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*
Department of Epidemiology and International Health, University of Alabama at Birmingham, Birmingham, AL 35294
Medical Research Council, Keneba, The Gambia, West Africa
**
Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011
Medical Research Council, Human Nutrition Research, Cambridge, U.K.
Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294
U.S. Department of Agriculture, Western Human Nutrition Research Center and Department of Nutrition, University of California, Davis, CA 95616
3To whom correspondence should be addressed. E-mail: gmarquis{at}iastate.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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150 µg retinol activity equivalents with (MF) or without (M) 5 g of fat, 5 d/wk for 4 mo or 60,000 µg of vitamin A (A) or placebo (P) capsule at baseline. After 4 mo, plasma ß-carotene was greater in both the M (P < 0.05) and MF (P = 0.07) groups compared with the P group. After controlling for baseline plasma retinol, elevated acute phase proteins and age, plasma retinol concentrations in the A and MF, but not M, groups were higher than in the P group at the end of the study (P < 0.01). Increases in retinol concentrations, however, were small in both groups. These results support the use of dietary supplementation with dried mangoes and a source of fat as one of several concurrent strategies that can be used to help maintain vitamin A status of children in developing countries where there is a severe seasonal shortage of carotenoid-rich foods.
KEY WORDS: • vitamin A deficiency • carotenoids • ß-carotene • mango • The Gambia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
); however, a more recent study found no effect of green leafy vegetables (4
). Fruits may be a better absorbed source of provitamin A carotenoids than green leafy vegetables. Mangoes, for example, are widely available and if solar-dried and stored properly could be a year-round source of vitamin A. Solar-dried mangoes maintain a high ß-carotene content (5
) and were found to be acceptable to young children and adults in Senegal (6
). In this study we therefore determined the effectiveness of solar-dried mangoes, with and without added fat, compared with a high-dose vitamin A capsule and a capsule placebo, in improving the vitamin A status of poor Gambian preschoolers over a 4-mo period when the home diet was low in vitamin A. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study was conducted during the dry season, November 1998 through April 1999, in a rural farming village of 1100 residents in The Gambia, West Africa. The World Health Organization listed The Gambia as a country with a high prevalence of subclinical vitamin A deficiency (7
). Food availability was seasonal; the diet was based on cereals with few fruits, vegetables or animal products. November through April represented the season when carotenoid intake in the home diet was lowest.
There were 218 children between the ages of 2.0 and 6.9 y living in the village, of whom 204 were measured for weight and height in the village; 14 were not measured because of parental (n = 13) or child (n = 1) refusal (Fig. 1
). Exclusion criteria included weight-for-height <2 SD below the National Center for Health Statistics (8
) mean (n = 11) and lack of a 3-d period free of illness immediately before study enrollment (n = 5). No child was allergic to mangoes, had received a vitamin A supplement within the past 4 mo or had clinical symptoms of vitamin A deficiency. Four eligible subjects were not recruited before the end of the enrollment period (November through December 1999), and the parents of eight children withdrew their consent before randomization to treatment, leaving 176 children to randomly assign to study groups.
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–11
), assuming an SD of 0.3 µmol/L, a 5% significance level and 80% power (12
). To allow for a dropout rate of 20%, the sample size was increased to 176 (44/group). Written consent to participate in the study was obtained from the children’s parents. The study was approved by the Medical Research Council scientific coordinating and ethics committees, The Gambia State Department of Health and the institutional review boards of the University of Alabama at Birmingham and Iowa State University.
Study interventions
The study was a four-cell design that included two treatment cells and two control cells. Using a daily block size of multiples of 4, a staff member not involved in data collection or analysis randomly assigned the children, stratifying by age group (2–4 y and 5–7 y), to one of the four study groups: mango (M),4 75 g of dried mango (n = 45); mango and fat (MF), 75 g of dried mango and 5 g of sunflower oil (n = 44); high-dose vitamin A (A), a single capsule of 60,000 µg of retinyl palmitate and 40 mg of 
-tocopherol that served as a positive control (n = 44); and placebo (P), a single capsule of 40 mg of -tocopherol that served as a negative control (n = 43). The food supplements were given 5 d/wk for 4 mo; the capsules were given once at baseline.
The assignment of the capsule treatments (A and P) was double blinded; neither the children nor the study staff knew which of the two treatments was given. Assignment to the different mango treatments (M and MF) was not truly double blinded because the presence of fat in the MF supplement could be noted on close inspection. However it is likely that it was masked as the children were unaware of differences and mothers, and the data collection and laboratory analysis staff were not told the children’s treatment assignment. It was not possible to mask whether the participant was in a capsule or mango treatment group because families inevitably made unsolicited comments about the study to the field workers (e.g., mothers would ask if their child could receive the mangoes if they had received the capsule). Nonmasked treatments may lead to information bias (13
). To minimize this bias, field workers were trained to collect the household data in a systematic fashion. In this present analysis, the surveillance data collected at the household level were used only to describe the population and were not used in the final analyses.
Preparation of dried mangoes. Solar-dried mangoes were obtained from the Institute De Technologie Alimentaire (Dakar, Senegal). Mangoes (Mangifera indica, local variety "amelie") were harvested when they were almost ripe and kept for 48 h at 30°C to ensure uniform ripeness and firmness. The mangoes were washed thoroughly and peeled, sliced to a 1- to 2-mm thickness and placed on clean trays inside the solar cabinet dryer where the temperature was between 45° and 55°C. The solar cabinet dryer consisted of a rectangular container insulated at its base and sides and covered with a double-layer transparent roof. Solar radiation was transmitted through the roof, absorbed on the blackened interior surfaces and trapped in solar panels that helped to maintain the interior temperature. Drying took place over 48 h with a final moisture content of 10–12%. The dried mangoes were then placed in clean polythene bags (50–75 g) that were closed tightly and heat sealed. The bags were packed in fiberboard carton boxes of 5 kg each and stored in a dry area at room temperature. The shelf-life of the dried mangoes was 4–6 mo.
Preparation and distribution of group treatments. Subjects in the capsule groups were given their capsules (Sight and Life, Hoffmann-La Roche, Basel, Switzerland) at the Medical Research Council’s Keneba clinic after baseline blood samples were collected. The neck of the capsule was cut, and the contents were squeezed into the child’s mouth. Staff documented that the subjects swallowed the contents of the capsules and observed them for 1 h after treatment. No child vomited. All children in the placebo group received a single high-dose capsule of vitamin A (60,000 µg of retinyl palmitate) at the end of the study. The subjects in the M and MF groups were given an initial mango treatment at the clinic immediately after the blood draw using the same preparation and feeding methodology as described later.
In the village supplementation center, the mangoes were rehydrated by adding 75 g of water to 75 g of dried mango and allowing the mixture to soak for 90 min in a covered bowl. The mango absorbed all of the water and became soft and chewy. For the MF group, 5 g of sunflower oil was added as a source of fat at the end of the 90 min. After 15 min, the mango and oil was mixed. The mango absorbed all of the oil. The rehydrated mango and bowl were weighed to a precision of 1 g on a food scale (model 8310; Soehnle-Waagen GMbH and Co. KG, Murrhardt, Germany), and then field workers observed the children consume the mangoes. After the child finished eating, the bowl was reweighed. There was no sharing of mangoes among the children. The children liked the mangoes; they formed a network and would collect each other to come to the supplementation center 5 d/wk. The children (6 M and 3 MF) who were absent during part of the period of supplementation were not supplemented for missed days.
Blood collection
Venous blood samples (2 mL) were collected into EDTA tubes at baseline and 2 and 4 mo to measure plasma retinol, ß-carotene and C-reactive protein (CRP). Baseline blood was drawn when the child was healthy and had been free of illness for 
3 d. All 176 children were successfully bled at baseline. At the 2-mo visit, 11 subjects were not bled because of refusal (n = 2), inability to successfully bleed the child (n = 1) or outmigration (n = 8). For the 4-mo visit, 7 children were not bled because of refusal (n = 5), outmigration (n = 1) or hospitalization of the child (n = 1). A blood smear was made for malarial parasites and hemoglobin concentrations measured by the oxyhemoglobin method (14
). All cases of malaria and anemia were treated by standard clinical protocols. The plasma samples of five subjects at baseline and two subjects at 2 mo were less than the minimum amount (0.1 mL) required for retinol and ß-carotene extraction and could not be analyzed.
Surveillance data
A food frequency questionnaire was used to collect data at baseline and on a weekly basis on the frequency and serving size of preformed vitamin A rich foods, carotenoid-rich foods and high fat foods (15
). To describe the morbidity patterns of this community, data were collected weekly through interviews with the mother concerning symptoms of diarrhea, respiratory and skin infections, fever and conjunctivitis. At baseline and every 4 wk, children’s height was measured to 0.1 cm with a portable stadiometer-karrimeter (CMS Manufacturing Equipment, London, U.K.), and they were weighed to 100 g, wearing only light clothing, using a portable digital solar scale (Seca 835, London, U.K.). For each time point, the mean value of three weights and three heights was reported. All anthropometry was completed by one person to ensure uniformity. Weights and heights were converted into weight-for-age (WAZ), weight-for-height (WHZ) and height-for-age (HAZ) Z-scores using Epi-Info version 6.0 software (Centers for Disease Control and Prevention, Atlanta, GA). Information was collected on family demographics, economic status, access to water and sanitation and cooking facilities in an interview with the child’s mother at baseline and at 4 mo.
Plasma analysis
The EDTA tubes were centrifuged at 3800 rpm for 15 min (International Equipment Company, Needham Heights, MA), and at least 1.6 mL of plasma was aspirated and frozen in duplicate at -80°C. The samples were protected from ultraviolet light by wrapping them in aluminum foil. At least 25 samples were placed in leak-proof secondary container (cyrostorage boxes, wrapped in thick plastic and heat sealed at both ends). At the time of shipping, the cyrostorage boxes were placed in a leak-proof shipping box: a Styrofoam ice chest packed with dry ice according to the International Air Transportation Association regulations. The duplicate samples were shipped separately on dry ice to the University of Alabama at Birmingham, where they were stored at -70°C for 4 mo until analysis at the Department of Nutrition Sciences. The extraction procedures by Prince and Frisoli (16
) and Bulux et al. (17
) worked for both retinol and ß-carotene and were selected for sample extraction. Briefly, under yellow light, the samples were thawed for 30 min; 0.25 mL of ethanol was added to 0.25 mL of plasma, mixed and vortexed for 20 s; 0.5 mL of HPLC grade hexane was added, mixed and vortexed and centrifuged at 16,000 x g for 2 min (International Equipment Company). The hexane layer was removed and saved. The precipitate was reextracted with 0.5 mL of hexane and saved. The two hexane layers were combined and evaporated to dryness in a Speedvac for 30 min (Automatic Environment Speedvac, Holbrook, NY). The precipitate was resuspended in 0.1 mL of HPLC solvent (mobile phase), and 0.02 mL of the sample was injected. HPLC analysis of retinol and ß-carotene (within-run and between-run coefficients of variation were 6% for retinol and 9% for ß-carotene) were measured in duplicate samples under yellow light by isocratic HPLC using a Spherisorb c-18 ODS 5-µm reverse-phase column (250 mm x 4.6 mm) (Alltech Associates, Deerfield, IL), 70:30:0.01 acetonitrile/methylene chloride/ß-hydroxytoluene mobile phase and UV detection at 325 and 450 nm, respectively. Concentrations were corrected for loss during processing using a ß-apocarotenal internal standard. Recovery was 95% for both analytes. Standard curves were created using purified retinol and ß-carotene (Sigma, St. Louis, MO) and validated using reference plasma pools (National Institutes of Standards and Technology, Gaithersburg, MD).
CRP was measured by radial immunodiffusion (RID) (The Binding Site, Birmingham, U.K.). A CRP concentration of >5.2 mg/L was considered elevated, indicating an activated acute phase response (18
). Two RID kits with CRP cutoff concentrations of 0.52–5.2 mg/L and 5.2–52 mg/L were used to measure CRP concentrations. Samples with CRP concentrations of >52 mg/L were diluted and repeated. Three standard calibrators were used to produce a linear calibration curve. Rings were allowed to develop to completion, which required a minimum diffusion time of 72 h. One calibration curve was used for three plates of the same batch. The neat calibrator was ran on the second and third plates to ensure all were performing correctly. The neat calibrator, medium and low calibrators and test samples were applied to the wells using a micropipette. For quality control, the neat calibrator was applied to each plate; its CRP concentration was measured each time and found to be the same. CRP concentrations were read off the calibration curve.
Blood collection was inadequate for some laboratory analyses; sample sizes varied by measurement and are indicated in the tables.
Carotenoid content of mango
Every month, three bags of mango were randomly selected and stored at -20°C. A total of 12 samples of mangoes (1 g was randomly selected per bag from 12 bags) were rehydrated, homogenized in 6 mL of ethanol, 1 mL of petroleum ether and 1 mL of KOH, incubated for 40 min at 70°C, cooled and extracted with hexane for analysis (19
,20
). One hundred grams of dried mango contained 197.6 ± 8.8 µg retinol activity equivalents (RAE) (173.6–231.9 µg RAE, assuming 1 µg RAE = 12 µg trans-ß-carotene). The sunflower oil did not contain preformed vitamin A or carotenoids as verified by the food label on the container and local food composition tables. Four hundred micrograms of RAE is the international recommended intake for vitamin A for 1- to 10-y-olds (21
).
Statistical analysis
The principal outcomes of this study were plasma retinol and ß-carotene concentrations at 2 and 4 mo. Because some blood samples were not available at each of the study time points, an "intention-to-treat" analysis was impossible. Analyses were completed on all children with blood samples at baseline and 2 and 4 mo, regardless of the total amount of mango supplement consumed. Descriptive analysis for continuous data included measures of central tendency and dispersion; for categorical data, frequency counts and proportions were obtained. Differences in categorical variables among treatment groups were assessed using a 
2 goodness-of-fit test. One-way ANOVA was used to ascertain group differences in continuous variables. A priori, it was decided that the analyses of retinol and ß-carotene outcomes should be adjusted for age and CRP concentrations because retinol concentrations normally increase with age and decrease with infection while ß-carotene concentrations increase with age (22
,23
). ANCOVA was used to estimate the effect of study group on final retinol and ß-carotene concentrations, controlling for age (2–4 and 5–7 y), CRP (<5.2 mg/L and 5.2 mg/L) and baseline plasma values. Group differences were made using Bonferroni post-hoc tests. Other biological and sociodemographic factors, including anthropometric indicators of nutritional status, were not significant determinants of the outcomes of interest and are not included. Unless stated otherwise, data are presented as mean ± SEM, tests were two-tailed and significance was P < 0.05. All analyses were conducted using SAS version 8 (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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There were 176 children seen at baseline. Follow-up data through 4 mo were collected on 170 (96.6%) subjects (Fig. 1)
. Six subjects were lost to follow-up because of refusal (n = 5) and migration (n = 1). There were no differences in any baseline characteristics (sociodemographic, illness, home diet and plasma retinol and ß-carotene concentrations) between the subjects who completed the study and those who were lost to follow-up. There also were no differences among the four treatment groups in any baseline characteristic (Table 1
). The overall treatment group effect, however, approached statistical significance for WHZ.
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At baseline, malaria affected about one fourth (50/172) of the children; about one third (65/172) of the children had elevated CRP (Table 2
). At the end of the study, malaria affected only 4 of 152 (2.4%) children, and 18 of 152 (10.7%) children had indications of elevated CRP. This expected drop reflected the seasonal conditions of the area (malaria cases peak during the rainy season, June through October, and decline thereafter) as well as the routine treatment of detected cases. Over the 4 mo, about one half of the children in the A (22/44) and P (24/43) groups and about one third of the children in the M (16/44) and MF (17/44) groups were seen at the clinic. There were no significant differences in any measure of illness (malaria, elevated CRP, number of days ill, number of clinic visits) during the follow-up period across treatment groups.
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There was seasonal variation in food availability. Intake of ß-carotene, preformed vitamin A and fat from the home diet was higher during the first 2 mo of the study compared with the last 2 mo (data not shown). The children did not replace their normal breakfast with the mango supplement as determined through the weekly food frequency. There were no differences in home diet among groups.
Ninety-nine percentage of the children were supplemented throughout each week. There was no difference in the total grams of mangoes eaten over 4 mo by the M and MF groups (7715 ± 284 versus 7500 ± 371 g, respectively).
Plasma ß-carotene status
ß-carotene concentrations differed by age but not by gender. Children between the ages of 5 and 7 y had higher plasma ß-carotene concentrations than did 2- to 4-y-olds at baseline (0.56 ± 0.05 versus 0.41 ± 0.04 µmol/L).
There were no group differences in ß-carotene concentrations at baseline or 2 mo (Table 3
). At 4 mo, ß-carotene concentrations were highest in the M and MF groups and lowest in the P group. After adjusting for baseline ß-carotene, CRP and age, the M group remained higher than the P group; the difference between MF and P groups also tended to be significant (P = 0.07).
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As with ß-carotene, retinol concentrations differed by age, not by gender. Compared with children between 2 and 4 y of age, 5- to 7-y-olds tended (P = 0.07) to have slightly higher retinol concentrations at baseline (0.62 ± 0.01 versus 0.64 ± 0.01 µmol/L).
Findings regarding plasma retinol concentrations by study group also were similar to those for ß-carotene (Table 3)
. There was no group difference in baseline or 2 mo. By the end of 4 mo, retinol concentrations differed across groups. The highest mean retinol concentrations were in the A and MF groups; the means, however, remained below 0.70 µmol/L, a cutoff that indicates an increased risk of inadequate liver stores (24
). These small differences remained after controlling for baseline retinol concentrations, CRP and age in the analysis, indicating that the 4-mo change in retinol was greatest in the MF and A groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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The 4-mo plasma retinol concentration in the A group was significantly higher than in the M and P groups. At first glance, this may be interpreted that there was a better response to retinol administered using high-dose capsules than dried mango. However, the total dosage of vitamin A administered differed among the three supplement groups. The vitamin A group received a total of 60,000 RE; the mango groups received approximately one fourth the dose of the vitamin A group, or about 15,000 µg of RAE, using the 12:1 ß-carotene/retinol conversion factor recommended for dietary ß-carotene (25
), including ß-carotene from mango (26
). The magnitude of the mean increase in plasma retinol in the MF group (0.04 µmol/L change) was at least as great as the A group (0.07 µmol/L change) when dosage was taken into account. At the time of the study design, the conversion factor for µg ß-carotene/µg retinol was 6:1. A dose of 75 g of dried mango was chosen to provide what was thought to be 75–100% of the Recommended Daily Allowance (depending on age) for vitamin A. Importantly, it also represented the amount that the young children could easily consume within a few minutes, and this portion size could be readily incorporated as a serving of fruit at a meal or as a supplemental snack.
We saw no increases in plasma ß-carotene concentrations at 2 mo in either mango group. This could be due to an inadequate length of time to increase ß-carotene concentrations using a low dose of vitamin A or to the residual effect of earlier infections that may have continued to reduce ß-carotene absorption. Not surprisingly, 2-mo plasma retinol concentrations among the mango groups also did not increase. Both mango groups had higher plasma ß-carotene concentrations at 4 mo than the other two treatment groups, indicating that ß-carotene was absorbed from the dried mango. This is consistent with results reported by Bates et al. (27
) showing a seasonal variation in plasma carotenoid and retinol concentrations of pregnant and lactating women that was attributed to the consumption of mangoes in May and June.
Surprisingly, there was no change in plasma retinol at 2 mo in the vitamin A group. Because the supplement was given only at baseline and there was a small increase at 4 mo, the unchanged 2-mo plasma concentration suggests that an unknown factor or factors (e.g., seasonal variation, undiagnosed infection, deficiencies in other nutrients) diminished the utilization of stored vitamin A, perhaps by decreasing retinol-binding protein synthesis (28
).
Tanumihardjo (29
) reviewed several vitamin A intervention trials to estimate the participants’ increase in vitamin A liver reserves, based on body weight, estimated liver weight, vitamin A dose, study duration and vitamin A losses. Using her assumptions of absorption and storage, we estimated that our total mango intervention (150 µg of RAE/d) would have provided a net increase in liver vitamin A of 0.039 µmol/g. This increase, along with the small change in plasma retinol concentrations, was between estimates from cited studies that demonstrated increases in vitamin A status indicators (0.05–0.1 µmol/g) and those that did not (0.007–0.01 µmol/g). Although the high-dose supplement provided an immediate net increase in liver vitamin A of 0.109 µmol/g, the stores would rapidly diminish if the home diet were insufficient to meet daily needs. The small increase in plasma retinol suggests that the high dose was insufficient to substantially improve vitamin A status for 4 mo.
Our findings are consistent with other randomized trials. Rahman et al. (30
) found 61% (n = 21) of Bangladeshi infants continued to be deficient despite receiving three 15-mg doses of vitamin A at monthly intervals. The authors attributed the lack of response to frequent respiratory infections, especially those accompanied by fever. The mean frequency of days ill with fever and cough in our study (3.5 d/mo) was similar to that of the deficient children (3.7 d/mo) and higher than the occurrence found among children with normal retinol concentrations (1.7 d/mo) in the Rahman et al. study.
The percent increase in children receiving a food supplement and who had normal plasma retinol concentrations in our study (13%) was lower than that reported (20%) in the Takyi et al. (31
) study in northern Ghana. This greater response in the Takyi study may be because the children 2–6 y of age were dewormed before supplementation, had lower baseline retinol concentrations (19.6% were deficient) and received more supplementation (assuming a 12:1 ß-carotene/retinol conversion, 200 µg of RAE of dark green leafy vegetables was served with an unreported amount of energy and protein, over a similar time period). Given the multiple components of this intervention, it is not possible to determine exactly which combinations (or if all) of components were necessary to produce the positive results.
Similarly the decrease in the proportion of children with marginal plasma retinol concentrations in the mango interventions (13%) was lower than changes (23%) reported by Tang et al. (32
). In the latter study in China, at baseline 39% of subjects had retinol concentrations of <1.05 µmol/L. Children received albendazole treatment for intestinal parasites and were supplemented for 10 wk with green-yellow (n = 22) or light-colored (n = 19) vegetables. Only the carotene-rich vegetable group, who consumed daily about 400 µg of RAE of ß-carotene from the supplement in addition to the usual diet containing 200 µg of RAE preformed vitamin A and almost 60 g of fat per child, increased their serum retinol concentrations.
Although the mean plasma retinol concentrations of our study children did not increase above 0.7 µmol/L, the plasma ß-carotene and retinol concentrations increased minimally with the mango treatments. The small improvement in vitamin A status suggests that a higher total dose is needed to compensate for low home dietary ß-carotene and retinol intake. Additional fat intake may also be necessary to increase ß-carotene absorption. Our findings support the use of dietary supplementation with dried mangoes and a source of fat as one of several strategies that could be used to maintain vitamin A status of children in developing countries where there is a significant seasonal shortage of carotenoid-rich foods. Larger studies are needed to determine the most effective and efficient dose of carotenoids and fat as well as what other concurrent interventions are needed to replete vitamin A status of young children.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Presented in part at Experimental Biology 2001 [Drammeh, B., Marquis, G. S., Stephensen, C. B. & Eto, I.2001A randomized fruit-based intervention study to improve vitamin A status of rural Gambian children. FASEB J. 15:A732 (abs.)] and Experimental Biology 2000 [Drammeh, B., Marquis, G. S. & Stephensen, C. B.2000Comparison of mangoes and vitamin A supplements to increase serum retinol in Gambian children. Late-breaking session. Abstract No. ALB190].
4 Abbreviations used: A, vitamin A; HAZ, height-for-age; M, mango; MF, mango and fat; P, placebo; WAZ, weight-for-age; WHZ, weight-for-height.
Manuscript received 28 May 2002. Initial review completed 3 July 2002. Revision accepted 23 September 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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