 25 µg, and ,1% from $ 25 µg (Allen, 2009). In flour fortification programs the usual intake is likely to be 1 2 µg/day, so absorption from fortified flour is usually estimated at around 50%. However, as exemplified by a national flour fortification

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in Cameroon described below, absorption from fortified food is possibly .50%—it may be more efficiently absorbed from repeated small doses consumed in flour or breads. Nevertheless, there is no evidence that recommended levels of B12 addition to flour will result in any safety problems. Further evidence for safety of high levels of B12 comes from the well-established practice of providing intramuscular injections of  1000 µg, and/or the daily consumption of oral doses of 500 1000 µg by people with B12 depletion. Elderly, who are at greater risk of vitamin B12 deficiency because of their difficulty in releasing and absorbing the vitamin from food, are recommended to consume a higher proportion from fortified foods or supplements, from which the vitamin is better absorbed as it is in the free form (Institute of Medicine, 2000). In the United States the plasma vitamin B12 of adults increased by 34 pmol/L on average for each doubling of intake in the range of 0 10 µg/day—28, 24, and 19 pmol/ L with each doubling of intake from supplements, fortified cereal, and other foods, respectively (Tucker et al., 2000). An algorithm to estimate vitamin B12 bioavailability, which takes intake into consideration, was developed by the European Food Standards Agency (EFSA): log absorption 5 0.7694 3 log intake 2 0.9614 (EFSA NDA Panel Panel on Dietetic Products Nutrition and Allergies, 2015). This equation does not consider the potentially more efficient absorption that could occur when intake is spread across a day, which is the usual situation when the source is fortified foods. When estimating the contribution of foods that are very rich in vitamin B12 (such as liver) we know that absorption from such sources will be closer to 10% rather than 50%. Thus we have suggested dividing the total amount of B12 in those foods by five (Jones et al., 2007; Heyssel et al., 1966) as a more realistic estimate of how much the diet would actually contribute to the amount of the vitamin absorbed. These approaches to estimating absorption efficiency from intake need further testing.

flour, assuming consumption of 75 100 g flour per day, to provide approximately the Estimated Average Requirement. The recommended fortification level fell to 20, 10, and 8 µg/kg for usual flour intakes of 75 149, 150 300, and .300 g/day average consumption per capita. These estimates were not based on experience, and from the results of the more recent Cameroon national fortification program described below it appears that these recommended levels of fortification can likely be reduced. At the time of the WHO/FAO report the cost of adding B12 at 20 µg/kg flour was US$0.85/MT, adding 0.21% to the cost of the flour. Due to the very small amounts of B12 required, the recommendation is that the vitamin “should be purchased in a diluted form (0.1%) with 100% active particles (i.e., all spray-coated with vitamin B12) and diluted 1:15 to 1:25 in a premix.” If the premix also contains iron at a known ratio relative to vitamin B12, analyzing the iron content in the final premix and product will provide an easier way to obtain an approximate estimate of the vitamin B12 content. It is assumed that the average coefficient of variation (CV) of the amount of vitamin B12 in fortified flour is 6 35% of the mean value, so there should be at least the mean 6 45% (35% 3 1.28 for 80% of the expected values). So if the average content is 20 µg/kg the expected allowable range is 10 30 µg/kg. Both the minimum and the maximum values should be enforced. Loss of B12 during storage of the flour is assumed to be 10%, but to be negligible in bread because it is rapidly consumed. The extraction rate of the flour will not affect absorption of the added vitamin. Cyanocobalamin is the recommended form of B12 for use as a fortificant, because the cyanocobalamin has been stabilized by adding a cyanide molecule. This is also the form used in supplements. Although cyanocobalamin has a red color, adding up to 10,000 µg/100 g flour does not produce a noticeable pink or red color (Strouts, 1998). Adding up to 1000 µg/100 g does not affect dough handling, fermentation rates, or subjective ratings of the quality of the bread.

26.6 EXPERT CONSENSUS ON RECOMMENDED VITAMIN B12 FORTIFICATION

26.7 EXPERIENCE WITH VITAMIN B12 FORTIFICATION

In 2008 a group of experts met under the auspices of The Flour Fortification Initiative, the World Health Organization (WHO), the Food and Agriculture Organization (FAO), and other international organizations to update recommendations on the fortification of flour with six nutrients, including, for the first time, vitamin B12 (Flour Fortification Initiative, 2008; Allen et al., 2010). Where vitamin B12 fortification is implemented, the recommendation from this report was to add 40 µg/kg

In spite of the clear evidence that the prevalence of B12 deficiency is high in populations with a low intake of animal source foods, there has been little experience with vitamin B12 fortification, especially on a large scale. In a small efficacy trial in The Netherlands, elderly were provided with bread fortified with 9.6 µg B12/day or unfortified bread, for 12 weeks. Serum B12 increased by 49% in the fortified group and no participants were B12 deficient after fortification compared to 8% initially

Efficacy and Safety of Vitamin B12 Fortification Chapter | 26

(Winkels et al., 2008). While the fortification level was higher than would be used in large-scale programs, the study demonstrated that B12 “survives” being added to flour and baked. The most well-designed and documented national vitamin B12 fortification program took place in Cameroon. In 2009 a representative national survey was conducted to determine the baseline prevalence of micronutrient deficiencies prior to implementation of a mandatory wheat flour fortification program. Vitamin B12 deficiency or depletion occurred in 29% of women and children under five in the South, 40% in the poorer North, and 11% in the cities (Yaounde/Douala) (Shahab-Ferdows et al., 2015). The government decided that vitamin A should be added to vegetable oil, and wheat should be fortified with iron, zinc, folic acid, and vitamin B12. The level of addition of vitamin B12 was 0.04 mg/kg, the amount that was estimated to be effective in the Flour Fortification Initiative consensus (Flour Fortification Initiative, 2008). This estimate was based on meeting the EAR of 2 µg/day and assumed an average wheat flour intake of ,75 g/day, which was the mean intake by consumers and nonconsumers of flour in the national survey. In 2012 the impact of the fortification program was evaluated, a year after mandatory fortification was implemented (Engle-Stone et al., 2017). This follow-up survey was conducted in the same season as the first. It was also representative but conducted only in the urban stratum, in Yaounde/Douala, in the same 15 clusters per city that were surveyed at baseline. While the sampling design was well thought out (and described in the publication) it is likely that not all the individuals were the same as in the first survey. Moreover, Yaounde/Douala had a lower prevalence of most micronutrient deficiencies including vitamin B12, than in the more rural North and South strata. Participants were 10 women of reproductive age and 10 children 12 59 months of age per cluster, for a total of 309 women and 309 children. In addition 2 to 4 lactating women were recruited per cluster to obtain samples of breast milk. Plasma vitamin B12 was analyzed in the remaining 50% (n 5 195) of stored samples from the baseline survey and all of the post-fortification samples (n 5 287). Prefortification 13% of these urban women and 12% of children had low plasma B12. The fortification program increased plasma B12 by  50% and mean breast milk B12 concentrations doubled, to unusually high levels. Plasma and breast milk B12 concentrations were correlated with the usual frequency of fortified flour consumption in both women and children, a relationship not seen in the prefortification samples. Approximately 10 g wheat flour was collected from households, or from local vendors because only about 8% of households had wheat flour at home (it was consumed

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mainly as purchased breads). This flour was analyzed later to confirm that the level of B12 addition was correct; the mean value was 0.038 µg/100 g, close to the target level. Food intake surveys found that the median and 75th percentile of usual flour intake was 31 and 66 g/day, while for the women these values were 24 and 79 g/day— lower in both groups than estimated from the baseline national survey. There are several lessons to be learned from the results of this national program. The baseline prevalence of B12 deficiency was higher in the North and South but the postevaluation was not done in those regions; possibly the benefit would have been even greater there. The program proved that B12 fortification can produce a large increase in plasma and breast milk B12 within a year. It is possible that the fortification level was higher than necessary— lower levels should be tested before programs are implemented, to lower cost. The very large increase in breast milk B12 is intriguing, as it was greater than has been observed in maternal B12 supplementation trials (Duggan et al., 2014; Siddiqua et al., 2015). It is possible that more frequent intake of small doses of B12 in foods made from the fortified flour was more efficiently absorbed than a larger dose in a supplement. Many other foods are fortified with vitamin B12 including breakfast cereals, nondairy milks, and meat substitutes that are used by vegetarians. On food labels the “Daily Value” of 6 µg for B12 is based on the RDA in 1968. When one cup of breakfast cereal fortified with 4.8 µg B12 was provided to US adults, mean plasma B12 concentration increased from 296 to 354 pmol/L in 12 14 weeks (Tucker et al., 2004). In Chile a program for feeding elderly people provided 1 mg of vitamin B12 in a pill or in a fortified milk drink, or a placebo, for 18 months. There was slightly but significantly greater improvement in plasma B12 and cB12 after providing the pill for 4 months than providing the same amount of B12 in the milk drink, with little change in the intervention groups between 4 and 18 months (Brito et al., personal communication).

26.8 CONCLUSIONS The compelling arguments for vitamin B12 fortification include; the high global prevalence of deficiency across the life span, the substantial number of confirmed and probable adverse consequences of deficiency especially in the perinatal period, and the need to prevent exacerbation of deficiency by folic acid fortification. There are no known risks of adverse effects on health or on the quality of fortified foods, and the cost is affordable. Hopefully there will be more national fortification programs like the one in Cameroon—one is ongoing in Tanzania. As with all fortification programs the prevalence of deficiency at

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baseline should be assessed to determine the need for the intervention and to provide a benchmark comparison during monitoring of the program. A strong recommendation for such a program would be to assess at least two biomarkers of B12 status if possible; to monitor the effects on neural tube defect births, and on cognition and anemia in the elderly; to measure effects on pregnant and lactating women and their infants, and on concentrations of the vitamin in breast milk.

REFERENCES Allen, L.H., 2008. Causes of vitamin B12 and folate deficiency. Food Nutr. Bull. 29 (2 Suppl), S20 S34. discussion S5-7. Allen, L.H., 2009. How common is vitamin B-12 deficiency? Am. J. Clin. Nutr. 89 (2), S693 S696. Available from: https://doi.org/ 10.3945/ajcn.2008.26947A [pii] 10.3945/ajcn.2008.26947A. Allen, L.H., 2012. B vitamins in breast milk: relative importance of maternal status and intake, and effects on infant status and function. Adv. Nutr. 3 (3), 362 369. Available from: https://doi.org/10.3945/ an.111.001172. Allen, L.H., 2012. Pros and cons of increasing folic acid and vitamin B12 intake by fortification. Nestle Nutr. Inst. Workshop Ser. 70, 175 183. Available from: https://doi.org/10.1159/000337775. Allen, L.H., Rosenberg, I.H., Oakley, G.P., Omenn, G.S., 2010. Considering the case for vitamin B12 fortification of flour. Food Nutr. Bull. 31 (1 Suppl), S36 S46. Allen, L.H., Miller, J.W., de Groot, L., Rosenberg, I.H., Smith, D.A., Refsum, H., et al., 2017. Biomarkers of Nutrition for Development (BOND): vitamin B-12 review. J. Nutr. Brito, A., Verdugo, R., Hertrampf, E., Miller, J.W., Green, R., Fedosov, S.N., et al., 2016. Vitamin B-12 treatment of asymptomatic, deficient, elderly Chileans improves conductivity in myelinated peripheral nerves, but high serum folate impairs vitamin B-12 status response assessed by the combined indicator of vitamin B-12 status. Am. J. Clin. Nutr. 103 (1), 250 257. Available from: https://doi. org/10.3945/ajcn.115.116509. Carmel, R., 2013. Diagnosis and management of clinical and subclinical cobalamin deficiencies: why controversies persist in the age of sensitive metabolic testing. Biochimie 95 (5), 1047 1055. Available from: https://doi.org/10.1016/j.biochi.2013.02.008. Deegan, K.L., Jones, K.M., Zuleta, C., Ramirez-Zea, M., Lildballe, D.L., Nexo, E., et al., 2012. Breast milk vitamin B-12 concentrations in Guatemalan women are correlated with maternal but not infant vitamin B-12 status at 12 months postpartum. J. Nutr. 142 (1), 112 116. Available from: https://doi.org/10.3945/jn.111.143917. Dror, D.K., Allen, L.H., 2008. Effect of vitamin B12 deficiency on neurodevelopment in infants: current knowledge and possible mechanisms. Nutr. Rev. 66 (5), 250 255. Available from: https://doi.org/ 10.1111/j.1753-4887.2008.00031.x. Duggan, C., Srinivasan, K., Thomas, T., Samuel, T., Rajendran, R., Muthayya, S., et al., 2014. Vitamin B-12 supplementation during pregnancy and early lactation increases maternal, breast milk, and infant measures of vitamin B-12 status. J. Nutr. 144 (5), 758 764. Available from: https://doi.org/10.3945/jn.113.187278.

EFSA NDA Panel (EFSA Panel on Dietetic Products Nutrition and Allergies, 2015. Scientific opinion on dietary reference values for cobalamin (vitamin B12). EFSA J. 13, 4150 4213. Engle-Stone, R., Nankap, M., Ndjebayi, A.O., Allen, L.H., ShahabFerdows, S., Hampel, D., et al., 2017. Iron, zinc, folate, and vitamin B-12 status increased among women and children in Yaounde and Douala, Cameroon, 1 year after introducing fortified wheat flour. J. Nutr. 147 (7), 1426 1436. Available from: https://doi.org/10.3945/ jn.116.245076. Fedosov, S.N., 2013. Biochemical markers of vitamin B12 deficiency combined in one diagnostic parameter: the age-dependence and association with cognitive function and blood hemoglobin. Clin. Chim. Acta 422, 47 53. Available from: https://doi.org/10.1016/j.cca.2013.04.002. Fedosov, S.N., Brito, A., Miller, J.W., Green, R., Allen, L.H., 2015. Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cutpoints. Clin. Chem. Lab. Med. Available from: https://doi.org/ 10.1515/cclm-2014-0818. Flour Fortification Initiative, 2008. Summary Report. Second Technical Workshop on Flour Fortification: Practical Recommendations for National Application. Atlanta, GA. Green, R., Allen, L.H., Bjorke-Monsen, A.L., Brito, A., Gueant, J.L., Miller, J.W., et al., 2017. Vitamin B12 deficiency. Nat. Rev. Dis. Primers 3, 17040. Available from: https://doi.org/10.1038/ nrdp.2017.40. Herrmann, W., Schorr, H., Obeid, R., Geisel, J., 2003. Vitamin B-12 status, particularly holotranscobalamin II and methylmalonic acid concentrations, and hyperhomocysteinemia in vegetarians. Am. J. Clin. Nutr. 78 (1), 131 136. Heyssel, R.M., Bozian, R.C., Darby, W.J., Bell, M.C., 1966. Vitamin B12 turnover in man. The assimilation of vitamin B12 from natural foodstuff by man and estimates of minimal daily dietary requirements. Am. J. Clin. Nutr. 18 (3), 176 184. Institute of Medicine, 2000. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. National Academies Press, Washington, D.C. Jones, K.M., Ramirez-Zea, M., Zuleta, C., Allen, L.H., 2007. Prevalent vitamin B-12 deficiency in twelve-month-old Guatemalan infants is predicted by maternal B-12 deficiency and infant diet. J. Nutr. 137 (5), 1307 1313. Miller, J.W., Garrod, M.G., Allen, L.H., Haan, M.N., Green, R., 2009. Metabolic evidence of vitamin B-12 deficiency, including high homocysteine and methylmalonic acid and low holotranscobalamin, is more pronounced in older adults with elevated plasma folate. Am. J. Clin. Nutr. 90 (6), 1586 1592. Available from: https://doi.org/ 10.3945/ajcn.2009.27514. Morris, M.S., Jacques, P.F., Rosenberg, I.H., Selhub, J., 2007. Folate and vitamin B-12 status in relation to anemia, macrocytosis, and cognitive impairment in older Americans in the age of folic acid fortification. Am. J. Clin. Nutr. 85 (1), 193 200. Selhub, J., Paul, L., 2011. Folic acid fortification: why not vitamin B12 also? Biofactors 37 (4), 269 271. Available from: https://doi.org/ 10.1002/biof.173. Selhub, J., Morris, M.S., Jacques, P.F., 2007. In vitamin B12 deficiency, higher serum folate is associated with increased total homocysteine and methylmalonic acid concentrations. Proc. Natl. Acad. Sci. U.S.A.

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104 (50), 19995 20000. Available from: https://doi.org/10.1073/ pnas.0709487104. Shahab-Ferdows, S., Anaya-Loyola, M.A., Vergara-Castaneda, H., Rosado, J.L., Keyes, W.R., Newman, J.W., et al., 2012. Vitamin B12 supplementation of rural Mexican women changes biochemical vitamin B-12 status indicators but does not affect hematology or a bone turnover marker. J. Nutr. 142 (10), 1881 1887. Available from: https://doi.org/10.3945/jn.112.165712. Shahab-Ferdows, S., Engle-Stone, R., Hampel, D., Ndjebayi, A.O., Nankap, M., Brown, K.H., et al., 2015. Regional, socioeconomic, and dietary risk factors for vitamin B-12 deficiency differ from those for folate deficiency in Cameroonian women and children. J. Nutr. 145 (11), 2587 2595. Available from: https://doi.org/10.3945/ jn.115.210195. Siddiqua, T.J., Allen, L.H., Raqib, R., Ahmed, T., 2014. Vitamin B12 deficiency in pregnancy and lactation: Is there a need for pre and post-natal supplementation? J. Nutr. Disorders Ther. 4, 142. Available from: https://doi.org/10.4172/2161-0509.1000142. Siddiqua, T.J., Ahmad, S.M., Ahsan, K.B., Rashid, M., Roy, A., Rahman, S.M., et al., 2015. Vitamin B12 supplementation during pregnancy and postpartum improves B12 status of both mothers and infants but vaccine response in mothers only: a randomized clinical trial in Bangladesh. Eur. J. Nutr. Available from: https://doi.org/ 10.1007/s00394-015-0845-x. Smith, A.D., 2007. Folic acid fortification: the good, the bad, and the puzzle of vitamin B-12. Am. J. Clin. Nutr. 85 (1), 3 5. Smith, A.D., Refsum, H., 2009. Vitamin B-12 and cognition in the elderly. Am. J. Clin. Nutr. 89 (2), S707 S711. Available from: https://doi.org/10.3945/ajcn.2008.26947D.

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Smith, A.D., Smith, S.M., de Jager, C.A., Whitbread, P., Johnston, C., Agacinski, G., et al., 2010. Homocysteine-lowering by B vitamins slows the rate of accelerated brain atrophy in mild cognitive impairment: a randomized controlled trial. PLoS One 5 (9), e12244. Available from: https://doi.org/10.1371/journal.pone.0012244. Strand, T.A., Taneja, S., Ueland, P.M., Refsum, H., Bahl, R., Schneede, J., et al., 2013. Cobalamin and folate status predicts mental development scores in North Indian children 12-18 mo of age. Am. J. Clin. Nutr. 97 (2), 310 317. Available from: https://doi.org/10.3945/ ajcn.111.032268. Strouts, B.L., 1998. Fortifying with vitamin B12 does not affect dough handling or bread color. Available at: http://www.sph.emory.edu/ wheatflour/inpractice.php. Tucker, K.L., Rich, S., Rosenberg, I., Jacques, P., Dallal, G., Wilson, P. W., et al., 2000. Plasma vitamin B-12 concentrations relate to intake source in the Framingham Offspring study. Am. J. Clin. Nutr. 71 (2), 514 522. Tucker, K.L., Olson, B., Bakun, P., Dallal, G.E., Selhub, J., Rosenberg, I.H., 2004. Breakfast cereal fortified with folic acid, vitamin B-6, and vitamin B-12 increases vitamin concentrations and reduces homocysteine concentrations: a randomized trial. Am. J. Clin. Nutr. 79 (5), 805 811. Wang, Z.P., Shang, X.X., Zhao, Z.T., 2012. Low maternal vitamin B(12) is a risk factor for neural tube defects: a meta-analysis. J. Matl. Fetal. Neonat. Med. 25 (4), 389 394. Available from: https://doi. org/10.3109/14767058.2011.580800. Winkels, R.M., Brouwer, L.A., Clarke, R., Katan, M.B., Verhoef, P., 2008. Bread cofortified with folic acid and vitamin B-12 improves the folate and vitamin B-12 status of healthy older people: a randomized, controlled trial. Am. J. Clin. Nutr. 88, 348 355.