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Department of Nutrition, The Pennsylvania State University, University Park, PA;
*
Department of Plant Biotechnology, National Institute of Agrobiological Sciences (NIAS), Kannondai 2–1-2, Tsukuba, Ibaraki 305-8602, Japan;
Department of Bio-Science, Central Research Institute of Electric Power Industry (CRIEPI), 1646 Abiko, Chiba 277-1194, Japan; and
**
CHORI (Children’s Hospital Oakland Research Institute), Oakland, CA
2To whom correspondence should be addressed. E-mail: its{at}psu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • ferritin • iron deficiency • rats • rice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). A sustainable solution to dietary iron deficiency is required to reduce significantly the prevalence of iron deficiency anemia in the Third World. Most programs to date have focused on supplementation and/or fortification. However, food supplementation programs are relatively expensive, noncompliance can be high and interactions between supplements and endogenous food components are complex (2
,3
). Fortification programs can be highly effective but again require an infrastructure for delivery and perhaps some targeting (2
,3
). A novel approach has been to bioengineer or to select by classical plant breeding protocol, staple crops that contain increased amounts of iron or decreased amounts of inhibitors of absorption (4
–7
).
Ferritin is the major source of iron in the early development of humans as well as other animals and plants (8
). Cellular concentrations of iron equivalent to >1011 times the solubility of the free Fe (III) ion are achieved with ferritin. We previously examined purified ferritin and soybean meal, in which much of the iron is in ferritin, to determine whether they could be sources of iron for treating iron deficiency in rats (9
). We showed that full recovery of anemia occurred after 28 d of treatment with any of the iron sources. This suggested that manipulating ferritin expression and other soluble components of seed iron in soybeans and possibly other seeds, may contribute to a solution to global problems of iron deficiency. In our continued exploration of the potential benefits of different forms of seed iron, we employed a similar research design utilizing "iron-improved rice" (either increased amounts of ferritin or increased amounts of iron) and compared this with an FeSO4 diet.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Sprague-Dawley rats (n = 50), 21 d old, were purchased from Harlan Industries (Indianapolis, IN) and maintained in an NIH-approved facility. The lights in the facility were turned off between 1900 and 0700 h and the room temperature was maintained at 25 ± 1°C. All studies were prereviewed and approved by The Pennsylvania State University Animal Care and Use Committee and were performed in full compliance with all institutional and governmental animal welfare standards.
Dehulled rice from three types of plants was used, i.e., Kitaake (KIT)3
variety, and FK11 and FK22, two transgenic plants derived from KIT that express soybean ferritin (Fig. 1
), which was targeted to the seeds (10
,11
). In the original study, the iron concentration of the whole seeds overexpressing ferritin was approximately 3X higher than the parent KIT rice (10
,11
), but the dehulled seeds used in this experiment had an iron concentration (µg/g dry weight) as follows: KIT, 4.2; FK11, 2.7; and FK22, 6.7; for the "embryos"/hulls, the iron concentration (µg/g dry weight) was: KIT, 71.21; FK11, 71.21; and FK22, 68.6. The hull contributes 
5% to the total seed weight, making the whole-seed iron concentration approximately (µg/g dry weight): KIT, 10.02; FK11, 8.5; and FK22, 11.6. Such results suggest that changes in growing conditions or the fraction of whole-seed iron contributed by the hulls or both influence the final rice seed iron concentration, which poses questions to be explored in the future.
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) for the entire study (42 d) starting at d 21 of age. Other rats were fed an iron-deficient (ID) diet for 2 wk [AIN 93G diet modified to contain a low amount of iron (1.3 mg Fe/kg diet)] and then divided into 5 different dietary groups such that there was an equal distribution of body weights and hemoglobin (Hb) levels at baseline. Rats were fed one of the following diets for the next 28 d: 1) low iron diet (1.3 mg Fe/kg diet; n = 11); 2) FeSO4 repletion diet (9.4 mg Fe/kg diet; n = 8); 3) KIT variety repletion diet (9.9 mg Fe/kg diet; n = 8); 4) FK22 variety repletion diet (bioengineered to contain increased amounts of ferritin: 8.3 mg Fe/kg diet; n = 6); and 5) FK11 variety repletion diet (bioengineered to contain increased amounts of ferritin: 9.9 mg Fe/kg diet; n = 7). The composition of the diets were as follows: CN diet, AIN 93G diet with ferric citrate as the iron source; FeSO4 diet, AIN 93G diet (modified to contain the same amount of iron as the rice diets) with FeSO4 as the iron source; FK11, FK22 and KIT diets (g/kg diet), rice (744); casein (148); oil (60); mineral mix without iron (33); vitamin mix (9.5); L-cystine (2.8); choline bitartrate (2.36); and tert-butylhydroquinone (0.013); ID diet, AIN 93G diet without iron.
All diets met the AIN93G recommended values for all nutrients except iron. The number of rats in each group was determined by the amount of rice meal available to us, but making sure that no group had fewer than 6 rats. The design of all repletion diets containing nearly equal amounts of iron allowed a direct computation of biological iron availability. For assessment of iron, diets were digested and analyzed for iron content on a flame atomic absorption spectrophotometer.
Blood was collected from the tail on d 14, 21, 28 and 35 of the study. On d 42, the rats were killed by exsanguination while under anesthesia (CO2). Blood was collected from the abdominal aorta, and livers and spleens were removed. Hemoglobin (Hb; procedure no. 525, Sigma Chemical, St. Louis, MO), hematocrit (Hct), plasma and tissue iron and total iron binding capacity were determined by standard methods (12
).
Values are expressed as the arithmetic means ± SEM. Statistical comparisons were made among the groups of rats for iron intake as well as iron status differences. ANOVA was used to compare data among groups followed by Tukey’s test as a post-hoc evaluation. Differences with a P-value 
0.05 were considered significant. Data were analyzed using SAS 8 (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) with the exception of the control and iron-deficient groups.
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) but were significantly greater than in the ID rats. Because the amount of dietary iron was insufficient to fully replete iron stores, the indices of iron transport to marrow did not differ between the iron repletion groups and the ID group (Table 1)
. Therefore, iron from rice was not differentially available for Hb production than iron from ferrous sulfate, and was used as efficiently.
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). In addition, rats fed the FK11 or KIT diets gained more weight than those fed the FeSO4 diet despite the same macronutrient and micronutrient intakes. The rats fed KIT rice had larger livers and 66% more iron in liver (P = 0.15) than rats fed FeSO4 (Table 2)
. The FK11 and KIT groups also had significantly higher concentrations of iron in the spleen compared with the FeSO4 group (Table 2)
. The greater weight gain in some rice repletion groups should have translated into a greater total blood volume and red cell mass (RCM) at the end of the refeeding period. Iron in the RCM was significantly higher in the rice and ferrous sulfate groups compared with the ID group at the end of the study. However, estimates of iron in the RCM at the end of the study did not differ among iron repletion groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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9 mg Fe/kg diet to rats in each of the groups. This amount of iron is not enough to completely reverse iron deficiency in 4 wk. However, the efficiency of iron incorporation into the RCM and storage iron does indicate directly the bioavailability of iron from the rice used in this study.
Earlier studies (13
–17
) showed ferritin to be generally a poor source of dietary iron. However, those studies were performed before some of the features of ferritin regulation were fully understood. Intrinsically labeled ferritin was generally produced by giving an animal a large bolus of iron followed by induction of an inflammatory state (14
,15
). This probably produced a "stress" ferritin not typical of the form that would normally be contained in foods. Also, the use of isotopically labeled ferric citrate to label ferritin extrinsically (17
) would have measured only a small fraction of the iron because the time frame for equilibration of the label with the "core" iron was too short.
Our data (Tables 1
, 2)
show that iron in rice has bioavailability equal to FeSO4, at the same concentrations. Moreover, when ferritin is overexpressed in rice, the iron remains bioavailable. Increasing iron in the seed without ferritin would be toxic to the plant. But the safety of iron in ferritin and the high amount of iron per molecule of ferritin (800–2000 Fe atoms/molecule) indicates that small changes in seed ferritin concentrations can have a large effect on iron concentrations when iron delivery to the seed is increased sufficiently. The equivalent effectiveness of iron in rice diets and FeSO4, in replenishing Hct, Hb concentration and liver iron concentrations emphasizes the importance of experiments to increase ferritin and iron in rice and other grains as an important but neglected tool to use in addressing global iron deficiency.
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FOOTNOTES |
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3 Abbreviations used: CN, control; Hb, hemoglobin; Hct, hematocrit; ID, iron deficient; KIT, kitaake; RCM, red cell mass.
Manuscript received 22 October 2001. Initial review completed 2 November 2001. Revision accepted 28 January 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. World Health Organization (1992) National Strategies for Overcoming Micronutrient Malnutrition, Document A45/3 1992 WHO Geneva, Switzerland. .
2. McGuire, J. (1993) Addressing micronutrient malnutrition. SCN News 9:1-10.
3. Yip, R. (1997) The challenge of improving iron nutrition: limitations and potentials of major intervention approaches. Eur. J. Clin. Nutr. 51:S16-S24.
4. Welch, R. M., House, W. A., Beebe, S. & Cheng, Z. (2000) Genetic selection for enhanced bioavailable levels of iron in bean (Phaseolus vulgaris L.) seeds. J. Agric. Food Chem. 48:3576-3580.[Medline]
5. Graham, R. D., Senadhira, D., Beebe, S., Iglesias, C. & Monasterio, I. (1999) Breeding for micronutrient density in edible portions of staple food crops: conventional approaches. Field Crops Res. 60:57-80.
6. Welch, R. M. & Graham, R. D. (1999) A new paradigm for world agriculture: meeting human needs. Productive sustainable, nutritious. Food Crops Res. 60:1-10.
7. Graham, R. D. & Welch, R. M. (19996) Breeding for Staple-Food Crops with High Micronutrient Density, Agricultural Strategies for Micronutrients, Working Paper 3 19996 International Food Policy Research Institute Washington, DC. .
8. Linder, M. C., Moor, J. R., Scott, L. E. & Munro, H. N. (1972) Prenatal and postnatal changes in the content and species of ferritin in rat liver. Biochem. J. 129:455-462.[Medline]
9. Beard, J. L., Burton, J. W. & Theil, E. C. (1996) Purified ferritin and soybean meal can be sources of iron for treating iron deficiency in rats. J. Nutr. 126:154-160.
10. Goto, F., Yoshihara, T., Shigemoto, N., Toki, S. & Takaiwa, F. (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat. Biotechnol. 17:282-286.[Medline]
11. Goto, F. & Yoshihara, T. (2001) Improvement of micronutrient contents by genetic engineering—development of high iron content crops. Plant Biotechnol 18:7-15.
12. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
13. Cook, J. D. (1980) Iron 1980:32-36 Churchill Livingstone New York, NY and 105–109.
14. Hussain, R., Walker, R. B., Laryisse, M., Clark, P. & Finch, C. A. (1965) Nutritive value of food iron. Am. J. Clin. Nutr. 16:464-471.[Abstract]
15.
Laryisse, M., Martinez-Torres, C., Renzy, M. & Leets, I. (1975) Ferritin iron absorption in man. Blood 45:689-698.
16. Hallberg, L. (1981) Bioavailability of dietary iron in man. Annu. Rev. Nutr. 1:123-147.[Medline]
17. Derman, D. P., Bothwell, T. H., Torrance, J. D., MacPhail, A. P., Bezwoda, W. R., Charlton, R. W. & Mayet, F.G.H. (1982) Iron absorption from ferritin and ferric hydroxide. Scand. J. Haematol. 29:18-24.[Medline]
18.
Pinero, D. J., Li, N. Q., Connor, J. R. & Beard, J. L. (2000) Variations in dietary iron alter brain iron metabolism in developing rats. J. Nutr. 130:254-263.
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