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BMC Pediatrics

, 19:334 | Cite as

Effects of dietary intervention on vitamin B12 status and cognitive level of 18-month-old toddlers in high-poverty areas: a cluster-randomized controlled trial

  • Xiaoyang Sheng
  • Junli Wang
  • Feng Li
  • Fengxiu Ouyang
  • Jingqiu MaEmail author
Open Access
Research article
Part of the following topical collections:
  1. Nutrition, diet, physical health and endocrinology

Abstract

Background

The local diet in high-poverty areas in China is mainly vegetarian, and children may be more vulnerable to vitamin B12 deficiency.

Objective

The aims of this study were to explore the vitamin B12 status of toddlers living in high-poverty areas of China and to observe the effects of different complementary foods on the vitamin B12 status and cognitive level of these toddlers.

Methods

The study was nested within a cluster-randomized controlled trial implemented in 60 administrative villages (clusters) of Xichou County in which infants aged 6 months old were randomized to receive 50 g/d of pork (meat group), an equi-caloric fortified cereal supplement (fortified cereal group) or local cereal supplement (local cereal group) for one year. At 18 months, a subsample of the 180 toddlers (60 from each group) was randomly tested for serum vitamin B12 and total homocysteine (tHcy) levels, and their neurodevelopment was evaluated.

Results

The median serum concentrations of vitamin B12 and tHcy were 360.0 pg/mL and 8.2 μmol/L, respectively, in children aged 18 months. Serum vitamin B12 concentrations less than 300 pg/mL were found in 62 (34.4%) children, and concentrations less than 200 pg/mL were found in 30 (16.7%) children. The median vitamin B12 concentration was significantly different among the three groups (P < 0.001). The highest vitamin B12 level was demonstrated in the fortified cereal group (509.5 pg/mL), followed by the meat group (338.0 pg/mL) and the local cereal group (241.0 pg/mL). Vitamin B12 concentration was positively correlated with the cognitive score (P < 0.001) and the fine motor score (P = 0.023) of the Bayley Scales of Infant Development, 3rd Edition (BSID III) screening test. Compared to the local cereal group, children in the meat group had higher cognitive scores (P < 0.05).

Conclusion

In poor rural areas of China, vitamin B12 deficiency in toddlers was common due to low dietary vitamin B12 intake. Fortified cereal and meat could help improve the vitamin B12 status of children and might improve their cognitive levels.

Trial registration

The larger trial in which this study was nested was registered at clinical trials.gov as NCT00726102. It was registered on July 31, 2008.

Keywords

Vitamin B12 Deficiency Toddler Complementary food Neurodevelopment 

Abbreviations

Hb

Hemoglobin

HcAZ

Head circumference-for-age z-score

LAZ

Length-for-age z-score

MCH

Mean corpuscular hemoglobin

MCHC

Mean corpuscular hemoglobin concentration

MCV

Mean corpuscular volume

MMA

Methylmalonic acid

tHcy

Total homocysteine

WAZ

Weight-for-age z-score

WLZ

Weight-for-length z-score

Background

Vitamin B12, also called cobalamin, is an essential water-soluble micronutrient found exclusively in animal-derived foods, such as meat, eggs, fish and milk, and it cannot be synthesized by the body. Vitamin B12 deficiency can cause hematological shortages, resulting in increased red cell mean corpuscular volume (MCV) and macrocytic anemia through the alteration of erythropoiesis [1]. Furthermore, vitamin B12 is necessary for the development and initial myelination of the central nervous system as well as for the maintenance of its normal function [2].

Vitamin B12 deficiency is a worldwide public health issue. People who consume a vegetarian diet or limit animal products may develop vitamin B12 deficiency. The prevalence of low Vitamin B12 status is high in low-income settings, especially in rapidly growing children with a high demand for vitamin B12. In North India, one-third of children aged 6 to 35 months had a plasma vitamin B12 concentration ≤ 200 pmol/L [3]. Recently, a cross-sectional household cluster survey revealed that 30.2% of infants and toddlers aged 6 to 23 months in two districts in Nepal had a vitamin B12 deficiency (serum vitamin B12 < 150 pmol/L) [4]. Another community-based, randomized, double-blind clinical trial in Nepal demonstrated that more than 50% of breastfed infants aged 6 to 11 months with a length-for-age z-score (LAZ) < − 1 have a vitamin B12 deficiency [5].

It is also known that vitamin B12 deficiency is one potential cause of adverse developmental outcomes. In recent years, neurodevelopment in children has been linked with vitamin B12 status in several studies. Recently, the vitamin B12 status of Nepalese infants showed positive associations with development and performance on social perception tasks and visuospatial abilities at 5 years of age [6]. Indian infants aged 12–18 months with a vitamin B12 deficiency presented with lower psychomotor and mental development scores compared with the scores of infants with higher vitamin B12 status [7].

To date, there have been no reports on the vitamin B12 status of infants and young children in China, especially in poor areas. Due to low socioeconomic status, the local diet is mainly vegetarian. Therefore, infants and toddlers may be more vulnerable to vitamin B12 deficiency. Beginning in March 2009, a large intervention trial was conducted in a poor rural area, Xichou County, located in Yunnan Province, China, in which the effect of meat was evaluated as the primary complementary food affecting the linear growth of toddlers between 6 and 18 months of age. In comparison, equi-caloric quantities of rice cereal or of micronutrient-fortified rice cereal were used as controls. Our substudy was a cross-sectional subsample nested within a large intervention trial.

In this substudy, our objectives were to determine 1) the levels of serum vitamin B12, total homocysteine (tHcy) and hemoglobin in toddlers at 18 months of age; 2) the relationship between cognition and motor development and vitamin B12 nutritional status of toddlers at 18 months of age; and 3) the efficacy of complementary foods, including micronutrient-fortified rice cereal and red meat, for improving vitamin B12 status.

Methods

Study design

The substudy was nested within a cluster-randomized, nonmasked, controlled efficacy trial conducted from March 2009 to December 2011 in a poor rural area, Xichou County, located in the Yunnan Province of China. Details of the study design have been described elsewhere [8, 9].

In the study areas, early complementary foods offered to infants are mainly various types of plant-based gruels made from cereal grains or starchy roots and tubers. Sixty administrative villages (clusters) in 9 districts in Xichou County were included in this study. Six-month-old infants were randomized to receive 50 g/d of pork (meat group), an equi-caloric micronutrient-fortified rice cereal-based supplement (fortified cereal group), or a local nonfortified rice cereal supplement (local cereal group) for one year [8, 9]. Approximately 20–30 infants were involved in each administrative village. Infants were identified at 3–5 months of age by their community doctors. Eligibility criteria for infants included term delivery without serious neonatal complications, absence of acute or chronic illness, healthy singleton status with birth weight > 2000 g, no metabolic or physical problems and being exclusively breastfed [8, 9].

Administering the intervention to participants and evaluating the compliance and morbidity of participants were performed by the specially trained community doctors in each of the small communities within each village. The fresh certified-safe lean pork was purchased weekly and was minced and accurately weighed into daily 50 g (80 kcal) aliquots containing 0.21 μg vitamin B12 and stored frozen until transported weekly to the district hospitals which were serving the meat group villages [8, 9]. The quantity of either micronutrient-fortified cereal (20 g, 80 kcal) or local cereal (20 g, 80 kcal) was designed to be equi-caloric to the daily supply of pork. The participants in the fortified cereal group received a commercial product (Nestle, fortified with vitamin B12, iron and zinc) and the vitamin B12 content in 20 g of the fortified cereal was 0.2 μg. Local rice cereal was made from a mixture of glutinous rice flour, white granulated sugar and honey, without vitamin B12 [8, 9]. Supplies of these control foods were provided weekly to the district hospitals in all participating villages, and the control foods were collected at these hospitals and distributed weekly by the participating community doctors [8, 9]. Participants in the meat group, fortified cereal group and local cereal group were encouraged to consume 50 g of red meat, 20 g of fortified rice cereal and 20 g of local rice cereal each day, respectively.

Furthermore, the participants were seen by the assessment team at the nearest hospital at baseline and at 3-month follow-up intervals. All anthropometric measurements, including length, weight, and head circumference at the age of 6 and 18 months, were measured using standardized procedures by the assessment team. The effect of supplementary food on linear growth and micronutrient status between 6 and 18 months of age was assessed. The operation manual for the study is shown in more detail in Additional file 1.

Participants

A total of 1465 infants completed the study at baseline (6 months), including 511 in the meat group, 465 in the fortified cereal group and 489 in the local cereal group. At 18 months, a total of 1316 children completed the study, including 461 in the meat group, 419 in the fortified cereal group and 436 in the local cereal group. Three children in the meat group and 15 children in the local cereal group did not provide blood samples. Finally, a total of 1298 children, without upper respiratory tract infection, diarrhea or other acute infections, provided blood samples. The flow of the participants through the study is shown in Fig. 1.
Fig. 1

Participant flow through the study

Blood sampling and biochemical assessment

Hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were measured in whole blood with an HC3000 auto-hematology analyzer on the day of blood sampling. Peripheral venous blood samples of 5 mL were collected by trained and experienced phlebotomists and put into a trace element-free, additive-free evacuated tube. Serum was divided into two aliquots and stored immediately at − 20 °C; then, they were transported frozen and stored at − 80 °C until serum vitamin B12 and tHcy analysis. The individuals who carried out the laboratory analyses were unaware of the participants’ group assignments.

Serum vitamin B12 was measured by an automatic ACCESS microparticle chemiluminescence immunoassay analyzer with a chemiluminescence immune assay (Beckman Coulter, Inc., USA). Serum tHcy was measured on a Hitachi 7600-120E automated biochemistry analyzer with enzymatic cycling assay (DiaSys Diagnostic Systems GmbH, Germany).

Subsample size

According to the vitamin B12 levels of children from poor areas reported in the literature [10, 11, 12], it is estimated that the average serum vitamin B12 concentration of children in this study would be 300 pg/mL in the meat group, 350 pg/mL in the fortified cereal group, and 250 pg/mL in the local cereal group, with the assumption of a standard deviation of 150 pg/mL. Based on the sample size calculation formula for a multisample study design [13], blood samples from 60 children in each group were required to determine significant differences among the three groups and to achieve a power of 90% at a 5% level of significance. A total of 180 blood samples were collected. Sixty was randomly selected from the meat group (cluster 1–20), 60 from the fortified cereal group (cluster 21–40) and 60 from the local cereal group (cluster 41–60) by two investigators who were not involved in the recruitment and data collection, with 3 blood samples from each cluster.

Neurocognitive testing

The cognitive scale and the fine motor and gross motor subtests of the Bayley Scales of Infant Development, 3rd Edition (BSID III) screening test were used to assess the infants’ development at 18 months of age [14]. The language domain was not performed because it was not applicable in the setting of Yunan, China. All children were assessed within a two-week window at approximately 18 months of age, and the assessments were administered either by a psychologist familiar with the test or by trained assistants/students. All assistants/students were graduate students majoring in pediatrics who received a two-month intensive training on the BSID III test in the Department of Children and Adolescents Health Care of Xin Hua Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Before the children were evaluated with the BSID III test, the evaluators had been living and working locally in rural Yunnan for several months. All evaluators were able to communicate with the participants and their parents in Mandarin and simple local dialects. Community sites were adapted and arranged to provide a standardized environment for the testing. When tests were administered, the mothers were present. If the child did not cooperate during the test, the evaluator would try again when the child was in a better mood. Each test administration took approximately 30 min. Double data entry was adopted, and the original paper copies of the data were retained for reverification.

Definitions

Xichou County in Yunnan Province has an average elevation of 1400 m above sea level, so anemia was defined as Hb < 115 g/L [9, 15].

According to the World Health Organization (WHO) [12, 16, 17], serum vitamin B12 concentrations of < 200 pg/mL, 200–300 pg/mL and > 300 pg/mL were used to classify individuals as deficient, marginally deficient, or adequate, respectively. However, stand-alone marker of serum vitamin B12 has been proven insufficient for the unequivocal diagnosis of vitamin B12 deficiency [18]. Therefore, a tHcy concentration of > 12 μmol/L was also used to obtain a correct diagnosis, based on the suggestions in the recent literature [1].

The weight-for-age z-score (WAZ), LAZ, weight-for-length z-score (WLZ) and head circumference-for-age z-score (HcAZ) were calculated according to the 2006 WHO Child Growth Standards using WHO Anthro 2011 software [19]. Underweight, stunting, and wasting were defined as WAZ < -2, LAZ < -2, and WLZ < -2, respectively.

Statistical analysis

All data were analyzed using descriptive and frequency statistics in SPSS 13.0 for Windows. The results are presented as the mean ± SD for normally distributed continuous variables or as the median (interquartile range, IQR) for data not normally distributed. Log transformations were used where residuals were skewed or exhibited nonconstant variance. Anthropometric variables, Hb level and subtest scores of the BSID III screening test were compared among groups by ANOVA. Not normally distributed data, including serum vitamin B12 and tHcy concentrations and maternal education duration, were compared after log transformations among groups by ANOVA. The LSD multiple comparison test was used for the post hoc test if the overall ANOVA had a P value less than 0.05. Chi-square tests were used for categorical variables. To address the clustering effect, the differences in serum vitamin B12 and tHcy concentrations (after log transformations) and cognitive score between the meat or fortified cereal groups and the local cereal group were analyzed using mixed-effect linear models, and the local cereal group was used as the reference. Outcomes were analyzed with villages as the random factor and group as the fixed factor. Comparisons were expressed as estimated differences with 95%CIs. Correlations between serum vitamin B12 and tHcy levels and the associations of vitamin B12 or tHcy levels with LAZ, WAZ, WLZ, HcAZ, Hb, MCV, MCH and MCHC were tested by Pearson correlation after log transformation. The associations between the subtest scores of the BSID III screening test (the cognitive score, the fine motor score or the gross motor score) and the serum vitamin B12 or tHcy concentrations were examined by a linear regression model on condition of the adjusting covariates (maternal education duration and birth weight) with “enter” as the regression method. All statistical tests were two-tailed and P values < 0.05 were considered statistically significant.

Results

Anthropometric outcomes of the subsample

A total of 180 blood samples, 60 from each group, were selected by a simple random sampling from 1298 blood samples stored at − 80 °C in Shanghai Key Laboratory of Children’s Environmental Health.

The mean age of toddlers in the meat group, fortified cereal group and local cereal group was 17.9 ± 0.1, 18.0 ± 0.2 and 17.9 ± 0.2 months, respectively. The percentages of males among the three groups were 51.7, 48.3 and 48.3%, respectively. The mean birth weights in the three groups were 3075.7 ± 380.2, 3039.6 ± 362.1 and 3005.7 ± 373.4 g, respectively, and the median maternal education duration in the three groups was 9(6–9), 9(6–9) and 9(6–9) years, respectively. There was no significant difference in birth weight and maternal education duration; furthermore, the LAZ, WAZ, WLZ and HcAZ at 18 months of age were similar among the three groups (all P > 0.05) (Table 1).
Table 1

Comparison of anthropometric outcomes and biochemical markers by intervention group at age 18 months

Variables

All subjects (n = 180)

Meat group (n = 60)

Fortified cereal group (n = 60)

Local cereal group (n = 60)

P value

Age (months)

17.9 ± 0.2a)

17.9 ± 0.1

18.0 ± 0.2

17.9 ± 0.2

0.001

Male [n(%)]

91 (50.6)

31 (51.7)

29 (48.3)

29 (48.3)

0.915

LAZ

−1.6 ± 1.0

−1.5 ± 1.0

−1.8 ± 0.9

− 1.6 ± 1.1

0.196

WAZ

−0.9 ± 0.9

−0.9 ± 0.9

−1.0 ± 0.9

−0.8 ± 1.0

0.417

WLZ

−0.2 ± 0.9

−0.3 ± 0.9

− 0.2 ± 0.8

−0.1 ± 0.9

0.361

HcAZ

−0.3 ± 0.9

−0.4 ± 0.8

− 0.3 ± 0.9

−0.3 ± 0.9

0.802

VitB12 (pg/mL)b)

360.0 (233.0~573.8)c)

338.0 (233.0~578.3)d)e)

509.5 (364.8~992.3)d)

241.0 (185.5~396.3)

< 0.001

tHcy (μmol/L)b)

8.2 (6.9~10.2)

8.0 (6.6~10.3)d)

7.4 (6.1~8.9)d)

9.2 (7.8~11.3)

< 0.001

Hb (g/L)

122.5 ± 11.8

123.3 ± 12.5

123.6 ± 9.9

120.7 ± 12.8

0.346

MCV (fL)

78.9 ± 6.1

79.7 ± 5.0

78.8 ± 6.2

78.3 ± 6.9

0.445

MCH (pg)

26.8 ± 2.5

27.1 ± 2.4

26.9 ± 2.5

26.3 ± 2.6

0.215

MCHC (g/L)

339.6 ± 19.2

340.5 ± 19.9

342.0 ± 21.9

336.5 ± 15.1

0.266

a) Mean ± SD (for all results in this format)

b) Data there were not normally distributed were compared after log transformations among the three groups by ANOVA

c) Median; interquartile range in parentheses (for all results in this format)

d) Compared with the local cereal group, P < 0.05

e) Compared with the fortified cereal group, P < 0.05

Vitamin B12 status and anemia

The median (IQR) concentration of serum vitamin B12 was 360.0 (233.0–573.8) pg/mL in all children at 18 months of age. Deficient (< 200 pg/mL) and marginally deficient (200–300 pg/mL) serum vitamin B12 concentrations were found in 62 (34.4%) children and 30 (16.7%) children were vitamin B12 deficient.

The median (IQR) level of serum tHcy was 8.2 (6.9–10.2) μmol/L in all children. Serum tHcy > 12 μmol/L was found in 21 (11.7%) children, and among them, 7 children also had serum vitamin B12 levels < 200 pg/mL, and another 7 children had serum vitamin B12 levels of 200–300 pg/mL.

The mean Hb level in all children was 122.5 ± 11.8 g/L. Anemia (Hb < 115 g/L) was found in 42 (23.3%) children, and among them, 14 children also had serum vitamin B12 levels < 300 pg/mL. There was no significant difference in Hb, MCV, MCH and MCHC among the meat group, fortified cereal group and local cereal group (P > 0.05) (Table 1).

Pearson correlation results suggested that serum vitamin B12 levels were negatively correlated with tHcy after log transformation (r = − 0.45, P < 0.001). There was no association between vitamin B12 level and the LAZ, WAZ, WLZ, HcAZ, Hb, MCV, MCH or MCHC (P > 0.05). The tHcy level was positively correlated with the WLZ after log transformation (r = 0.15, P = 0.039), but there was no correlation between the tHcy level and the LAZ, WAZ, HcAZ, Hb, MCV, MCH or MCHC (P > 0.05).

Effects of different food interventions on vitamin B12 status

The median vitamin B12 concentration was significantly different among the three groups (P < 0.001). The fortified cereal group had the highest level (509.5 pg/ml), followed by the meat group (338.0 pg/ml) and the local cereal group (241.0 pg/mL) (Table 1). Compared with the local cereal group, toddlers in the meat group (estimated difference 0.16, 95% CI: 0.06~0.26, P = 0.002) and fortified cereal group (estimated difference 0.35, 95% CI: 0.26~0.45, P < 0.001) had higher vitamin B12 concentrations, according to the mixed-effect linear models after log transformations.

Among 62 children whose vitamin B12 concentration was < 300 pg/mL, there were 22 (36.7%) in the meat group, 3 (5.0%) in the fortified cereal group and 37 (61.7%) in the local cereal group. The difference between the three groups was statistically significant (χ2 = 42.86, P < 0.001).

There was a significant difference in the serum tHcy levels among the three groups (P < 0.001). The serum tHcy levels in both the meat group and fortified cereal group were lower than the levels in the local cereal group (P < 0.05) (Table 1). Compared with the local cereal group, toddlers in the meat group (estimated difference − 0.08, 95% CI: − 0.13~ − 0.02, P = 0.005) and fortified cereal group (estimated difference − 0.12, 95% CI: − 0.18~ − 0.07, P < 0.001) had lower tHcy concentrations, according to the mixed-effect linear models after log transformations.

Among 21 children whose tHcy level > 12 μmol/L, 7 (11.7%) were in the meat group, 3 (5.0%) were in the fortified cereal group and 11 (18.3%) were in the local cereal group. There was no difference among the three groups (χ2 = 5.18, P > 0.05). Serum tHcy > 12 μmol/L and vitamin B12 < 300 pg/mL were found in 14 (7.8%) toddlers. Among them, 7 toddlers had a vitamin B12 level of < 200 pg/mL.

Vitamin B12 status and neurocognitive development

Because 9 children were unable to cooperate with staff to complete the test, including 2 in the meat group, 5 in the fortified cereal group and 2 the in local cereal group, 171 children completed the BSID III screening test at 18 months of age.

There were significant differences in the cognitive score of the BSID III screening test among the three groups (P = 0.020). Compared to the local cereal group, children in both the meat group and fortified cereal group had higher cognitive scores (P < 0.05) (Table 2). The mixed-effect linear models showed that the meat group had higher cognitive scores than the local cereal group (estimated difference 0.99, 95% CI: 0.21~1.76, P = 0.013). The cognitive score of the fortified cereal group was also higher than that of the local cereal group, but the difference was not statistically significant (estimated difference 0.78, 95% CI: − 0.00~1.57, P = 0.051).
Table 2

Comparison of the BSID III screening test scores at age 18 months by intervention group

Variables

All subjects (n = 171)

Meat group (n = 58)

Fortified cereal group (n = 55)

Local cereal group (n = 58)

Statistics

P value

Cognitive score

21.0 ± 2.0a)

21.3 ± 1.9b)

21.2 ± 2.0b)

20.4 ± 2.0

F = 4.02

0.020

Fine motor score

18.2 ± 1.1

18.0 ± 1.1

18.3 ± 1.1

18.3 ± 1.0

F = 1.63

0.199

Gross motor score

20.1 ± 1.2

20.1 ± 1.4

20.0 ± 1.1

20.2 ± 1.1

F = 0.67

0.513

a) Mean ± SD (for all results in this format)

b) Compared with the local cereal group, P < 0.05

In 171 children, serum vitamin B12 concentrations were positively correlated with the cognitive score (beta = 2.15, SE = 0.55, P < 0.001) and the fine motor score (beta = 0.71, SE = 0.31, P = 0.023), according to a multiple linear regression analysis.

Discussion

This study is the first to report the vitamin B12 status of 18-month-old toddlers in a poor rural area of China. Due to the low economic level, the diet of the local people in the study areas is mainly vegetarian. Vitamin B12 content in the local rice-based complementary foods was too low to meet the physiological needs of infants and young children. Therefore, local populations, especially infants and young children, are likely at high risk for vitamin B12 deficiency. In the population subsample, serum vitamin B12 concentrations < 300 pg/mL were found in 34.4% of toddlers, and concentrations of < 200 pg/mL were found in 16.7% of toddlers. In addition, toddlers with serum vitamin B12 concentrations < 300 pg/mL were comprised up to 61.7% of the local cereal group. Our results suggested that the vitamin B12 status of toddlers in the study area is poor, similar to that in other developing countries [3, 4, 5].

Vitamin B12 status was the best in the fortified cereal group, followed by the meat group and local cereal group. Both the daily 50 g of lean pork and 20 g of fortified cereal contained approximately 0.2 μg of vitamin B12, and there was no vitamin B12 supplement in the local cereal. As a result, during the one-year follow-up, the intake of vitamin B12 in the meat group and fortified cereal group should have been higher than that in the local cereal group. In addition, though vitamin B12 from fortified cereal is directly consumed, meat may lose some of its vitamin B12 during the cooking process. Therefore, the actual vitamin B12 intake in toddlers in the fortified cereal group may be higher than that in the meat group, which may explain why the vitamin B12 status of toddlers in the fortified cereal group was better than that in the meat group. At present, according to the WHO, the recommended nutrient intake (RNI) for vitamin B12 in children aged 0–3 years is as follows: 0.4 μg/d for ages 0–6 months, 0.5 μg/d for ages 7–12 months, and 0.9 μg/d for ages 1–3 years [20]. Therefore, even in the meat group and fortified cereal group, the intake of vitamin B12 from the intervention foods after the age of 12 months was still far below the recommended intake, which led to the prevalence of vitamin B12 deficiency among toddlers in this study.

Vitamin B12 is extremely important for the protection of nerve cells. Vitamin B12 deficiency may have adverse outcomes through a variety of metabolic pathways that can alter energy use and reduce the production of neurotransmitters and myelin. Myelin is the main component of white matter in the brain and is essential for nerve conductivity. Disorders in myelination decrease the conduction velocity of multiple systems both in the central and peripheral nervous systems [21]. Therefore, the correlation between vitamin B12 status and the neurodevelopment of young children in poor areas deserves our attention. In our study, vitamin B12 status was found to be significantly correlated with the cognitive score and the fine motor score of the BSID III screening test, which was similar to the results of other studies [6, 7]. The results suggested that vitamin B12 status was closely related to the neurodevelopment of young children. Furthermore, compared with the local group, the toddlers in the meat group and the fortified cereal group had higher vitamin B12 intake and higher cognitive scores, suggesting that although the vitamin B12 intake from the intervention foods in this study was low, the serum vitamin B12 level was still improved to some extent, thus improving children’s cognitive levels. In this study, compared to the fortified cereal group, toddlers in the meat group had lower vitamin B12 levels but higher cognitive scores. In addition to vitamin B12, meat is also rich in zinc and iron, all of which are linked to cognitive development in children. It is therefore necessary to increase the intake of animal-based food in infants in poor areas starting at the age of 6 months. It is also recommended that local cereal be replaced with micronutrient-fortified rice cereal as an initially introduced complementary food.

Serum vitamin B12 concentration is the most commonly used marker of vitamin B12 status. However, the functional markers tHcy and methylmalonic acid (MMA) have been established as useful indicators of vitamin B12 status and may be more sensitive indicators of mild vitamin B12 deficiency [6]. In view of the limitations of MMA, such as the high cost of analysis, the need for gas chromatography mass spectrometry, and especially in developing countries, the possibility of concentrations being increased by bacterial overgrowth [17], in this study, both serum vitamin B12 and tHcy were selected as the biological basis for the assessment of the vitamin B12 status of toddlers. The metabolic conversion of tHcy to methionine is inhibited if the coenzyme methionine synthase is not saturated with vitamin B12, leading to the accumulation of tHcy [18]. The results of this study showed that the serum vitamin B12 level of young children was negatively correlated with tHcy. However, the percentage of individuals with vitamin B12 deficiency screened with criteria of serum tHcy > 12 μmol/L and vitamin B12 < 300 pg/mL was 7.8%, significantly lower than that screened by using serum vitamin B12 (< 300 pg/mL) alone (34.4%). The cutoff of tHcy which was used to determine vitamin B12 deficiency in young children was considered different from that of older children or adults [1]. Therefore, how to better judge the vitamin B12 status of infants and young children is worth further study.

In this study, no correlation was found between serum vitamin B12 and Hb, MCV, MCH or MCHC levels in toddlers. Animal products are not only the only source of vitamin B12 but also the main dietary source of iron. Therefore, inadequate intake of animal-based foods can cause vitamin B12 deficiency as well as iron deficiency. As a result, due to the simultaneous deficiency of both, the hematological manifestation induced by vitamin B12 deficiency, macrocytic anemia, may not be apparent.

The role of vitamin B12 in nucleic acid and protein synthesis determines its effect on the growth and development of infants and young children. Recently, a randomized, controlled double-blind trial found that linear and ponderal growth of North Indian children aged 6 to 35 months improved significantly after vitamin B12 supplementation [3]. From the age of 6 months, infants in the study areas were given dietary interventions, such as meat or fortified cereal, but the growth retardation of the local children was still obvious. Our previous studies have found that local children may have subclinical enteritis, induced by frequent exposure to the pathogens found in poor hygienic conditions, which may impair their linear growth [9, 22]. Therefore, more factors should be taken into consideration for improvement of children’s nutritional status in poor rural areas.

There are some limitations in the present study. First, the biomarkers of vitamin B12 status were not detected at baseline due to ethical issues, so we did not observe the changes in the aforementioned indexes from 6 to 18 months. Therefore, this study lacks a deep and comprehensive assessment of the effects of various nutritional interventions on vitamin B12 status. Second, the study is a subsample nested within a clustered randomized trial, and higher sampling error may exist. In addition, the nonblind design may have generated bias when the subjects were enrolled. These biases may make the research conclusions deviate from the actual situation. Third, the BSID III screening test was used to assess the infants’ development at 18 months of age and was probably the best developmental test for this age group. However, the BSID III had poor predictive ability for intelligence quotient and school achievement later in life. Finally, the determination of serum vitamin B12 by chemiluminescence is susceptible to matrix effects and antibody specificity, which may affect the accuracy of detection results.

Conclusions

Vitamin B12 is an essential micronutrient for children’s growth and neurodevelopment, and it only exists in animal products. In poor rural areas of China, vitamin B12 deficiency in young children was more common and was associated with low dietary vitamin B12 intake. Micronutrient-fortified rice cereal and meat could help improve the nutritional status of vitamin B12 in children and might improve their cognitive level.

Notes

Acknowledgments

We thank Professor K Michael Hambidge from the University of Colorado, the principal investigator of the study, for his guidance. We thank the staff of Xichou County Maternal and Nutrition Services for their generous and high-quality support in enrolling community doctors, training hospital directors, and purchasing fresh pork and for their principal role in data collection.

Authors’ contributions

XYS participated in the design, analysis and drafting of the paper. JLW and FL participated in the acquisition and analysis of the data. FXOY participated in the analysis of the data. JQM participated in the design and analysis of the research, revision of the paper and had primary responsibility for the final content of the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (No. 81302446, No. 81172686, No. 81703249 and No. 81673178) and the Thrasher Research Fund (TRF 200708–029). The funding sources had no involvement in the preparation of the manuscript or in the collection, analysis or interpretation of the data.

Ethics approval and consent to participate

Infants were enrolled after parents or main caregivers who were the legal guardian of the minors provided written informed consent. The study protocol was approved by the Ethics Committee of Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine (No. 08–0768) and the study was conducted in accordance with the revised Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflicts of interest.

Supplementary material

12887_2019_1716_MOESM1_ESM.pdf (188 kb)
Additional file 1. (Nutrition Monitoring of Young Children, Third Version). (PDF 187 kb)

References

  1. 1.
    Rizzo G, Laganà AS, Rapisarda AM, La Ferrera GM, Buscema M, Rossetti P, et al. Vitamin B12 among vegetarians: status, assessment and supplementation. Nutrients. 2016;8 12). pii: E767. doi: 10.3390/nu8120767. PMID 27916823.CrossRefGoogle Scholar
  2. 2.
    Hunt A, Harrington D, Robinson S. Vitamin B12 deficiency. BMJ. 2014;349:g5226.  https://doi.org/10.1136/bmj.g5226. PMID 25189324.CrossRefPubMedGoogle Scholar
  3. 3.
    Strand TA, Taneja S, Kumar T, Manger MS, Refsum H, Yajnik CS, et al. Vitamin B-12, folic acid, and growth in 6- to 30-month-old children: a randomized controlled trial. Pediatrics. 2015;135(4):e918–26.  https://doi.org/10.1542/peds.2014-1848. PMID 25802345.CrossRefPubMedGoogle Scholar
  4. 4.
    Ng'eno BN, Perrine CG, Whitehead RD, Subedi GR, Mebrahtu S, Dahal P, et al. High prevalence of vitamin B12 deficiency and no folate deficiency in young children in Nepal. Nutrients. 2017; 9(1). pii: E72. doi:  https://doi.org/10.3390/nu9010072. PMID 28106733.CrossRefGoogle Scholar
  5. 5.
    Chandyo RK, Ulak M, Kvestad I, Hysing M, Shrestha M, Ranjitkar S, et al. Cobalamin and folate status among breastfed infants in Bhaktapur, Nepal Nutrients 2018; 10(5). pii: E639. doi:  https://doi.org/10.3390/nu10050639. PMID 29783689.CrossRefGoogle Scholar
  6. 6.
    Kvestad I, Hysing M, Shrestha M, Ulak M, Thorne-Lyman AL, Henjum S, et al. Vitamin B-12 status in infancy is positively associated with development and cognitive functioning 5 y later in Nepalese children. Am J Clin Nutr 2017; 105(5):1122–1131. doi:  https://doi.org/10.3945/ajcn.116.144931. PMID 28330909.CrossRefGoogle Scholar
  7. 7.
    Obeid R, Murphy M, Solé-Navais P, Yajnik C. Cobalamin status from pregnancy to early childhood: lessons from global experience. Adv Nutr 2017; 8(6):971–979. doi:  https://doi.org/10.3945/an.117.015628. PMID 29141978CrossRefGoogle Scholar
  8. 8.
    Tang M, Sheng XY, Krebs NF, Hambidge KM. Meat as complementary food for older breastfed infants and toddlers: a randomized, controlled trial in rural China. Food Nutr Bull 2014; 35(4 Suppl):S188–S192. doi:  https://doi.org/10.1177/15648265140354S304. PMID 25639137.CrossRefGoogle Scholar
  9. 9.
    Ma J, Sun Q, Liu J, Hu Y, Liu S, Zhang J, et al. The effect of Iron fortification on Iron (Fe) status and inflammation: a randomized controlled trial. PLoS One 2016; 11(12):e0167458. doi:  https://doi.org/10.1371/journal.pone.0167458. PMID 27923057.CrossRefGoogle Scholar
  10. 10.
    Sally P, Stable, Robert H. Vitamin B12 deficiency as a worldwide problem. Annu Rev Nutr 2004; 24:299–326. doi:  https://doi.org/10.1146/annurev.nutr.24.012003.132440. PMID: 15189123CrossRefGoogle Scholar
  11. 11.
    Animal source foods to improve micronutrient nutrition and human function in developing countries. Proceedings of a conference. June 24-26, 2002. Washington, DC, USA. J Nutr. 2003; 133(11 Suppl 2):3875S-4061S. Doi:  https://doi.org/10.1093/jn/133.11.3875S. PMID 14714275.CrossRefGoogle Scholar
  12. 12.
    Jones KM, Ramirez-Zea M, Zuleta C, Allen LH. Prevalent vitamin B12 deficiency in twelve-month-old Guatemalan infants is predicted by maternal B12 deficiency and infant diet. J Nutr 2007; 137(5):1307–1313. doi:  https://doi.org/10.1093/jn/137.5.1307. PMID 17449597.CrossRefGoogle Scholar
  13. 13.
    Chow S-C, Shao J, Wang H. In: Taylor & Francis Inc: Boca Raton, FL, United States, editor. Sample size calculations in clinical research. 2nd ed; 2007. p. 70–1. ISBN-13: 9781584889823.CrossRefGoogle Scholar
  14. 14.
    Bayley N. Bayley scales of infant and toddler development screening test. 3rd ed. San Antonio, TX, USA: Harcourt Assessment; 2006.Google Scholar
  15. 15.
    Sullivan KM, Mei Z, Grummer-Strawn L, Parvanta I. Haemoglobin adjustments to define anaemia. Tropical Med Int Health. 2008;13(10):1267–71.  https://doi.org/10.1111/j.1365-3156.2008.02143.x. PMID 18721184.CrossRefGoogle Scholar
  16. 16.
    De Benoist B. Conclusions of a WHO technical consultation on folate and vitamin B12 deficiencies. Food Nutr Bull 2008; 29(2 Suppl): S238–S244. doi:  https://doi.org/10.1177/15648265080292S129. PMID 18709899.CrossRefGoogle Scholar
  17. 17.
    Allen LH. How common is vitamin B-12 deficiency? Am J Clin Nutr. 2009;89(2):693S–6S.  https://doi.org/10.3945/ajcn.2008.26947A. PMID 19116323.CrossRefPubMedGoogle Scholar
  18. 18.
    Kvammen JA, Thomassen RA, Eskerud MB, Rugtveit J, Henriksen C. Micronutrient status and nutritional intake in 0- to 2-year-old children consuming a Cows' Milk exclusion diet. J Pediatr Gastroenterol Nutr 2018; 66(5):831–837. doi:  https://doi.org/10.1097/MPG.0000000000001942. PMID 29481443.CrossRefGoogle Scholar
  19. 19.
    World Health Organization Anthro for personal computers, version 3.2.2, software for assessing growth and development of the world's children. Geneva: WHO; 2011. Available online: http://www.who.int/childgrowth/software/en/.
  20. 20.
    World Health Organization. Food and agricultural Organization of the United Nations. Vitamin and mineral requirements in human nutrition. 2nd ed. Geneva, Switzerland: WHO; 2004. http://apps.who.int/iris/bitstream/handle/10665/42716/9241546123.pdf?ua=1 Google Scholar
  21. 21.
    Black MM. Effects of vitamin B12 and folate deficiency on brain development in children. Food Nutr Bull. 2008;29(2 Suppl):S126–31 PMID: 18709887.CrossRefGoogle Scholar
  22. 22.
    Liu JR, Sheng XY, Hu YQ, Yu XG, Westcott JE, Miller LV, et al. Fecal calprotectin levels are higher in rural than in urban Chinese infants and negatively associated with growth. BMC Pediatr 2012; 12:129. doi:  https://doi.org/10.1186/1471-2431-12-129. PMID: 22917269

Copyright information

© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Department of Children and Adolescents Health Care, Xin Hua Hospital, School of MedicineShanghai Jiao Tong University, Shanghai Institute for Pediatric Research, MOE-Shanghai Key Laboratory of Children’s Environmental HealthShanghaiChina
  2. 2.Ministry of Education and Shanghai Key Laboratory of Children’s Environmental Health, Xin Hua Hospital, School of MedicineShanghai Jiao Tong UniversityShanghaiChina
  3. 3.Shanghai Institute for Pediatric Research, Xin Hua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Key Laboratory of Pediatric Gastroenterology and NutritionShanghaiChina

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