Effect of time of initiation and dose of prenatal iron and folic acid supplementation on iron and folate nutriture of Korean women during pregnancy

¹ From the Department of Food and Nutrition, College of Human Ecology, Chonnam National University, Gwangju, Korea

² Presented in part at Experimental Biology 2003, San Diego, CA, April 13.

³ Supported by a grant of the Korea Health 21 R and D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-B-22000-0158).

⁴ Reprints not available. Address correspondence to H-S Lim, Department of Food and Nutrition, College of Human Ecology, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju, 500-757, South Korea. E-mail: limhs{at}chonnam.ac.kr.

ABSTRACT  
Background: In Korea, it is customary to prescribe iron and folic acid supplements to pregnant women after the 20th wk of gestation; however, little evidence exists to support this practice.

Objective: The objective was to determine the effects of time of initiation and dose of prenatal iron and folic acid supplementation on the iron and folate nutriture of Korean women during pregnancy.

Design: A total of 131 pregnant women were placed into 1 of 5 experimental groups, either the control group or 1 of 4 supplemented groups. The supplemented groups varied by time of initiation, which was either during the first trimester or at week 20 of gestation, and by dose of iron and folic acid supplements provided, which consisted of either 30 mg Fe plus 175 µg folic acid or 60 mg Fe plus 350 µg folic acid. All supplemented groups continued supplementation until delivery.

Results: Improvements in iron and folate nutriture were highly dependent on when the supplement program was initiated, but both supplement doses were equally effective. In contrast, the influence of folic acid supplementation on maternal folate status was not as pronounced as was the influence of iron supplementation on iron status.

Conclusion: In pregnant Korean women, initiating iron and folic acid supplementation earlier during pregnancy may prevent the deterioration of iron and folate nutriture more than does increasing supplement doses in later stages of pregnancy.

Key Words: Iron • folic acid • homocysteine • pregnancy • supplementation

INTRODUCTION  
Iron requirements during pregnancy are not easily fulfilled through diet alone; thus, many countries recommend that pregnant women take iron supplements (1–4). In Korea, it is customary to prescribe iron supplementation after the 20th wk of gestation, although this is not yet a national public health policy. However, little evidence exists to support this practice for Korean women. More than 80% of pregnant Korean women receive iron supplements after the 20th wk of gestation; nevertheless, their iron nutriture tends to decline as pregnancy progresses (5, 6). This indicates that the amount or the duration of iron supplementation might not be sufficient. Blot et al (7) and a United Nations Subcommittee on Nutrition (1) suggested that pregnant women with iron deficiency take more iron or start iron supplementation earlier. Such advice may be especially appropriate for Korean women because the incidence of anemia is relatively high in women of childbearing age (8, 9).

Folate, similar to iron, is not easily provided through diet alone (10). Folic acid supplementation is recommended for women who are planning to become pregnant as well as for pregnant women (11) because folate nutriture at periconception is critical for preventing neutral tube defects (NTDs) (12, 13) and because low serum folate concentrations during pregnancy are associated with preterm delivery and low birth weight (14–16). In Korea, >80% of women begin folic acid supplementation after the 20th wk of pregnancy (17), although it is not a national public health policy. When supplementation is initiated at week 20, it cannot be expected to prevent NTDs or to maintain adequate folate status during early pregnancy because Korean women of childbearing age typically have poor folate nutriture (18) and maternal serum folate in early pregnancy tends to be low (19, 20). Folate deficiency can lead to hyperhomocysteinemia (21), which is a known risk factor for NTDs (22, 23); however, plasma total homocysteine (tHcy) concentrations seem to decrease during normal pregnancy (24, 25). This pregnancy-related decline might be explained by hemodilution (25, 26), endocrine-based mechanisms (27), or folic acid supplementation (25), but how folic acid supplementation affects maternal plasma concentrations of tHcy is still undetermined.

It has been reported that iron plus folic acid supplementation is more effective than iron supplementation alone for the treatment of anemia during pregnancy (28). Most pregnant women in Korea take iron and folic acid supplements together. Accordingly, we designed this study to evaluate longitudinally the effects of prenatal iron plus folic acid supplementation on the iron and folate nutriture of Korean women during pregnancy and to determine either the optimal supplement dosage or the ideal time for initiating supplementation.

SUBJECTS AND METHODS  
Subjects
The subjects for the present study were recruited from among pregnant women who visited a hospital and a health center in Gwangju, Korea, for prenatal care from April 2000 to January 2002. A total of 154 women in their first trimester of pregnancy participated voluntarily. The purposes and procedures of the study were explained to each subject, and all subjects provided written informed consent. Twenty-three subjects who were unable or unwilling to complete the study (25) were excluded: 6 women wanted to change their group assignments, 9 women did not want to have blood samples taken, 5 women had miscarriages, and 3 women changed their addresses. The research protocol was approved by the Research Ethics Committee of Chonnam National University Hospital.

Protocol
The subjects were assigned to 1 of 5 groups (Table 1): control (n = 20 subjects), early single-dose supplemented (ES; n = 24 subjects), early double-dose supplemented (ED; n = 31 subjects), late single-dose supplemented (LS; n = 27 subjects), and late double-dose supplemented (LD; n = 29 subjects). The control group received no supplement, the ES and LS groups received 30 mg Fe plus 175 µg folic acid/d, and the ED and LD groups received 60 mg Fe plus 350 µg folic acid/d. The subjects in the ES and ED groups received the supplements beginning at the first trimester (
View this table:
TABLE 1. Experimental groups and general characteristics of the subjects¹

 
Blood sampling and analytic methods
Venous blood samples from fasting subjects were collected once during each trimester ( ± SD: 39.3 ± 1.0 wk). Five indexes of iron nutriture were evaluated: hemoglobin concentrations and hematocrit were measured with an automated hematology analyzer (Coulter STKS; Beckman Coulter, Fullerton, CA), serum ferritin concentrations were measured with the use of a Coat-A Count ferritin ¹²⁵I immunoradiometric assay kit (Diagnostic Products Corp, Los Angeles, CA) and with a gamma counter (Packard Cobra II; Perkin-Elmer, Wellesley, MA), serum soluble transferrin receptor (sTfR) concentrations were assayed with an immunoenzymatic method with the use of an IDeA sTfR kit (Orion Diagnostica, Espoo, Finland) and a microplate reader (ELX 808; Bio-Tec Instruments Inc, Winooski, VT), and the sTfR:ferritin ratio was calculated from these values. Three indexes of folate nutriture were measured: folate concentrations in both serum and erythrocytes were assessed with a microbiological method (29) with the use of Lactobacillus casei (ATCC 7469; ATCC, Manassas, VA) on the same microplate reader, and plasma tHcy concentrations were measured by HPLC (model 501; Waters Corporation, Milford, MA) according to the method of Araki and Sako ( Statistical methods
All statistical analyses were performed with the use of SPSS version 10.0 (SPSS Inc, Chicago, IL). Mean (±SD) differences between the 5 groups (control, ES, ED, LS, and LD) were analyzed with a 2-factor analysis of variance with the Tukey’s test. A one-factor repeated-measures analysis of variance with the Tukey’s test was used to make comparisons between the 4 periods (the first, second, and third trimesters and delivery). Mean (±SD) changes in iron and folate indexes from the first trimester to delivery between the 5 groups were compared with the use of a one-factor analysis of variance with the Tukey’s test. A P value < 0.05 was chosen as the level of significance.

RESULTS  
General characteristics of the subjects
As shown in Table 1, the mean (±SD) age of the subjects was 28.3 ± 3.4 y, the mean (±SD) parity number was 0.7 ± 0.7 (47% of the subjects were primiparous), the mean (±SD) height was 159.7 ± 5.0 cm, and the mean (±SD) prepregnancy weight was 52.8 ± 5.7 kg. Although educational backgrounds, housing conditions, and household income levels are not presented, most of the subjects were in the middle to lower-middle class and lived in a large city. No significant differences in the above general characteristics were observed between the 5 groups.

Iron nutriture indexes
As presented in Table 2, the initial hemoglobin and hematocrit values were significantly different in the groups despite the random placements of the subjects. The hemoglobin concentrations in the ES group were significantly lower than in the other groups except for the ED group, and hematocrit values in the ES group were significantly lower than in the LD and control groups. As pregnancy progressed, the changes in these 2 indexes were different in the groups. In the control group, both hemoglobin concentrations and hematocrit declined by the second trimester, decreased more by the third trimester, and had not recovered at delivery. In the ES and ED groups, hemoglobin concentrations declined by the second trimester, did not decrease any more by the third trimester, and had returned to the initial concentrations at delivery. However, hematocrit did not change significantly in the ES and ED groups throughout the entire pregnancy. In the LS and LD groups, both hemoglobin concentrations and hematocrit declined by the second trimester, similar to the control group, but had not declined more by delivery. At delivery, hemoglobin concentrations in all supplemented groups, except the LS group, were higher than in the control group, and hematocrit in the ES, ED, and LD groups was greater than in the control group. No interaction effects of dose and time of initiation of supplementation on hemoglobin concentrations or hematocrit were observed at any period. However, the time of supplement initiation affected hemoglobin and hematocrit values at delivery.

View this table:
TABLE 2. Hemoglobin concentrations and hematocrit values for the subjects during pregnancy¹

 
The respective percentage changes in hemoglobin and hematocrit values from the first trimester to delivery were as follows: –21.9% and –21.4% in the control group, –0.8% and 6.5% in the ES group, –3.1% and –2.4% in the ED group, –15.9% and –15.0% in the LS group, and –7.4% and –12.8% in the LD group. The declines in hemoglobin and hematocrit values in the ES and ED groups were significantly smaller than in the control group. Additionally, hematocrit in the LS group was reduced significantly more than in the ES and ED groups.

As presented in Table 3, the initial values of serum ferritin and sTfR and the sTfR:ferritin ratio were not significantly different between the groups; however, the changes in these 3 indexes during pregnancy were different between the groups. In the control group, ferritin concentrations had decreased by the second trimester, worsened by the third trimester, and remained at the decreased concentration until delivery. Furthermore, both sTfR concentrations and the sTfR:ferritin ratio had increased by the third trimester, and sTfR concentrations had increased even more by delivery. In the ES and ED groups, no significant differences were observed in ferritin concentrations and the sTfR:ferritin ratio throughout the study period. However, sTfR concentrations increased by the third trimester but decreased to initial concentrations by delivery. In the LS and LD groups, a decline in ferritin concentrations and an increase in both sTfR concentrations and the sTfR:ferritin ratio occurred by the second trimester, similar to the control group, but then remained stable. Ferritin concentrations in the ES and ED groups were higher than in the control, LS, and LD groups at the second trimester and higher than only the control group at the third trimester and delivery. At the third trimester, the sTfR:ferritin ratios in the ES and ED groups were lower than in the control group at the third trimester, and the sTfR concentration and sTfR:ferritin ratio in all supplemented groups were lower than in the control group at delivery. No significant interaction effects of supplement dose and time of initiation on serum ferritin concentrations, sTfR concentrations, or the sTfR:ferritin ratio were observed at any period. However, the time of supplement initiation, but not the supplement dose, affected serum ferritin concentrations and the sTfR:ferritin ratio at all periods and affected sTfR concentrations at the third trimester and delivery.

View this table:
TABLE 3. Serum concentrations of ferritin and serum transferrin receptor (sTfR) and the ferritin:sTfR ratio of the subjects during pregnancy¹

 
The percentage changes from initial values to delivery for ferritin were –65.4% in the control group, –18.5% in the ES group, –22.3% in the ED group, –45.7% in the LS group, and –40.2% in the LD group. The percentage changes for sTfR concentrations were 65.0% in the control group, –9.8% in the ES group, –5.0% in the ED group, 29.7% in the LS group, and 10.5% in the LD group. The percentage changes for the sTfR:ferritin ratio were 376.9% in the control group, 10.0% in the ES group, 19.8% in the ED group, 138.7% in the LS group, and 85.1% in the LD group. The changes in sTfR concentrations and the sTfR:ferritin ratio, but not the change in ferritin concentrations, of all supplemented groups were smaller than were the changes in the control group.

Folate nutriture indexes
As presented in Table 4, the initial folate concentrations in serum and erythrocytes and plasma tHcy concentrations were not significantly different between the groups. However, the changes in folate concentrations during pregnancy were different between the groups. Serum folate concentrations in the control group declined significantly at delivery. In contrast, no decline in serum folate concentrations was observed in any of the supplemented groups. Additionally, serum folate concentrations increased significantly at delivery in the 2 double-dose supplemented groups (ED and LD). Erythrocyte folate concentrations, however, did not change significantly in any group, including the control group, during the entire study period. Serum folate concentrations in the ES and ED groups were higher than in the control, LS, and LD groups at the second trimester, and the serum folate concentrations in all supplemented groups were greater than in the control group at the third trimester and delivery. No significant interaction effects of dose and time of initiation of supplementation on serum and erythrocyte folate concentrations were observed at any period. The time of supplement initiation, but not the supplement dose, affected serum folate concentrations only at the second trimester.

View this table:
TABLE 4. Serum folate, erythrocyte folate, and plasma total homocysteine (tHcy) concentrations of the subjects during pregnancy¹

 
The respective percentage changes in serum and erythrocyte folate concentrations from the first trimester to delivery were –25.5% and –26.5% in the control group, 25.4% and –6.0% in the ES group, 28.1% and 3.8% in the ED group, 22.0% and –2.3% in the LS group, and 38.2% and 8.0% in the LD group. No significant differences in the percentage changes in folate concentrations were observed between the groups.

As shown in Table 4, the initial plasma tHcy values were not significantly different between the groups and did not change significantly in any of the groups throughout the entire study. No interaction effects of dose and time of initiation of supplementation were observed on plasma tHcy concentrations at any period. Additionally, neither time of initiation nor dose of supplementation affected plasma tHcy concentrations at any period. The percentage changes in plasma tHcy concentrations from the first trimester to delivery were 9.1% in the control group, –13.0% in the ES group, –15.7% in the ED group, –9.1% in the LS group, and –16.7% in the LD group. No significant differences were observed in the percentage changes in plasma tHcy concentrations between the groups.

DISCUSSION  
In the nonsupplemented women (the control group), hemoglobin concentrations, hematocrit, and especially ferritin concentrations decreased at the second trimester and declined more at the third trimester, sTfR concentrations deteriorated at the third trimester and worsened more at delivery, and the sTfR:ferritin ratio increased at the third trimester and was maintained until delivery. When supplementation began early in pregnancy (week 9.1, in the ES and ED groups), hemoglobin concentrations recovered to the initial concentration by delivery after a decrease at the second trimester, sTfR concentrations were restored by delivery after worsening at the third trimester, and hematocrit, ferritin concentrations, and the sTfR:ferritin ratio did not change significantly during the entire pregnancy. When supplementation was initiated later in pregnancy (week 20, in the LS and LD groups), hemoglobin and ferritin concentrations and hematocrit were reduced by the second trimester but then remained stable until delivery. Because it is common for hemoglobin concentrations and hematocrit to decrease after the 8th wk of gestation (31), the time chosen for the early initiation of iron supplementation in this study, ie, 9.1 ± 2.3 wk, might not be early enough to prevent the decline in iron nutriture that occurred during the second trimester. It was previously reported that iron plus folic acid supplementation from the 5th wk of gestation resulted in maternal hemoglobin concentrations and hematocrit that were higher than those in the population not receiving supplements by the 12th wk of gestation (31). Our finding that doubling the dose of iron had little effect on the indexes of iron nutriture is consistent with the report by Aaseth et al (32), which showed that supplementation with 15 mg Fe/d from the 10th wk of gestation until delivery resulted in a smaller reduction in hemoglobin concentrations than did supplementation with 100 mg Fe/d when started later in pregnancy. Thus, early initiation of iron supplementation in pregnant women may be more effective in preventing a decrease in iron nutriture than increasing the iron dosage later.

Hemodilution (33) is a possible explanation for the decrease in hemoglobin concentrations, hematocrit, and ferritin concentrations that was observed in the control group and in the 2 late-supplemented groups (LS and LD) during the second and third trimesters. However, this decrease did not occur in the 2 early-supplemented groups (ES and ED); hematocrit and ferritin concentrations did not change significantly, and the sTfR concentration, which is a specific marker of iron deficiency in pregnancy (34), was raised at the third trimester point. Therefore, the decreases in hemoglobin concentrations, hematocrit, and ferritin concentrations observed in the control, LS, and LD groups could not be explained by only hemodilution. The increase in sTfR concentrations in all groups at the third trimester was consistent with results of previous studies (6, 34, 35). The results of the present study suggest that hemodilution might have little effect on the indexes of iron nutriture and indicate that an iron deficiency may be primarily responsible for decreases in iron nutriture. The ratio of sTfR:ferritin, one of the most sensitive markers of iron deficiency (35), at delivery was 4.8 times that at the first trimester in the control group and 2.4 and 1.9 times that at the first trimester in the 2 late supplemented groups (LS and LD, respectively); in contrast, the ratio of sTfR:ferritin at delivery was only 1.1 and 1.2 times that at the first trimester in the 2 early supplemented groups (ES and ED, respectively).

Serum folate concentrations improved only in the 2 double-dose supplemented groups (ED and LD) and only at delivery. However, at the second trimester, the subjects in the ES and ED groups had higher serum folate concentrations than did the subjects who were not supplemented at that time point (the control, LS, and LD groups). At the third trimester, the women in all supplemented groups had greater serum folate concentrations than did the women in the control group. Erythrocyte folate concentrations did not change significantly during the entire study period in all groups, including the control group. However, the subjects who were not supplemented (the control group) had lower erythrocyte folate concentrations at delivery than did the women who were supplemented, regardless of the dose and time of initiation. These observations indicate that folic acid supplementation may cause folate nutriture to recover rapidly and that 175 µg supplemental folic acid/d may be sufficient to maintain adequate folate status during pregnancy, although 350 µg folic acid/d might additionally enhance folate nutriture. Folate intake is significantly correlated with circulating folate concentrations (14). We previously showed that maternal serum folate concentrations were significantly enhanced from 7.4 to 14.0 ng/mL after supplementation with 43 mg Fe and 665 µg folic acid/d from the 20th wk of pregnancy (17, 36). Mahomed (37) also reported that routine supplementation with folic acid and iron during pregnancy increased or maintained maternal folate concentrations in both serum and erythrocytes. The results of the present study also suggest that the time of folic acid supplementation might not be important for maintaining adequate folate status throughout pregnancy, because folate status recovered quickly after supplementation began. However, it is essential to begin folic acid supplementation earlier in pregnant women who are folate deficient before pregnancy or to protect against NTDs.

Plasma tHcy concentrations decrease during normal pregnancy (21, 24–27). Although the lack of prepregnancy data was a limitation of this study, we found that plasma tHcy concentrations did not change significantly from the first trimester until delivery in any group. These results imply that plasma tHcy concentrations may not always decrease during pregnancy, and whether this occurs might be related to a woman’s protein and folate nutriture before pregnancy. Walker et al (25) reported that the decline in plasma tHcy concentrations occurred in association with the physiologic decline in serum albumin concentrations during pregnancy as well as with supplementation of 1 mg folic acid/d. Folic acid supplementation during pregnancy could reduce the risk of obstetric complications related to high concentrations of homocysteine (25).

The present study convincingly showed the positive effects of iron plus folic acid supplementation on maternal nutriture of both iron and folate. Moreover, our results indicated that optimal results may depend more on the time that supplementation is initiated than on the amount supplemented, especially for iron status. In conclusion, pregnant women should be advised to take iron and folic acid supplements earlier than the time currently recommended in Korea to prevent the deterioration of iron and folate nutriture during pregnancy.

ACKNOWLEDGMENTS  
H-SL participated in the study design, interpretation, and manuscript preparation. J-IL performed the biochemical analyses of iron status and the statistical analyses and drafted the manuscript. J-AL contributed to the data collection and performed the biological analyses of folate status. None of the authors had any conflicts of interest.

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