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American Journal of Clinical Nutrition, Vol. 88, No. 2, 448-457, August 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Folate status and homocysteine response to folic acid doses and withdrawal among young Chinese women in a large-scale randomized double-blind trial 1,2,3,4

Ling Hao, Quan-He Yang, Zhu Li, Lynn B Bailey, Jiang-Hui Zhu, Dale J Hu, Bo-Lan Zhang, J David Erickson, Le Zhang, Jacqueline Gindler, Song Li and Robert J Berry

1 From the National Reference Laboratory on Reproductive and Child Health, Ministry of Health and National Center for Maternal and Infant Health, Peking University Health Science Center, Beijing, China (LH, ZL, J-HZ, LZ, and SL); the National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA (Q-HY, DJH, JDE, and RJB); the Food Science and Human Nutrition Department, University of Florida, Gainesville, FL (LBB); the Maternal and Child Health Institute, Xianghe County, Hebei Province, China (B-LZ); the National Center for Preparedness, Detection, and Control of Infectious Diseases, Division of Global Migration and Quarantine and the Thailand MOPH-US CDC Collaboration, Bangkok, Thailand (JG). LH and Q-HY contributed equally to the study

2 The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

3 Supported by a cooperative agreement with the Centers for Disease Control and Prevention.

4 Reprints not available. Address correspondence to Z Li, National Center for Maternal and Infant Health, Peking University Health Science Center, Room 101, Research Center Building, 38 College Road, Haidian District, Beijing 100083, PRC. E-mail: lizhu3699{at}gmail.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: There are no large randomized trials of the effect of folic acid dosing regimens on blood folate and homocysteine concentrations.

Objective: We aimed to evaluate the changes in folate and homocysteine concentrations in response to different folic acid doses and to withdrawal in young women not exposed to other sources of folic acid.

Design: Women (n = 1108) were randomly assigned to 1 of 6 intervention groups for which daily intakes of folic acid for 6 mo were 100 µg 1 time/d, 25 µg 4 times/d, 400 µg 1 time/d, 100 µg 4 times/d, 4000 µg 1 time/d, or 4000 µg 1 time/wk. Plasma and red blood cell folate and homocysteine concentrations were measured at baseline; at 1, 3, and 6 mo; and 3 mo after the discontinuation of folic acid.

Results: Folate and homocysteine concentrations were not different at baseline between the groups who had the same daily intake of folic acid as a single dose or multiple doses (P = 0.058). Plasma folate concentrations plateaued at 3 mo with 108% (95% CI: 97.7%, 120%), 259% (95% CI: 240%, 279%), 460% (95% CI: 417%, 503%), and 142% (95% CI: 123%, 162%) observed increases for the folic acid groups receiving 100, 400, and 4000 µg/d and 4000 µg/wk, respectively. The rate of reduction in folate concentrations during the 3 mo after cessation of folic acid was dose-dependent—higher intakes were associated with faster reductions.

Conclusions: Changes in folate and homocysteine concentrations were unaffected by different dosing schedules. After folic acid cessation, blood folate declined rapidly, which indicated that the intervention-enhanced folate status was rapidly diminished.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maternal use of periconceptional folic acid supplements or fortified foods reduces the risk of neural tube defects (NTDs) (1, 2). A population-based cohort study in China showed that maternal use of 400 µg folic acid/d alone reduced the risk of NTDs by up to 85% (2). In the United States, folic acid supplements are recommended for all women of childbearing age, and the US Food and Drug Administration (FDA) mandated folic acid fortification to reduce the risk of NTDs (3, 4). Policies of mandatory folic acid fortification in the United States, Canada, and Chile have led to significant reductions in NTD prevalence (5-8), and this public health measure is now being considered in many other countries (9).

Blood folate concentration among the US population increased dramatically after fortification, and several studies have suggested that the observed increases may be due in part to the consumption of fortified foods containing folic acid in amounts higher that those required by FDA (10-14). There has been considerable discussion about the optimal intake of folic acid in both supplementation and fortification programs (15-19). It is likely that the reduced NTD risk afforded by folic acid is due to improved maternal folate status; however, there are few data comparing changes in folate status in response to chronic consumption of specific amounts of folic acid in fortified foods relative to the consumption of supplemental folic acid (20-22).

Although a few studies examined the reduction of folate concentration after cessation of folic acid supplements (23, 24), this change has not been evaluated in a large-scale, population-based study. The rate of reduction of folate concentration after intervention could have significant public health policy implications related to NTD risk reduction, because women may become pregnant after discontinuing folic acid supplementation. In the United States, most pregnancies are unplanned, and women of reproductive age who take supplements do so on an irregular basis (25).

The present randomized population-based study is the first to determine the timing and dose-response relation between folic acid intake and blood folate concentration and the effects of cessation of supplements on folate concentration. The objectives of the present trial were 1) to compare the blood folate and homocysteine response to daily intakes of folic acid administered as either a single dose (simulating supplementation) or divided (4 times/d) daily doses (simulating fortification), 2) to evaluate the rate of change in blood folate and homocysteine concentrations in response to different folic acid quantities and dosing schedules, 3) to examine the dose-response relation between folic acid intake and blood folate and homocysteine concentrations; and 4) to examine the magnitude and rate of reduction of folate and homocysteine concentrations after cessation of supplementation.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Setting and participants
The intervention trial was conducted in 6 townships of Xianghe County, Hebei Province, in northern China, near Beijing. Eligible women included residents of the townships who were not anemic (hemoglobin > 12 g/dL) or vitamin B-12 deficient (plasma vitamin B-12 concentration > 148 pmol/L). Given the safety consideration that high doses of folic acid (eg, 4000 µg folic acid/d) exceed the Tolerable Upper Level of folic acid intake as recommended by the Institute of Medicine (IOM) (26) and have the potential to mask the diagnosis of anemia in persons with vitamin B-12 deficiency, vitamin B-12–deficient women were excluded. Additional eligibility criteria included no chronic disease (eg, hypertension, diabetes, cancer, or epilepsy); no supplement use within the past 3 mo; no routine use of prescription medication; no current pregnancy or breastfeeding; delivery of an infant 2–4 y before the initiation of the study; no plans to become pregnant within the next 9 mo; and use of an intrauterine device (IUD) for contraception. A total of 2084 women were contacted, of whom 1702 (81.7%) agreed to participate; these women underwent baseline assessments of hematologic and vitamin status. Of this group, 206 (12.1%) anemic women and 305 (17.9%) vitamin B-12–deficient women were excluded. Fifty-two women were both anemic and vitamin B-12 deficient at baseline, and those women were referred to physicians for evaluation, diagnosis, and treatment. In addition, women who described themselves as chronically ill, who had a diagnosed chronic disease, or who were planning to become pregnant within the next 9 mo were excluded (n = 31). The remaining 1108 women (24–42 y old) were randomly assigned to 1 of 6 intervention groups in which daily intakes of folic acid for 6 mo were 100 µg 1 time/d, 25 µg 4 times/d, 400 µg 1 time/d, 100 µg 4 times/d, 4000 µg 1 time/day, or 4000 µg 1 time/wk (Table 1Go).


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  		TABLE 1 Dosing schedule and pill composition for 6 folic acid intervention groups

 
All participants provided oral informed consent. This study was approved by the Centers for Disease Control and Prevention (CDC) Institutional Review Board (IRB) and the Ethical Committee on Biomedical Research Involving Human Subjects, Peking University, Health Science Center, Beijing, People's Republic of China.

Study design
A double-blind randomization was used to assign equal numbers of women to each of the 6 folic acid intervention groups. A placebo group was not included because the repeated-measures design of the study allowed each subject to serve as her own control. The study was designed to have 80% power to detect a 15% difference in means with an SD of 45% and at a significance level of 0.05. It was estimated that {approx}160 participants would be needed for each group, and that {approx}15% of participants would drop out during follow-up; therefore, 185 women were required for each group. Women were issued an identification card linking their identity with assigned supplements, which were given out weekly in each subject's home by trained village physicians, who also monitored the subject's health status and compliance with the pill-taking protocol. Each subject filled out a questionnaire daily to provide information regarding any adverse physical symptoms that may have occurred. The prevalence of self-reported adverse events (eg, nausea, vomiting, diarrhea, constipation, dizziness, or headache) did not differ significantly across folic acid intervention groups at any time during follow-up, which suggested that the folic acid doses were unlikely to be associated with the prevalence of self-reported adverse events during the trial.

Blood collection procedures
Fasting blood samples were collected at the time of enrollment and at the end of 1, 3, 6, and 9 mo. Blood was collected in tubes with EDTA (Vacutainer; Becton Dickinson, Franklin, NJ) for red blood cell (RBC) folate, plasma folate, vitamin B-12, and homocysteine concentrations. The plasma was separated by centrifugation (4 °C, 2000 x g, 15 min) and frozen at –20 °C within 1 h of collection. All specimens were transported on dry ice to the central laboratory of the Institute of Reproductive and Child Health, Peking University, and stored at –70 °C until analyzed.

Biochemical measurements of blood folate and homocysteine concentrations
Plasma and RBC folate concentrations were measured by a microbiological assay (Lactobacillus casei) by using the microtiter technique described by O'Broin et al (27). Plasma homocysteine measurements were carried out by using HPLC with fluorometric detection (28). Plasma vitamin B-12 was measured in duplicate samples by using the Quantaphase II radioassay (Bio-Rad Laboratories, Hercules, CA).

The intraassay and interassay CVs for these assays were <9% for folate, <8% for plasma homocysteine, and <7% for vitamin B-12 concentrations. Before the initiation of sample analysis, a large number of control plasma samples with high, medium, and low folate concentrations and a set of control samples with high, medium, and low plasma homocysteine concentrations were prepared for analysis. During this pilot testing phase, these quality-control (QC) samples were analyzed repeatedly over the course of several weeks to determine the value for each QC sample. The mean ± 2SD and interassay and intraassay CV% for the full concentration range of folate and homocysteine also were calculated, and the upper and lower limits for the QC monitoring graph were then determined. During the sample analysis phase of the study, {approx}300 assays/wk were performed for both folate and homocysteine for each subject at each time-point. Within each assay, for the entire duration of the study, replicate pooled plasma control samples at high, medium, and low concentrations were analyzed to validate each folate and homocysteine value. Throughout the entire study, these blinded controls were included in all assay runs to ensure data quality and comparability. Established upper or lower limits (mean ± 2SD) on the QC monitoring graph were used to monitor any discrepancies or drift over time that would have triggered retesting. Such trends were not observed, and the methods and resultant data for folate and homocysteine were quite stable. The QC monitoring system for this study ensured that the observed time trends were real and verified that the changes over time were not due to variations within the assays for either folate or homocysteine.

Statistical analysis
Because the distributions of folate and homocysteine concentrations were skewed to the right, their logarithms were used in the analysis. The geometric means and 95% CIs were calculated for folate and homocysteine concentrations. To account for the correlated measurements during follow-up, we used the generalized linear mixed model for repeated measures for all primary analyses. The outcome variables were plasma, RBC folate, and homocysteine concentrations at each follow-up time. The model included baseline folate or homocysteine measures, 6 intervention groups, time (repeated measures), and interaction between intervention group and time as fixed effects (29). Toeplitz covariance structure was selected for the analysis on the basis of –2 log likelihood and the information criteria of Akaike and Schwarz, as discussed by Littell et al (30). The mixed model for repeated-measure analysis was restricted to 0-, 1-, 3-, and 6-mo follow-up measures. Consecutive differences in folate and homocysteine concentration during follow-up were tested for each intervention group; they defined the nonsignificant change in concentration from one follow-up to the next as evidence of reaching a plateau in changes in concentration. We used the t test to identify the plateau with Bonferroni correction. To compare the folate and homocysteine concentrations at month 9 (3 mo after cessation of folic acid) with the baseline concentrations, we used analysis of variance (ANOVA), in which the 9-mo and baseline measurements were used as an outcome variable and a covariate, respectively (29). Assuming a linear reduction in folate and homocysteine concentrations after the cessation of supplements, the average monthly percentage changes in concentrations were estimated. To adjust for the possible effects of seasonal variation in folate intake on blood folate and homocysteine concentrations during the follow-up, we included the covariates in the model to indicate whether the follow-up for each participant was in the high (June–November) or low (December–May) folate seasons (31). The differences in folate and homocysteine concentrations between the adjusted and unadjusted results for seasonality were <3%, and therefore we presented the unadjusted results.

We calculated the percentage change in folate and homocysteine concentrations at 6 mo for women receiving 100 and 400 µg folic acid/d relative from the concentrations at 6 mo among women taking 4000 µg folic acid/d [which was taken as a maximum or a minimum (for homocysteine) concentration]. For timing and percentage change analysis, the 100 µg 1 time/d group and the 25 µg 4 times/d group were combined, as were the 400 µg/d group and the 100 µg 4 times/d group, because there were no significant differences in folate and homocysteine concentration at follow-up between these groups. For the 43 missing data points among a total of 4004 observations for 1, 3, and 6 mo, the "last observation carry-forward" technique was applied to fill in these missing values (32). Multiple comparisons were accounted for by calculating the Bonferroni-adjusted P value. PROC MIXED was used for the repeated-measures analysis in SAS software (version 9.1; SAS Institute Inc, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 1108 women were randomly assigned to 1 of 6 intervention groups. Women in the groups were similar with regard to baseline characteristics. On average, women took >95% of all pills during the 24-wk study (Table 2Go). The 107 women who dropped out at month 1 were evenly distributed among the groups (Figure 1Go).


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  		TABLE 2 Baseline characteristics of the participants1

 

Figure 1
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  		FIGURE 1. Flow diagram of the women from Xianghe County, Hubei Province, China, who participated in the randomized trial.

 
Effects of different daily intakes and dosing schedules of folic acid
Mean folate and homocysteine concentrations for groups who had the same daily intake of folic acid (100 and 400 µg/d) according to different dosing schedules are presented in Table 3Go. Blood folate and homocysteine concentrations were similar in groups with the same total intake for each of the measurements during the 6-mo period of folic acid supplementation (Table 3Go). The interactions between follow-up time and the same daily folic acid intake groups were not significant except for plasma folate concentration among 100 µg folic acid doses (P < 0.05). At 3 mo, 100-µg folic acid doses taken 4 times/d resulted in a 14.2% (95% CI: 6.8%, 27.1%) higher plasma folate concentration than that observed in the group that took 400 µg/d at 3 mo (adjusted P = 0.058). Results with or without the last observation carry-forward technique for missing data were virtually identical, and therefore the results with the estimates for missing data were presented.


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  		TABLE 3 Estimated geometric means (and 95% CI) of folate and homocysteine concentrations among women receiving the same amount of folic acid by different dosing schedules1

 
Timing and effect of stopping supplements
Plasma folate concentration plateaued after 3 mo of pill taking in all groups (Table 4Go). Plasma folate concentrations among women who took 100 µg/d, 400 µg/d, 4000 µg/d and 4000 µg/wk were 108% (95% CI: 97.7%, 120%), 259% (95% CI: 240%, 279%), 460% (95% CI: 417%, 503%), and 142% (95% CI: 123%, 162%) higher, respectively, at 6 mo than at baseline. The increase in plasma folate concentrations during the trial was nonlinear: >80% of the increase in concentration took place during the first month. In the 4000 µg/d group, the maximum plasma folate concentration was reached at 3 mo, and the concentration then decreased between 3 and 6 mo (Table 4Go).


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  		TABLE 4 Estimated geometric mean (and 95% CI) folate and homocysteine concentrations by different folic acid–dosing schedules and different follow-up times1

 
The increase in plasma folate concentrations at 6 mo among women whose folic acid dose was 100 or 400 µg/d was 23.5% and 55.8% of the maximum increase in concentration (maximum as measured by 4000 µg/d at 6 mo), respectively. Assuming a linear rate of reduction, plasma folate concentration decreased 1.9, 6.0, 12, and 3.1 nmol · L–1 · mo–1 up to 3 mo after cessation of folic acid in the 100 µg/d, 400 µg/d, 4000 µg/d and 4000 µg/wk groups, respectively. However, 3 mo after folic acid cessation, plasma folate concentrations were significantly higher than baseline in all intervention groups. At 9 mo, the concentrations in women who took folic acid doses of 400 and 4000 µg/d were significantly (adjusted P < 0.05) higher than those in women who took folic acid doses of 100 µg/d and 4000 µg/wk, respectively (Table 4Go).

RBC folate concentration had not plateaued at 6 mo, although the rate of increase in concentration was less from 3 to 6 mo than that from 1 to 3 mo (Table 4Go). At 6 mo, RBC folate concentrations were 27.7% (95% CI: 22.9%, 32.7%), 71.8% (95% CI: 65.2%, 78.4%), 137% (95% CI: 124%, 150%), and 45.7% (95% CI: 37.5%, 53.9%) higher than those at baseline in the 100 µg/d, 400 µg/d, 4000 µg/d, and 4000 µg/wk groups, respectively. In contrast to plasma folate concentration, the increase in RBC folate concentration was approximately linear from baseline to the 3-mo follow-up: the average monthly increase in concentration was 21.5, 104, 248, and 57 nmol/L in the 100 µg/d, 400 µg/d, 4000 µg/d, and 4000 µg/wk groups, respectively (Table 4Go).

The percentage change in RBC folate concentrations was similar to that of plasma folate concentrations: doses of 100 and 400 µg folic acid/d achieved 20.1% and 52.7% of the maximum increase in concentration, respectively. RBC concentrations decreased 45.8, 103.4, 192, and 74.4 nmol · L–1 · mo–1 during the 3 mo after folic acid cessation in the 100 µg/d, 400 µg/d, 4000 µg/d, and 4000 µg/wk groups, respectively. At 3 mo after folic acid cessation, RBC folate concentrations returned to a level equivalent to the concentrations after 1 mo of intervention in all groups, and they remained significantly higher than baseline only among women receiving 400 and 4000 µg folic acid/d (Table 4Go). However, at 9 mo of follow-up, the RBC folate concentrations among women who took 4000 µg/d were significantly (adjusted P < 0.001) higher than those among women who took 400 µg/d, and the concentration in the 400 µg/d group was significantly higher than the concentrations in the 100 µg/d and 4000 µg/wk (adjusted P < 0.001 and P = 0.054, respectively) groups. The difference in RBC folate concentrations at 9 mo between 100 µg/d and 4000 µg/wk was not significant (adjusted P = 0.841) (Table 4Go).

The decline in homocysteine concentrations from month 3 to month 6 of follow-up became nonsignificant in all intervention groups (Table 4Go). Homocysteine concentrations were 4.5% (95% CI: 1.6%, 7.5%), 16.9% (95% CI: 14.2%, 19.3%), 22.1% (95% CI: 18.8%, 25.55) and 9.2% (95% CI: 5.0%, 13.2%) lower at the 6-mo follow-up than at baseline in the 100 µg/d, 400 µg/d, 4000 µg/d, and 4000 µg/wk groups, respectively. The decline in homocysteine concentration at 6 mo associated with different doses of folic acid intake appeared to be nonlinear: the 100 and 400 µg/d groups achieved 17.6% and 76.5%, respectively, of the maximum reduction in homocysteine (as measured by 4000 µg/d at 6 mo). After folic acid had been stopped for 3 mo, homocysteine concentrations returned to baseline among women receiving folic acid doses of 100 µg/d and 4000 µg/wk, and they remained significantly lower than baseline among women receiving 400 and 4000 µg/d (Table 4Go).

Changes in homocysteine concentrations after 1 mo of folic acid supplementation
Homocysteine concentrations differed between folic acid intake groups after 1 mo of supplementation. At the 1-mo follow-up, compared with baseline concentrations, homocysteine concentrations increased significantly among women receiving 100 µg of folic acid/d and 4000 µg of folic acid/wk (adjusted P < 0.001), decreased significantly among women receiving 4000 µg/d (adjusted P < 0.001), and was unchanged among women taking 400 µg/d (Table 4Go). To examine the differences in homocysteine concentration related to folic acid intakes at 1 mo, an additional analyses stratified by quartiles of baseline homocysteine concentration was conducted (Figure 2Go); these analyses indicated that, in response to folic acid intakes of 100 µg/d or 4000 µg/wk, the observed increase in homocysteine from baseline to 1 mo occurred irrespective of baseline homocysteine concentration, although some increases were not statistically significant. In contrast, baseline homocysteine concentrations did appear to influence the initial (baseline to 1 mo) response to 400 and 4000 µg/d, because homocysteine increased significantly in the lowest quartiles (adjusted P < 0.01), did not change in the second quartiles of baseline homocysteine, and then shifted to a different pattern of decrease as baseline homocysteine concentrations increased (Figure 2Go).


Figure 2
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  		FIGURE 2. Geometric x (and 95% CI) plasma homocysteine concentrations (µmol/L) at baseline and at 1-mo follow-up by baseline quartiles (low = <25th percentile; 2 = ≥25th and <50th percentile; 3 = ≥50th and <75th percentile; and high = ≥75th percentile) of homocysteine concentration (µmol/L) and different folic acid–dosing groups. *P < 0.01, **P < 0.001 (both: based on pair-wise t tests). P values for interactions between the follow-up time and quartiles of baseline homocysteine concentrations were 0.0020, <0.0001, <0.0001, and 0.3696 for the 100 µg/d, 400 µg/d, 4000 µg/d, and 4000 µg/wk folic acid intervention groups, respectively, based on F statistics. P values were adjusted by Bonferroni correction for multiple comparisons. All tests were 2-tailed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The design of this investigation provided the opportunity to evaluate changes in blood indexes during folic acid intervention and after the termination of the intervention. Our findings suggested that changes in blood folate and homocysteine concentrations were insensitive to different dosing schedules of the same daily amount of folic acid intake. There was a nonlinear dose-response relation between folic acid intake and blood folate and homocysteine concentrations, and a weekly dose of 4000 µg folic acid was relatively ineffective in increasing blood folate concentration. The effects of folic acid on blood folate diminished relatively rapidly after cessation of folic acid intervention, and the predicted NTD risk reduction associated with supplemental folic acid intake would be rapidly lost after supplementation was halted.

Besides the reduction of NTD risk, several studies suggested that folic acid intake may be associated with other benefits (33-37) and also with risks, especially the risks of certain types of cancer (34, 38-40). In terms of potential adverse effects of folic acid supplementation, this short-term intervention study was not designed to assess safety endpoints, and the assessment was limited to self-reported monitoring. The exclusion of vitamin B-12–deficient persons reduced the possibility of misdiagnosis or masking of a vitamin B-12 deficiency (26). Because the issue of whether folic acid reduces or increases the risk of certain conditions is very complex (39, 41) and is strongly related to dose, as in the possible masking of vitamin B-12 deficiency (42), this study provides extremely important information that allows a better understanding of the complex relations between folic acid intake and folate concentrations in the body.

In all intervention groups, plasma folate concentrations plateaued between 3 and 6 mo, whereas the RBC folate concentrations did not plateau, even after 6 mo of folic acid administration; similar findings have been reported by other investigators (23, 43). Previous studies have suggested that RBC blood folate concentrations increase linearly as folic acid intake increases (22, 44). Although the relation may be approximately linear at lower folic acid intakes (eg, <1000 µg/d)(22, 44), the data from those studies suggest that the whole dose-response curve might be nonlinear. Our results indicated that 100 µg folic acid/d was associated with an increase of {approx}24% of maximum in folate concentration (measured by 4000 µg/d); in response to a 400 µg folic acid/d), the increase was {approx}56% of maximum concentration. At the 6-mo follow-up, different dosing schedules of the same daily intake of folic acid resulted in nearly identical changes in folate and homocysteine concentrations.

Surveys have provided evidence that approximately one-quarter to one-third of women of reproductive age in the United States take a daily vitamin containing the recommended 400 µg folic acid (45). It was of interest in the current study to determine how a larger intake (ie, 4000 µg) taken weekly would affect folate status and homocysteine concentrations as compared with lower daily intakes. The 4000 µg/wk regimen (571 µg/d average intake) was not nearly as effective as 400 µg/d, which is consistent with the findings of other studies (46). The ineffectiveness of the weekly high-dose folic acid regimen may be explained by the mechanisms by which intracellular folate concentrations are regulated (47) and the saturable process of folate reabsorption from glomerular filtrate by proximal renal tubular cells (48, 49).

Blood folate concentrations declined relatively rapidly after the cessation of folic acid supplementation. However, at 3 mo after the cessation of the folic acid intervention, plasma folate concentrations remained significantly higher than baseline in all intervention groups, a finding that is consistent with previous reports (23, 50). The rapid decline in RBC folate concentrations after cessation of the folic acid intervention appeared to be dose-dependent, and the concentrations at 3 mo after intervention were significantly higher than those at baseline only among women with higher folic acid intakes (400 and 4000 µg/d). The rate of RBC folate decline during the 3 mo after supplementation was greater in the higher supplementation groups, which is associated with higher RBC folate concentrations during the intervention. The daily folate catabolic rates are predicted to range from {approx}0.5% to 1.0%, depending on the daily folate intake: higher catabolic rates are associated with higher intakes. This provides an explanation for the greater rate of cellular folate decline in association with the higher folic acid intake (51). Our results suggest that the effect of folic acid on NTD risk reduction may diminish relatively rapidly after the cessation of folic acid supplementation. These findings therefore support folic acid fortification as potentially a more effective long-term intervention to prevent NTDs than is folic acid supplementation.

Our results suggested that a maximum reduction in homocysteine concentrations in the study population was {approx}22% (measured by an intake of 4000 µg folic acid/d at 6 mo compared with concentrations at baseline), which was consistent with the findings of other studies (44, 50, 52-54). The 100 and 400 µg folic acid/d doses were associated with an estimated 4% and 17% reduction in homocysteine concentration, respectively.

Our findings of a significant increase in homocysteine from baseline to 1 mo among women with folic acid intakes of 100 µg/d and 4000 µg/wk and of no change or a decrease in homocysteine concentrations in response to intakes of 400 or 4000 µg/d are different from the findings reported from 2 previous investigations of the effect of a daily intake of 100 µg folic acid on homocysteine response during the first month (44, 55). Our results suggest that the paradoxical changes in homocysteine concentration from baseline to 1 mo after starting folic acid supplementation were both baseline homocysteine- and folic acid–dose-dependent. In response to folic acid supplementation of 100 µg/d or 4000 µg/wk, homocysteine concentrations increased significantly from baseline to 1 mo. A dose-response relation was evidenced by the fact that the decrease in homocysteine concentration among women who took 4000 µg folic acid/d was significantly greater than that among women who took 400 µg folic acid/d. This study is the first to provide definitive evidence that the initial (≤1 mo) elevation in homocysteine concentrations is both baseline homocysteine- and folic acid–dose-dependent and that it is less likely to be attributed only to interindividual variation or regression to the mean. The large sample size of the present study and the inclusion of multiple folic acid doses strengthen the conclusion.

In summary, the findings of this trial among women from a region of northern China with previously high NTD rates are important to efforts to evaluate optimal folic acid supplementation and fortification strategies. Future research from this collaborative population-based surveillance system will also help elucidate the gene x environment interactions related to the metabolism of folate and other vitamins in connection with optimal maternal-child nutrition and health.


   ACKNOWLEDGMENTS
 
We thank Wang Shi-qiang, Wu Ai-jun, Liu Xue-hai, Zou Fu, and Liu Rui-bo from XiangHe County, Hebei Province, China, for their assistance in the coordination of this trial. We are grateful to the staff of the Maternal and Child Health Institute of Xianghe County, Hebei province, China—Yang Shi-zeng, Zhang Bo-lan, Yao Xiao-yun, Yang Qing, Wang Zhen-xia, Wang Zhao-xiu, Li Jing, Wu He-ming, Wang Bao-xing, Qin Jing, Gao Jing-ping, Cheng Zhi-lian, Chen Jing, Zhang Yan-hui, Liu Hai-Xia, Shan Song-mei, Huang Hao, Li Yan-ling, Li Chun-yan, Wang Li-xin, Chuai Li-jun, and Jiang Wei-min—and to staff members from the 6 participating township hospitals—Yang Hong-hai, Gao Xiu-rong, Liu Yu-bao, Xiao Shu-qin, Wei Feng-yan, Sun Hong-xia, Liu Chun-xia, Dong Jian-jun, Zhang Lian-ru, Yuan Hong-mei, Liang Xue-mei, Wu Li-hua, Guo Guang, Wang De-hong, Huang Peng, Xu Feng-nan, Han Jing, Wen Zhan-jun, Zhang Li, Chen Jian-ling, Wu Rong-cai, Li Jing-xin, Zhang He-lin, Ma Ling-yun, Zhang Yan-ping, Chen Zhi-ling, Liu Xue-feng, Luo Yu-liang, Meng Guang-xing, Wang Hui-jun, and Feng Yan—for their assistance in the collection of data and of blood samples in the trial. We are also grateful to the village doctors from 98 participating villages for delivering weekly packages of folic acid pills to the participants and for monitoring their health status and compliance with the pill-taking protocol. We thank all of the women who participated in this clinical trial. We thank Mary E Cogswell (National Center on Birth Defects and Developmental Disabilities, CDC) for her helpful comments with regard to the manuscript.

The authors’ responsibilities were as follows: LH, Q-HY, and RJB: full access to all of the data in the study and responsibility for the integrity of the data and the accuracy of the data analysis; JDE, ZL, RJB, and JG: study concept and design; Q-HY, LH, LBB, DJH, and RJB: analysis and interpretation of the data; LH, ZL, B-LZ, and JHZ: acquisition of the data; LH and JHZ: laboratory analysis of the data; Q-HY: statistical expertise and writing the draft of the manuscript; LH, Q-HY, LBB, DJH, JG, and RJB: critical revision of the manuscript for important intellectual content; B-LZ, SL, and LZ: administrative, technical, or material support; and LH, JG, and B-LZ: study supervision. None of the authors had a personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication November 20, 2007. Accepted for publication May 12, 2008.





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