Abstract
Folate and vitamin B-12 (B-12) are essential for normal brain development. Few studies have examined the relationship of maternal folate and B-12 status during pregnancy to offspring cognitive function. To test the hypothesis that lower maternal plasma folate and B-12 concentrations and higher plasma homocysteine concentrations during pregnancy, are associated with poorer neurodevelopment, cognitive function was assessed during 2007-2008 among 536 children (aged 9-10 y) from the Mysore Parthenon birth cohort. Maternal folate, B-12 and homocysteine concentrations were measured in stored plasma samples taken at 30±2 wk gestation. The children’s cognitive function was measured using 3 core tests from the Kaufman Assessment Battery and additional tests measuring learning ability, long-term storage/retrieval, attention and concentration, visuo-spatial and verbal abilities. During pregnancy 4% of mothers had low folate concentrations (<7 nmol/L), 42.5% had low B-12 concentrations (<150 pmol/L) and 3% had hyperhomocysteinemia (>10 μmol/L). There was a 0.1-0.2 SD increase in the children’s cognitive scores per SD increase in maternal folate concentration (p<0.001 for all tests). The associations with learning ability and long-term storage/retrieval, visuo-spatial ability, attention and concentration were independent of maternal age, BMI, parity, the parents’ education, socio-economic status, rural/urban residence, religion, the child’s gestational age, birth size, sex and the children’s size, educational level and folate and B-12 concentrations at 9.5 y. There were no consistent associations of maternal B-12 and homocysteine concentrations with childhood cognitive performance.
Conclusions
In this Indian population higher maternal folate, but not vitamin B-12 concentrations during pregnancy, predicted better childhood cognitive ability.
Introduction
Folate and vitamin B-12 are important micronutrients essential for neurodevelopment and function in the antenatal and early postnatal period (1-3). The folate and vitamin B-12 supply to the growing fetus depends on maternal folate and vitamin B-12 status (4-6). Deficiencies of folate and vitamin B-12 are prevalent across the globe (7). A recent review has reported that circulating levels of vitamin B-12 and total homocysteine (tHcy), a functional measure of folate or vitamin B-12 status, drop during normal pregnancy (8). Folate deficiency has been reported in Indians (9-11). Vitamin B-12 deficiency is common in Indians including pregnant women and children (9-13) and has been attributed mainly to vegetarian diet, with or without malabsorption due to intestinal infections (14). Deficiency of these nutrients during pregnancy has been linked to fetal growth restriction and neural tube defects (8, 15-18).
Literature reporting associations between maternal folate status during pregnancy and later cognitive ability in the offspring are few, and limited mainly to animal studies (19, 20). Studies in rodents have shown associations of maternal folate deficiency with structural brain abnormalities (19) and poor post-natal learning ability in the offspring (20). In humans, maternal megaloblastic anemia has been associated with delayed or abnormal infant development (3) and poor mental development have been reported in children of genetically susceptible mothers with low dietary folate intakes (21). In relation to vitamin B-12 status during pregnancy, there are case reports of delayed neurodevelopment in infants born to mothers with pernicious anemia or among strict vegetarians (1, 2, 22, 23). Two recent studies, from Mexico and India, have reported poor cognitive outcomes in children of vitamin B-12 deficient mothers (21, 24).
There is recent interest in potential interactions between folate and vitamin B-12 status. Among elderly US individuals, higher folate status in the presence of vitamin B-12 deficiency was associated with impaired cognitive function and anemia (25). A study in India has reported that children of mothers with high folate and low vitamin B-12 status in pregnancy had increased insulin resistance (13). A recent trial in Nepal reported a reduced risk of metabolic syndrome in children of mothers supplemented during pregnancy with folic acid and vitamin A compared with children of control mothers who received vitamin A alone. This benefit was not apparent in children of mothers who received multiple micronutrients (which included folic acid and vitamin B-12) suggesting the possibility of nutrient interactions (26).
We have examined associations between maternal plasma folate, vitamin B-12 and tHcy concentrations in pregnancy and cognitive performance in 9-10 y old children from the Mysore Parthenon Study, India (27, 28). Our main objective was to test the hypothesis that low maternal folate and/or vitamin B-12 concentrations are associated with lower offspring cognitive ability, independent of socio-economic factors and the child’s size and current vitamin B-12 and folate status. We have also examined the associations of maternal plasma folate, vitamin B-12 and tHcy status, across the whole range of concentrations, with offspring cognitive ability.
Materials and Methods
Study population
The Mysore Parthenon study is a prospective birth cohort study initiated in 1997-1998 mainly to examine the incidence and determinants of gestational diabetes mellitus in India and its short and long-term effects (27, 28). Eight hundred and thirty women booking consecutively into the antenatal clinic at the Holdsworth Memorial Hospital (HMH), Mysore, India and satisfying the eligibility criteria (no history of diabetes before pregnancy, planning to deliver at HMH, and having a singleton pregnancy of <32 wk gestation) participated in the study. Six hundred and seventy four women delivered their babies at HMH (81% of the participants). Excluding 7 stillborn babies, and 4 with major congenital anomalies, detailed newborn anthropometry was performed on 663 normal live born babies according to a standard protocol, within 72 h of birth, as reported previously (27, 28). Excluding 25 children who died, and 8 with major medical problems, 630 healthy children were followed up with repeat anthropometry, annually till the age of 5 y and every 6 mo thereafter.
Maternal folate and Vitamin B-12 concentrations
Maternal micronutrient (folate, vitamin B-12 and tHcy) concentrations were measured using stored plasma samples. Maternal blood samples collected at 30±2 wk of gestation were immediately centrifuged after venepuncture and frozen at −80°C within 1 h in light proof boxes and remained at or below that temperature throughout their time in storage (8 y). Children’s blood samples collected at 9.5 y and stored in a similar way were analyzed within a year of collection to measure folate and vitamin B-12 concentrations. Laboratory analyses were performed using microbiological assays (Wallac Victor 1420 (PerkinElmer life sciences, Turku, Finland) at the Diabetes Research Centre, KEM Hospital, Pune, India. Plasma vitamin B-12 was measured using a colistin sulfate-resistant strain of L. leichmanii (29), plasma folate was measured using a chloramphenicol-resistant strain of L. casei (30) and plasma tHcy was measured by fluorescence polarization immunoassay (Abbott, IL, USA) (31). Intra- and inter-assay CV were <8% for the folate and vitamin B-12 assays and for the tHcy assay, between-day CV were <3%. Low folate and vitamin B-12 status were defined as concentrations <7 nmol/L (32) and <150 pmol/L (33) respectively and hyperhomocysteinemia as a concentration >10μmol/L (34).
Folic acid and vitamin B-12 supplementation
General practitioners and obstetricians routinely prescribe folic acid and/or multivitamin supplements to pregnant women. We collected data on the intake of multivitamin supplements at the time of recruitment, but not subsequently, and therefore no information is available on their use when blood samples were collected or at term. Approximately 70% of women were recruited at <24 wk gestation and 30% were recruited between 24-32 wk.
Study sample for cognitive function assessment
At 9-10 y of age (September 2007-May 2008) children were invited for assessment of their cognitive function. Of the 630 children, 61 were unwilling, 17 had moved away from Mysore and 10 were untraceable. The remaining 542 (86%) underwent cognitive testing. Six children were excluded because maternal folate and vitamin B-12 concentrations were not available. The current analysis is restricted to 536 children (boys, n=259, girls, n=277) (Supplemental Fig 1).
Tests of cognitive function
The cognitive measures consisted of a series of neuropsychological tests applicable for use in school aged children and related to specific cognitive domains (memory, attention, fluid reasoning) consistent with the Carroll model (35). They included 3 core tests from the Kaufman Assessment Battery for children (36) and additional tests (37-40) that underwent extensive adaptation to the local cultural context and the adapted version was validated (41, 42). These cognitive tests covered the domains of learning, long-term memory and retrieval ability (Atlantis), short-term memory (Word order), reasoning ability (Pattern reasoning), language production (Verbal fluency), visuo-spatial ability (Kohs’ block design) and visuo-motor processing speed and coordination, attention and concentration (coding-Wechsler Intelligence Scale for Children-III (WISC-III)). The descriptions of these cognitive tests are summarized in Supplemental Table 1. All the tests were administered to each child in a single session of 60 to 90 min at the Epidemiology Research Unit, HMH, in separate rooms, free from distraction, by one of 2 trained masters’ level child psychologists (unaware of the maternal folate and vitamin B-12 status of study children) in the local Kannada language.
Covariates and confounders
We considered the following as important covariates and potential confounding variables: ‘Parental factors’ included maternal age, parity, BMI and height in pregnancy, maternal and paternal educational attainment (completed y), rural/urban residence and current socio-economic status (SES), assessed using the Standard of Living Index (43). None of the mothers had ever smoked or consumed alcohol. ‘Infant factors’ included the child’s sex, gestational age at birth, newborn weight and head circumference. ‘Child factors’ included current age, BMI, head circumference, educational level and folate and vitamin B-12 concentrations, at the time of cognitive testing.
The research ethics committee of the HMH approved the study and informed verbal consent was obtained from parents and children.
Statistical methods
Variables with skewed distributions were either log transformed (maternal BMI, vitamin B-12, tHcy and Kohs block design score) or square root transformed (pattern reasoning score). Scores of cognitive tests and maternal folate, vitamin B-12 and tHcy concentrations were z-standardized to facilitate interpretation of regression models. To describe the current BMI and height of the children with reference to international standards we derived their SD score using the WHO growth reference. Comparisons of means and percentages between groups were made using t tests and chi-square tests, where appropriate. Correlations between maternal folate, vitamin B-12 and tHcy, and the children’s current folate and vitamin B-12, were examined using Pearson’s correlation coefficients. Associations of covariates and confounders with maternal folate and vitamin B-12 concentrations (exposure) and cognitive scores (outcomes) were initially examined using multiple linear regression adjusting for sex and current age. For categorical covariates/confounders (religion), the largest category was used as the reference and tests of general association (Wald) were performed. Associations of maternal folate and vitamin B-12 concentrations (as binary variables (low compared to normal concentrations) and as continuous variables) with cognitive scores were then analyzed using multiple linear regression adjusting for covariates/confounders that were significantly associated with either maternal folate and vitamin B-12 concentrations or cognitive outcomes. A series of models was considered to examine whether any associations found were acting through socio-economic factors, fetal growth and/or the child’s current size and vitamin B-12 and folate status. Interaction terms were used to test for differences in associations between boys and girls, and to examine interactions between maternal folate and vitamin B-12 concentrations, in relation to cognitive scores. Data presented in the text include percentages, mean ± SD, and median (inter quartile range) values, correlation coefficients (r) and regression coefficients (β) (95% CI). For all tests, p<0.05 was considered significant. Stata (version10.0, Stata corporation, Texas, USA) was used for all analyses.
Results
Characteristics of the study cohort are summarized in Table 1. During pregnancy 22 (4%) women had low folate concentrations while 228 (42.5 %) had low vitamin B-12 concentrations and 18 (3.4%) had hyperhomocysteinemia. At 9.5 y <1% of the children had low folate and 2.6% had low vitamin B-12 concentrations. Folate and vitamin B-12 concentrations and the prevalence of low concentrations were not statistically significantly different between mothers whose children were tested for cognitive function and mothers whose children did not take part (data not shown).
Table 1.
General characteristics of the study cohort1
| Variable | Boys n=259 |
Girls n=277 |
All n=536 |
|---|---|---|---|
| Maternal characteristics in Pregnancy | |||
| Age (y) | 24.0 ± 4.2 | 23.8 ± 4.2 | 23.9 ± 4.2 |
| Parity - 0 - No (%) | 125 (48.3) | 147 (53.1) | 272 (50.8) |
| 1 - No (%) | 88 (34.0) | 88 (31.8) | 176 (32.8) |
| 2or more – No (%) | 46 (17.8) | 42 (15.2) | 88 (16.4) |
| Height (cm) | 154.1 ± 5.5 | 154.5 ± 5.3 | 154.3 ± 5.4 |
| Body mass index (kg/m2) | 22.9 (21.0, 25.6) | 23.5 (21.0, 26.5) | 23.2 (21.0, 26.1) |
| Plasma folate concentration (nmol/L) | 34.1 ± 19.2 | 35.4 ± 19.3 | 34.7 ± 19.2 |
| Low folate concentration –No (%) | 11 (4.3) | 11 (4.0) | 22 (4.1) |
| Plasma vitamin B-12 concentration (pmol/L) | 183.0 (126.0, 226.0) | 153.0 (123.0,207.0) | 162.5 (124.0,220.0) |
| Low vitamin B-12 concentration –No (%) | 98 (37.8) | 130 (46.9) | 228 (42.5) |
| Plasma homocysteine concentration (μmol/L) | 6.0 (5.0, 6.9) | 6.0 (5.1, 7.1) | 6.0 (5.1, 7.0) |
| Hyperhomocysteinemia – No (%) | 8 (3.1) | 10 (3.7) | 18 (3.4) |
| Children’s Characteristics | |||
| Tests of cognitive function | |||
| Atlantis (score) | 67.5 ± 17.8 | 67.6 ± 16.7 | 67.6 ± 17.2 |
| Word order (score) | 16.1 ± 2.5 | 16.7 ± 2.6 | 16.4 ± 2.5 |
| Pattern reasoning (score) | 9.0 (4.0, 13.0) | 11.0 (6.0, 14.0) | 10.0 (5.0, 14.0) |
| Verbal fluency (score) -Animals | 11.9 ± 3.2 | 12.2 ± 3.4 | 12.0 ± 3.3 |
| -First names | 14.7 ± 4.1 | 17.4 ± 5.3 | 16.1 ± 4.9 |
| Kohs block design (score) | 76.9 (63.7, 88.0) | 76.2 (63.1, 88.4) | 76.6 (63.3, 88.4) |
| Coding-WISC-III2 (score) | 30.1 ± 7.5 | 35.0 ± 8.0 | 32.7 ± 8.1 |
| At birth | |||
| Gestational age (wk) | 39.1 ± 1.7 | 39.5 ± 1.5 | 39.3 ± 1.6 |
| Birthweight (kg) | 2.908 ± 0.471 | 2.840 ± 0.437 | 2.873 ± 0.454 |
| Head circumference (cm) | 34.1 ± 1.4 | 33.5 ± 1.3 | 33.8 ± 1.4 |
| At the time of testing | |||
| Age (y) | 9.7 ± 0.3 | 9.7 ± 0.3 | 9.7 ± 0.3 |
| Body mass index (kg/m2) | 14.5 ± 1.7 | 14.6 ± 2.0 | 14.6 ± 1.8 |
| Body mass index WHO SD score | −1.3 ± 1.2 | −1.2 ± 1.2 | |
| Height (cm) | 131.1 ± 5.5 | 130.3 ± 6.0 | 130.7 ± 5.8 |
| Height WHO SD score | −0.6 ± 0.8 | −0.8 ± 0.9 | |
| Head circumference (cm) | 50.7 ± 1.4 | 50.5 ± 1.5 | 50.6 ± 1.4 |
| Plasma vitamin B-12concentration (pmol/L) | 338.9 ± 160.1 | 351.3 ± 169.5 | 345.4 ± 165.0 |
| Low vitamin B-12 concentration -No (%) | 5 (2.1) | 8 (3.1) | 13 (2.6) |
| Plasma folate concentration (nmol/L) | 28.6 ± 15.3 | 29.1 ± 14.8 | 28.8 ± 15.0 |
| Low folate concentration –No (%) | 0 (0.0) | 1 (0.4) | 1 (0.2) |
| Parents socio-economic status | |||
| Standard of living index (score) | 36.1 ± 7.9 | 36.3 ± 8.6 | 36.2 ± 8.3 |
| Maternal education –No (%) | |||
| <10 completed y | 106 (41.1) | 88 (31.8) | 194 (36.3) |
| −10 completed y | 74 (28.7) | 91 (32.9) | 165 (30.8) |
| >10 completed y | 78 (30.2) | 98 (35.4) | 176 (32.9) |
| Paternal education –No (%) | |||
| <10 completed y | 105 (40.7) | 100 (36.1) | 205 (38.3) |
| −10 completed y | 92 (35.7) | 116 (41.9) | 20 (38.9) |
| >10 completed y | 61 (23.6) | 61 (22.0) | 122 (22.8) |
| Residence - Rural –No (%) | 77 (29.7) | 70 (25.3) | 147 (27.4) |
| - Urban –No (%) | 182 (70.3) | 207 (74.7) | 389 (72.6) |
| Religion - Hindu3 - No (%) | 145 (56.0) | 164 (59.2) | 309 (57.7) |
| Muslim4 - No (%) | 90 (34.8) | 92 (33.2) | 182 (34.0) |
| Christian5 – No (%) | 24 (9.2) | 21 (7.6) | 45 (8.4) |
Values are mean ± SD or median (Inter quartile range) or No (%)
Wechsler intelligence scale for children-III
Maternal folate and Vitamin B-12 deficiency among Hindus (3.2% and 50.5% respectively)
Maternal folate and Vitamin B-12 deficiency among Muslims (6.0% and 30.2% respectively)
Maternal folate and Vitamin B-12 deficiency among Christians (2.2% and 37.8% respectively)
Girls scored better than boys in tests of word order (short-term memory) (p=0.01), pattern reasoning (planning and fluid reasoning, p=0.004), verbal-fluency-names (broad retrieval ability and speed and flexibility of verbal thought process; p<0.0001) and coding-Wechsler intelligence scale for children-III (attention and concentration; p<0.0001) (Table 1). Approximately 5% of the children were in the 3rd y at school, 38% in 4th y, 50% in 5th y and 5% in 6th y (this variation in a sample of children of the same age arises because of different admission guidelines in different schools and was not generally due to children being held back for poor academic performance). One percent of mothers were illiterate, approximately 35% had only received primary school education; 50% had completed secondary school education, and 14% were graduates or postgraduates and/or professionals. Corresponding figures for fathers were 3%, 35%, 39% and 23% respectively. Approximately 73% of the families were from urban areas and 27% from rural areas.
Associations of maternal plasma folate and vitamin B-12 concentrations with covariates and confounders
Higher maternal folate concentration was associated with lower parity, larger child’s size at birth and 9.5 y and higher SES and parental education (Table 2). The prevalence of low folate concentrations was higher among Muslim women (6.0%, mean ± SD folate=28.5 ± 17.9 nmol/L) than Hindus (3.2%, 37.9 ± 19.1) and Christians (2.2%, 38.4 ± 19.9). The mother’s folate concentration was positively correlated with her vitamin B-12 concentrations (r=0.1, p=0.015) and with the child’s folate at 9.5 y (r=0.2, p<0.0001).
Table 2.
Associations of covariates or confounders with maternal plasma folate and vitamin B-12 concentrations.
|
|
|||
|---|---|---|---|
| Maternal folate | Maternal vitamin B-12 | Maternal homocysteine | |
|
|
|||
| Covariates/confounders | β (95% CI) | ||
|
|
|||
| nmol/L | pmol/L | μmol/L | |
| Maternal age (y) | 0.18 (−0.20, 0.57) | −0.004 (−0.01, 0.004) | 0.004 (−0.002, 0.01) |
| Maternal parity (0, 1 and ≥2) | −3.8 (−5.7, -1.87)*** | 0.003 (−0.04, 0.04) | 0.01(−0.02, 0.03) |
| Maternal BMI in pregnancy (kg/m2) | 2.75 (−8.26, 13.76) | −0.56 (−0.79, −0.33)*** | 0.05 (−0.10, 0.21) |
| Birthweight (kg) | 3.89 (0.28, 7.50)* | −0.01(−0.09, 0.06) | −0.08 (−0.13, −0.03)** |
| Head circumference at birth (cm) | 1.29 (0.10, 2.49)* | −0.02 (−0.04, 0.01) | −0.01(−0.03, 0.002) |
| Child’s BMI at 9.5 y (kg/m2) | 1.14 (0.25, 2.03)* | −0.01(−0.03, 0.01) | −0.0004 (−0.01, 0.01) |
| Child’s head circumference at 9.5 y (cm) | 2.11 (0.97, 3.26)*** | −0.01 (−0.04, 0.01) | −0.002 (−0.02, 0.01) |
| Standard of living index (score) | 0.54 (0.35, 0.73)*** | 0.0004 (−0.004, 0.005) | −0.002 (−0.004, 0.001) |
| Maternal education (completed y) | 1.05 (0.58, 1.51)*** | −0.007 (−0.02, 0.003) | 0.001 (−0.006, 0.008) |
| Paternal education (completed y) | 0.96 (0.60, 1.32)*** | −0.007 (−0.01, 0.001) | 0.001 (−0.004, 0.006) |
| Residence (urban 0, rural 1) | −1.79 (−5.5, 1.88) | −0.05 (−0.13, 0.03) | −0.01 (−0.06, 0.04) |
| Religion: Hindu v Muslim | −9.3 (−12.8, −5.9)***,† | 0.12 (0.05, 0.20)**, † | −0.11 (−0.16, -0.06)***, † |
| Hindu v Christian | 0.62 (−5.26, 6.50) | 0.13 (0.004, 0.26)*, † | −0.06 (−0.14, 0.03) |
β is the effect size on exposure (maternal plasma folate, vitamin B-12 and homocysteine concentrations) per unit change in covariates/confounders derived using multiple linear regression adjusted for the child’s sex and current age, and using all variables as continuous
P values (* p<0.05; ** p<0.01; p<0.001***) derived using multiple linear regression adjusted for the child’s sex and current age
P value <0.01 derived using a test of general association (Wald) among categories of religion, adjusted for the child’s sex and current age
As already reported (44) there was an inverse association between maternal vitamin B-12 and maternal BMI in pregnancy (Table 2). Hindu women had a higher prevalence of low vitamin B-12 concentrations (50.5%, median (inter quartile range) 149 (117, 209) pmol/L) than Muslims (30.2%, 180 (135, 224)) and Christians (37.8%, 186 (134, 229)). Maternal vitamin B-12 correlated with the child’s vitamin B-12 concentration at 9.5 y (r=0.2, p<0.0001).
Maternal tHcy concentration was inversely correlated with maternal folate (r=-0.3, p<0.0001) and vitamin B-12 (r=-0.2, p<0.0001). Offspring birthweight decreased with increasing maternal tHcy concentrations (p=0.003) (Table 2). Compared to Hindu women, Muslim women had higher tHcy concentrations.
At the time of recruitment 156 (29%) women reported taking multivitamin supplements containing both folic acid and vitamin B-12. Of these 89 (57%) were recruited at <24 wk gestation and 67 (43%) between 24-32 wk gestation. Forty-eight women (9%) were taking folic acid alone of whom 38 (79%) were recruited below 24 wk gestation and 10 (21%) between 24-32 wk gestation. There was no significant association of supplement status at recruitment, either before 24 wk or between 24-32 wk, with folate and vitamin B-12 concentrations at 30±2 wk of gestation.
Associations of cognitive outcomes with covariates and confounders
Cognitive scores tended to be lower in children of mothers of higher parity and increase with increasing maternal age and children’s birth size (Table 3). The children’s current BMI and head circumference, parental educational level and SES were strongly positively related to all the cognitive outcomes. The cognitive ability of urban children was significantly better than rural children. Muslim children performed less well compared to other children in tests of pattern reasoning, verbal fluency, Kohs block design (visuo-spatial ability) and coding. The children’s current folate, but not vitamin B-12, concentration was positively associated with all the cognitive outcomes except word order and coding. Coding test scores were positively related to the children’s current educational level (Table 3).
Table 3.
Association of covariates or confounders with children’s cognitive function score.
| Atlantis | Word order | Pattern reasoning | Verbal fluency animals | Verbal fluency 1st names | Kohs block design | Coding-WISC-III1 | |
|---|---|---|---|---|---|---|---|
|
|
|||||||
| Covariates/confounders | β (95% CI) |
||||||
| Maternal age (y) | 0.35 (0.01, 0.70)* |
0.05 (−0.003, 0.10) |
0.03 (0.01, 0.06)** |
0.04 (−0.03, 0.11) |
0.03 (−0.07, 0.12) |
0.08 (0.003, 0.01)** |
0.16 (0.01, 0.31)* |
| Maternal parity (0, 1 and ≥2) | −2.2 (−3.9, −0.50)* |
−0.35 (−0.60, −0.10)** |
−0.16 (−0.27, −0.05)** |
−0.40 (−0.73, −0.07)* |
−0.36 (−0.83, 0.11) |
−0.01 (−0.04, 0.01) |
0.006 (−0.75, 0.76) |
| Maternal BMI in pregnancy (kg/m2) | 4.35 (−5.48,14.20) |
0.05 (−1.40, 1.50) |
0.28 (−0.33, 0.89) |
0.61 (−1.29, 2.50) |
−0.51 (−3.21, 2.18) |
0.09 (−0.03, 0.22) |
3.67 (−0.65, 7.99) |
| Birthweight (kg) | 3.66 (0.43, 6.88)* |
0.46 (−0.02, 0.93) |
0.24 (0.04, 0.44)* |
0.52 (−0.11, 1.14) |
0.10 (−0.078, 0.99) |
0.07 (0.03, 0.12)*** |
1.34 (−0.08, 2.76) |
| Head circumference at birth (cm) | 1.65 (0.59, 2.72)** |
0.22 (0.06, 0.37)** |
0.08 (0.01, 0.14)* |
0.14 −0.07, 0.34) |
0.14 (−0.16, 0.43) |
0.03 (0.01, 0.04)*** |
0.35 (−0.12, 0.83) |
| Child’s BMI at 9.5 y (kg/m2) | 1.77 (0.99, 2.55)*** |
0.19 (0.08, 0.31)** |
0.11 (0.06, 0.16)*** |
0.32 (0.17, 0.47)*** |
0.32 (0.10, 0.54)** |
0.01 (0.001, 0.02)* |
0.69 (0.34, 1.04)*** |
| Child’s head circumference at 9.5 y (cm) | 2.49 (1.48, 3.50)*** |
0.39 (0.24, 0.54)*** |
0.15 (0.09, 0.21)*** |
0.37 (0.17, 0.57)*** |
0.39 (0.11, 0.67)** |
0.03 (0.01, 0.04)*** |
0.96 (0.51, 1.41)*** |
| Child’s current educational level (y) | 0.57 (−1.8, 3.03) |
0.32 (−0.04, 0.68) |
0.14 (−0.008, 0.30) |
0.10 (0.37, 0.58) |
0.47 (−0.21, 1.14) |
0.02 (−0.01, 0.05) |
1.21 (0.14, 2.29)* |
| Child’s folate at 9.5 y (nmol/L) | 0.11 (0.01, 0.21)* |
0.01 (−0.002, 0.03) |
0.01 (0.003, 0.02)** |
0.02 (0.01, 0.04)* |
0.06 (0.03, 0.08)*** |
0.002 (0.0003, 0.003)* |
0.03 (−0.02, 0.07) |
| Child’s vitamin B-12 at 9.5 y (pmol/L) | 0.001 (−0.08, 0.01) |
0.0003 (−0.001, 0.002) |
−0.0002 (−0.001, 0.0003) |
−0.001 (−0.002, 0.001) |
−0.001 (−0.003, 0.002) |
0.00001 (−0.0001, 0.0001) |
−0.004 (−0.01, 0.0003) |
| Standard of living index (score) | 0.46 (0.29, 0.64)*** |
0.07 (0.04, 0.09)*** |
0.03 (0.02, 0.05)*** |
0.08 (0.05, 0.12)*** |
0.11 (0.06, 0.15)*** |
0.01 (0.004, 0.008)*** |
0.19 (0.11, 0.26)*** |
| Maternal education (completed y) | 1.02 (0.61, 1.44)*** |
0.20 (0.14, 0.26)*** |
0.08 (0.06, 0.11)*** |
0.19 (0.11, 0.27)*** |
0.24 (0.13, 0.36)*** |
0.02 (0.01, 0.02)*** |
0.43 (0.25, 0.62)*** |
| Paternal education (completed y) | 0.89 (0.57, 1.21)*** |
0.12 (0.08, 0.17)*** |
0.07 (0.05, 0.09)*** |
0.14 (0.08, 0.21)*** |
0.18 (0.09, 0.27)** |
0.01 (0.008, 0.02)*** |
0.35 (0.21, 0.50)*** |
| Residence (urban 0, rural 1) | 6.8 (3.62, 10.07)*** |
0.70 (0.22, 1.18)** |
0.34 (0.14, 0.54)** |
0.08 (−0.56, 0.71) |
0.03 (−0.87, 0.93) |
0.07 (0.03, 0.11)** |
0.67 (−0.77, 2.11) |
| Religion: Hindu v Muslim | −1.86 (−4.99, 1.26) |
−0.23 (−0.70, 0.23) |
−0.40 (−0.60, -0.21)***, † |
−1.51 (−2.10, -0.92) ***, † |
−2.99 (−3.82, -2.17) ***, † |
−0.05 (−0.09, 0.01) **, † |
−2.46 (−3.82, −1.10) ***, † |
| Hindu v Christian | 8.03 (2.69,13.40) **, † |
0.61 (−0.19, 1.40) |
0.40 (0.08, 0.73) *, † |
1.16 (0.15, 2.16) *, † |
0.22 (−1.19, 1.63) |
0.11 (0.05, 0.18) **, † |
2.74 (0.41, 5.07) *, † |
Wechsler intelligence scale for children-3rd edition β is the effect size on cognitive scores per unit change in covariates/confounders, derived using multiple linear regression adjusted for the child’s sex and current age, and using all variables as continuous
P values (* p<0.05; ** p<0.01; *** p<0.001) derived by multiple linear regression adjusted for the child’s sex and current age
P value <0.01 derived using a test of general association (Wald) among categories of religion, adjusted for the child’s current age and sex
Associations of maternal plasma folate and vitamin B-12 concentrations with cognitive outcome
Cognitive scores were lower in the small group of children whose mothers had low folate status compared with children of women with normal status, though none of the differences were statistically significant (Table 4). However, there were positive associations, across the whole range of maternal folate concentrations, with scores for all the cognitive outcomes (adjusted for sex and current age (model 1)) (Table 4). All these associations, except for word order and pattern reasoning, were reduced but remained statistically significant after further adjustment for SES, parent’s education, rural/urban residence, religion, parity, maternal pregnancy age and BMI, gestation and child’s birthsize (model 2). Further adjusting for the children’s current size, educational level and folate and vitamin B-12 concentrations at 9.5 y (model 3) showed independent associations of maternal folate with the cognitive measures of learning ability and long-term storage and retrieval, visuo-spatial ability and attention and concentration.
Table 4.
Associations between maternal plasma folate and vitamin B-12 concentrations during pregnancy and children’s cognitive performance1
| Atlantis | Word Order | Pattern Reasoning | Verbal Fluency Animals |
Verbal Fluency First Names |
Koh’s block design | Coding-WISC-III2 | ||
|---|---|---|---|---|---|---|---|---|
| n | ||||||||
| Maternal folate status | Score | |||||||
| Normal (>7 nmol/L) | 514 | 67.9 ± 17.0 | 16.4 ± 2.5 | 10.0 (5.0, 14.0) | 12.1 ± 3.3 | 16.1 ± 4.9 | 76.9 (63.7, 88.3) | 32.7 ± 8.1 |
| Low (<7 nmol/L) | 22 | 60.7 ± 20.4 | 16.4 ± 3.0 | 5.0 (3.0, 13.0) | 11.2 ± 4.0 | 17.5 ± 5.5 | 71.0 (57.2, 90.3) | 31.3 ± 7.8 |
| β3 (95% CI) | ||||||||
| Model 1 | −0.40 (−0.83, 0.03) | 0.01 (−0.41, 0.44) | −0.37 (−0.80, 0.05) | −0.25 (−0.68, 0.18) | 0.32 (−0.10, 0.73) | −0.22 (−0.65, 0.21) | −0.10 (−0.50, 0.30) | |
| Model 2 | −0.33 (−0.74, 0.08) | 0.04 (−0.37, 0.45) | −0.32 (−0.72, 0.08) | −0.21 (−0.63, 0.22) | 0.33 (−0.07, 0.74) | −0.20 (−0.61, 0.24) | −0.13 (−0.53, 0.26) | |
| Model 3 | −0.32 (−0.73, 0.09) | 0.12 (−0.31, 0.54) | −0.23 (−0.64, 0.19) | −0.13 (−0.56, 0.31) | 0.22 (−0.19, 0.64) | −0.14 (−0.57, 0.28) | −0.02 (−0.38, 0.42) | |
|
Maternal folate in quartiles
nmol/L |
Score | |||||||
| ≤17.4 | 134 | 63.5 ± 17.3 | 16.1 ± 2.3 | 8.0 (4.0, 13.0) | 11.1 ± 3.3 | 14.9 ± 4.7 | 74.0 (62.0, 85.2) | 30.4 ± 7.6 |
| 17.5-33.9 | 134 | 67.3 ± 17.1 | 16.2 ± 2.4 | 9.0 (4.0, 14.0) | 12.1 ± 3.2 | 15.9 ± 4.4 | 73.1 (57.7, 83.4) | 32.6 ± 8.3 |
| 34-50.6 | 134 | 66.9 ± 16.8 | 16.7 ± 2.5 | 10.0 (6.0, 14.0) | 12.3 ± 3.1 | 16.6 ± 4.9 | 77.2 (64.8, 88.7) | 33.4 ± 8.1 |
| ≥50.7 | 134 | 72.7 ± 16.4 | 16.8 ± 2.9 | 11.0 (7.0, 15.0) | 12.6 ± 3.5 | 16.9 ± 5.4 | 81.9 (71.0, 92.6) | 34.3 ± 8.1 |
| β4 (95% CI) | ||||||||
| Model 1 | 0.17 (0.08, 0.25)*** | 0.10 (0.02, 0.18)* | 0.16 (0.07, 0.24)*** | 0.17 (0.09, 0.26)*** | 0.15 (0.07, 0.24)*** | 0.17 (0.08, 0.25)*** | 0.17 (0.09, 0.25)*** | |
| Model 2 | 0.10 (0.01, 0.18)* | 0.01(−0.07, 0.10) | 0.06 (−0.03, 0.14) | 0.10 (0.01, 0.19)* | 0.09 (0.003, 0.17)* | 0.09 (0.005, 0.18)* | 0.12 (0.04, 0.20)** | |
| Model 3 | 0.10 (0.01, 0.19)* | −0.001 (−0.09, 0.09) | 0.05 (−0.03, 0.14) | 0.06 (−0.03, 0.15) | 0.08 (−0.003, 0.17) | 0.10 (0.01, 0.19)* | 0.10 (0.02, 0.18)* | |
| Maternal vitamin B-12 status | Score | |||||||
| Normal (>150 pmol/L) | 308 | 66.3 ± 17.5 | 16.3 ± 2.5 | 9.0 (4.0, 13.0) | 11.8 ± 3.4 | 15.7 ± 4.5 | 76.8(63.6, 87.7) | 32.3 ± 8.0 |
| Low (<150 pmol/L) | 228 | 69.3 ± 16.7 | 16.6 ± 2.6 | 10.5 (6.0, 14.0) | 12.4 ± 3.2 | 16.7 ± 5.4 | 76.5 (63.1, 89.3) | 33.2 ± 8.2 |
| β3 (95% CI) | ||||||||
| Model 1 | 0.17 (−0.001, −0.34) | 0.12 (−0.05, 0.29) | 0.14 (−0.03, 0.31) | 0.20 (0.02, 0.37)* | 0.16 (−0.004, 0.33) | 0.01 (−0.16, 0.19) | 0.06 (−0.10, 0.22) | |
| Model 2 | 0.15 (−0.02, 0.32) | 0.09 (−0.09, 0.26) | 0.06 (−0.11, 0.23) | 0.15 (−0.02, 0.33) | 0.09 (−0.08, 0.26) | −0.06 (−0.23, 0.12) | 0.01 (−0.16, 0.17) | |
| Model 3 | 0.12 (−0.05, 0.30) | 0.03 (−0.15, 0.22) | 0.03 (−0.14, 0.21) | 0.19 (0.01, 0.37)* | 0.09 (−0.09, 0.27) | −0.06 (−0.24, 0.12) | −0.007 (−0.18, 0.16) | |
|
Maternal vitamin B-12 in quartiles
pmol/L |
Score | |||||||
| ≤124 | 138 | 69.6 ± 16.9 | 16.5 ± 2.6 | 10.5 (6.0, 15.0) | 12.4 ± 3.3 | 16.6 ± 4.0 | 76.6 (63.0, 88.3) | 33.2 ± 7.8 |
| 125-162 | 130 | 69.0 ± 17.1 | 16.5 ± 2.5 | 10.0 (7.0, 13.0) | 12.2 ± 3.4 | 16.3 ± 5.5 | 73.4 (62.5, 87.8) | 33.3 ± 8.8 |
| 163-220 | 134 | 64.6 ± 16.8 | 16.3 ± 2.4 | 9.0 (5.0, 13.0) | 11.5 ± 2.8 | 15.4 ± 4.3 | 77.2 (66.3, 88.9) | 31.8 ± 8.3 |
| >220 | 134 | 67.2 ± 17.7 | 16.3 ± 2.6 | 10.0 (4.0, 14.0) | 12.2 ± 3.7 | 16.1 ± 4.7 | 77.2 (62.5, 88.4) | 32.4 ± 7.6 |
| β4 (95% CI) | ||||||||
| Model 1 | −0.08 (−0.17, 0.004) | −0.03 (−0.11, 0.06) | −0.07 (−0.16, 0.01) | −0.04 (−0.13, 0.04) | −0.04 (−0.12, 0.05) | −0.01 (−0.10, 0.07) | −0.04 (−0.12, 0.04) | |
| Model 2 | −0.08 (−0.17, 0.0004) | −0.02 (−0.10, 0.07) | −0.05 (−0.13, 0.03) | −0.03 (−0.12, 0.06) | −0.003 (−0.09, 0.08) | 0.01 (−0.08, 0.09) | −0.03 (−0.11, 0.06) | |
| Model 3 | −0.08 (−0.17, 0.009) | 0.007 (−0.08, 0.10) | −0.03 (−0.12, 0.06) | −0.05 (−0.14, 0.04) | −0.006 (−0.09, 0.08) | 0.02 (−0.07, 0.11) | −0.02 (−0.11, 0.06) | |
Values are mean ± SD or median (inter quartile range) unless otherwise stated
Wechsler Intelligence Scale for Children-3rd edition
β is the effect size (SD) on outcome (cognitive scores) per unit change in maternal folate or vitamin B-12 status derived by multiple linear regression
β is the effect size (SD) on outcome (cognitive scores) per SD increase in maternal plasma folate and vitamin B-12 concentration derived by multiple linear regression
P values (* p<0.05; ** p<0.01; *** p<0.001) derived by multiple linear regression
Model 1 adjusted for child’s sex and age at the time of study
Model 2 model 1 parameter + gestational age, SES, parent’s education, religion, rural/urban residence, parity, maternal pregnancy age and BMI, child’s newborn weight and head circumference
Model 3 adjusted for model 2 parameters + children’s current head circumference, BMI, education and vitamin B-12 and folate concentrations at 9.5 y
Lower maternal vitamin B-12 status was associated with higher verbal-fluency scores (Table 4). There were no associations between maternal vitamin B-12 concentrations across the whole range and the cognitive test scores (Table 4). There were no associations between maternal tHcy concentrations and most of the cognitive test scores (data not shown). Higher maternal tHcy concentrations was associated with higher scores for verbal fluency-first names (β=0.10; 95% CI: 0.02, 0.19; p=0.02 in the fully adjusted model).
There were no significant interactions between maternal folate and vitamin B-12 groups in relation to cognitive scores. All the cognitive test scores tended to increase with increasing thirds of folate concentrations in children of mothers with low as well as normal vitamin B-12 status (data not shown).
There were no differences in maternal folate and vitamin B-12 effects on cognitive scores between boys and girls except for the association of maternal folate with verbal fluency-first names which was stronger among girls than boys (p for interaction=0.003).
Discussion
To our knowledge, no previous study has examined associations between maternal plasma folate, vitamin B-12 and tHcy concentrations during pregnancy and cognitive performance in the children, in a large unselected sample of healthy mothers and children. We found that in our sample, there was a high prevalence of low vitamin B-12 concentrations (43%), while few women had low folate concentrations (4%). Higher maternal folate concentration was associated with better cognitive performance in the children. This effect occurred across the whole range of maternal folate concentrations, with no apparent threshold at the level used to define deficiency. The associations with learning ability and long-term storage and retrieval, visuo-spatial ability and attention and concentration were independent of all the confounding factors measured and of the children’s current folate and vitamin B-12 status. There were no consistent associations between maternal vitamin B-12 or tHcy concentrations and cognitive ability. There were no interactions between maternal folate and vitamin B-12 groups in relation to cognitive scores.
Strengths of the study were that in a large sample of children, we measured a battery of cognitive function tests specifically adapted for, and validated in, a South Indian population and also collected data on a range of important confounding factors. A limitation was that maternal micronutrient assays were performed using plasma samples stored for 8 y. However, vitamin B-12 and folate have been shown to be stable following long-term storage at lower temperatures (45). The interpretation of concentrations of these vitamins in pregnancy is complicated because of haemodilution, raised glomerular filtration rate and complex physiological storage mechanisms for both vitamins (especially vitamin B-12) resulting in lower concentrations of these vitamins during pregnancy (8, 46). There is no universally agreed cut-off value to define deficiency in pregnancy; however our definition has been used in earlier studies (47). Other limitations were a lack of data on maternal diet and use of folate and vitamin B-12 supplements at the time of sample collection, and a lack of information on parental IQ and the home environment.
A high prevalence of low vitamin B-12 concentrations has been observed in earlier studies in India (9-13). Consistent with these, 43% of women in our study had low vitamin B-12 status. Animal products are the main dietary source of vitamin B-12, and the lowest concentrations were found among Hindus who are mainly lacto-vegetarians (with milk and milk products being the main non-vegetarian food source). In spite of being non-vegetarians, 30% Muslims and 37% Christians also had low vitamin B-12 concentrations, probably due to poor economic conditions. In comparison to rural women in Pune, India, fewer Mysore women had low vitamin B-12 concentrations (Mysore 43% v Pune 71%) (13). This is probably due to a higher proportion of Muslim women, higher consumption of non-vegetarian food and possibly higher intakes of fermented foods like Idli, Dosa and Yogurt, which are commonly eaten in southern India and which favor bacterial vitamin B-12 production. Few (4%) women had low folate concentrations; the lowest concentrations were found among Muslims, who are mainly non-vegetarians with low vegetable intakes. Apart from dietary factors, adiposity (higher BMI) has been shown to be associated with lower micronutrient concentrations, possibly due to disruption in absorption, higher excretion, fat sequestration, increased catabolism and lower dietary intakes (48,49). Our finding of no significant associations between intake of vitamin supplements and vitamin concentrations are possibly because of lack of complete information on supplement intake as our study was not originally designed to study maternal folate and vitamin B-12 status and we recorded intake of vitamin supplements only at the time of recruitment and among women recruited between 24-32 wk gestation very few were taking supplements. Women who were on supplements in early pregnancy might have stopped taking them by 30 wk and women who were not on supplements at recruitment may have been prescribed them later in pregnancy.
We found that the children’s cognitive performance increased with increasing maternal folate concentrations across the whole range. Although cognitive scores were lower in children of mothers with low compared with normal folate concentrations, these differences were not statistically significant, probably because of a lack of statistical power, as low folate status group was small. A recent study from Birmingham, Alabama, reported no association between maternal folate status at 37 wk gestation and mental or psychomotor development of the children at 5 y (50). In this study, 14.0% of mothers had ‘poor folate’ status (plasma folate concentrations ≤11.0 nmol/L). It was carried out among socially disadvantaged families and the author concluded that the severity of socio-economic factors overwhelmed any effect of maternal folate status in the first 5 y of life. Animal studies examining maternal folate status during pregnancy and neurobehavioral development in the offspring have reported a positive relationship (19, 20). Rats born to dams fed on a diet low in folate during pregnancy, and reared on the same diet post-natally, demonstrated poor maze learning ability (20), and adverse effects on the developing brain were found among rats born to folate deficient dams (19). Data from human studies are limited. A case study reported abnormal or delayed development in infants born to mothers with severe folate deficiency (megaloblastic anemia) during pregnancy (3). Another recent study, based on maternal first trimester dietary data, found that a low dietary intake of folate (<400 μg/day) was associated with a lower mental development index only among children of genetically susceptible mothers (carriers of the TT genotype (MTHFR677C>T)) (21). Thus most of the published literature on this topic has examined the effects of maternal deficiency. If the associations seen in our study are causal, our data suggests that, in terms of cognitive development, the concentration used to define folate deficiency is set too low.
In our study, neither maternal vitamin B-12 status (low versus normal) nor the range of vitamin B-12 concentrations was associated with cognitive performance in the children except that verbal ability scores were higher in children of mothers with low vitamin B-12 compared to children of mothers with normal concentrations. Data on maternal vitamin B-12 deficiency and childhood cognitive development come mainly from case reports of maternal deficiency and small observational studies. Infants of mothers with untreated anemia and a strict vegetarian lifestyle were irritable, anorexic and failed to thrive (1, 2, 22, 23). In the Netherlands, infants of macrobiotic mothers had delayed motor and language development compared to infants of omnivores (23), and scored lower at age 12 y, even though their current diet contained the recommended daily intake of vitamin B-12 (51). A recent study in Mexico reported that deficient maternal dietary intakes of vitamin B-12 in the first trimester were associated with impaired mental development in early childhood (21). Comparison of our study with these studies is difficult due to differing age groups of children and different measures of nutritional status. In contrast to our findings, a recent study in Pune, India reported that children of mothers with very low plasma vitamin B-12 concentrations during pregnancy (<77pmol/L) performed poorly in tests of sustained attention and short-term memory compared to children of mothers with high B12 concentrations (>224pmol/L)(24). None of our mothers had such extreme low vitamin B-12 concentrations, which may explain the difference in findings.
In our study cognitive scores were positively related to maternal folate concentrations even in children of mothers with low vitamin B-12 concentrations. A study in an elderly population in the USA (25) reported that normal/high folate status in individuals with vitamin B-12 deficiency was associated with anemia and cognitive impairment. It was suggested that folate may have damaging effects on cognitive function in individuals with low vitamin B-12 status. We did not find evidence for such an effect in our children.
There are few data on maternal homocysteine and offspring cognitive function. Consistent with our findings of a positive association between maternal homocysteine and the children’s verbal fluency, a recent study in the USA reported that, at age 5 y, children of mothers with high plasma tHcy (>7.0μmol/L; 8.4%) at 26 wk of gestation had better manual dexterity than children of mothers with normal homocysteine (50). In contrast, an animal study in Turkey, demonstrated poor memory and maze learning ability in adult offspring born to rats with methionine induced hyperhomocystinemia during pregnancy (52).
Mechanisms linking folate and vitamin B-12 status with neurocognitive development and function are not well understood. Shared metabolism between folate and vitamin B-12 means that deficiency of one vitamin may alter the metabolism of the other. Possible mechanism by which deficiencies could interfere with brain development include delayed myelination or demyelination of nerves; altering methionine synthesis from homocysteine; imbalance in tissue levels of neurotransmitters (neurotrophic and neurotoxic cytokines); and/or accumulation of lactate in brain cells (1, 2, 22). The functional consequences could vary depending on the specific nutrient deficiency and its timing relative to the processes of development, which starts in early gestation and continues throughout childhood.
In conclusion, in this Indian population, we found that maternal plasma folate concentrations were mainly in the ‘normal’ range, but strongly predicted cognitive function in the children, independently of a range of potential confounding factors. The data suggest that folate may be required not only for the prevention of neural tube defects but also for optimal early brain growth and cognitive development. It also suggests that, in terms of neurodevelopment, current definitions of folate deficiency may be set too low. In contrast, although many mothers had low vitamin B-12 concentrations, maternal vitamin B-12 and homocysteine concentrations were largely unrelated to the children’s cognitive function. It is possible that only extremely low maternal vitamin B-12 status may result in cognitive impairment, and/or that this population has adapted over centuries to low intakes of vitamin B-12. Our findings add to a very small literature on this topic and more studies from different populations are required.
Supplementary Material
Supplemental Figure 1: Flow chart of the study participants.
Supplemental Table 1. Description of the cognitive tests used in the study
Abbreviations
- HMH
Holdsworth Memorial Hospital
- SES
Socio-economic status
- tHcy
Total Homocysteine
Footnotes
Authors’ Contributions: SRV: Designed and conducted research, data analysis and interpretation of the data and drafted the manuscript. GVK: Recruited the mothers, carried out maternal and newborn measurements and assisted in the manuscript preparation. AKW: Assisted in statistical analysis, data interpretation and drafting of the manuscript. AVK, KS and SM: Helped in designing cognitive function assessment and also in manuscript preparation. CSY: Helped in interpretation of the data and drafting of the manuscript. CHDF: Designed the baseline and follow-up study, contributed to the data interpretation, drafting of the manuscript and had primary responsibility for final content. All authors read and approved the final manuscript.
Financial Support: The Wellcome Trust grant 079877/Z/06/Z; MRC grant G0400519 (ID no.71108)
Author Disclosure: S.R. Veena, G.V. Krishnaveni, K. Srinivasan, A.K. Wills, S. Muthayya, A.V. Kurpad, C.S. Yajnik and C.H.D. Fall, no conflicts of interest.
Supplementary Materials: Supplemental Figure 1 and Supplemental Table 1 are available as online; Supporting Material with the online posting of this paper at http://jn.nutrition.org
Literature Cited
- 1.Ramakrishna T. Vitamins and brain development. Physiol Res. 1999;48:175–187. [PubMed] [Google Scholar]
- 2.Black MM. Effects of vitamin B12 and folate deficiency on brain development in children. Food Nutr Bull. 2008;29:S126–131. doi: 10.1177/15648265080292S117. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Benton D. Micronutrient status, cognition and behavioral problems in childhood. Eur J Nutr. 2008;3:S38–50. doi: 10.1007/s00394-008-3004-9. [DOI] [PubMed] [Google Scholar]
- 4.Allen LH. Vitamin B12 metabolism and status during pregnancy, lactation and infancy. Adv Exp Med Biol. 1994;352:173–186. doi: 10.1007/978-1-4899-2575-6_14. [DOI] [PubMed] [Google Scholar]
- 5.Obeid R, Munz W, Jäger M, Schmidt W, Herrmann W. Biochemical indexes of the B vitamins in cord serum are predicted by maternal B vitamin status. Am J Clin Nutr. 2005;82:133–139. doi: 10.1093/ajcn.82.1.133. [DOI] [PubMed] [Google Scholar]
- 6.Bjørke Monsen AL, Ueland PM, Vollset SE, Guttormsen AB, Markestad T, Solheim E, Refsum H. Determinants of cobalamin status in newborns. Pediatrics. 2001;108:624–630. doi: 10.1542/peds.108.3.624. [DOI] [PubMed] [Google Scholar]
- 7.McLean E, de Benoist B, Allen LH. Review of the magnitude of folate and vitamin B12 deficiencies worldwide. Food Nutr Bull. 2008;29:S38–51. doi: 10.1177/15648265080292S107. [DOI] [PubMed] [Google Scholar]
- 8.Varela-Moreiras G, Murphy MM, Scott JM. Cobalamin, folic acid, and homocysteine. Nutr Rev. 2009;67:S69–72. doi: 10.1111/j.1753-4887.2009.00163.x. [DOI] [PubMed] [Google Scholar]
- 9.Taneja S, Bhandari N, Strand NA, Sommerfelt H, Refsum H, Ueland PM, Schneede J, Bahl R, Bhan MK. Cobalamine and folate status in infants and young children in a low to middle income community in India. Am J Clin Nutr. 2007;86:1302–1309. doi: 10.1093/ajcn/86.5.1302. [DOI] [PubMed] [Google Scholar]
- 10.Pathak P, Kapil U, Yajnik CS, Kapoor SK, Dwivedi SN, Singh R. Iron, folate, and vitamin B12 stores among pregnant women in a rural area of Haryana state, India. Food Nutr Bull. 2007;28:435–438. doi: 10.1177/156482650702800409. [DOI] [PubMed] [Google Scholar]
- 11.Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE, Orning L, Guttormsen AB, Joglekar A, Sayyad MG, et al. Hyperhomocysteinemia and elevated methylmalonic acid indicate a high prevalence of cobalamin deficiency in Asian Indians. Am J Clin Nutr. 2001;74:233–241. doi: 10.1093/ajcn/74.2.233. [DOI] [PubMed] [Google Scholar]
- 12.Yajnik CS, Deshpande SS, Lubree HG, Naik SS, Bhat DS, Uradey BS, Deshpande JA, Rege SS, Refsum H, et al. Vitamin B12 deficiency and hyperhomocysteinemia in rural and urban Indians. J Assoc Physicians India. 2006;54:775–782. [PubMed] [Google Scholar]
- 13.Yajnik CS, Deshpande SS, Jackson AA, Refsum H, Rao S, Fisher DJ, Bhat DS, Naik SS, Coyaji KJ, et al. Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study. Diabetologia. 2008;51:29–38. doi: 10.1007/s00125-007-0793-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Antony AC. Vegetarianism and vitamin B-12 (cobalamin) deficiency. Am J Clin Nutr. 2003;78:3–6. doi: 10.1093/ajcn/78.1.3. [DOI] [PubMed] [Google Scholar]
- 15.Molloy AM, Kirke PN, Brody LC, Scott JM, Mills JL. Effects of folate and vitamin B12 deficiencies during pregnancy on fetal, infant, and child development. Food Nutr Bull. 2008;29:S101–111. doi: 10.1177/15648265080292S114. [DOI] [PubMed] [Google Scholar]
- 16.Refsum H. Folate, vitamin B12 and homocysteine in relation to birth defects and pregnancy outcome. Br J Nutr. 2001;85:S109–113. [PubMed] [Google Scholar]
- 17.Muthayya S, Kurpad AV, Duggan CP, Bosch RJ, Dwarkanath P, Mhaskar A, Mhaskar R, Thomas A, Vaz M, et al. Low maternal vitamin B12 status is associated with intrauterine growth retardation in urban south Indians. Eur J Clin Nutrition. 2006;60:791–801. doi: 10.1038/sj.ejcn.1602383. [DOI] [PubMed] [Google Scholar]
- 18.Molloy AM, Kirke PN, Troendle JF, Burke H, Sutton M, Brody LC, Scott JM, Mills JL. Maternal vitamin B12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic Acid fortification. Pediatrics. 2009;123:917–923. doi: 10.1542/peds.2008-1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Craciunescu CN, Brown EC, Mar MH, Albright CD, Nadeau MR, Zeisel SH. Folic acid deficiency during late gestation decreases progenitor cell proliferation and increases apoptosis in fetal mouse brain. J Nutr. 2004;134:162–166. doi: 10.1093/jn/134.1.162. [DOI] [PubMed] [Google Scholar]
- 20.Whitley JR, O’Dell BL, Hogan AG. Effect of diet in maze learning in second-generation rats; folic acid deficiency. J Nutr. 1951;45:153–160. doi: 10.1093/jn/45.1.153. [DOI] [PubMed] [Google Scholar]
- 21.Del Río Garcia C, Torres-Sánchez L, Chen J, Schnaas L, Hernández C, Osorio E, Portillo MG, López-Carrillo L. Maternal MTHFR 677C>T genotype and dietary intake of folate and vitamin B(12): their impact on child neurodevelopment. Nutr Neurosci. 2009;12:13–20. doi: 10.1179/147683009X388913. [DOI] [PubMed] [Google Scholar]
- 22.Dror DK, Allen LH. Effect of vitamin B12 deficiency on neurodevelopment in infants: current knowledge and possible mechanisms. Nutr Rev. 2008;66:250–255. doi: 10.1111/j.1753-4887.2008.00031.x. [DOI] [PubMed] [Google Scholar]
- 23.Black MM. Micronutrient deficiencies and cognitive functioning. J Nutr. 2003;133:S3927–3931. doi: 10.1093/jn/133.11.3927S. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Bhate V, Deshpande S, Bhat D, Joshi N, Ladkat R, Watve S, Fall C, de Jager CA, Refsum H, et al. Vitamin B12 status of pregnant Indian women and cognitive function in their 9-year-old children. Food Nutr Bull. 2008;29:249–254. doi: 10.1177/156482650802900401. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Selhub J, Morris MS, Jacques PF, Rosenberg IH. Folate-vitamin B-12 interaction in relation to cognitive impairment, anemia, and biochemical indicators of vitamin B-12 deficiency. Am J Clin Nutr. 2009;89:702S–706S. doi: 10.3945/ajcn.2008.26947C. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Stewart CP, Christian P, Schulze KJ, Leclerq SC, West KP, Jr, Khatry SK. Antenatal micronutrient supplementation reduces metabolic syndrome in 6- to 8-year-old children in rural Nepal. J Nutr. 2009;139:1575–1581. doi: 10.3945/jn.109.106666. [DOI] [PubMed] [Google Scholar]
- 27.Hill JC, Krishnaveni GV, Annamma I, Leary SD, Fall CHD. Glucose tolerance in pregnancy in South India: Relationships to neonatal anthropometry. Acta Obstet Gynecol Scand. 2005;84:159–165. doi: 10.1111/j.0001-6349.2005.00670.x. [DOI] [PubMed] [Google Scholar]
- 28.Krishnaveni GV, Hill JC, Veena SR, Leary SD, Saperia J, Chachyamma KJ, Karat SC, Fall CH. Truncal adiposity is present at birth and in early childhood in South Indian children. Indian Pediatrics. 2005;42:527–538. [PubMed] [Google Scholar]
- 29.Kelleher BP, Walshe KG, Scott JM, O’Broin SD. Microbiological assay for vitamin B12 with use of a colistin-sulfate-resistant organism. Clin Chem. 1987;33:52–54. [PubMed] [Google Scholar]
- 30.Horne DW, Patterson D. Lactobacillus casei microbiological Assay of Folic Acid Derivatives in 96-Well Microtiter plates. Clin Chem. 1988;34:2357–2359. [PubMed] [Google Scholar]
- 31.Shipchandler MT, Moore EG. Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer. Clin Chem. 1995;41:991–994. [PubMed] [Google Scholar]
- 32.Clarke R, Grimley EJ, Schneede J, Nexo E, Bates C, Fletcher A, Prentice A, Johnston C, Ueland PM, et al. Vitamin B12 and folate deficiency in later life. Age Ageing. 2004;33:34–41. doi: 10.1093/ageing/afg109. [DOI] [PubMed] [Google Scholar]
- 33.Kelleher BP, Broin SD. Microbiological assay for vitamin B12 performed in 96-well microtitre plates. J.Clin. Pathol. 1991;44:592–595. doi: 10.1136/jcp.44.7.592. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Refsum H, Smith AD, Ueland PM, Nexo E, Clarke R, McPartlin J, Johnston C, Engbaek F, Schneede J, et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin Chem. 2004;50:3–32. doi: 10.1373/clinchem.2003.021634. [DOI] [PubMed] [Google Scholar]
- 35.Carroll JB. Human cognitive abilities: A survey of factor-analytic studies. Cambridge University Press; New York: 1993. [Google Scholar]
- 36.Kaufman AS, Kaufman LN. Kaufman Assessment Battery for Children. Second Edition: Manual AGS Publishing; Circle Pines, MN: 2004. [Google Scholar]
- 37.Korkman M, Kemp SL, Kirk U. Effects of age on neurocognitive measures of children ages 5 to 12: A cross-sectional study on 800 children from the United States. Dev Neuropsychol. 2001;20:331–354. doi: 10.1207/S15326942DN2001_2. [DOI] [PubMed] [Google Scholar]
- 38.Kohs SC. Intelligence measurement: a psychological and statistical study based upon the Block-design test. Macmillan; New York: 1923. [Google Scholar]
- 39.Wigg CM, Duro LA. The Koh’s block tests as an important instrument to investigate the visuo-spatial impairments in myotonic dystrophy. Part I. Quantitative and qualitative analysis. Arq Neuropsiquiatr. 1999;57:547–555. doi: 10.1590/s0004-282x1999000400002. [DOI] [PubMed] [Google Scholar]
- 40.Wechsler D. Manual for the Wechsler Intelligence Scale for Children. 3rd ed Psychological Corporation; San Antonio, TX: 1991. [Google Scholar]
- 41.Malda M, van de Vijver FJR, Srinivasan K, Transler C, Sukumar P, Rao K. Adapting a cognitive test for a different cultures: An illustration of qualitative procedures. Psychol Sci Quarterly. 2008;50:451–468. [Google Scholar]
- 42.Malda M, van de Vijver FJR, Srinivasan K, Transler C, Sukumar P. Travelling with cognitive tests: testing the validity of a KABC-II adaptation in India. 2009. Assessment doi:10.1177/1073191109341445 (Epub ahead of print) [DOI] [PubMed]
- 43.International Institute for Population Sciences (IIPS) and Operations Research Centre (ORC) Macro . National Family Health Survey (NFHS-2), India 1998-1999. IIPS; Maharashtra, Mumbai: 2001. [Google Scholar]
- 44.Krishnaveni GV, Hill JC, Veena SR, Bhat DS, Wills AK, Karat CL, Yajnik CS, Fall CH. Low plasma vitamin B(12) in pregnancy is associated with gestational ‘diabesity’ and later diabetes. Diabetologia. 2009;52:2350–2358. doi: 10.1007/s00125-009-1499-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Ocke MC, Schrijver J, Obermann-de Boer GL, Bloemberg BP, Haenen GR, Kromhout D. Stability of blood (pro)vitamins during four years of storage at −20 degrees C: Consequences for epidemiological research. J Clin Epidemiol. 1995;48:1077–1085. doi: 10.1016/0895-4356(94)00232-f. [DOI] [PubMed] [Google Scholar]
- 46.Murphy MM, Molloy AM, Ueland PM, Fernandez-Ballart JD, Schneede J, Arija V, Scott JM. Longitudinal study of the effect of pregnancy on maternal and fetal cobalamin status in healthy women and their offspring. J Nutr. 2007;137:1863–1867. doi: 10.1093/jn/137.8.1863. [DOI] [PubMed] [Google Scholar]
- 47.Milman N, Byg KE, Bergholt T, Eriksen L, Hvas AM. Cobalamine status during normal pregnancy and postpartum: a longitudinal study comprising 406 Danish women. Eur J Haematol. 2006;76:521–525. doi: 10.1111/j.0902-4441.2006.t01-1-EJH2550.x. [DOI] [PubMed] [Google Scholar]
- 48.Kimmons JE, Blanck HM, Tohill BC, Zhang J, Kahn LK. Associations between body mass index and the prevalence of low micronutrient levels among US adults. MedGenMed. 2006;8:59. [PMC free article] [PubMed] [Google Scholar]
- 49.Mahabir S, Ettinger S, Johnson L, Baer DJ, Clevidence BA, Hartman TJ, Taylor PR. Measures of adiposity and body fat distribution in relation to serum folate levels in postmenopausal women in a feeding study. Eur J Clin Nutr. 2008;62:644–650. doi: 10.1038/sj.ejcn.1602771. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Tamura T, Goldenberg RL, Chapman VR, Johnston KE, Ramey SL, Nelson KG. Folate status of mothers during pregnancy and mental and psychomotor development of their children at five years of age. Pediatrics. 2005;116:703–708. doi: 10.1542/peds.2004-2189. [DOI] [PubMed] [Google Scholar]
- 51.Louwman MW, van Dusseldorp M, van de Vijver FJ, Thomas CM, Schneede J, Ueland PM, Refsum H, van Staveren WA. Signs of impaired cognitive function in adolescents with marginal cobalamin status. Am J Clin Nutr. 2000;72:762–769. doi: 10.1093/ajcn/72.3.762. [DOI] [PubMed] [Google Scholar]
- 52.Baydas G, Koz ST, Tuzcu M, Nedzvetsky VS, Etem E. Effects of maternal hyperhomocysteinemia induced by high methionine diet on the learning and memory performance in offspring. Int J Dev Neurosci. 2007;25:133–139. doi: 10.1016/j.ijdevneu.2007.03.001. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental Figure 1: Flow chart of the study participants.
Supplemental Table 1. Description of the cognitive tests used in the study