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4 Institute of Human Nutrition, Medical College of Qingdao University, 266021 Qingdao, China; 5 Division of Human Nutrition, Wageningen University, 6700 EV Wageningen, The Netherlands; 6 People's Hospital of Liaocheng, 7 College of Liaocheng Technology, and 8 Service Center for Children and Women, Shen County, Shandong Province, 252000 China
* To whom correspondence should be addressed. E-mail: aiguom502{at}hotmail.com.
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ABSTRACT |
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 TOP ABSTRACT IntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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105 g/L] were randomly assigned to 4 groups, all receiving 60 mg/d iron and 400 µg/d folic acid. The iron+folic acid (IF) group (n = 93) served as reference, the iron+folic acid+retinol group (IFA) (n = 91) was treated with 2000 µg retinol, the iron+folic acid+riboflavin group (IFB) (n = 91) with 1.0 mg riboflavin, and the iron+folic acid+retinol+riboflavin group (IFAB) (n = 91) with retinol and riboflavin. After the 2-mo intervention, the Hb concentration increased in all 4 groups (P < 0.001). The increase in the IFAB group was 5.4 ± 1.1 g/L greater than in the IF group (P < 0.001). The reduced prevalence of anemia (Hb < 110g/L) and ID anemia were significantly greater in the groups supplemented with retinol and /or riboflavin than in the IF group. Moreover, gastrointestinal symptoms were less prevalent in the IFA group than in the IF group (P < 0.05) and improved well-being was more prevalent in the groups receiving additional retinol and/or riboflavin than in the IF group (P < 0.05). Thus, a combination of iron, folic acid, retinol, and riboflavin was more effective than iron plus folic acid alone. Multimicronutrient supplementation may be worthwhile for pregnant women in rural China.
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Introduction |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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There is a low prevalence of parasitic infection in the rural population (hookworm, 0.65%; ascaris lumbricoides 5.16%) in recent years, and this has never been considered as an important factor of anemia in rural China (10). In fact, retinol and riboflavin deficiencies tend to coexist in anemic pregnant women in developing countries or impoverished settings (11). A total of 44% of women were deficient in at least 1 B vitamin (9). Biochemical evidence of riboflavin deficiency was documented during the third trimester of pregnancy (12). Serum concentration of retinol <0.70 µmol/L is associated with an increased risk of preterm delivery and maternal anemia (13). Supplementation with iron and riboflavin enhances dark adaptation response to vitamin A-fortified rice in iron-deficient, pregnant, night-blind Nepali women (6). Retinol or riboflavin plus iron supplementation could improve hematological status better than iron alone (14). In a previous unpublished study in 2001 (A. Ma, X. Chen, and E. Schouten, unpublished data), we observed that 55% of pregnant women suffered from anemia and the mean vitamin A intake was only 54% of the Chinese recommended daily allowance. Riboflavin deficiency was widely prevalent among pregnant women. This prompted us to perform the present study, with the objective to investigate the efficacy of vitamin A and/or riboflavin supplementation in addition to iron and folic acid on hematologic status, subjective well-being, and the occurrence of possibly iron-related gastrointestinal symptoms.
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Subjects and Methods |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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105 g/L; therefore, 178 pregnant women did not meet the inclusion criteria, and another 89 women with Hb 105 g/L refused to take any "drugs" during pregnancy. Finally, 366 women remained (Hb >80 and 105 g/L, no dietary supplements during 2 mo, no abnormal pregnancy response, and receiving 400 µg/d folic acid). They were allocated to the 4 intervention groups in the order of enrollment. Registration numbers 1, 5, 9, 13, etc. were allocated to 1 group. The 4 treatments were randomly assigned to the groups and the key for the treatment groups was kept in a sealed envelope with a person unaware of the treatments in the Institute. IF (n = 93) as a reference group was supplemented with 60 mg iron as ferrous sulfate, the IFA group (n = 91) with 2 mg retinol (as retinyl palmitate) and 60 mg iron, the IFB group (n = 91) with 1.0 mg riboflavin and 60 mg iron, and the IFAB group (n = 91) with 2000 µg retinol, 1.0 mg riboflavin, and 60 mg iron daily (Supplemental Fig. 1). The sample size was calculated based on difference between the groups, not on interaction effects. The IFA and IFB groups increased by 3 g/L (
)compared with the IF group and an additional increase of 3 g/L compared with the IFA and IFB groups. Considering a 5% significance level and a power of 0.80, the total required number of subjects for the study was 340 anemic pregnant women (85 subjects for each group). The capsules were colored red, yellow, green, and blue and manufactured by Hurun. Trial participants and the research team were unaware of the treatment assignment. The treatment groups were revealed after the analysis of the primary outcomes. After giving written consent, the pregnant women had a baseline interview to obtain data on dietary habits, alcohol intake, and tobacco use and information on household socioeconomic status. Information on well-being, gastrointestinal side effects, appetite, and physical functions were assessed at the end of the treatment period using the Short Form 36 (15), a self-administered questionnaire that was translated from English to Chinese (16).
During the trial, a village nurse was responsible for recruiting subjects and distributing the supplements. After ascertaining their eligibility, consenting women were enrolled in the study to receive their allocated supplements daily for 2 mo. Women were home-visited once each week by the village nurse to replenish supplements and to monitor compliance by counting and recording the number of supplements taken. Village nurses provided counseling about possible side effects of taking the supplement. The mean number of capsules taken was 62 in the IF group, 60 in the IFA group, 61 in the IFB group, and 62 in IFAB group during the 2 mo.
The study was approved by the ethical review committee of the Medical College of Qingdao University. Written consent was given by each subject at the start of the trial.
Sample collection and laboratory analyses. Samples of blood (5 mL) and urine (10 mL) were collected from subjects and transported on dry-ice and stored at –80°C until analysis. The baseline and final samples were analyzed in duplicate during the same analysis.
Plasma retinol concentrations were measured using HPLC (17). Folic acid in plasma was measured by radioimmunoassay method. The nutritional status of riboflavin was determined by the urine riboflavin:creatinine ratio and we measured the erythrocyte glutathione reductase activity coefficient for assessing riboflavin status (18).
Hb concentration was measured using HemoCue. Measurements of serum ferritin were performed by radioimmunoassay (19), as described by the manufacturer (North Biological Technology Institute). The cutoff value for mild to moderate anemia was Hb 105 g/L in the second and third trimesters of pregnancy. The erythrocyte protoporphyrin (EP) was measured with a hematofluorometer. The soluble transferrin receptor (sTfR) assay was performed using a commercial kit (R&D Systems). According to the instructions of the kit, the central 95th percentile of the reference distribution of sTfR concentration was 4.0 to 9.1 mg/L. The cutoff value of ID was >9.1 mg/L. All sTfR assays were performed in duplicate. Plasma iron concentrations were analyzed by atomic absorption spectrometry on an Analyst 3100 Analyzer (Perkin Elmer Life Sciences). Total iron-binding capacity (TIBC) was determined by a turbidimetric method.
Statistical analysis.
Categorical data are presented as frequencies, such as prevalence of anemia, whereas continuous data are presented as means ± SD or means ± SEM. Baseline iron, folate, and anemia status of pregnant women were compared across treatment groups. The differences in mean change during the intervention period among intervention groups and reference group and 95% (CI) were estimated for hematologic indicators and vitamin levels using a general linear model ANOVA. Prevalence ratios (PR) and 95% CI for mild to moderate anemia (Hb <105 g/L), all anemia (Hb <110 g/L) and IDA (Hb <110 g/L and ferritin <12 µg/L), and for well-being and gastrointestinal symptoms were estimated using a binomial model. Because there was no indication of interaction between retinol and riboflavin in 2 x 2 factorial analysis (P = 0.702 for Hb, P = 0.777 for ferritin, P = 0.746 for sTfR), we calculated variables for each treatment (retinol and riboflavin) using 1-way ANOVA followed by Bonferroni correction. Therefore, the effects on changes in Hb concentration, ferritin, and sTfR were then assessed by including them in a general linear model. A 
of 0.05 was considered significant.
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Results |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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The groups did not differ in age, gestational stage, parity, hematological status, or vitamin levels at baseline (P > 0.05) (Supplemental Table 1). After the 2-mo trial, there were significant increases of Hb, plasma ferritin, and iron concentrations, and decreases in sTfR, EP, and TIBC in all 4 groups (Table 1).
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Discussion |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Compared with the few similar studies conducted so far (14,20), ours had a relatively large study population. It was conducted under difficult circumstances in a poor rural part of China, where nutritional problems, especially among pregnant women, are frequent. However, compliance was excellent, because study subjects were motivated by the offer of free medical care and all women were weekly visited by village nurses, who counted leftover capsules, provided new supplies, and gave support in case of any problems or questions related to the study.
Unfortunately, there was a small proportion (6.6%) of women who dropped out after randomization. This drop-out and the underlying reasons were evenly distributed over the groups. Due to the restriction on the amount of blood that we were allowed to draw, we had missing data on a number of hematologic variables for 
80 participants. These were reasonably evenly distributed over the groups and the remaining groups were still balanced with respect to important characteristics.
The quality of the supplements was assured by storage at –20°C and micronutrient levels were examined every 3 mo and showed no appreciable changes. Although the 4 treatment capsules were different colors, participants and study personnel remained unaware of the treatments, because there was no need to reveal the code before the main analyses had been conducted. In our trial, most subjects only knew their own supplement, which was provided by their village nurse. Because they lived in a remote area, contacts with other trial participants were rare.
Riboflavin deficiency may be one of the most common vitamin deficiencies in regions, like rural areas in China, where diets are predominantly rice based and contain insufficient milk, meat, fish, and fresh fruit and vegetables (21). The riboflavin combined with iron was also considered to be beneficial to anemic pregnant women (14) and significantly reduced the prevalence of riboflavin deficiency and IDA (6). Our results further confirmed the positive effect of the combination of retinol and riboflavin on anemia in Chinese pregnant women living in rural areas (22,23).
We found evidence for an additive effect of retinol and riboflavin on hematologic parameters and anemia in addition to iron and folic acid. This may be explained by the coexisting deficiencies of these micronutrients in our population. The effects of retinol and riboflavin appeared to be additive and we did not find evidence for interaction. This may indicate that the beneficial effects of each of the vitamins on erythropoiesis are independent. Retinol plays an important role in reducing anemia and improving iron status, enhancing iron utilization by stimulating erythropoiesis and iron metabolism by raising mean red cell volume, plasma iron concentration, and TIBC (23,24). Improvement of poor appetite was observed in 42.2% of the IFB group and in 41.0% of IFAB group. Riboflavin is thought to improve iron uptake and absorption by improving the gastrointestinal function (25) and better appetite, which may have resulted in significantly enhanced concentration of serum iron (26). Consistently, women who took riboflavin had better appetites (subjectively) than those who did not take any riboflavin. At the same time, riboflavin is also necessary for synthesis of the globin component of Hb (27).
In our study, the combination of iron and folic acid already had a strong beneficial effect, so the study was designed to demonstrate possible additional efficacy of retinol and/or riboflavin. Such additional benefits are expected to be modest.
In conclusion, these women often have poor nutritional status, lacking sufficient dietary intake of multiple micronutrients. Surprisingly, almost none of the subjects took dietary supplements, including iron. Our study showing additional benefits of vitamins in tackling the anemia problem has important public health implications. It underlines the need for a comprehensive nutritional policy for this target population. Besides improving their diet, multimicronutrient supplementation may be worthwhile for pregnant women in rural China.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: A. G. Ma, E. G. Schouten, F. Z. Zhang, F. J. Kok, F. Yang, D. C. Jiang, Y. Y. Sun, and X. X. Han, no conflicts of interest.
3 Supplemental Figure 1 and Supplemental Tables 1–3 are available with the online posting of this paper at jn.nutrition.org.
9 Abbreviations used: EP, erythrocyte protoporphyrin; Hb, hemoglobin; ID, iron deficiency; IDA, iron deficiency anemia; IF, iron and folic acid; IFA, iron+ folic acid + retinol; IFAB, iron +folic acid+ retinol+ riboflavin; IFB, iron+ folic acid + riboflavin; PR, prevalence ratio; sTfR, soluble transferrin receptor; TIBC, total iron-binding capacity.
Manuscript received 18 February 2008. Initial review completed 30 March 2008. Revision accepted 21 July 2008.
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LITERATURE CITED |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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