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The National Institute of Nutrition, Hanoi, Vietnam;
* The Institute of Research for Development, Montpellier, France;
ILSI Center for Health Promotion of Japan, Tokyo, Japan;
** Eastern Virginia Medical School, Norfolk, VA; and
Iowa State University, Ames, IA
3To whom correspondence should be addressed. E-mail: vanthuy63{at}yahoo.com.
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ABSTRACT |
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KEY WORDS: • NaFeEDTA • fortification • fish sauce • iron deficiency
Iron deficiency (ID)4 is the most common nutritional deficiency disorder worldwide, affecting an estimated 3.5 billion individuals (1). The risk is highest for children and women of childbearing age. There are major health consequences including lower birth weight in infants born to women suffering from iron deficiency during pregnancy, impaired psychomotor development in infancy with effects on later cognitive function, an increased prevalence and duration of upper respiratory infections in children, and reduced physical work performance at all ages (2–5).
Iron deficiency is particularly prevalent in Vietnam and other developing countries in which cereal staples, rich in inhibitors of iron absorption such as phytates, are the major energy source. Food fortification is considered to be the most cost-effective strategy for combating nutritional iron deficiency in population groups that buy one or more commonly eaten food items (6). It is an attractive strategy because consumer compliance is ensured; it can be both inexpensive to initiate and sustainable (6). However, it depends greatly on the selection of a suitable food vehicle. The ideal vehicle is a dietary item that is consumed daily in relatively consistent quantities by the individuals at greatest risk for nutritional iron deficiency. It must also be feasible to add sufficient bioavailable iron to the vehicle without changing its storage properties or the color and taste of meals in which it is eaten. Fish sauce is a suitable vehicle in Vietnam. It is a traditional condiment that is consumed daily with most meals in all regions. Fish sauce can be fortified with NaFeEDTA. It does not oxidize the lipids or precipitate the peptides in the sauce and remains stable for at least 12 mo if protected from light (7). No degradation of NaFeEDTA was observed after storage in Vietnamese fish sauce for 364 d if the fish sauce was protected from direct exposure to natural or artificial sunlight. Furthermore, we demonstrated recently (8) that monitored consumption of NaFeEDTA-fortified fish sauce [10 mL containing 0.18 mmol (10 mg) Fe/d] for 6 d/wk for 6 mo was highly efficacious in correcting iron deficiency in a group of women of childbearing age who suffered from iron deficiency anemia (IDA).
The present study was designed to evaluate the effectiveness of supplying NaFeEDTA-fortified fish sauce [9 mmol (500 mg)/Fe/L] at the household level for reducing the prevalence of nutritional iron deficiency. Although the fish sauce was provided for the whole household, iron status was assessed only in women of childbearing age. Women and children are most likely to benefit from mass fortification. However, parents in this community were not willing to allow their children to participate in the study protocol that required blood samples to be obtained.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The 2 different batches of fish sauce were produced for the study every 3 mo by a commercial fish sauce supplier (Cat Hai Fish Sauce). One batch was prepared without added iron. NaFeEDTA (9 mmol Fe/L, Akzo Nobel Chemicals) was added to the other. The fish sauce was then stored in 0.5-L polyethylene bottles wrapped in paper to protect the NaFeEDTA from light during storage. The iron content of the fish sauce was monitored by measuring its concentration by atomic absorption spectroscopy (General Office of Standard and Quality Control, Ministry of Science, Technology and Environment, Hanoi, Vietnam) in 3 randomly selected bottles from each batch, one filled at the beginning of the bottling step, one in the middle, and one at the end.
Data collection was carried out 4 times during the study period, at baseline (T0), 6 mo (T6), 12 mo (T12), and 18 mo (T18). Height (Microtoise, precision 0.1 cm) and weight (electronic scale, precision 0.1 kg) were measured. Blood samples (4 mL venous blood) were collected in EDTA-coated vacutainers between 0800 and 1100 h at the Commune Health Centers, kept cool, and transported to the laboratory at the Micronutrient Department of Research and Application at the National Institute of Nutrition in Hanoi within 8 h. Hemoglobin was determined on whole blood by the cyanomethemoglobin method within 12 h using Sigma diagnostic kits (Sigma). A 3-level commercial quality control material (Dia-HT-1, 2, 3, Diamed) was analyzed together with all samples. Samples were analyzed in duplicate and the analysis repeated if the results differed by >5%. Plasma was then separated after centrifugation at 5000 x g for 10 min at 4°C. Aliquots (n = 5) of each plasma sample were stored at –70°C for later analysis of serum ferritin (SF), C-reactive protein (CRP), and serum retinol (SR). SF was determined using an ELISA technique (Ramco). Quality control was monitored by analyzing 10% of the samples in duplicate. The CV was 4.7%. CRP was measured by an ELISA method (Diagnostic Systems Laboratories). Anemia was defined as hemoglobin < 120 g/L and ID as SF < 12 µg/L (9). A CRP > 10 mg/L was used to indicate the presence of inflammation or infection (10).
SR, food consumption, and intestinal parasites were evaluated in the same subsample of women (in a random subsample of 
40% of the subjects in each group at T0, T12, and T18), 119 in Group C and 116 in Group F. SR was analyzed by HPLC in the Micronutrient Department of Research and Application at the National Institute of Nutrition in Hanoi. Dietary intake was estimated by 24-h recall. Conversion to nutrient intake was based on food composition tables for Vietnamese foods (11). Infection with gastrointestinal parasites was monitored. Fecal samples were collected and prepared for microscopic analysis using the Kato-Katz methodology.
The principal hypothesis was that women in the fortified group would have higher iron stores than women in the control group at the end of the study. Sample size was estimated on the basis of an increase in body iron store as the primary outcome indicator. Subjects in the iron-fortified group were expected to have a body iron store that would be 1 mg/kg higher than that of the control group by the end of the study period, using the transferrin receptor:SF ratio method published by Cook et al. (12) to calculate body iron storage size. The SD for body iron was estimated to be 3 mg/kg. The estimated group sample size for a CI of 95% and a power of 90% was 188. The sample size was increased by 50% to allow for dropouts because of the length of the study and its field setting. A total of 576 women (n = 288/group) were enrolled. However, due to technical problems the serum transferrin receptor levels were considered to be unreliable. Therefore, they are not reported. Estimated body iron stores described in the discussion are based on an earlier algorithm published by the same investigators that used only SF (9).
Data were analyzed using SAS version 8.2 (SAS) on an intention-to-treat basis. SF values and parasite egg counts were not normally distributed. Therefore logarithmic transformation was used for statistical inferences. Results for hemoglobin are presented as means and 95% CI. Results for SF are given as geometric means and 95% CI. Results for parasite egg counts are given as geometric means and 95% CI for infected individuals. In general, the effect of fortification was assessed using linear mixed models that included an additional variance component to account for correlation of individuals in the same village. Overall differences between the 2 groups were tested using a repeated-measures ANOVA, implemented by using SAS PROC MIXED with an unstructured within-subject correlation matrix and an additional correlation of subjects within villages. The differences between groups at individual times were tested using a linear mixed model with 2 variance components, one for village and one for individual within village. Intraclass correlation coefficients were computed for each individual response from these variance components. Within-individual change from baseline to 12 mo or to 18 mo was analyzed employing the linear mixed model used for individual responses. This approach generalizes a paired t test to account for additional village-village variability. Comparisons of the prevalence of anemia, SF < 12 µg/L, SR < 0.7 µmol/L, and the prevalence of parasite infections were tested by adjusting 
2 statistics for overdispersion due to village-village variation. The overdispersion factor was estimated by the ratio of residual deviance to residual df. Change in prevalence from baseline was tested using McNemar’s test, adjusted for overdispersion. All available data points at each of the sampling times were used in the analyses. Multivariate ANOVA was used to compare the baseline characteristics of individuals who were missing data at T18 with those for whom data were available. There was no significant difference (P = 0.14). It was therefore assumed that dropout occurred at random. Data from individuals who continued in the study were used to draw conclusions about all study participants.
The protocol of the study was approved by the Scientific Committee of the National Institute of Nutrition and Ministry of Health, Hanoi, Vietnam. All women who volunteered for the evaluation protocol were informed orally and in writing about the study and gave their written informed consent. Women who had a hemoglobin concentration < 80 g/L at any blood collection were withdrawn from the study and referred immediately to the health center for treatment.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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8.0%, Table 2). SR levels were significantly higher in subgroup C at T18, but the groups did not differ at T0 and T12. There was also no correlation between baseline hemoglobin concentration or SF and SR (data not shown).
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The 2 groups did not differ with respect to hemoglobin or SF at the start of the trial (T0, Table 3). There was a progressive rise in hemoglobin and SF in Group F with no significant change in Group C. Analysis of the group x time interaction using a repeated-measures test for each response revealed a significant effect of fortification on hemoglobin (P = 0.039) and log SF (P < 0.0001). Mean hemoglobin was significantly higher in Group F compared with Group C at 12 and 18 mo. SF was significantly higher after 6, 12, and 18 mo (Table 3). There was a fall in the prevalence of anemia and ID in Group F. The difference in the prevalence of anemia between Group F and Group C was significant at T18 (T12 P = 0.1, T18 P = 0.015, Fig. 2). On the other hand, there was a significant difference in the prevalence of ID at T6, T12, and T18 (P < 0.0001, Fig. 2).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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25% of the women were anemic. Dietary analysis revealed a low iron intake from a cereal-based diet that provided, on average, only 50% of the Recommended Dietary Allowance for women of childbearing age (14). Vitamin A deficiency may have had a minor contributory role, but low SR levels were present in 8.0% of the women surveyed. Malarial transmission does not occur in the Red River delta. Helminth infections including hookworm were detected in >90% of the subjects at the beginning of the study and persisted in many women throughout the period of the trial. Hookworm would be expected to have the largest effect on iron nutrition. However, only a small number of women suffered from hookworm infections; with very few exceptions, the hookworm loads were too low to have affected iron status (13). Finally, inflammatory disorders played a relatively insignificant role because CRP levels were within normal limits in all of the women. In summary, an inadequate intake of bioavailable dietary iron is likely the primary cause of iron deficiency in the Red River delta, making this region an ideal setting for dietary iron fortification. The low prevalence of iron deficiency in industrialized countries such as the United States is thought to be due in part to the consumption of iron-fortified foods (6). However, until recently, evidence demonstrating that mass fortification is an effective strategy for improving iron status has been lacking in developing countries (6,15), and iron deficiency remains the most common nutritional disorder in the world (16). Several potential contributory reasons have been identified, but one of the most important may be the use of iron compounds that are poorly absorbed. Fortification of staple cereal flour is the intervention method most commonly employed, and elemental iron powders that may not be adequately bioavailable are used frequently (17).
NaFeEDTA fortification was shown to have a positive effect on iron status (8,18–21). The results of the trial described here confirm and extend the conclusions from these earlier reports and demonstrate that the fortification of fish sauce with NaFeEDTA at a level of 9 mmol Fe/L was very effective in markedly reducing the prevalence of iron deficiency and improving body iron status in Vietnamese women of childbearing age living in the Red River delta. There were significant improvements in hemoglobin and SF in Group F. Both hemoglobin and SF were significantly higher in Group F than in Group C after 12 mo of fortification. Fortification also led to a marked reduction in the prevalence of anemia and ID after 18 mo. Because there were no significant changes in Group C and a striking difference in SF between the 2 groups after 12 mo of fortification, the improvement is undoubtedly attributable to fortification.
Most of the improvement in iron status occurred within the first 12 mo in the present trial (Table 3, Fig. 2). This agrees with the predictions made by Hallberg et al. (22) for the expected time course of adjustments in iron status after a change in bioavailable iron intake. A 12-mo period should therefore be adequate for future fortification trials. Surprisingly, the prevalence of anemia was still 16.2% at T12 although almost all of the women had already accumulated iron stores (SF < 12 µg/L in 5.5%); prevalence declined to 8.5% at T18. We cannot explain this observation, but it suggests that one or more factors other than iron deficiency had a role. It also illustrates the importance of using specific iron indicators to evaluate the response to a fortification intervention. Many fortification trials are conducted over a 6- to 12-mo period. Had the trial relied on hemoglobin measurements alone and been planned as a 12-mo intervention, an erroneous conclusion that the fortification level was too low might have been reached.
At the end of the study, the iron stores of the women were modestly higher than those reported for women of childbearing age in the United States. The 5th, 50th, and 95th percentile values for SF were 13, 70, and 230 µg/L, respectively. These values are 86, 67, and 19% higher than those reported for women aged 31–50 y in the United States in the 3rd National Health and Nutrition Examination Survey (14). The calculated iron stores for the women at 5th, 50th, and 95th percentiles are 14 mg, 306 mg, and 512 mg, respectively (9).
This study does not meet all of the criteria for a rigorous effectiveness trial because the fish sauce was supplied free of charge, rather than merely being made available in the market. It does, however, provide important information. The introduction of the fortification step was feasible and did not require a major modification of the manufacturing plant or affect the commercial production of the fish sauce. A simple quality control system was adequate to ensure the effectiveness of the fortified product. We measured only iron content in the fortified product. When commercial fortification is implemented, it will be necessary to ensure that the added iron is in the form of NaFeEDTA. The fortified fish sauce was well accepted by the target population over an 18-mo period. In fact, the mean consumption for all individuals in all villages was 18 mL/(person · d), which is higher than the national average of 16 mL/(person · d) recorded in a recent survey (23). Presumably this occurred because the family purchasing power was no longer a constraint to fish sauce use. Finally the fish sauce was used in the customary manner, i.e., merely added to the communal family food. No effort was made to ensure that the women of childbearing age received a specified quantity.
Although the effect of additional cost was not evaluated in the current study, a national iron fortification program is being initiated in Vietnam. Low- and medium-quality fish sauce will be fortified with NaFeEDTA at a level of 7 mmol Fe/L. The added cost of fortification is estimated to be US$ 0.11/(person · y), based on an average fish sauce consumption of 4.6 L/y (12.6 mL/d). It will increase the cost of the fish sauce by 8%. The Vietnamese National Fortification Alliance has concluded that consumers will absorb this additional cost if it is passed on gradually and that adequate public and private funding sources are available for surveillance, quality control monitoring, and social marketing.
The results of the current trial were important in justifying and planning the national fortification strategy being initiated in Vietnam. They could be used for designing similar strategies in other Southeast Asian countries in which fish sauce or soy sauce is consumed regularly. NaFeEDTA was shown to be equally compatible with and stable in Thai fish sauce and soy sauce (7). Moreover, soy sauce fortified with NaFeEDTA was efficacious in improving the iron status of children in China (21).
In conclusion, this trial demonstrates that the provision of free fish sauce fortified with NaFeEDTA (9 mmol Fe/L) at the household level significantly improved iron status and reduced the prevalence of anemia in women of reproductive age in a rural area of Vietnam. Additional studies are warranted to demonstrate its commercial sustainability.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Funded by the Nippon Foundation through the support of Project IDEA of the International Life Sciences Institute Research Foundation/Center for Health Promotion (ILSI RF/CHP).
4 Abbreviations used: CRP, C-reactive protein; ID, iron deficiency; Group C, control group receiving unfortified fish sauce; Group F, fortified group receiving fish sauce fortified with NaFeEDTA; IDA, iron deficiency anemia; data collection times: T0, baseline, T6, 6 mo, T12, 12 mo, T18, 18 mo; SF, serum ferritin; SR, serum retinol.
Manuscript received 15 April 2005. Initial review completed 23 May 2005. Revision accepted 6 August 2005.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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