Association between vitamin B₁₂ intake and EURRECA's prioritized biomarkers of vitamin B₁₂ in young populations: a systematic review

Abstract

Objective

To review evidence on the associations between vitamin B₁₂ intake and its biomarkers, vitamin B₁₂ intake and its functional health outcomes, and vitamin B₁₂ biomarkers and functional health outcomes.

Design

A systematic review was conducted by searching electronic databases, until January 2012, using a standardized strategy developed in the EURRECA network. Relevant articles were screened and sorted based on title and abstract, then based on full text, and finally included if they met inclusion criteria. A total of sixteen articles were included in the review.

Setting

Articles covered four continents: America (n 4), Europe (n 8), Africa (n 1) and Asia (n 3).

Subjects

Population groups included healthy infants, children and adolescents, and pregnant and lactating women.

Results

From the total number of 5815 papers retrieved from the initial search, only sixteen were eligible according to the inclusion criteria: five for infants, five for children and adolescents, and six for pregnant and lactating women.

Conclusions

Only one main conclusion could be extracted from this scarce number of references: a positive association between vitamin B₁₂ intake and serum vitamin B₁₂ in the infant group. Other associations were not reported in the eligible papers or the results were not provided in a consistent manner. The low number of papers that could be included in our systematic review is probably due to the attention that is currently given to research on vitamin B₁₂ in elderly people. Our observations in the current systematic review justify the idea of performing well-designed studies on vitamin B₁₂ in young populations.

Nutrition plays an important role in the programming of health across the lifespan, especially during the earliest periods, because of short- and long-term consequences in the absence of appropriate nutrition⁽¹⁾. There are biological substances which keep homeostasis to prevent adverse health outcomes like vitamin B₁₂. In recent years, only a few studies have focused on the relationship between low vitamin B₁₂ intake and cognitive function, megaloblastic anaemia or growth in young populations.

Across Europe, current reference values for vitamin B₁₂ intake vary for infants from 0·3–0·5 to 1·5 μg/d depending on whether they are 3 or 9 months old, respectively⁽²⁾, from 0·8 to 3·0 μg/d for children and adolescents⁽³⁾ and from 1·5 to 4·0 μg/d for pregnant and lactating women^{(4, 5)}. The range of ages, values and terminology used for recommendations differ between European countries. However, the underlying concepts could be equivalent to: the RDA (Recommended Dietary Allowance, which is the daily dietary intake level of a nutrient considered sufficient to meet the requirements of nearly all (97–98 %) healthy individuals in each life stage and gender group), the AI (Adequate Intake, which is an estimation of the lowest intake level that seems sufficient for almost all people in a group) and the acceptable range (which is defined as the range of intakes high enough to avoid deficiency and low enough to avoid toxic effects). For vulnerable population groups such as those represented herein, nutrient requirements are generally obtained from data extrapolated from the adult ANR (Average Nutrient Requirement, which is the estimated average or median requirement of a specific nutrient in a population)⁽⁶⁾.

In Western countries, the dietary intake of vitamin B₁₂ among children, adolescents and adults is usually higher than the average requirement for vitamin B₁₂. For instance, the Spanish study EnKid showed that the 2–24-year-old population had a mean daily vitamin B₁₂ intake of 8·2 μg (males) and 6·8 μg (females)⁽⁷⁾. However, data from the Framingham Offspring Study suggest that suboptimal vitamin B₁₂ status occurs at intakes exceeding the recommended intakes⁽⁸⁾ and raise the question of whether the current recommended intakes for vitamin B₁₂ are adequate to promote a normal vitamin B₁₂ status⁽⁹⁾ and influence the occurrence of several health outcomes^{(8, 10–12)}.

The preferred approach to define the requirement takes into account the level of intake at which functioning is optimal. This implies that both preventing deficiencies as well as reducing the risk of developing other chronic disorders have to be taken into account^{(13, 14)}.

In order to provide up-to-date and evidence-based micronutrient reference values across Europe, it is important to assess the micronutrient status for different population groups⁽¹⁵⁾ through its preferred biomarkers or functional health outcomes⁽¹⁶⁾. The use of a biomarker that reflects changes in micronutrient status can facilitate the understanding of the relationships between dietary micronutrient intake and status or health outcomes (Fig. 1). The best tools to provide this information are dose–response and repletion–depletion studies, but they are rarely carried out.

Fig. 1 Intake–status–health relationships relevant for deriving reference values: 1 = intake–health relationship; 2 = intake–status relationship; 3 = status–health relationship

The aim of the present paper is to systematically review dose–response evidence from randomized controlled trials (RCT), prospective cohort and cross-sectional studies on the association of vitamin B₁₂ with its main biomarkers, and also with its main health outcomes in infants, children, adolescents and pregnant and lactating women. The ultimate goal would be to provide micronutrient reference intake values for vitamin B₁₂ in the aforementioned population groups.

Methods

The current systematic review on vitamin B₁₂ in young populations and pregnant and lactating women was performed within the framework of EURRECA (www.eurreca.org) and has focused on one of the prioritized relationships set by the network⁽¹⁷⁾ as illustrated in Fig. 1.

Search methods for identification of studies

To find the search strategy terms and the criteria for exclusion/inclusion papers, data on vitamin B₁₂⁽¹⁷⁾ were first reviewed. A multiple-database searching in MEDLINE, Embase (both on Ovid) and the Cochrane Library CENTRAL was carried out until 17 February 2009. The general search strategy included terms on study designs in humans AND (intake or status) AND (vitamin B₁₂). The search terms included both MeSH terms and words to be found in the title or abstract. The initial search yielded 5815 references after exclusion of duplicates. Reference lists of six relevant review articles^(18–23) were checked also to identify potentially relevant references that were not yet collected. This search did not yield any other references.

In January 2012 the search was repeated to retrieve other possible relevant papers. This search retrieved 596 new papers.

Criteria for the consideration of studies

Studies had to fulfill the following criteria to be included in the review:

1. 1. Investigate the possible relationships between vitamin B₁₂ intake, its biomarker levels or the selected health outcomes, following the structure available in Fig. 1;

2. 2. Provide vitamin B₁₂ from supplements, fortified foods or natural dietary sources;

3. 3. Be observational studies (prospective cohort, nested case–control or cross-sectional, the latter for intake–status associations only) or intervention studies (only RCT);

4. 4. Be performed in human subjects from birth to 18 years or pregnant or lactating women;

5. 5. Include apparently healthy subjects.

Results on adults and the elderly in studying these relationships are reported elsewhere.

Accepted dietary assessment methods to include the paper were: (i) validated FFQ/dietary history; and (ii) 24 h recall/food records/diary measures for at least 2 d.

Serum/plasma vitamin B₁₂, methylmalonic acid (MMA) and holotranscobalamin (HoloTC)⁽²⁴⁾ were the biomarkers included as the most robust and sensitive biomarkers identified through earlier research activities in the EURRECA network^{(25, 26)}.

The health outcomes chosen were those most relevant for the population group (based on public health reports and the scientific literature, i.e. current evidence of a relationship and the number of preliminary search hits from online databases) and not recently and thoroughly covered by a similar review. Health outcomes differed between population groups:

1. 1. Neurodevelopment and megaloblastic anaemia for infants;

2. 2. Megaloblastic anaemia, growth and cognitive function for children and adolescents;

3. 3. Fetal malformations and fetal growth for fetuses;

4. 4. Megaloblastic anaemia and pre-eclampsia for mothers.

Collection of papers

The results of the searches were combined in EndNote XII (Thompson Reuters). References were screened based on title and abstract. They were then sorted by population group: (i) infants, (ii) children and adolescents and (iii) pregnant and lactating women; and by relationship following the analytical model: (i) intake–health (I-H), (ii) intake–status (I-S), (iii) status–health (S-H) and (iv) intake–status–health (I-S-H).

Selection of studies

Once papers were screened based on title and abstract and sorted by population group, those selected were again screened based on full text by obtaining them electronically, as photocopies or reprints, according to the predefined criteria. The reasons for exclusion and the name of the reviewer were registered in the EndNote library. One hundred and seventeen potentially relevant references were considered for inclusion based on full text review; characteristics of the 101 references excluded are shown in Table 1. Figure 2 shows the flowchart of the selection steps for the populations reviewed herein. If language expertise existed in the review team, articles written in languages other than English could be included.

Table 1 Characteristics of excluded studies

RCT, randomized controlled trial; S, status; H, health; I, intake.

Fig. 2 Selection of studies for the current systematic review

Data extraction

Data from papers identified as relevant were extracted to characterize studies and to facilitate meta-analysis. Data were entered into an Access database specifically developed for EURRECA.

Quality check controls

For alignment and quality control, at the start of each step two independent reviewers screened 10 % of the references in duplicate. Any discrepancies at this step were discussed before proceeding with the rest of the references.

Assessment of risk of bias in included studies

To exclude major sources of bias, internal validity of the relevant studies was assessed. The criteria used were adapted from the Cochrane Handbook⁽²⁷⁾. The criteria for RCT were based on: method of sequence generation and allocation; blinding; potential funding bias; number of participants at start; drop-outs and reasons for dropping out; dose check; dietary intake data reported; and similarity of most and least exposed groups at baseline. For longitudinal studies the criteria were based on: drop-outs adequate and outcome data complete; funding; lack of other potential threats to validity; control for confounders; and assessment of exposure adequacy. For cross-sectional studies the criteria were based on: funding; lack of other potential threats to validity, such as those related to the specific study design used or related to differences in baseline characteristics of participants; confounders; and assessment of exposure adequacy.

Results

The systematic search retrieved sixteen relevant papers. Table 2 summarizes the characteristics and results of these studies.

Table 2 Main characteristics of the studies selected in the systematic review by study population group

IUGR, intra-uterine growth retardation; FA, folic acid supplement intake; IM, intramuscular(ly); SES, socio-economic status; RCT, randomized controlled trial; HoloTC, holotranscobalamin; MMA, methylmalonic acid; IQR, interquartile range; sem, standard error of measurement; SGA, small-for-gestational age; AOR, adjusted odds ratio.

Infants

Two out of five selected papers were RCT^{(28, 29)} and three were observational studies (one cross-sectional⁽³⁰⁾ and two longitudinal studies^{(31, 32)}). In all these studies the association between intake and status (I-S) was reported, except for one longitudinal study⁽³²⁾. In both RCT, the intervention groups^{(28, 29)} received vitamin B₁₂ through intramuscular injection: once per month during the first 4 months (100 μg/month) in one study⁽²⁹⁾ and in the other⁽²⁸⁾ the injected amount was only once (400 μg). In the RCT from Worthington-White et al.⁽²⁹⁾, serum levels were significantly increased after the intervention (either with or without folate supplementation) at each point of the measurements. In that study, the dose–response association between injected vitamin B₁₂ and levels of biomarkers was not estimated.

In the RCT from Bjorke-Monsen et al.⁽²⁸⁾, the intervention was the strongest predictor of changes for all blood indices (regression coefficient = 183 for serum vitamin B₁₂ and regression coefficient = −0·70 for MMA). Four months after delivery, the median (range) of serum vitamin B₁₂ was 421 (291–497) pmol/l and 240 (162–337) pmol/l for the intervention and placebo groups, respectively; corresponding values for MMA were 0·2 (0·15–0·43) pmol/l and 0·51 (0·23–1·55) pmol/l.

In the Guatemalan cross-sectional study⁽³⁰⁾, mean intake of vitamin B₁₂ was 3·1 μg/d for mothers and 2·2 μg/d for infants at the age of 12 months and the accompanying mean (sd) plasma vitamin B₁₂ concentration in mothers and infants was 114·4 (9·2) g/l and 262·2 (163·5) pmol/l, respectively. The plasma vitamin B₁₂ concentrations of the infants were correlated with maternal concentrations and they were also positively associated with infant B₁₂ intake from complementary foods (r = 0·16, P < 0·0001).

In the longitudinal study by Hay et al.⁽³¹⁾, the results were divided between breast-fed (n 104) and non-breast-fed (n 115) infants: the mean intake of vitamin B₁₂ was 1·4 (95 % CI 1·3, 1·6) μg/d for breast-fed infants excluding the intake from breast milk and 2·4 (95 % CI 2·1, 2·6) μg/d for the non-breast-fed infants. In that study, the selected biomarkers were measured at the age of 12 months. Mean (95 % CI) serum vitamin B₁₂, HoloTC and MMA were 343 (319, 369) pmol/l, 54 (49, 60) pmol/l and 0·22 (0·20, 0·25) μmol/l, respectively, for breast-fed infants, and 397 (372, 424) pmol/l, 76 (70, 83) pmol/l and 0·20 (0·19, 0·22) μmol/l, respectively, for non-breast-fed infants. Infants who were breast-fed at the age of 12 months had significantly lower serum vitamin B₁₂ and HoloTC and higher MMA than those who were not breast-fed at the same age. In that study, total vitamin B₁₂ intake from complementary foods was positively associated with serum vitamin B₁₂ (r = 0·15 and P = 0·030) and HoloTC (r = 0·25 and P = 0·001).

The longitudinal study by Dagnelie et al.⁽³²⁾ was the only one studying the relationship between vitamin B₁₂ intake and health, specifically psychomotor development, in spite of the status also being stated in the paper. However, they were not related with intakes or health outcomes. The results were divided between infants following a specified macrobiotic diet and those following an omnivorous one. Mean vitamin B₁₂ intakes were significantly higher in the omnivorous group (2·9 (sd 1·3) μg/d) in comparison to the macrobiotic group (0·3 (sd 0·2) μg/d; P < 0·001). These differences could be also be shown in the scores obtained in the psychomotor development test in the areas of gross motor development (for which the mean difference in standard deviations between feeding groups was −0·48) and speech and language development (for which the mean difference in standard deviations between feeding groups was −0·42), with a P value of 0·04 and 0·03, respectively. Despite these differences in health outcomes obtained between feeding groups, the authors did not study an association between vitamin B₁₂ intakes and differences in scores in psychomotor tests; for this reason, these results cannot be attributed only to the obtained difference in vitamin B₁₂ intakes.

The results of the four studies evaluating the I-S relationship showed that the status of vitamin B₁₂ biomarkers is significantly and positively associated with vitamin B₁₂ consumption (ingested or injected). The strength of this association was stated in almost all of the studies, with the exception of one RCT⁽²⁹⁾ in which the regression coefficient was not given. The limited availability of I-H data in infants did not allow for drawing any conclusions.

Children and adolescents

For the children and adolescents group, we identified four cross-sectional studies^{(33–35, 37)} and one RCT⁽³⁶⁾. Two out of three cross-sectional studies were conducted with children^{(33, 37)}, one study⁽³⁴⁾ was carried out among adolescents and one⁽³⁵⁾ included both children and adolescents. In three cross-sectional studies^(33–35), vitamin B₁₂ intake and plasma vitamin B₁₂ was described. However, searching for an association between intake and status was not the purpose of the studies. Only in the study by Hay et al.⁽³⁷⁾, performed in Norwegian children, was vitamin B₁₂ intake shown to be significantly and positively associated (r = 0·21, P < 0·05) with HoloTC. In that study, serum vitamin B₁₂ and MMA were also measured; however, no association with them was found.

In the RCT by Gewa et al.⁽³⁶⁾, the targeted population group was children and the studied relationship was I-H. The authors discovered that children with a daily high intake of vitamin B₁₂ gained a significant 0·24 more points in the Digit Span-forward test (as part of the entire cognitive test) than others with a low intake level, considering intakes of vitamin B₁₂ predictors of the Digit Span-forward test.

Pregnant and lactating women

Regarding the pregnant and lactating women group, six prospective observational studies were included^(38–43). Four of them studied the relationship between status and health outcomes in the fetus (intra-uterine growth retardation (IUGR), small for gestational age (SGA) and growth in general)^{(38, 40, 42, 43)}. In Lindblad et al.'s study⁽⁴⁰⁾, the results suggested that in infants with normal birth weight, cord blood levels of vitamin B₁₂ were correlated with maternal levels of serum vitamin B₁₂. However these correlations were weaker when infants had IUGR. In the study by Baker et al.⁽³⁸⁾, serum vitamin B₁₂ levels in mothers were not associated with the risk of SGA infants. However, in Muthayya et al.'s study⁽⁴²⁾ women in the lowest tertile for serum vitamin B₁₂ concentration during each of the trimesters of pregnancy had significantly higher risk of delivering IUGR infants. In this last study, a correlation between vitamin B₁₂ intake and status was also reported in all three trimesters. In Takimoto et al.'s study⁽⁴³⁾, maternal vitamin B₁₂ status, assessed in the third trimester of the pregnancy, was not associated with gestational weight, weight, length or head circumference of infants at delivery or at 1 month after delivery. Two studies^{(39, 41)} described longitudinal changes in vitamin B₁₂ biomarkers through pregnancy, I-S being the main relationship examined. In one longitudinal study⁽³⁹⁾, vitamin B₁₂ intake in pregnant women was not associated with serum vitamin B₁₂. In the study by Morkbak et al.⁽⁴¹⁾, which is the only selected study on pregnant women, in spite of there being three different supplementation groups, as there were no significant differences between them, results were presented for all three groups together. No change was observed in serum vitamin B₁₂ throughout the study period, whereas a significant decrease was observed for HoloTC from baseline to the 9th month.

The observed I-S and S-H relationships were not consistent and further conclusions cannot be extracted.

Quality of included studies

Table 3 summarizes the method used to assess the quality of the included studies. Only three studies had a high risk of bias^{(29, 33, 36)}. Five studies had a moderate risk of bias^{(28, 31, 34, 35, 38)} and eight studies reflect low risk of bias^{(30, 32, 37, 39–43)}. The most repeated reason for risk of bias across the studies was an inadequate explanation about the drop-outs and an inadequate assessment of exposure (method to assess vitamin B₁₂ intakes).

Table 3 Assessment of methodological quality of included randomized controlled trials, longitudinal and cross-sectional studies

Discussion

From 5815 identified papers, only sixteen were suitable to be included in the review according to EURRECA's eligibility criteria. From these, five papers focused only on descriptions of vitamin B₁₂ intakes and biomarkers without any stated association. Because of the small number of eligible papers included in the review, only a few main conclusions can be drawn for the specific population groups studied.

Infants

In this population group, vitamin B₁₂ (ingested or injected) was significantly and positively associated with vitamin B₁₂ biomarkers. Serum vitamin B₁₂ was investigated in all four studies. The evidence, however, was not sufficient for HoloTC (only one study⁽³¹⁾) or MMA (two studies^{(28, 31)}, while in one study⁽³¹⁾, associations were not found for MMA).

In this population group, the two included interventions were performed through injection of vitamin B₁₂. Although injection could be a more reliable method of intervention, in general oral administration is better tolerated in the absence of neurological problems^{(44, 45)}. Moreover, it should be noted that exposure to vitamin B₁₂ via oral supplements or intramuscular injections is very different as e.g. bioavailability issues are different.

Children and adolescents

Among the four cross-sectional studies^{(33–35, 37)} included in this population group, the only available finding was the positive association between children's intake of vitamin B₁₂ and serum HoloTC in one of them. In that study, serum vitamin B₁₂ and MMA were also investigated without any obtained association. The other three cross-sectional studies did not look for any association.

In the RCT of Gewa et al.⁽³⁶⁾, it was demonstrated that higher vitamin B₁₂ intakes are associated with higher scores in one part of a cognitive test. However, only one study represents very limited data from which to extract a clear conclusion and in this respect, drawing conclusions may not be justified.

Pregnant and lactating women

Regarding the S-H relationships searched for in this group, as well as for I-S ones, no conclusions can be drawn due to the discrepancies in the results. One study⁽⁴²⁾ showed an association between status and fetal growth and three^{(38, 40, 43)} showed no association. On the other hand, due to the heterogeneity shown in results regarding I-S relationships in all five included studies, it is possible to conclude that intake of vitamin B₁₂ in pregnant and lactating women is not related to vitamin B₁₂ concentration in their blood. This fact can be derived from the vitamin B₁₂ gradient in the placenta, between the fetus and the mother. During pregnancy, vitamin B₁₂ had been noted to decrease in mothers but not its transport molecules. Such an observation of the placenta facilitating the transport of a critical nutrient (as occurs with vitamin B₁₂) for fetal growth and development when the mother is deficient is another revelation of how important the placenta is in maintaining the development of the fetus^{(46, 47)}. In the other four studies^(38–41) there were no significant or relevant associations present.

Use of biomarkers in studies

One of the currently open questions regarding vitamin B₁₂ is to determine the best biomarker to assess its status. In the present review, data were insufficient to draw conclusions about the effectiveness of serum HoloTC or MMA as a biomarker of vitamin B₁₂ status (only one study showed a positive association between vitamin B₁₂ intakes and HoloTC in children⁽³⁷⁾). However, MMA and HoloTC are more sensitive markers for vitamin B₁₂ deficiency than plasma vitamin B₁₂⁽⁴⁸⁾ by reflecting sudden changes in vitamin B₁₂ homeostasis, whereas plasma vitamin B₁₂ seems to reflect the accumulation of vitamin B₁₂⁽⁴⁹⁾. On the other hand, they are extremely variable in these periods of life, making difficult their interpretation⁽³¹⁾. Moreover, due to the ability of serum/plasma vitamin B₁₂ to describe the status of vitamin B₁₂ through time, without being influenced by punctual intake, serum/plasma vitamin B₁₂ is the most common biomarker to assess vitamin B₁₂ status.

Cognitive function

One of the constraints to the lack of data in the research on vitamin B₁₂ intake and cognitive function is that even detailed examinations are not sufficiently accurate to detect developmental delays in young infants. However, reports on short- and long-term neurological effects related to vitamin B₁₂ deficiency in young infants demonstrate the importance of an adequate vitamin B₁₂ status during the first months of life⁽²⁸⁾. Vitamin B₁₂ is also suggested to be related with neurocognitive function in school-aged children⁽⁵⁰⁾. In the present systematic review, two papers on this topic suggested this association (one in infants, the other in children). However, in the infants study⁽³²⁾, the differences in scores in psychomotor tests were associated with type of diet (macrobiotic or omnivorous) and not with intake of vitamin B₁₂ (however, the authors found significant differences in vitamin B₁₂ intakes between diet groups).

Megaloblastic anaemia

Although being selected as a relevant health outcome for infants and children and adolescents, no paper on megaloblastic anaemia was finally included. However, some bibliography has reported megaloblastic anaemia as a typical symptom of vitamin B₁₂ deficiency, usually as a consequence of previous maternal vitamin B₁₂ deficiency⁽⁵¹⁾. Absence of included studies investigating this outcome suggests the low quality of reporting of the available studies, which were mostly old case reports.

No studies were found for megaloblastic anaemia in pregnant and lactating women. The explanation for no revealed hits could be that the literature about megaloblastic anaemia in this vulnerable group is linked mostly to intake and status of folate rather than the intake and status of vitamin B₁₂⁽⁵²⁾.

Growth

Of four papers focusing on fetal/infant growth (SGA, IUGR or general growth) in pregnant and lactating women, as only one has shown a positive association, no clear conclusion can be extracted in this regard.

Fetal malformations

The literature reveals that neural tube defects are the most common fetal malformation linked to deficiency of vitamin B₁₂ in mothers⁽⁵³⁾. However, due to the strict inclusion criteria of the present systematic review, no studies on this topic were included.

Maternal pre-eclampsia

This health outcome was mentioned in only one of the longitudinal studies in the pregnant and lactating women group. However, there was no significant difference in vitamin B₁₂ status among mothers who suffered pre-eclampsia compared with mothers without pre-eclampsia⁽⁴⁰⁾. In another similar study, no significant differences were observed in both maternal and fetal serum vitamin B₁₂ between a severe pre-eclampsia group v. mild pre-eclampsia and control groups⁽⁵⁴⁾.

Conclusions

The current systematic review emphasizes a number of knowledge gaps in the field of vitamin B₁₂ research for young populations and pregnant and lactating women, derived from the scarcity and the low quality of available studies.

One of the reasons for this scarce literature on vitamin B₁₂ in young population groups could be that mild vitamin B₁₂ deficiency is more prevalent among elderly people in association with a number of chronic diseases⁽⁵⁵⁾.

There is also evidence that vitamin B₁₂ deficiency is uncommon in young populations, unless they belong to a vegan community, or live in a developing area, or have a congenital malabsorption syndrome⁽⁵⁶⁾. However, the prevalence in younger groups may be higher than formerly recognized⁽⁵⁷⁾.

RCT with enough power and varying doses of dietary intakes and duration of supplementation are required in order to establish vitamin B₁₂ recommendations for young populations. Further studies to correlate serum/plasma vitamin B₁₂, MMA and HoloTC and also to explore vitamin B₁₂ adequacy in young age groups are needed.