Folate Status of Mothers During Pregnancy and Mental and Psychomotor Development of Their Children at Five Years of Age
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《小儿科》
Department of Nutrition Sciences
Department of Obstetrics and Gynecology
Department of Pediatrics
Department of Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama
ABSTRACT
Objective. There are limited data relating folate nutritional status of mothers during pregnancy to mental and psychomotor development of their offspring. Using an existing data set from a study on the effect of prenatal zinc supplementation on child neurodevelopment, we evaluated the association between folate nutritional status of mothers during pregnancy and neurodevelopment of their children.
Methods. Maternal blood folate and total homocysteine (tHcy) concentrations were measured at 19, 26, and 37 weeks of gestation. At a mean of 5.3 years of age, 355 black children with low-socioeconomic background were given 6 tests: Differential Ability Scales, Visual and Auditory Sequential Memory, Knox Cube Test, Gross Motor Scale, and Grooved Pegboard. The scores of the tests between the 2 groups of mothers with poor versus adequate folate nutritional status classified by blood folate or tHcy concentrations were compared.
Results. There were no differences in the test scores of neurodevelopment between the 2 groups.
Conclusion. Folate nutritional status of mothers in the later half of pregnancy assessed by plasma and erythrocyte folate and plasma tHcy concentrations had no impact on neurodevelopment of their children at age 5. It is unknown whether our findings in a low-socioeconomic population can be readily extrapolated to other populations.
Key Words: folate homocysteine pregnancy mothers mental and psychomotor development children
Abbreviations: tHcy, total homocysteine
Folate plays important roles in amino acid metabolism, de novo purine and thymidylate biosynthesis, and formate oxidation.1 Adequate folate nutritional status is important for normal fetal growth,2,3 and periconceptional folic acid supplementation is effective in reducing the risk for neural-tube defects.4,5 Mental retardation has been recognized as 1 of the distinct clinical features of inborn errors of folate metabolism since their early identification in the 1960s.6–8 However, the mechanism is not well understood as to how altered folate metabolism is associated with mental retardation.9 It is unknown whether the direct cause of mental retardation is the result of the imbalance in folate derivatives in the brain or other metabolic disturbance secondary to the defect of folate-dependent enzymes. Furthermore, the deprivation of certain forms of folate may be the cause of mental retardation in such cases.
Studies on the association between prenatal folate nutritional status and neurodevelopment are scarce. In 1951, Whitley et al10 reported that rats born to dams that were fed a low-folate diet and reared on the same diet had inferior maze-learning ability compared with those with adequate folate nutritional status. Craciunescu et al11 recently reported that maternal folate deficiency during days 11 to 17 of gestation in the mouse leads to the decreased progenitor cell replication and increased apoptotic cells in the fetal forebrain. They concluded that the growing brain is vulnerable to maternal folate deficiency during the critical time of its development; therefore, prenatal folate deficiency may adversely affect neurodevelopment in later life. To our knowledge, only 1 human study has been performed to evaluate the association between prenatal folate nutritional status and neurodevelopment in infants. In 1974, Gross et al12 reported that infants who were born to mothers with severe folate deficiency during pregnancy showed abnormal or delayed neurodevelopment compared with those who were born to mothers without the deficiency. We hypothesized that prenatal folate nutritional status determines mental and psychomotor development later in life in humans.
In the early 1990s, we conducted a trial to evaluate the effect of zinc supplementation during pregnancy on fetal growth in black women. In this trial, although a positive effect of zinc supplementation was found on birth weight and head circumference of newborns, there was no effect of zinc supplementation on mental and psychomotor development of children at 5 years of age.13,14 In the present study, we evaluated the association of prenatal folate nutritional status of mothers with neurodevelopment of their children using existing developmental test scores.14 Maternal folate nutritional status was assessed by plasma folate concentrations, a measure of recent folate intake and the measurement directly related to placental folate transfer to the fetus, and erythrocyte folate concentrations, a measure of a long-term folate intake. We also measured plasma total homocysteine (tHcy) concentrations, a functional measure of folate nutritional status.
METHODS
Participants
The study was approved by the Institutional Review Board for Human Use at the University of Alabama at Birmingham. The mothers of the children who were evaluated in the present study were chosen from 589 black women in a prenatal zinc supplementation trial performed between 1990 and 1993 in the Birmingham, Alabama, area.13,15 All mothers received prenatal care at the local public health clinics and were eligible for the Special Supplemental Nutrition Program for Women, Infants, and Children. In the trial, 294 mothers received a daily oral dose of 25 mg of elemental zinc and the remaining 286 received placebo from 19 weeks of gestation until delivery. All mothers were provided a daily multivitamin/mineral tablet that contained folic acid (400 μg, pteroylglutamic acid), vitamin B12 (2.0 μg, cyanocobalamin), vitamin B6 (3.0 mg, pyridoxine), vitamin B2 (2.0 mg, riboflavin), and other vitamins or minerals but not zinc (Mission Prenatal; Mission Pharmacal, San Antonio, TX). On the basis of the pill counts, the percentage of mothers who did not comply with taking the supplements was 22%.13 Fewer than 5% of this population breastfed their infants. Zinc supplementation resulted in significantly greater birth weight and head circumference of infants who were born to mothers who received zinc than those who received placebo.13 Approximately 5 years later, we recruited 355 of 589 mother–child pairs to evaluate neurodevelopment of children (178 girls and 177 boys) and found that prenatal zinc supplementation had no effect on the developmental test scores.14 Because there were no differences in any of these scores by zinc supplementation group and because in a preliminary analysis zinc supplementation had no association with any of the measures of folate nutritional status used in this study, we combined the supplemented and unsupplemented women into a single group for additional analysis. In the study presented here, we used the same developmental test scores and the values of folate and plasma tHcy concentrations in maternal blood samples that were obtained during the original trial. Because of the insufficient amount of certain samples, laboratory values were not available for all mothers.
Mental and Psychomotor Tests
Mother–child pairs were tested at a mean of 5.3 years after delivery by experienced evaluators with no knowledge of the children’s birth status or maternal conditions at delivery. Mothers received 3 tests, 2 to evaluate their cognitive function, including the Wide Range Achievement Test and the Peabody Picture Vocabulary Test, and the Home Screening Questionnaire, which provided a measure of learning resources in the house.16–18 Children took 6 tests to assess their neurodevelopment: (1) Differential Ability Scales (nonverbal, verbal, and general conceptual ability [IQ]), a standardized intelligence test for assessing the cognitive abilities; (2) Visual Sequential Memory, a test for visual memory span; (3) Auditory Sequential Memory, a test for auditory memory span; (4) Knox Cube, an index of attention span and short-term memory; (5) Gross Motor Scale, a test for gross-motor development; and (6) Grooved Pegboard (dominant and nondominant hands), a test for manipulative dexterity.19–24 The rationale for selecting these tests was described previously.14 Because there was no effect of prenatal zinc supplementation on neurodevelopment of children, the results were combined regardless of whether their mothers received zinc in the original trial.
Maternal Blood Samples and Laboratory Analyses
Nonfasting blood samples were obtained at 19, 26, and 37 weeks of gestation using evacuated tubes that contained heparin (Vacutainer; Becton Dickinson, Rutherford, NJ).13 Immediately after phlebotomy, tubes were refrigerated to avoid obtaining falsely high tHcy values. Before centrifugation to separate plasma, a portion of whole blood was removed for hematocrit measurement, and another portion was mixed with 57 mmol/L ascorbic acid solution for erythrocyte folate assay. All samples were stored at –70°C until analyzed.
Before the folate assay was performed, whole-blood lysate was incubated at 37°C for 30 minutes to allow plasma folate conjugate to hydrolyze polyglutamyl folates in erythrocytes.25 Both plasma and whole-blood folate concentrations were determined by microbiologic assay using Lactobacillus rhamnosus (formerly known as L casei; ATCC 7469), and the day-to-day coefficient of variation of this assay was <10% using pooled human plasma samples.25 Erythrocyte folate concentrations were calculated on the basis of the values of hematocrit and plasma and whole-blood folate. Plasma tHcy concentrations were measured by an high-performance liquid chromatography–fluorescent method, and the interassay coefficient of variation for this assay was 10%.26 Plasma tHcy is considered to be a functional indicator of the nutritional status of folate along with vitamin B12, vitamin B6, and vitamin B2, and these 4 B-group vitamins play critical roles in Hcy metabolism.27 Because of limited resources at the time of analysis, plasma tHcy was measured only for samples obtained at 26 and 37 weeks of gestation. Folate analysis was completed within 4 weeks after blood drawing, and tHcy was assayed within 2 years after the completion of the original trial.
Classification of Mothers on the Basis of Folate and tHcy Values
The mothers were divided into normal and poor folate status on the basis of plasma folate and erythrocyte folate concentrations and tHcy concentrations. The low-folate group was defined by having a value of <11.0 nmol/L or 430 nmol/L for plasma and erythrocyte folate, respectively. These values were selected because they are generally accepted cutoffs for distinguishing between normal and inadequate folate nutritional status.28 If the folate concentrations were below the cutoffs, it is likely that the mothers did not regularly take a multivitamin/mineral tablet that contained 400 μg of folic acid. The high-tHcy group was defined by having plasma tHcy >7.0 μmol/L at each gestational time point. This arbitrary cutoff was chosen because plasma tHcy concentrations are generally low in pregnancy, and in our previous study, <30% of pregnant women with a similar socioeconomic background had tHcy values >7.0 μmol/L.29 It is likely that the mothers who had tHcy >7.0 μmol/L did not take the supplements.
Statistical Analyses
Data are presented as mean ± SD, when appropriate. The differences in the test scores between the low- or normal-folate group and high- or normal-tHcy groups were evaluated using t test and 2, when appropriate. The test scores were compared using linear regression analyses after the factors that could influence test results were adjusted for. These factors included birth weight, gestational age at birth, gender, maternal age, BMI, smoking status, alcohol and illicit drugs use, and scores of Peabody Picture Vocabulary Test and the Home Screening Questionnaire.
RESULTS
The mean age of all mothers combined was 23.7 ± 5.7 years, and they delivered infants at a mean gestational age of 38.7 ± 2.7 weeks with a mean birth weight of 3190 ± 634 g.14 As previously reported, the mean scores of the Home Screening Questionnaire (37.4 ± 6.9), Wide Range Achievement Test (43.8 ± 7.7), and Peabody Picture Vocabulary Test (73.1 ± 11.9) were similar to those observed in low-socioeconomic, inner-city populations.14
To determine whether women had consistent measures of folate status, we tested the correlation between the different measures of folate nutritional status at each time point. For each of the measures (plasma and erythrocyte folate and plasma tHcy concentrations), the correlation coefficients for the same measure at 3 time points (19, 26, 37 weeks) were generally 0.6 to 0.7, with a high degree of significance. The correlation coefficients between different measures at the same time point ranged from 0.3 to 0.6 and also were highly significant. Of the correlations between different measurements at different time points, all but 1 were significant, and most had values ranging from 0.3 to 0.6 as well. Thus, the measurements of folate nutritional status within individual women tended to be consistent.
Mean plasma folate concentrations of all mothers combined declined slightly from 35 nmol/L at 19 weeks to 34.7 and 32.5 nmol/L at 26 and 37 weeks of gestation, respectively, and the decline was significant (P = .0015). In contrast, mean erythrocyte folate concentrations steadily and significantly increased from 873 nmol/L at 19 weeks to 1070 nmol/L and 1096 nmol/L at 26 and 37 weeks of gestation, respectively (P < .0001). Mean plasma tHcy concentrations significantly increased from 5.0 μmol/L at 26 weeks to 5.6 μmol/L at 37 weeks of gestation (P < .001). We classified mothers into low versus normal plasma and erythrocyte folate groups using cutoffs of 11.0 nmol/L and 440 nmol/L, respectively, and high versus normal tHcy groups using a cutoff of 7.0 μmol/L. The distribution of mothers by each measure of poor folate status in each time point is shown in Table 1. These percentages were markedly smaller than 22% of participants who were judged as noncompliant in taking multivitamin/mineral tablets by the pill counts,13 indicating that the participants were not overclassified to the low-folate group.
Our participants could potentially have none to 9 abnormal measures of folate nutritional status (3 indices at 3 time points minus 1 tHcy at 19 weeks gestation). To determine whether the children of women with more consistently abnormal measures of folate status had different neurodevelopmental scores, we divided the population into 4 categories (0, 1, 2, and 3 or more abnormal measures). As can be seen in Table 6, there were no significant differences in the neurodevelopmental scores among the children of women with a range of folate nutritional status.
DISCUSSION
We found that folate nutritional status of mothers during the second half of pregnancy was not significantly associated with most measures of mental and psychomotor development of their children at 5.3 years of age. In this study, folate nutritional status was assessed by measuring the concentrations of plasma and erythrocyte folate and plasma tHcy. Our finding is not consistent with the findings of Gross et al,12 who reported that infants who were born to mothers with megaloblastic anemia as a result of severe folate deficiency during pregnancy had delayed neurodevelopment. The most likely explanation of this discrepancy is that our participants who were classified into the low-folate or high-tHcy groups had no overt clinical signs of folate deficiency, such as megaloblastic anemia, indicating that the levels of folate deficiency were not comparable. Considered with findings in animal studies,10,11 it may be that the degree of maternal folate deficiency during pregnancy must be sufficiently severe to have a negative impact on the brain at a critical window of the time for its development. Another explanation for the failure to find an association between maternal folate nutritional status and children’s neurodevelopment may be that they were so environmentally and educationally deprived that an effect of prenatal folate nutritional status, if any, was overwhelmed during the first 5 years of life. The negative effect of low-socioeconomic status on mental development has been well established, and is consistent with the finding of the overall mean IQ of 82 in our children. Therefore, it is unclear whether our findings are readily extrapolated to other populations with different socioeconomic backgrounds.
Additional studies are warranted to evaluate the association of maternal folate nutritional status during pregnancy to neurodevelopment of other populations of children in countries where no folic acid fortification is mandated. Our original study took place before the 1998 US mandate of folic acid fortification of enriched grain products to reduce the risk for neural-tube defects,30 and a similar mandate became effective later in Canada and Chile.31,32 Because this fortification program significantly improved folate nutritional status in these countries,32–34 it is nearly impossible to perform studies such as the one presented here.
We unexpectedly observed that maternal Peabody Picture Vocabulary Test scores were significantly higher in the high-tHcy group (37 weeks of gestation) than in the normal-tHcy group (P = .01), and a similar trend was seen at 26 weeks of gestation, although it was not significant (P = .23). These data are not in the same direction as other reports showing that elevated plasma tHcy concentrations may be associated with central atrophy of the brain in an elderly population or those with Alzheimer’s disease.35,36 Furthermore, elevated plasma tHcy concentrations or folate deficiency in growing animals have been associated with abnormal brain histology.37,38 The reason for the discrepancy between our findings and those of others is unknown. However, again, it may simply be that in this population, the degree of folate deficiency was not severe.
In summary, we found no effect of maternal folate nutritional status during the second half of pregnancy on mental and psychomotor development of children at 5 years of age. Our findings are not consistent with the negative effects of prenatal folate deficiency on the development of the brain in experimental animals and in children who are born to mothers with severe folate deficiency during pregnancy. However, it is unknown whether our finding can be extended to other populations because our participants had a relatively deprived economic status, which may have influenced the outcome of our evaluation.
ACKNOWLEDGMENTS
This work was supported in part by grants from the National Institute of Child Health and Human Development/National Institutes of Health (HD32901 to Dr Ramey) and the Agency for Health Care Policy Research (contract DHHS 282-92-0055 to Dr Goldenberg).
We extend our appreciation to Amanda Deason, Psychology Division, Sparks Clinics, Civitan International Research Center, who coordinated contact with families for administration of the child and mother assessments, and to the children and their families for participating in this study.
FOOTNOTES
Accepted Dec 30, 2004.
Tsunenobu Tamura, MD, Department of Nutrition Sciences, 455 Webb, University of Alabama at Birmingham, Birmingham, AL 35294. E-mail tamurat@uab.edu
No conflict of interest declared.
Dr Ramey’s current affiliation is Georgetown University Center on Health and Education, Georgetown University, Washington, DC.
REFERENCES
Wagner C. Biochemical role of folate in cellular metabolism. In: Bailey LB, ed. Folate in Health and Disease. New York, NY: Marcel Dekker; 1995:23–42
Baumslag N, Edelstein T, Metz J. Reduction of incidence of prematurity by folic acid supplementation in pregnancy. Br Med J. 1970;i :16 –17
Tamura T, Goldenberg RL, Freeberg LE, Cliver SP, Cutter GR, Hoffman HJ. Maternal serum folate and zinc concentrations and their relationships to pregnancy outcome. Am J Clin Nutr. 1992;56 :365 –370
MRC Vitamin study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet. 1991;338 :131 –137
Czeizel AE, Dudás I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992;327 :1832 –1835
Arakawa T, Ohara K, Kudo Z, Tada K, Hayashi T, Mizuno T. Hyperfolic-acidemia with formiminoglutamic-aciduria following histidine loading. Suggested for a case of congenital deficiency of formiminotransferase. Tohoku J Exp Med. 1963;80 :370 –382
Arakawa T, Tamura T, Higashi T, et al. Formiminotransferase deficiency syndrome associated with megaloblastic anemia responsive to pyridoxine or folic acid. Tohoku J Exp Med. 1968;94 :3 –16
Lanzkovsky P, Erlandson ME, Bezan AI. Isolated defect of folic acid absorption associated with mental retardation and cerebral calcification. Blood. 1969;34 :452 –465
Rosenblatt DS, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism. In: Scriver CR, Beaudet AL, Valle D, et al, eds. The Metabolic & Molecular Basis of Inherited Disease. 8th ed. New York, NY: McGraw-Hill; 2001:3897–3933
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
Craciunescu CN, Brown EC, Mar M-H, 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
Gross RL, Newberne PM, Reid JVO. Adverse effects on infant development associated with maternal folic acid deficiency. Nutr Rep Intern. 1974;10 :241 –248
Goldenberg RL, Tamura T, Neggers Y, et al. The effect of zinc supplementation on pregnancy outcome. JAMA. 1995;274 :463 –468
Tamura T, Goldenberg RL, Ramey SL, Nelson KG, Chapman VR. Effect of zinc supplementation to pregnant women on the mental and psychomotor development of their children at 5 y of age. Am J Clin Nutr. 2003;77 :1512 –1516
Tamura T, Goldenberg RL, Johnston KE, DuBard M. Maternal plasma zinc concentrations and pregnancy outcome. Am J Clin Nutr. 2000;71 :109 –113
Cooms CE, Gay EC, Fandal AW, Ker C, Frankenburg WK. The Home Screening Questionnaire, Reference Manual. Denver, CO: Denver Developmental Materials; 1981
Jastak S, Wilkinson GS. Wide Range Achievement Test (Revised). Wilmington, DE: Jastek Associates; 1984
Dunn LM, Dunn LM. Peabody Picture Vocabulary Test (Revised). Pine Circle, MN: American Guidance Service; 1981
Elliott CD. Differential Ability Scales. Cleveland, OH: Psychological Corporation; 1983
Kirk SA, McCarthy JJ, Kirk WD. Illinois Test of Psycholinguistic Abilities–Revised. Champaign, IL: University of Illinois Press; 1968
Stone M, Wright B. Knox’s Cube Test. Chicago, IL: Stoelting Company; 1980
Folio MR, Fewell RF. Peabody Developmental Motor Scales and Activity Cards. Allen, TX: DLM Teaching Resources; 1983
Wilson BC, Lacoviello JM, Wilson JJ, Risucci D. Purdue Pegboard performance of normal children. J Clin Neuropsychol. 1982;4 :19 –26
Keyser DJ, Sweetland RC, eds. Test Critiques. Vols. I, V, and VI, Kansas City, MO: Test Corporation of America; 1984, 1986, and 1987
Tamura T. Microbiological assay of folates. In: Picciano MF, Stokstad ELR, Gregory JF III, eds. Folic Acid Metabolism in Health and Disease. New York, NY: Wiley-Liss; 1990:121–137
Tamura T, Johnston KE, Bergman SM. Homocysteine and folate concentrations in blood from patients treated with hemodialysis. J Am Soc Nephrol. 1996;7 :2414 –2418
Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem. 1990;1 :228 –237
Herbert V. Development of human folate deficiency. In: Picciano MF, Stokstad ELR, Gregory JF III, eds. Folic Acid Metabolism in Health and Disease. New York, NY: Wiley-Liss; 1990:195–210
Pagán K, Hou J, Goldenberg RL, Cliver SP, Tamura T. Mid-pregnancy serum homocysteine and B-vitamin concentrations and fetal growth. Nutr Res. 2002;22 :1133 –1141
Food and Drug Administration. Food standards: amendment of standards of identity for enriched grain products to require addition of folic acid. Fed Reg. 1996;61 :8781 –8797
Ray JG, Meier C, Vermeulen MJ, Boss S, Wyatt PR, Cole DEC. Association of neural tube defects and folic acid food fortification in Canada. Lancet. 2002;360 :2047 –2048
Hertrampf E, Cortés F, Erickson JD, et al. Consumption of folic acid–fortified bread improves folate status in women of reproductive age in Chile. J Nutr. 2003;133 :3166 –3169
Lawrence JM, Petitti DB, Watkins M, Umekubo MA. Trends in serum folate after food fortification. Lancet. 1999;354 :915 –916
Jacques PF, Selhub J, Bostom AG, Wilson PWF, Rosenberg IH. The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N Engl J Med. 1999;340 :1449 –1454
Sachdev PS, Valenzuela M, Wang XL, Looi JCL, Brodaty H. Relationship between plasma homocysteine levels and brain atrophy in healthy elderly individuals. Neurology. 2002;58 :1539 –1541
Morris MS. Homocysteine and Alzheimer’s disease. Lancet Neurol. 2003;2 :425 –428
Chen Z, Karaplis AC, Ackerman SL, et al. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet. 2001;10 :433 –443
Lee H, Kim HJ, Kim J-m, Chang N. Effects of dietary folic acid supplementation on cerebrovascular endothelial dysfunction in rats with induced hyperhomocysteinemia. Brain Res. 2004;996 :139 –147(Tsunenobu Tamura, MD, Rob)
Department of Obstetrics and Gynecology
Department of Pediatrics
Department of Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama
ABSTRACT
Objective. There are limited data relating folate nutritional status of mothers during pregnancy to mental and psychomotor development of their offspring. Using an existing data set from a study on the effect of prenatal zinc supplementation on child neurodevelopment, we evaluated the association between folate nutritional status of mothers during pregnancy and neurodevelopment of their children.
Methods. Maternal blood folate and total homocysteine (tHcy) concentrations were measured at 19, 26, and 37 weeks of gestation. At a mean of 5.3 years of age, 355 black children with low-socioeconomic background were given 6 tests: Differential Ability Scales, Visual and Auditory Sequential Memory, Knox Cube Test, Gross Motor Scale, and Grooved Pegboard. The scores of the tests between the 2 groups of mothers with poor versus adequate folate nutritional status classified by blood folate or tHcy concentrations were compared.
Results. There were no differences in the test scores of neurodevelopment between the 2 groups.
Conclusion. Folate nutritional status of mothers in the later half of pregnancy assessed by plasma and erythrocyte folate and plasma tHcy concentrations had no impact on neurodevelopment of their children at age 5. It is unknown whether our findings in a low-socioeconomic population can be readily extrapolated to other populations.
Key Words: folate homocysteine pregnancy mothers mental and psychomotor development children
Abbreviations: tHcy, total homocysteine
Folate plays important roles in amino acid metabolism, de novo purine and thymidylate biosynthesis, and formate oxidation.1 Adequate folate nutritional status is important for normal fetal growth,2,3 and periconceptional folic acid supplementation is effective in reducing the risk for neural-tube defects.4,5 Mental retardation has been recognized as 1 of the distinct clinical features of inborn errors of folate metabolism since their early identification in the 1960s.6–8 However, the mechanism is not well understood as to how altered folate metabolism is associated with mental retardation.9 It is unknown whether the direct cause of mental retardation is the result of the imbalance in folate derivatives in the brain or other metabolic disturbance secondary to the defect of folate-dependent enzymes. Furthermore, the deprivation of certain forms of folate may be the cause of mental retardation in such cases.
Studies on the association between prenatal folate nutritional status and neurodevelopment are scarce. In 1951, Whitley et al10 reported that rats born to dams that were fed a low-folate diet and reared on the same diet had inferior maze-learning ability compared with those with adequate folate nutritional status. Craciunescu et al11 recently reported that maternal folate deficiency during days 11 to 17 of gestation in the mouse leads to the decreased progenitor cell replication and increased apoptotic cells in the fetal forebrain. They concluded that the growing brain is vulnerable to maternal folate deficiency during the critical time of its development; therefore, prenatal folate deficiency may adversely affect neurodevelopment in later life. To our knowledge, only 1 human study has been performed to evaluate the association between prenatal folate nutritional status and neurodevelopment in infants. In 1974, Gross et al12 reported that infants who were born to mothers with severe folate deficiency during pregnancy showed abnormal or delayed neurodevelopment compared with those who were born to mothers without the deficiency. We hypothesized that prenatal folate nutritional status determines mental and psychomotor development later in life in humans.
In the early 1990s, we conducted a trial to evaluate the effect of zinc supplementation during pregnancy on fetal growth in black women. In this trial, although a positive effect of zinc supplementation was found on birth weight and head circumference of newborns, there was no effect of zinc supplementation on mental and psychomotor development of children at 5 years of age.13,14 In the present study, we evaluated the association of prenatal folate nutritional status of mothers with neurodevelopment of their children using existing developmental test scores.14 Maternal folate nutritional status was assessed by plasma folate concentrations, a measure of recent folate intake and the measurement directly related to placental folate transfer to the fetus, and erythrocyte folate concentrations, a measure of a long-term folate intake. We also measured plasma total homocysteine (tHcy) concentrations, a functional measure of folate nutritional status.
METHODS
Participants
The study was approved by the Institutional Review Board for Human Use at the University of Alabama at Birmingham. The mothers of the children who were evaluated in the present study were chosen from 589 black women in a prenatal zinc supplementation trial performed between 1990 and 1993 in the Birmingham, Alabama, area.13,15 All mothers received prenatal care at the local public health clinics and were eligible for the Special Supplemental Nutrition Program for Women, Infants, and Children. In the trial, 294 mothers received a daily oral dose of 25 mg of elemental zinc and the remaining 286 received placebo from 19 weeks of gestation until delivery. All mothers were provided a daily multivitamin/mineral tablet that contained folic acid (400 μg, pteroylglutamic acid), vitamin B12 (2.0 μg, cyanocobalamin), vitamin B6 (3.0 mg, pyridoxine), vitamin B2 (2.0 mg, riboflavin), and other vitamins or minerals but not zinc (Mission Prenatal; Mission Pharmacal, San Antonio, TX). On the basis of the pill counts, the percentage of mothers who did not comply with taking the supplements was 22%.13 Fewer than 5% of this population breastfed their infants. Zinc supplementation resulted in significantly greater birth weight and head circumference of infants who were born to mothers who received zinc than those who received placebo.13 Approximately 5 years later, we recruited 355 of 589 mother–child pairs to evaluate neurodevelopment of children (178 girls and 177 boys) and found that prenatal zinc supplementation had no effect on the developmental test scores.14 Because there were no differences in any of these scores by zinc supplementation group and because in a preliminary analysis zinc supplementation had no association with any of the measures of folate nutritional status used in this study, we combined the supplemented and unsupplemented women into a single group for additional analysis. In the study presented here, we used the same developmental test scores and the values of folate and plasma tHcy concentrations in maternal blood samples that were obtained during the original trial. Because of the insufficient amount of certain samples, laboratory values were not available for all mothers.
Mental and Psychomotor Tests
Mother–child pairs were tested at a mean of 5.3 years after delivery by experienced evaluators with no knowledge of the children’s birth status or maternal conditions at delivery. Mothers received 3 tests, 2 to evaluate their cognitive function, including the Wide Range Achievement Test and the Peabody Picture Vocabulary Test, and the Home Screening Questionnaire, which provided a measure of learning resources in the house.16–18 Children took 6 tests to assess their neurodevelopment: (1) Differential Ability Scales (nonverbal, verbal, and general conceptual ability [IQ]), a standardized intelligence test for assessing the cognitive abilities; (2) Visual Sequential Memory, a test for visual memory span; (3) Auditory Sequential Memory, a test for auditory memory span; (4) Knox Cube, an index of attention span and short-term memory; (5) Gross Motor Scale, a test for gross-motor development; and (6) Grooved Pegboard (dominant and nondominant hands), a test for manipulative dexterity.19–24 The rationale for selecting these tests was described previously.14 Because there was no effect of prenatal zinc supplementation on neurodevelopment of children, the results were combined regardless of whether their mothers received zinc in the original trial.
Maternal Blood Samples and Laboratory Analyses
Nonfasting blood samples were obtained at 19, 26, and 37 weeks of gestation using evacuated tubes that contained heparin (Vacutainer; Becton Dickinson, Rutherford, NJ).13 Immediately after phlebotomy, tubes were refrigerated to avoid obtaining falsely high tHcy values. Before centrifugation to separate plasma, a portion of whole blood was removed for hematocrit measurement, and another portion was mixed with 57 mmol/L ascorbic acid solution for erythrocyte folate assay. All samples were stored at –70°C until analyzed.
Before the folate assay was performed, whole-blood lysate was incubated at 37°C for 30 minutes to allow plasma folate conjugate to hydrolyze polyglutamyl folates in erythrocytes.25 Both plasma and whole-blood folate concentrations were determined by microbiologic assay using Lactobacillus rhamnosus (formerly known as L casei; ATCC 7469), and the day-to-day coefficient of variation of this assay was <10% using pooled human plasma samples.25 Erythrocyte folate concentrations were calculated on the basis of the values of hematocrit and plasma and whole-blood folate. Plasma tHcy concentrations were measured by an high-performance liquid chromatography–fluorescent method, and the interassay coefficient of variation for this assay was 10%.26 Plasma tHcy is considered to be a functional indicator of the nutritional status of folate along with vitamin B12, vitamin B6, and vitamin B2, and these 4 B-group vitamins play critical roles in Hcy metabolism.27 Because of limited resources at the time of analysis, plasma tHcy was measured only for samples obtained at 26 and 37 weeks of gestation. Folate analysis was completed within 4 weeks after blood drawing, and tHcy was assayed within 2 years after the completion of the original trial.
Classification of Mothers on the Basis of Folate and tHcy Values
The mothers were divided into normal and poor folate status on the basis of plasma folate and erythrocyte folate concentrations and tHcy concentrations. The low-folate group was defined by having a value of <11.0 nmol/L or 430 nmol/L for plasma and erythrocyte folate, respectively. These values were selected because they are generally accepted cutoffs for distinguishing between normal and inadequate folate nutritional status.28 If the folate concentrations were below the cutoffs, it is likely that the mothers did not regularly take a multivitamin/mineral tablet that contained 400 μg of folic acid. The high-tHcy group was defined by having plasma tHcy >7.0 μmol/L at each gestational time point. This arbitrary cutoff was chosen because plasma tHcy concentrations are generally low in pregnancy, and in our previous study, <30% of pregnant women with a similar socioeconomic background had tHcy values >7.0 μmol/L.29 It is likely that the mothers who had tHcy >7.0 μmol/L did not take the supplements.
Statistical Analyses
Data are presented as mean ± SD, when appropriate. The differences in the test scores between the low- or normal-folate group and high- or normal-tHcy groups were evaluated using t test and 2, when appropriate. The test scores were compared using linear regression analyses after the factors that could influence test results were adjusted for. These factors included birth weight, gestational age at birth, gender, maternal age, BMI, smoking status, alcohol and illicit drugs use, and scores of Peabody Picture Vocabulary Test and the Home Screening Questionnaire.
RESULTS
The mean age of all mothers combined was 23.7 ± 5.7 years, and they delivered infants at a mean gestational age of 38.7 ± 2.7 weeks with a mean birth weight of 3190 ± 634 g.14 As previously reported, the mean scores of the Home Screening Questionnaire (37.4 ± 6.9), Wide Range Achievement Test (43.8 ± 7.7), and Peabody Picture Vocabulary Test (73.1 ± 11.9) were similar to those observed in low-socioeconomic, inner-city populations.14
To determine whether women had consistent measures of folate status, we tested the correlation between the different measures of folate nutritional status at each time point. For each of the measures (plasma and erythrocyte folate and plasma tHcy concentrations), the correlation coefficients for the same measure at 3 time points (19, 26, 37 weeks) were generally 0.6 to 0.7, with a high degree of significance. The correlation coefficients between different measures at the same time point ranged from 0.3 to 0.6 and also were highly significant. Of the correlations between different measurements at different time points, all but 1 were significant, and most had values ranging from 0.3 to 0.6 as well. Thus, the measurements of folate nutritional status within individual women tended to be consistent.
Mean plasma folate concentrations of all mothers combined declined slightly from 35 nmol/L at 19 weeks to 34.7 and 32.5 nmol/L at 26 and 37 weeks of gestation, respectively, and the decline was significant (P = .0015). In contrast, mean erythrocyte folate concentrations steadily and significantly increased from 873 nmol/L at 19 weeks to 1070 nmol/L and 1096 nmol/L at 26 and 37 weeks of gestation, respectively (P < .0001). Mean plasma tHcy concentrations significantly increased from 5.0 μmol/L at 26 weeks to 5.6 μmol/L at 37 weeks of gestation (P < .001). We classified mothers into low versus normal plasma and erythrocyte folate groups using cutoffs of 11.0 nmol/L and 440 nmol/L, respectively, and high versus normal tHcy groups using a cutoff of 7.0 μmol/L. The distribution of mothers by each measure of poor folate status in each time point is shown in Table 1. These percentages were markedly smaller than 22% of participants who were judged as noncompliant in taking multivitamin/mineral tablets by the pill counts,13 indicating that the participants were not overclassified to the low-folate group.
Our participants could potentially have none to 9 abnormal measures of folate nutritional status (3 indices at 3 time points minus 1 tHcy at 19 weeks gestation). To determine whether the children of women with more consistently abnormal measures of folate status had different neurodevelopmental scores, we divided the population into 4 categories (0, 1, 2, and 3 or more abnormal measures). As can be seen in Table 6, there were no significant differences in the neurodevelopmental scores among the children of women with a range of folate nutritional status.
DISCUSSION
We found that folate nutritional status of mothers during the second half of pregnancy was not significantly associated with most measures of mental and psychomotor development of their children at 5.3 years of age. In this study, folate nutritional status was assessed by measuring the concentrations of plasma and erythrocyte folate and plasma tHcy. Our finding is not consistent with the findings of Gross et al,12 who reported that infants who were born to mothers with megaloblastic anemia as a result of severe folate deficiency during pregnancy had delayed neurodevelopment. The most likely explanation of this discrepancy is that our participants who were classified into the low-folate or high-tHcy groups had no overt clinical signs of folate deficiency, such as megaloblastic anemia, indicating that the levels of folate deficiency were not comparable. Considered with findings in animal studies,10,11 it may be that the degree of maternal folate deficiency during pregnancy must be sufficiently severe to have a negative impact on the brain at a critical window of the time for its development. Another explanation for the failure to find an association between maternal folate nutritional status and children’s neurodevelopment may be that they were so environmentally and educationally deprived that an effect of prenatal folate nutritional status, if any, was overwhelmed during the first 5 years of life. The negative effect of low-socioeconomic status on mental development has been well established, and is consistent with the finding of the overall mean IQ of 82 in our children. Therefore, it is unclear whether our findings are readily extrapolated to other populations with different socioeconomic backgrounds.
Additional studies are warranted to evaluate the association of maternal folate nutritional status during pregnancy to neurodevelopment of other populations of children in countries where no folic acid fortification is mandated. Our original study took place before the 1998 US mandate of folic acid fortification of enriched grain products to reduce the risk for neural-tube defects,30 and a similar mandate became effective later in Canada and Chile.31,32 Because this fortification program significantly improved folate nutritional status in these countries,32–34 it is nearly impossible to perform studies such as the one presented here.
We unexpectedly observed that maternal Peabody Picture Vocabulary Test scores were significantly higher in the high-tHcy group (37 weeks of gestation) than in the normal-tHcy group (P = .01), and a similar trend was seen at 26 weeks of gestation, although it was not significant (P = .23). These data are not in the same direction as other reports showing that elevated plasma tHcy concentrations may be associated with central atrophy of the brain in an elderly population or those with Alzheimer’s disease.35,36 Furthermore, elevated plasma tHcy concentrations or folate deficiency in growing animals have been associated with abnormal brain histology.37,38 The reason for the discrepancy between our findings and those of others is unknown. However, again, it may simply be that in this population, the degree of folate deficiency was not severe.
In summary, we found no effect of maternal folate nutritional status during the second half of pregnancy on mental and psychomotor development of children at 5 years of age. Our findings are not consistent with the negative effects of prenatal folate deficiency on the development of the brain in experimental animals and in children who are born to mothers with severe folate deficiency during pregnancy. However, it is unknown whether our finding can be extended to other populations because our participants had a relatively deprived economic status, which may have influenced the outcome of our evaluation.
ACKNOWLEDGMENTS
This work was supported in part by grants from the National Institute of Child Health and Human Development/National Institutes of Health (HD32901 to Dr Ramey) and the Agency for Health Care Policy Research (contract DHHS 282-92-0055 to Dr Goldenberg).
We extend our appreciation to Amanda Deason, Psychology Division, Sparks Clinics, Civitan International Research Center, who coordinated contact with families for administration of the child and mother assessments, and to the children and their families for participating in this study.
FOOTNOTES
Accepted Dec 30, 2004.
Tsunenobu Tamura, MD, Department of Nutrition Sciences, 455 Webb, University of Alabama at Birmingham, Birmingham, AL 35294. E-mail tamurat@uab.edu
No conflict of interest declared.
Dr Ramey’s current affiliation is Georgetown University Center on Health and Education, Georgetown University, Washington, DC.
REFERENCES
Wagner C. Biochemical role of folate in cellular metabolism. In: Bailey LB, ed. Folate in Health and Disease. New York, NY: Marcel Dekker; 1995:23–42
Baumslag N, Edelstein T, Metz J. Reduction of incidence of prematurity by folic acid supplementation in pregnancy. Br Med J. 1970;i :16 –17
Tamura T, Goldenberg RL, Freeberg LE, Cliver SP, Cutter GR, Hoffman HJ. Maternal serum folate and zinc concentrations and their relationships to pregnancy outcome. Am J Clin Nutr. 1992;56 :365 –370
MRC Vitamin study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet. 1991;338 :131 –137
Czeizel AE, Dudás I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med. 1992;327 :1832 –1835
Arakawa T, Ohara K, Kudo Z, Tada K, Hayashi T, Mizuno T. Hyperfolic-acidemia with formiminoglutamic-aciduria following histidine loading. Suggested for a case of congenital deficiency of formiminotransferase. Tohoku J Exp Med. 1963;80 :370 –382
Arakawa T, Tamura T, Higashi T, et al. Formiminotransferase deficiency syndrome associated with megaloblastic anemia responsive to pyridoxine or folic acid. Tohoku J Exp Med. 1968;94 :3 –16
Lanzkovsky P, Erlandson ME, Bezan AI. Isolated defect of folic acid absorption associated with mental retardation and cerebral calcification. Blood. 1969;34 :452 –465
Rosenblatt DS, Fenton WA. Inherited disorders of folate and cobalamin transport and metabolism. In: Scriver CR, Beaudet AL, Valle D, et al, eds. The Metabolic & Molecular Basis of Inherited Disease. 8th ed. New York, NY: McGraw-Hill; 2001:3897–3933
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
Craciunescu CN, Brown EC, Mar M-H, 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
Gross RL, Newberne PM, Reid JVO. Adverse effects on infant development associated with maternal folic acid deficiency. Nutr Rep Intern. 1974;10 :241 –248
Goldenberg RL, Tamura T, Neggers Y, et al. The effect of zinc supplementation on pregnancy outcome. JAMA. 1995;274 :463 –468
Tamura T, Goldenberg RL, Ramey SL, Nelson KG, Chapman VR. Effect of zinc supplementation to pregnant women on the mental and psychomotor development of their children at 5 y of age. Am J Clin Nutr. 2003;77 :1512 –1516
Tamura T, Goldenberg RL, Johnston KE, DuBard M. Maternal plasma zinc concentrations and pregnancy outcome. Am J Clin Nutr. 2000;71 :109 –113
Cooms CE, Gay EC, Fandal AW, Ker C, Frankenburg WK. The Home Screening Questionnaire, Reference Manual. Denver, CO: Denver Developmental Materials; 1981
Jastak S, Wilkinson GS. Wide Range Achievement Test (Revised). Wilmington, DE: Jastek Associates; 1984
Dunn LM, Dunn LM. Peabody Picture Vocabulary Test (Revised). Pine Circle, MN: American Guidance Service; 1981
Elliott CD. Differential Ability Scales. Cleveland, OH: Psychological Corporation; 1983
Kirk SA, McCarthy JJ, Kirk WD. Illinois Test of Psycholinguistic Abilities–Revised. Champaign, IL: University of Illinois Press; 1968
Stone M, Wright B. Knox’s Cube Test. Chicago, IL: Stoelting Company; 1980
Folio MR, Fewell RF. Peabody Developmental Motor Scales and Activity Cards. Allen, TX: DLM Teaching Resources; 1983
Wilson BC, Lacoviello JM, Wilson JJ, Risucci D. Purdue Pegboard performance of normal children. J Clin Neuropsychol. 1982;4 :19 –26
Keyser DJ, Sweetland RC, eds. Test Critiques. Vols. I, V, and VI, Kansas City, MO: Test Corporation of America; 1984, 1986, and 1987
Tamura T. Microbiological assay of folates. In: Picciano MF, Stokstad ELR, Gregory JF III, eds. Folic Acid Metabolism in Health and Disease. New York, NY: Wiley-Liss; 1990:121–137
Tamura T, Johnston KE, Bergman SM. Homocysteine and folate concentrations in blood from patients treated with hemodialysis. J Am Soc Nephrol. 1996;7 :2414 –2418
Finkelstein JD. Methionine metabolism in mammals. J Nutr Biochem. 1990;1 :228 –237
Herbert V. Development of human folate deficiency. In: Picciano MF, Stokstad ELR, Gregory JF III, eds. Folic Acid Metabolism in Health and Disease. New York, NY: Wiley-Liss; 1990:195–210
Pagán K, Hou J, Goldenberg RL, Cliver SP, Tamura T. Mid-pregnancy serum homocysteine and B-vitamin concentrations and fetal growth. Nutr Res. 2002;22 :1133 –1141
Food and Drug Administration. Food standards: amendment of standards of identity for enriched grain products to require addition of folic acid. Fed Reg. 1996;61 :8781 –8797
Ray JG, Meier C, Vermeulen MJ, Boss S, Wyatt PR, Cole DEC. Association of neural tube defects and folic acid food fortification in Canada. Lancet. 2002;360 :2047 –2048
Hertrampf E, Cortés F, Erickson JD, et al. Consumption of folic acid–fortified bread improves folate status in women of reproductive age in Chile. J Nutr. 2003;133 :3166 –3169
Lawrence JM, Petitti DB, Watkins M, Umekubo MA. Trends in serum folate after food fortification. Lancet. 1999;354 :915 –916
Jacques PF, Selhub J, Bostom AG, Wilson PWF, Rosenberg IH. The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N Engl J Med. 1999;340 :1449 –1454
Sachdev PS, Valenzuela M, Wang XL, Looi JCL, Brodaty H. Relationship between plasma homocysteine levels and brain atrophy in healthy elderly individuals. Neurology. 2002;58 :1539 –1541
Morris MS. Homocysteine and Alzheimer’s disease. Lancet Neurol. 2003;2 :425 –428
Chen Z, Karaplis AC, Ackerman SL, et al. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition. Hum Mol Genet. 2001;10 :433 –443
Lee H, Kim HJ, Kim J-m, Chang N. Effects of dietary folic acid supplementation on cerebrovascular endothelial dysfunction in rats with induced hyperhomocysteinemia. Brain Res. 2004;996 :139 –147(Tsunenobu Tamura, MD, Rob)