More than 20 years after the publication of the breakthrough study that revolutionized supplementation policies worldwide, the Medical Research Council Vitamin Study (1), today it is widely recognized that folates deficiency during the periconceptional period can contribute to the development of Neural Tube Defects (NTDs) in the fetus or even affect/impact the newborn with incapacitant conditions such as spina bifida, anencephalia or even heart defects (2, 3). In addition, the availability of the omega-3 long-chain polyunsaturated fatty acid docosahexaenoic, also needed for normal brain development, might be limited by an impaired one-carbon metabolism (4) for which folates, vitamins B₁₂ and B₆ are key nutritional regulators. Vitamin B₆ and folates are closely working toward maintenance of essential developmental and adult processes (5). In fact, vitamin B₆ active form, pyridoxal 5′-phosphate, regulate folates levels and it is a necessary cofactor for enzymes involved in the folates metabolism (Figure 1) (6, 7). Therefore, vitamin B₆, folates, and vitamin B₁₂ deficits during child growth might additionally compromise brain development and cognitive function in the future (8). Moreover, the American Academy of Pediatrics has issued a statement advocating a focus on key nutrients for brain development, such as folic acid, vitamin B₆ and B₁₂, among others (9).

Folates is the generic term that encompasses several vitamers with the activity of pteroylglutamic acid or vitamin B₉ (10), that plays a crucial role in the metabolism of the developing child. Its functions include red blood cell formation and DNA synthesis through the one-carbon cycle (11). Folic acid (FA) is the synthetic form that has a higher bioavailability and which is commonly used in dietetic supplements and fortified food products such as wheat flour (e.g., mandatory fortification) or breakfast cereals (e.g., mainly, voluntary fortification) (12). Like folates, vitamin B₆ encompasses a neuroprotective role being a cofactor for glutathione and decreasing the brain’s susceptibility to oxidative stress-based damage (13) and its deficiency contributes to neurodegeneration (14). Vitamin B₁₂ or cyanocobalamin is also involved in red blood cell formation (11) but while folic acid is effective in the treatment of pernicious and other macrocytic anemias (15), vitamin B₁₂ can prevent megaloblastic anemia (16). The interconnection of these vitamins within the one-carbon cycle has been the subject of several studies in the past and still at present, although there is great controversy regarding populations at risk for their deficiency, of which infants are a crucial group to follow up. On the other hand, a strong reason for concern amongst public health authorities about excessive FA intakes has been unmetabolized FA in non-target groups as it was shown that it could accelerate or promote cancer, specifically colorectal (17). Thus, the European Union has not implemented mandatory fortification of any food product and only voluntary fortification takes place. Previous research by Samaniego-Vaesken et al. (18) described the typology and level of fortification available in the Spanish market: most products being ready-to eat breakfast cereals and confectionery products (usually high in added sugars and salt). However, other products such as FA fortified milk and beverages were also frequent. Unfortunately, consumption of these products remains unknown as we lack updated information of fortified food intakes by the Spanish population, including toddlers and children. A study by Egan et al. (19) in Ireland (a voluntary fortification market very similar to the Spanish one in terms of types of products and levels of fortification) showed that voluntary FA fortification levels of food products in Ireland were declining since 2014, and that this might pose implications for FA intakes.

Paradoxically, up to date we did not have a representative sample of the pediatric population like the one provided by the EsNuPi study [Estudio Nutricional en Población Infantil Española (Nutritional Study in Spanish Pediatric Population)] performed in Spanish children aged one to <10 years old (20) in order to update and improve the knowledge on micronutrient intakes related to the methionine cycle.

The novelty of the present work relies on data of micronutrients related to the methionine cycle that has not been previously published elsewhere and does not contain excessive overlap with previously published data from the ESNUPI study. Intakes of these vitamins have not been published in a sample of this age range (one to <10 years old) which is representative of the Spanish population.

Therefore, there is an urgent need for a better and more updated knowledge of the intakes of vitamins related to the methionine cycle in the Spanish pediatric population in order to prevent and/or delay the adverse effects derived from inadequate intakes in this stage of life. In fact, in the present study our objective was to assess folates intakes alongside vitamins B₆ and B₁₂ and to describe their major dietary contributors as well as to study the differences between infants who usually consume adapted milks and those consuming all types of milk.

A total of 1,448 children between the ages of 1 to <10 years (49.7% girls and 50.3% boys) completed the study. Their anthropometric, sociodemographic, and physical activity characteristics have been previously published (24, 28).

It is acknowledged that poor nutritional status, especially suboptimal status of folates, and vitamins B₆ and B₁₂ can affect brain function and cognitive performance (29), namely by perturbing one-carbon metabolism, and this can take place in early as well as in later life (30). Children of school age need adequate cognition to obtain higher academic performance (31). As a matter of fact, cognitive processes are substantially related with brain function, physiology and structure (32) and, therefore, its development can be highly dependent on nutrition, physical activity, and social and economic status. For this reason, we studied folates together with vitamins B₆ and B₁₂, to find their intakes and major dietary contributors and also to describe the differences between infants at school age who usually consume adapted milks and those consuming all types of milk. In fact, at the light of the results from the present study, we observed a suitable intake of vitamins B₆ and B₁₂ but not for folates amongst Spanish children. In turn, we observed that children consuming adapted milk products had higher folates compliance.

Back in 2008, comprehensive reviews published by Black (8) and Molloy (33) examined the mechanisms linking folates and vitamin B₁₂ deficiency to abnormal behaviors and development in infants (disruptions to myelination and inflammatory processes), as well as the effects resulting from these deficiencies. Prevention of folates and vitamin B₁₂ deficiencies is pivotal to this age group and should mostly rely on dietary sources as supplementation is only left to specific situations such as strict vegetarian or vegan children who should supplement B₁₂ (34). Vitamin B₁₂ sources are almost exclusively obtained from the animal kingdom and, therefore, these groups might develop deficiencies if not adequately supplemented. Indeed, a child who is deficient in vitamin B₁₂, will continue to suffer from deficiency symptoms if it is not diagnosed early in infancy. Therefore, school-aged children with B₁₂ deficiency could be affected by altered motor development, cognitive disorders, and speech and language skills impairment (35). Folates which play a key role in the metabolism of the developing child (11) and vitamin B₁₂ deficiency could result in irreversible damage, such as growth stunting (36). In fact, there is also a growing interest in comparing breast and formula feeding in early infancy and their impact later in life, particularly concerning developing diseases such as obesity, hypertension, hyperlipidemia, and diabetes mellitus (37). It is important to highlight that breast milk and/or infant formula are the only sources of nutrition during early infancy and should provide suitable quantities of energy, water, and all essential nutrients (38).

The magnitude of the folate inadequacy in this study in the SRS group was 27%. It was higher than in the study conducted by Serra-Majem et al. (39), in which when assessing the percentage of population predicted to have inadequate intakes of folates using a probability approach, boys 2–5 years had 2.5% and those 6–9 years had 1.7%; however, girls 2–5 years showed 10.1% and those 6–9 years just 2.1%, and the National Dietary Survey on the Child and Adolescent Population in Spain (ENALIA) (40) their estimates showed that in general, water-soluble vitamin intakes were adequate but in the case of folate >50% of the participants older than 9 years had intakes below the specific sex-age estimated average requirement (EAR). Conversely, the degree of folate inadequacy in the children consuming adapted milk had a higher level of compliance with folates recommendations (93.3%) than those in the reference group, except from early ages (1 to <3 years), who had a high level of compliance with recommendations regardless of the type of milk consumed. Nevertheless, it is important to note that dietary folates intakes amongst infants have been previously assessed in Spain, but not specifically targeting the groups studied within the EsNuPI Study (1 to <10 years), and therefore comparison remains limited (39, 41). In addition, the difference could be related to the difference in the use of different methodologies. Nevertheless, data on dietary folate intake in school-age children at a European level show that there are large proportions of this population that do not meet folate recommendations. Indeed, it is striking that a recent review of nutrient intakes from national dietary surveys of 21 surveys in 18 countries in three regions: two of five Northern European countries (Denmark and Norway); 10 out of 17 Western European countries (Germany, Austria, Belgium, Spain, France, Ireland, Italy, the Netherlands, Portugal and the United Kingdom) and six out of 31 Central and Eastern European countries (Bulgaria, Cyprus, Slovenia, Estonia, Latvia and Turkey), the only age group over 3 years with an adequate total folate intake were Irish children aged 13–14 (42). It has earlier been found that an increase of 10% in folate intake from food could result in a 6% increase in serum folate (43). Therefore, nutritional policy-making bodies should consider the need for fortification of foods with folic acid (FA) targeted to this age segment, review the RDIs and formulate specific recommendations to ensure that school-aged children have adequate intake of this micronutrient. However, these strategies will require careful monitoring to ensure effectiveness. Since, Lewis et al. (44) reported that after fortification in the USA, 26% of this population exceeded the tolerable upper limit. Likewise, Pfeiffer et al. (45) estimated that 43% of children up to 5 years of age had an elevated serum folate concentration. Furthermore, in Spain, we have shown that excesses are a common practice in the fortification of FA in breakfast cereals and dairy products, since total folate values were clearly higher than those declared by manufacturers in most cases (18, 46).

This study found that the prevalence of adequacy for vitamin B₁₂, among school-aged children in Spain, based on the European PRI was highly compliant by all population groups (>95% compliance), being only significantly higher in the adapted milk group (AMS) in children aged 1 to 3 years vs. the SRS group, which is consistent with findings from other national or international dietary surveys of children where vitamin B₁₂ intakes have also met the recommended nutrient intakes (RNI) for vitamin B₁₂ at school age (39, 41, 42). The high vitamin B₁₂ adequacy noted in Spanish children may be explained by the dietary patterns followed by the Spanish population, which nowadays includes a high intake of animal products, increasingly distant from the traditional Mediterranean diet (47, 48).

Furthermore, a high proportion of this population group have adequate intakes of vitamin B₆. This is consistent with previous Spanish study, which have showed mean intakes of 1.4 and 1.6 mg/day for vitamin B₆ in males (2–5 years and 6–9 years, respectively) and female had intake for this vitamin of 1.3 and 1.5 mg/day (2–5 years and 6–9 years, respectively) (39). More recently, in the ANIBES study (41) the youngest age-groups evaluated were those 9–12 years, so only a limited comparison could be undertaken. In addition, 89.7% of children in the ANIBES met the European recommendations for vitamin B₆ (49).

Comprehensive information on food sources is key for understanding the strengths/weaknesses as well as the quality of the diet of children from the Spanish population. The major contributors of vitamin B₆ for children from our study were milk and dairy products, followed by fruits and meat and meat products. Also, it is noteworthy that the contribution of folates from milk and dairy products in the AMS was significantly higher than in the SRS. Indeed, although milk and dairy products are the main sources of macro-and micronutrients and contribute to the general quality of the diet in both children and adults (50–52), it appears that the infant formulas, follow-on milk formulas, or toddler’s milk formulas consumption may contribute more effectively to achieving the PRIs in the infant population. In addition, in the present study the AMS also showed milk and dairy products as the major B₁₂ contributors but conversely, egg products were the second group as compared to the SRS were meat and meat products. In fact, it is worth remembering that restrictive diets such as vegetarian or vegan encompass a high risk of inadequate intake owing to the exclusion and/or limitation of foods of animal origin. Even more importantly in this stage of exponential growth and development, vitamin B₁₂ deficiency amongst these key population groups has to be taken under consideration by public health authorities (53). It has been estimated that in Europe, the prevalence of vegetarian diets ranges from 1.2 to 1.5% of the population in Portugal and Spain, to about 7% in the United Kingdom and 10% in Germany, although the proportion of vegans is substantially lower (1–3%) all over Europe (54). No specific data are available for infants and children, nonetheless, it is acknowledged that vitamin B₁₂ status can be compromised if supplementation is not provided for these children under a strictly vegans diets.

Our data can be compared with those acquired in other European studies in which the proportion of vitamin B₁₂ and folates obtained from milk and dairy products was significant. For example, milk and dairy products were the major contributors to Greek school children aged 9–13 years (55). In the multi-ethnic population of the Head Start Mothers study (USA), high milk/low sweetened beverage intakes were associated with higher mean intakes of numerous vitamins and minerals, including folates and B₆ (56). In Australian preschool children, one-carbon metabolism nutrients (methionine, folates, vitamins B₂, B₆, and B₁₂) intake was also found highest from dairy products (57).

It is worth underlining and something worrisome that according to our results major folate sources, like vegetables and green-leaf as well as legumes are scarce. Indeed, for all age groups, milk and dairy products was the main folate contributor. Noteworthy the SRS had higher vegetable consumption that the AMS, specially at younger ages. However, in both groups fruit intake was similar.

The results showed that higher compliance with folates recommendations was achieved by children consuming adapted milk products. Finally, across all population groups there was a high compliance with B₁₂ and vitamin B₆ PRI (>95%) based on the European recommendations, being significantly higher in the adapted milk group (AMS) in children aged 1–3 years vs. the SRS group for vitamin B₁₂. Likewise, the prevalence of adequacy for vitamin B₆ estimated by the EFSA criteria was significantly higher in AMS, independently of age. In conclusion, children would largely benefit from healthier dietary patterns increasing the consumption of milk and dairy products, since these may contribute significantly to these vitamin requirements of the infant population. Nevertheless, there is a great need to improve young children’s diet by including high folate sources that contribute to an adequate intake of this vitamin.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by University of Granada Ethical Committee (No. 659/CEIH/2018), and registered in ClinicalTrials.gov (Unique Protocol ID: FF01/2019). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

GV-M and ÁG: funding acquisition, project administration, and supervision. PR-A, MS-M, ÁH-R, GV-M, and ÁG: investigation. MS-V, TP, ÁG, and GV-M: methodology. TP and MS-V: writing—original draft. TP, MS-V, PR-A, MS-M, ÁH-R, ÁG, and GV-M: writing—review and editing. All authors have read and agreed to the published version of the manuscript.