![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
4 Department of Food Science and 5 Department of Animal Science, Cornell University, Ithaca, NY 14853; 6 Robert W. Holley Center for Agriculture and Health, Agricultural Research Service, USDA, Ithaca, NY 14853; and 7 Centro Internacional de Agricultura Tropical, Cali, Colombia
* To whom correspondence should be addressed. E-mail: ddm2{at}cornell.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
|
Introduction |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
The common bean (Phaseolus vulgaris L.), one of the staple food crops targeted for nutritional enhancement by HarvestPlus, is a grain legume (6). It provides significant quantities of protein and energy and is a source of vitamins and minerals including Fe (7). The common bean is an attractive candidate for biofortification, because there is genetic variability of Fe concentration and therefore it is possible to breed for significant increases in Fe concentrations in beans (8). Also, Fe concentrations in beans are high relative to other crops and therefore beans can deliver substantial amounts of Fe. Bean genotypes with high Fe concentrations delivered more absorbed Fe to rats than genotypes with lower concentrations of Fe (8).
Breeders at Centro Internacional de Agricultural Tropical, Cali, Columbia, developed a biofortified bean that contains >100 µg Fe/g bean, a substantial increase over standard beans (S. Beebe, unpublished observations). However, an increase in Fe concentration in beans or other staple food crops may not necessarily translate into a proportional increase in absorbed Fe, because genotypes with high Fe concentrations may also have increased (or decreased) concentrations of Fe absorption inhibitors or enhancers. Therefore, it is necessary to measure the amount of bioavailable Fe as well as the concentration of Fe in these new Fe-enhanced crops. Beans are usually consumed with foods that are high in starch such as maize, rice, wheat, sweet potatoes, or cassava. For example, black beans are consumed with maize in Guatemala, southern Mexico, and Haiti. The objective of this study was to compare the capacities of biofortified and standard common beans to deliver Fe for hemoglobin (Hb) synthesis in Fe-deficient pigs.
|
Materials and Methods |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
1 kg beans: 1.5 L water) and cooked in an autoclave at 121°C for 30 min. Following cooking, they were freeze-dried and ground to a coarse powder for incorporation into diets. Maize-based diets (Table 1) containing the beans were formulated to meet current recommendations for nutrient requirements of swine (9). Diets had no supplemental Fe but were adequate in protein, essential fatty acids, vitamins, and minerals for pigs of this age. Pigs were fed the experimental diets for 5 wk. Blood samples (100 µL) were collected weekly from the ear vein using micro-hematocrit heparinized capillary tubes (Fisherbrand).
|
25°C) with a light:dark cycle of 12:12 h, pigs were fed 2 times per day, had free access to water, and were checked daily. The groups were randomly assigned to the treatment diets at age 4 wk and maintained on these diets for 5 wk. Feed intakes were measured daily. Fe intakes were calculated from feed intakes and Fe concentration in the diets. Body weights and Hb concentrations were measured weekly.
Fe bioavailability was calculated as Hb repletion efficiency (HRE) (10):
 |
where Hb Fe = total body Hb Fe. Hb Fe was calculated from Hb concentrations and estimates of blood volume based on body weight (a blood volume of 67 mL/kg body weight was assumed) (11):
Hb Fe (mg) = body weight (kg) x 0.067 L blood/kg x Hb (g/L blood) x 3.35 mg Fe/g Hb.
Fe intakes were calculated from feed intake data and Fe concentrations in the feed.
Hb measurements. Blood Hb concentrations were determined spectrophotometrically using the cyanmethemoglobin method (Pointe Scientific) following the kit manufacturer's instructions.
Phytate analyses in beans and diets. Phytate was extracted from 250 mg of a lyophilized diet sample by suspending in 10 mL of 1.25% H2SO4 for 2 h. Supernatants of the extracts were diluted 1:10 with deionized water and 10-µL aliquots were analyzed by HPLC. A Dionex Liquid Chromatograph system (AS50 Autosampler) equipped with a conductivity detector (model ED50) and a gradient pump (GS50) was used. An IonPac AG11 guard column and an IonPac AS11 column (4 x 250 mm) were used. The mobile phases were 200 mmol/L NaOH (carbonate-free) and deionized water, running at a flow rate of 1 mL/min. PeakNet 6.40 software was used to process the chromatographic data (Dionex).
Polyphenol concentrations in the beans and diets. Samples (1.5 g) of the cooked beans and the diets were extracted in 4 mL of acidified methanol (methanol and 1.0 mol/L HCl, 85:15, v:v). The samples were shaken for 2 h, vortexed to mix, and centrifuged at 12,800 x g; 10 min. Total phenol content in the supernatants was measured as previously described (12). Briefly, to 0.125 mL of the supernatant, 0.5 mL of deionized water and 0.125 mL of the Folin-Ciocalteau reagent were added. The mixture was allowed to stand at room temperature for 5 min and then 0.125 mL of aqueous Na2CO3 (7% wt:v) was added. The final volume of the mixture was adjusted to 3 mL with deionized water and was allowed to stand for 60 min at room temperature. The absorbance was measured at 760 nm against a reagent blank. The amount of total phenolics was expressed as gallic acid equivalents (µg/g diet).
Statistical analysis. One-tailed Student's t tests were performed to compare differences between means using the JMP software (SAS Institute). Values were considered significantly different at P < 0.05. Values in the text are means ± SEM.
|
Results |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
Total phenolic concentration in the beans and bean-maize–based diet samples. The total phenolic concentrations in the bean samples [gallic acid equivalents (µg/g of bean; n = 3)] were 299.0 ± 11.7 µg/g in the high Fe beans and 366.9 ± 6.8 µg/g in the standard Fe beans. Total phenolic concentrations did not differ between the standard (113.9 ± 9.5 µg/g) and high Fe (112.7 ± 8.4 µg/g) bean diets (Table 1).
Growth rates, Hb, and HRE. The pigs grew well over the 5-wk study. Feed intakes and body weights did not differ at any time throughout the study. Fe intakes were consistently higher in the high Fe group due to the higher Fe concentration in the high Fe diet (Table 2). Hb concentrations were higher in the high Fe group than in the standard Fe group after wk 1 of the 5-wk study, but the difference was significant only on d 28 (Table 2). HRE values did not differ between treatments (Table 2). However, the increase in total body Hb Fe from the beginning of the study to the end of wk 5 was greater in the high Fe group [429 ± 24 mg (7.7 ± 0.4 µmol)] than the standard Fe group [361 ± 33 mg (6.4 ± 0.4 µmol)] (P = 0.034; Table 2).
|
|
Discussion |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
The main objective of the present study was to compare the capacities of biofortified and standard beans to deliver Fe for Hb synthesis in Fe-deficient pigs. The pigs receiving the high Fe bean diet gained significantly more Hb Fe than the piglets on the diet containing standard beans. This result clearly shows that Fe-biofortified beans can enhance Fe status in Fe-deficient pigs even when fed as part of a complete diet, where the difference in Fe concentration between the diets was only 12 µg/g and the feeding period was only 5 wk. In a similar study, Schaffer et al. (13) compared the effects of high Fe (13.4 µg/g) and low Fe (2.2 µg/g) rice on Fe status indices in early weaned piglets. At the end of the 33-d feeding trial, none of the indices differed. A likely reason for the lack of effect in this study is that both diets were extremely deficient in Fe; Hb concentrations at the end of the 33-d period were only 45 g/L. In contrast, Haas et al. (14) demonstrated that Fe-biofortified rice improved Fe stores in Fe-deficient but not anemic Filipino women in a 9-mo efficacy trial, even though Fe concentrations in the polished rice they used were extremely low (3.2 µg/g for the high Fe rice and 0.57 µg/g for the control rice). The longer feeding duration in the Haas study (14) and lower Fe requirements in adult women compared with early weaned pigs may explain why they found an effect whereas Schaffer et al. (13) did not.
We chose the piglet as a model for Fe bioavailability studies because of similarities in gastrointestinal anatomy and physiology between pigs and humans. Pigs, like humans, are truly omnivorous and digestive and metabolic processes in pigs are similar to those in humans (15). Moreover, pigs readily consume monotonous diets that can be formulated to simulate human diets that are common in resource-poor regions of the world. Also, Fe deficiency develops rapidly in young pigs unless they are given i.m. Fe injections shortly after birth (16).
We chose to use the increase in body Fe in the pigs as an index of Fe absorption rather than use isotope tracers because of concerns that have been raised about the validity of single meal isotope tracer studies for estimating Fe bioavailability (17,18). To do this, we needed to accurately measure the accumulation of Fe in the animal over an extended feeding period. Unfortunately, we did not have access to an antibody against pig ferritin and therefore we were unable to assess Fe stores in our pigs. We reasoned that if we could keep our pigs moderately anemic, then their Fe stores should be minimal and we could use Hb Fe as a reasonable index of absorbed Fe. This assumption is based on the concept that storage Fe is almost completely depleted before Fe deficiency anemia develops (19). We attempted to "titrate" the Fe status of the pigs so that they would be Fe-deficient anemic, but not severely so, at the start of the study. This was accomplished by reducing the usual amount of Fe given by i.m. injection to the newborn pigs. Piglets of this age grow extremely rapidly and therefore have very high Fe requirements that cannot be met by diet alone. Mean Hb concentrations in both groups at the start of the feeding period were 105 g/L. Hb concentrations were maintained at this level throughout the study in the group receiving biofortified beans but fell slightly in the standard bean group (Table 2).
Baker and DeMaeyer (20) define nutritional anemia as "... a condition in which the Hb concentration is below the level that is normal, for a given individual, due to deficiency of one or more of the nutrients required for hemopoiesis, and conversely, as a condition in which the Hb concentration can be raised by increasing the amount of nutrient(s) absorbed." The WHO (1) defines anemia as a Hb concentration < 110 g/L for children 6–59 mo and <115 g/L for children aged 5–11 y. Cut-off points for anemia for pigs have not been clearly established, but some estimates have been suggested. In a 1980 review, Zimmerman (16) wrote that, according to the NRC, Hb concentrations 
100 g/L are normal for pigs, 90 g/L is the minimum for optimal performance, 80 g/L is borderline anemic, and 60 g/L will result in retarded growth. However, these values are based on growth performance and not on normal levels as defined by Baker and DeMaeyer (20). Zimmerman (16) went on to state that absorption of 59Fe begins to increase at a Hb concentration < 110 g/L, suggesting that pigs with Hb concentrations < 110 are Fe deficient. More recently, Rincker et al. (21) reported on a 35-d feeding trial where weanling piglets had initial Hb concentrations of 110 g/L. Basal diets were supplemented with either 0, 25, 50, 100, or 150 mg Fe/kg diet as ferrous sulfate. The concentration of supplemental Fe was related to the Hb concentration (P = 0.001), suggesting that Hb concentrations of 107 g/L were not normal, i.e. they could be raised by increasing the amount of absorbed Fe. Therefore, we are confident that our pigs, with Hb concentrations of 105 g/L, were anemic and that most of the absorbed Fe went to Hb synthesis and not to stores.
The pig model used in this study has its limitations for predicting Fe bioavailability to humans. Growing pigs must retain 20 mg Fe/kg weight gain (16). Our pigs gained 0.5 kg/d, so they needed to absorb 10 mg/d Fe to meet requirements. The requirement for absorbed Fe for children under 5 y is only 0.6 mg/d or 3% of the pig's daily requirement. Thus, one might argue that pigs are not a good model for the human, because they grow so much faster and their Fe requirements are so much higher. On the other hand, an advantage of the pig is that the length of the feeding period required for obtaining meaningful results is proportionally shorter than with humans because of their rapid growth. Another difference between pigs and humans is that the small intestine in pigs is longer than the small intestine in humans (15). This may give the pig a comparative advantage in Fe absorption because of the greater intestinal surface area. However, when expressed on a per-kilogram body weight basis, intestinal lengths are similar at 0.1 m intestinal length per kg body weight (22).
We conclude that the biofortified beans are a promising vehicle for increasing intakes of bioavailable Fe in human populations where beans are a dietary staple and that black beans contain relatively high levels of bioavailable Fe. An efficacy trial comparing biofortified and standard beans in a human population is needed to confirm the findings reported here.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Author disclosures: E. Tako, J. M. Laparra, R. P. Glahn, R. M. Welch, X. G. Lei, S. Beebe, and D. D. Miller, no conflicts of interest.
3 Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
Manuscript received 20 August 2008. Initial review completed 24 September 2008. Revision accepted 24 November 2008.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
|---|
1. WHO. Iron deficiency anemia assessment, prevention and control. A guide for program managers. Geneva: WHO/NDH; 2001. p. 15–21.
2. Baltussen R, Knai C, Sharan M. Iron fortification and iron supplementation are cost-effective interventions to reduce iron deficiency in four subregions of the world. J Nutr. 2004;134:2678–84.
3. Tontisirin K, Nantel G, Bhattacharjee L. Food-based strategies to meet the challenges of micronutrient malnutrition in the developing world. Proc Nutr Soc. 2002;61:243–50.[Medline]
4. Lynch SR. The impact of iron fortification on nutritional anaemia. Best Pract Res Clin Haematol. 2005;18:333–46.[Medline]
5. Nestel P, Bouis HE, Meenakshi JV, Pfeiffer W. Biofortification of staple food crops. J Nutr. 2006;136:1064–7.
6. Salunkhe DK, Desaphande SS. Food of plant origin: production, technology and human nutrition. New York: Van Nostrand Reinhold; 1991.
7. Geil PB, Anderson JW. Nutrition and health implications of dry beans: a review. J Am Coll Nutr. 1994;13:549–58.[Abstract]
8. Welch RM, House WA, Beebe S, Cheng Z. Genetic selection for enhanced bioavailable levels of iron in bean (Phaseolus vulgaris L.) seeds. J Agric Food Chem. 2000;48:3576–80.[Medline]
9. NRC. Nutrition requirements of swine. 10th ed. Washington, DC: National Academy Press; 1998.
10. Howard L, Buchowski M, Wang B, Miller DD. Bioavailability of electrolytic iron in fortified infant cereal determined by hemoglobin repletion in piglets. Nutr Res. 1993;13:287–95.
11. Yasuda K, Roneker KR, Miller DD, Welch RM, Lei XG. Supplemental dietary inulin affects bioavailability of iron present in corn and soybean meal to young pigs. J Nutr. 2006;136:3033–8.
12. Dewanto V, Wu X, Adom K, Hai Lui R. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem. 2002;50:3010–4.[Medline]
13. Schaffer S, Pallauf J, Krawinkel MB. Impact of feeding high- iron rice on plasma iron, hemoglobin and red blood cell variable of early-weaned piglets. Ann Nutr Metab. 2004;48:109–17.[Medline]
14. Haas JD, Beard JL, Murray-Kolb LE, del Mundo AM, Felix A, Gregorio GB. Iron biofortified rice improves the iron stores of non-anemic Filipino women. J Nutr. 2005;135:2823–30.
15. Patterson JK, Lei X, Miller DD. The pig as an experimental model for elucidating the mechanisms governing dietary influence of mineral absorption. Exp Biol Med (Maywood). 2008;233:651–64.
16. Zimmerman DR. Iron in swine nutrition. National feed ingredient association literature review on iron in animal and poultry nutrition. Des Moines (IA): National Feed Ingredient Association; 1980.
17. Cook JD, Reddy SR. Effect of ascorbic acid intake on non-heme iron absorption from a complete diet. Am J Clin Nutr. 2001;73:93–8.
18. Cook JD, Dassenko SA, Lynch SR. Assessment of the role of non-heme iron availability in iron balance. Am J Clin Nutr. 1991;54:717–22.
19. Yip R. Iron. In: Bowman BA, Russell RM, editors. Present knowledge in nutrition. 8th ed. Washington, DC: International Life Sciences Institute Press; 2001. p. 311–28.
20. Baker SJ, DeMaeyer EM. Nutritional anemia: its understanding and control with special reference to the work of the World Health Organization. Am J Clin Nutr. 1979;32:368–417.
21. Rincker MJ, Hill GM, Link JE, Rowntree JE. Effects of dietary iron supplementation on growth performance, hematological status and whole body mineral concentrations of nursery pigs. J Anim Sci. 2004;82:3189–97.
22. Emmans GC, Kyriazakis I. Growth and body composition. In: Kyriazakis I, editor. Quantitative biology of the pig. Oxon (UK): CAB International; 1999. p. 181–98.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
