2006 Annual Report
1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Important food crops do not provide enough bioavailable levels of several essential micronutrients (e.g., iron, zinc, selenium, vitamin A, etc.) to meet the needs of certain population groups dependent on them for their nutriture, which has resulted in micronutrient deficiencies occurring in over three billion people globally (mostly resource-poor women, infants and children). For example, worldwide there are now over 3 billion people deficient in iron and 200 million people are known to be vitamin A deficient. It is estimated that there may be as many zinc deficient people as there are iron deficient people because the primary sources of these nutrients are the same foods. However, currently there is no clinical test to determine the extent of zinc deficiency globally. Additionally, some important food crops (e.g., durum wheat grain) can contain potentially harmful levels of heavy metals (e.g., cadmium) that can depress the bioavailability of the essential micronutrients - iron and zinc, increasing the frequency of micronutrient malnutrition in at risk populations. Furthermore, unacceptable levels of heavy metals in food crops that exceed international limits set by the Codex Alimentarius Commission can restrict their sale in international markets. Thus, ways need to be found to increase the bioavailable levels of micronutrients, and lower the levels of harmful heavy in staple food crops to improve the health of people and maintain international markets for the sale of farmers' products.
This project has three specific goals:.
1)increase the density of bioavailable Fe and Zn in edible portions of staple food crops through identifying the criteria needed for selecting for or engineering high yielding genotypes having these characteristics,.
2)identify factors (antinutrients, promoter substances, nutrient interactions) that affect trace mineral bioavailability in foods and ascertain the key mechanisms of action, and.
3)determine the processes controlling Cd accumulation in grain elucidating the physiological functions of genes that confers low grain-Cd-accumulation to durum wheat.
The research to be undertaken falls under National Program Codes 107 - Human Nutrition Requirements and Intake, and 108 - Food Composition and Food Safety addressing goals 3.1.2 and 3.1.3 as described in the National Program Action Plan. Specifically, these include:
126.96.36.199. Determine the mechanisms of plant accumulation of toxic amounts of heavy metals. 188.8.131.52. Develop management strategies, and assist plant breeders to produce crops with reduced toxic metal accumulation. 184.108.40.206. Use cell culture and animal models to determine the biological activity, mechanism(s) of action, and health effects of food components modified by classical breeding and biotechnology. 220.127.116.11. Develop in vitro methodologies that function as screening tools to measure nutrient bioavailability including biological systems (e.g., in vitro digestion/cell culture) and non-biological systems (e.g., mathematical modeling and algorithms).
This research project empowers scientists with knowledge needed to understand the ways in which food crops control the accumulation of bioavailable micronutrients, health promoting factors and toxic metals. The knowledge obtained can be used to genetically modify food crops or change cultural practices in ways that enhance human nutrition and health protecting consumers, shielding farmers from trade restrictions being imposed by international regulatory bodies on plant food commodities and providing new crops with nutritional quality traits that can improve public health and increase small farm profitability in niche markets.
2.List by year the currently approved milestones (indicators of research progress)
Year 1 (FY 2004)
Test and rank select Fe-dense genotypes of rice, wheat, maize, and beans for bioavailable Fe using an in vitro Caco-2 model.
Identify factors affecting Fe bioavailability using an in vitro Caco-2 model.
Develop an in vitro Caco-2 model for determining Zn bioavailability from plant foods.
Establish and map the distribution of trace elements in soils of major durum production in North Dakota.
Determine effects of field landscape location on trace elements in soils and durum grain in North Dakota.
Determine the role of Cd-phytochelatin complexes on Cd accumulation in durum wheat grain.
Determine the mechanisms for Cd compartmentalization in durum wheat roots.
Year 2 (FY 2005)
Test most promising genotypes of maize, wheat and beans using a pig model in collaboration with Drs. Xingen Lei and Dennis Miller (Cornell University) to verify Caco-2 cell model results.
Continue to identify factors affecting Fe bioavailability using an in vitro Caco-2 model.
Begin application of Zn bioavailability model to test Zn-dense genotypes of beans, rice, wheat and maize, or explore contingencies.
Prepare technical bulletin of maps and data for trace elements in soils of northern North Dakota.
Prepare manuscript comparing uptake and root-shoot Cd-phytochelatin partitioning in durum wheat.
Begin investigations on PC enzymes and on studies of Cd-PC transport across the tonoplast of cortical root cells.
Year 3 (FY 2006)
Test most promising Fe and Zn dense genotypes of maize and wheat using a pig model validating data from the in vitro Caco-2 model.
Use results on metal speciation studies to initiate identifying promising lines of staple food crops having high levels of bioavailable Fe.
Develop protocal for in vito human trial testing the bioavailable Fe in the most promising bean genotype in collaboration with the Grand Forks Human Nutrition Research Center in Grand Forks, ND.
Continue practical applications, or exploration of contingencies for the in vitro Caco-2 cell model for Zn bioavailability.
Publish technical bulletin on trace elements in soils of ND, and evaluate the distribution of Soil-Zn in the region and prepare manuscript of findings.
Investigate mechanisms controlling Cd re-translocation from durum wheat roots to shoots.
Year 4 (FY 2007)
Begin in vitro Caco-2 cell model testing of Fe bioavailability in sweet potato and cassava and begin human trial of iron and zinc bioavailability in elite genotypes of rice and beans.
Summarize results of factors affecting iron bioavailability form staple plant foods and share data with the CGIAR cooperating Centers (IRRI, CIAT, CIMMYT, and IFPRI).
Continue Human trial on bioavailable Fe and Zn in elite genotypes of beans.
Continue practical applications of Zn bioavailability Caco-2 cell model and evaluate results.
Initiate molecular phase of research on Cd accumulation in durum wheat grain and attempt to clone and confirm function of low-Cd gene in durum wheat.
Year 5 (FY 2008)
Begin human trials of bioavailable Fe in elite lines of rice in collaboration with the Grand Forks Human Nutrition Research Center, Grand Forks, ND.
Publish the results concerning the identity of factors affecting Fe bioavailability from staple plant foods.
Publish results of the bioavailability of Fe in rice, wheat, beans, and wheat.
Report results of bioavailable Zn in rice, wheat, beans and maize to cooperating CGIAR Centers (IRRI, CIMMYT, CIAT and IFPRI) and publish papers concerning results.
Use cloned low-Cd gene in wheat transformation system to develop a low-Cd durum wheat transformant for using in plant breeding programs.
4a.List the single most significant research accomplishment during FY 2006.
Identifying the maize genes that control kernel Fe level and bioavailability. Genes in maize controlling both kernel-Fe concentration and kernel-Fe bioavailability in maize were identified using the in vitro digestion Caco-2 cell model. Results using intermated recombinant inbred maize lines (B73 and Mo17) and composite interval mapping (CIM) for quantitative trait loci (QTLs) indicated traits for kernel-Fe concentration mapped to 3 chromosomes (1, 2,.
5)while traits for kernel-Fe bioavailability mapped to 3 different chromosomes (3, 6, 9). Identification of QTLs for these traits will greatly reduce the time needed to breed new lines of maize higher in bioavailable kernel-Fe levels. NP 107 Action Plan 18.104.22.168 and 22.214.171.124.
4b.List other significant research accomplishment(s), if any.
4c.List significant activities that support special target populations.
In-house "D" Project:
We have completed a follow-up studies comparing in vitro Caco-2 cell Fe bioavailability results with a piglet model and human subjects using white beans and red beans. Generally, the Fe bioavailability determined using the three models (Caco-2 cell, pig and human) did not agree. The human studies used extrinsically labeled bean meals and Fe adequate subjects while the pig model used hemoglobin repletion assays, anemic pigs and the pigs were equilibrated on bean meals for one week before testing Fe bioavailability. These factors could account for the differences in results obtained between the models. Further studies are being planned to test this hypothesis.
We continue research on producing more of the meat factor isolates from fish muscle to allow further testing and characterization of the active compounds. This project is still being significantly delayed in the past year due to retraining of personnel at our lab and at our collaborator's lab in ERRC. We now have a postdoctoral associate performing mass spectral analysis of fractions and generating fractions for isolation and purification by Dr. Arland Hotchkiss at the ERRC.
Currently, we are still isolating and identifying polyphenol inhibitors in colored seed coats of beans and trying to isolate and identify promoter substances in white bean seed coats and in orange flesh sweet potato and yellow cassava.
An in vitro Caco-2 cell model for Zn bioavailability was been pursued using cell metallothionein as a proxy for Zn bioavailability. However, these studies used only ZnCl2 and did not investigate intrinsic Zn in foods. Upon further investigation we found that in the presence of foods, very little food-Zn was bioavailable to the Caco-2 cells in our in vitro digestion model. We tested a broad range of food samples and could not generate significant measurable levels of Zn uptake in the cells. We therefore conclude that measurement of Zn bioavailability in foods via the Caco-2 cell model system is not useful for studies of Zn bioavailability. Apparently, Caco-2 cells only absorb free ionic Zn (i.e., Zn2+) from digestates. We advocate the use of an animal model for screening studies of Zn bioavailability in staple food crops.
In an effort to understand the basis for differential Cd translocation from roots to shoots in near-isogenic lines of durum wheat that differ in grain Cd concentrations, we have examined Cd fluxes among compartments within roots of both isolines. The results of Cd efflux analysis experiments show that there are no discernable differences between isolines in movement of Cd out of vacuoles, cytoplasm or cell walls into external root bathing solutions, whereas xylem contents expressed from excised shoots show higher Cd concentrations in the high Cd-accumulating isoline.
This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the Department of Crop and Soil Science, Cornell University entitled "Bioavailability and geographic distribution of nutritionally important elements in crops and Soils" 1907-42520-003-01S. This agreement terminated on May 15, 2006. Thus, there was little activity or expenditure of funds under this cooperative agreement during the FY2006. A technical bulletin for farmers, agricultural advisors, and researchers is being prepared by a retired ARS scientist (Dr. Wendell Norvell). The manuscript submitted to GEODERMA has been accepted for publication comparing four geostatistical methods for handling skewed geographic information for soil zinc in northern North Dakota. This study has demonstrated benefits also from concurrent use of correlated characteristics of soil pH and organic carbon content in predicting soil zinc by cokriging. Collaborating in this research were Drs. Jay Wu and Steve DeGloria (Cornell University), and David Hopkins (NDSU).
This report serves to document research conducted under a Trust Fund Type Cooperative Agreement between ARS and Kraft Foods entitled "Nutritional quality of fortified foods and their contribution to diets of developed regions" 1907-42520-003-03T. This agreement was continued into FY2006 under a co-cost extension, and no research was conducted or planned as we were awaiting a decision from our cooperators (Kraft Foods, Inc.) regarding further funding. They have since declined to renew the agreement, and therefore, this project has been terminated and a final report was written and submitted. In the previous reporting period, the significant research of this project was summarized in the following statements. Due to its chemical and organoleptic properties, encapsulated micronized ferric phosphate (MFP) may be a suitable form of Fe fortificant for process cheese. Using the in vitro digestion/Caco-2 cell culture model, iron availability from encapsulated MFP was evaluated in process cheese, and in the combination of the MFP plus a rice, fish and vegetable meal typical of Southeast Asia. The results indicate that Fe from the MFP cheese was moderately available and was enhanced by the presence of ascorbic acid in the meal, thus it represents a promising form of fortification for this food product. Various forms of encapsulated NaFeEDTA were also evaluated in a snack food such as chocolate chip cookies. In general, NaFeEDTA was found to be a good Fe fortificant in the cookies, however the chocolate chips were found to be strong inhibitors of Fe availability. Although the cookies are considered a snack food, the addition of NaFeEDTA to the food does appear to increase its nutritional value by providing more bioavailable Fe.
This report serves to document research conducted under a General Assistance Type Cooperative Agreement between ARS and the Department of Food Science at Cornell University entitled "Bioavailability of Fe and Zn in biofortified crops" 1907-42520-003-07G. Experiments were performed to determine the bioavailability of iron in two wheat varieties that differ in their inulin levels (one high and one low inulin wheat variety) using a pig model in cooperation with Drs. Dennis Miller (Department of Food Science) and Xingen Lei (Dept. Animal Science) at Cornell University. Previous pig model studies, carried out in 2004 and early 2005, showed that pigs fed a maize-soy based diet supplemented with 4% inulin absorbed significantly more iron from the diet (as determined by iron incorporation into hemoglobin; hemoglobin repletion efficiency) compared to pigs fed the same diet not supplemented with inulin. Unfortunately, the wheat lines provided by our cooperators at the University of Adelaide, S. Australia were found to contain similar amounts of inulin and therefore, the experiment did not provide any useful information. Studies also compared iron bioavailability from colored and white beans fed to pigs with the same bean varieties screened for iron bioavailability using an in vitro Caco-2 cell model in order to further validate the Caco-2 cell model. Results showed significant differences between the models in the amount of bioavailable Fe in the beans lines studied. Further research is underway to ascertain the reason for these differences. This research was supported, in part from HarvestPlus funds.
This report serves to document research conducted under a Reimbursable Fund Type Cooperative Agreement between ARS, and the IFPRI and the CIAT entitled "Biofortified crops for improved human nutrition" 1907-42520-003-08R. Research was extended on Fe absorption enhancing properties of prebiotics with a focus on mechanisms underlying this enhancing effect. Anemic piglets at age 5 weeks were allocated to a standard corn-soy diet (control) or the same diet supplemented with 4% inulin. After 7 weeks on this diet, the animals were killed and sections of the small intestine and colon were quickly removed. Enterocytes were harvested by scraping with a glass slide and frozen in liquid nitrogen. Using primers designed to represent DMT-1, DcytB, ferroportin, and ZnT-1, RT-PCR analyses were performed to quantify the expression of the respective genes encoding for these proteins. Concentrations of m-RNA encoding for DMT-1, DcytB, ferroportin, and ZnT-1 in duodenal samples were significantly higher in the inulin group (p < 0.05). In colon samples, DMT-1 and ferroportin were significantly upregulated. These results suggest that up-regulation of genes encoding for Fe and zinc membrane transporters is a possible mechanism whereby inulin enhances Fe and possibly zinc absorption. Portions of this research were funded by Harvest Plus.
This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Food Science at Cornell University entitled "Improving the micronutrient bioavailability of staple foods, food ingredients and food products" 1907-42520-003-09S. Our primary goal for this past year was to extend our work on the iron absorption enhancing properties of prebiotics with a focus on mechanisms underlying this enhancing effect. Anemic piglets at age 5 weeks were allocated to a standard corn-soy diet (control) or the same diet supplemented with 4% inulin. After 7 weeks on this diet, the animals were killed and sections of the small intestine and colon were quickly removed. Enterocytes were harvested by scraping with a glass slide and frozen in liquid nitrogen. Using primers designed to represent DMT-1, DcytB, ferroportin, and ZnT-1, RT-PCR analyses were performed to quantify the expression of the respective genes encoding for these proteins. Concentrations of m-RNA encoding for DMT-1, DcytB, ferroportin, and ZnT-1 in duodenal samples were significantly higher in the inulin group (p < 0.05). In colon samples, DMT-1 and ferroportin were significantly upregulated. These results suggest that up-regulation of genes encoding for iron and zinc membrane transporters is a possible mechanism whereby inulin enhances iron and possibly zinc absorption. A manuscript describing this work is nearly ready for submission to a journal. Portions of this research were funded by Harvest Plus.
We also continued our work on the regulation of iron absorption from NaFeEDTA that we reported on last year. Specifically, we studied the uptake of iron from NaFeEDTA, FeSO4, and FeCl3 by Caco-2 cells in the presence or absence of ascorbic acid (AA) and bathophenanthroline disulfonic acid (BPDS). Uptake of iron from all 3 iron sources was similar in the absence of ascorbic acid. Adding ascorbic acid at a 5:1 molar excess compared to iron increased uptake by 5.4, 5.1, and 2.8-fold for FeSO4, FeCl3, and NaFeEDTA, respectively. The smaller effect of AA on uptake from NaFeEDTA may be related to the higher solubility of NaFeEDTA, and/or the strong binding affinity of EDTA for Fe3+. Adding BPDS to the media inhibited uptake from all three iron sources equally. BPDS binds Fe2+ but not Fe3+. These results suggest that uptake of iron from all 3 sources follows a common pathway, i.e. the iron is released from complexes or salts, reduced to Fe2+, and transported into the cell via DMT-1. Taken together, these results suggest that uptake of iron from NaFeEDTA by intestinal enterocytes is regulated similarly to uptake from iron salts. Portions of this work were supported by the USDA/NRI Competitive Grant Program (#58-1907-0-033).
We also continued our studies designed to determine the effects of bread baking on the bioavailability of iron from flour fortified with elemental iron powders. Our hypothesis was that elemental iron powders get oxidized during bread baking to either ferrous or ferric iron, thereby enhancing the bioavailability of the elemental iron (it has been reported that the bioavailability of elemental iron powders to human subjects is approximately 50% that of FeSO4). We baked 3 large batches of bread, one using flour fortified with hydrogen reduced (HR) iron powder, one with unfortified flour, and a 3rd with flour fortified with FeSO4. The bread was dried and mixed into corn-based swine diets. For the diet containing the unfortified bread, hydrogen reduced iron was added to the diet during mixing, i.e. after baking. The diets were fed to anemic pigs in a hemoglobin repletion trail. Hemoglobin repletion efficiencies, an index of iron bioavailability, were 7.5%, 8.7%, and 18.7% for the pigs on the HR iron diet (iron added after baking), HR iron diet (iron added prior to baking), and FeSO4 diets, respectively. The difference between the 2 HR diets was not significant but iron bioavailability from the FeSO4 diet was significantly higher than from either of the HR diets. Therefore, our hypothesis was not supported. We conclude that bread baking does not enhance the bioavailability of elemental iron powders.
5.Describe the major accomplishments to date and their predicted or actual impact.
Micronutrient malnutrition afflicts over 3 billion people worldwide especially resource-poor women, infants and children in both developing and developed countries. Iron deficiency alone affects over 2 billion people worldwide. Staple food crops (whole grains and edible legume seeds) contain substances (e.g., phytate and polyphenolics) that inhibit the utilization of iron and other micronutrients in these crops to people that are dependent on these foods for sustenance and are thought to be primarily responsible for about 50% of iron deficiency in the world. Ways need to be found to counteract the inhibitory effects of these staple food crop substances on micronutrient bioavailability. We have been studying the potential beneficial effects of certain prebiotics on iron bioavailability from staple food crops. The non-digestible carbohydrate, inulin (a fructooligosaccharide) is such a prebiotic that stimulates the proliferation of certain beneficial bacteria (i.e., Bifidobacteria and Lactobacilli) in the hind gut (i.e., colon). We hypothesized that the fermentation of inulin in the hind gut would enhance iron bioavailability in the presence of inhibitory substances from staple plant foods by making the hind gut an absorptive surface for iron absorption, and by the action of hindgut microorganism on degrading the dietary inhibitors. In two weanling pig experiments, maize-soybean diets (containing 0.8% phytate) supplemented with 4% inulin significantly improved iron bioavailability (as measured by hemoglobin repletion efficiency) compared to the same diets without inulin added. Current research is focused on the mechanisms of action of how inulin effects iron bioavailability. This research was performed in cooperation with Drs. Dennis Miller (Department of Food Science) and Xingen Lei (Department of Animal Science) at Cornell University. Milestone 2a. Continue to identify factors affecting Fe bioavailability. ARS National Program Action Plan 107 [Objective 3.1.3, Nutritious Plant and Animal Products (Components 6 & 7)]. NP 108; Action Plan Component. Year 3 (FY2006), milestone - 1.
6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Results on the bioavailability of Fe in promising genotypes of bean, wheat, maize, sweet potato and cassava using an in vitro Caco-2 cell model are being provided to cooperating CGIAR Centers (i.e., CIMMYT, CIAT, IITA & CIP). Results were provided as a ranked list of genotypes in order of bioavailable Fe normalized using a standard control genotype. These results will be used by the Centers to advance genotypes in their breeding programs to improve the bioavailable Fe density in the diets of resource-poor people dependent on these staples for their nutriture globally.
7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Dr. Welch gave an invited keynote address titled "Harvesting Health – the Future of Agriculture" at the 15th International Plant Nutrition Colloquium held in Beijing, China from Sept. 14-19, 2005.
Dr. Welch gave an invited seminar to the faculty and students in the College of Environmental and Resource Sciences at the University of Hangzhou, China titled "Linking Agriculture to Human Nutrition" on Sept. 21, 2005.
Dr. Welch gave an invited presentation titled "Agriculture and Human Health: Soil Fertility and Plant Nutrition Interactions" at the FarmTech 2006 Global Perspectives-Local Knowledge meeting held in Edmonton, Alberta, Canada from Jan. 25-27, 2006.
Dr. Welch gave an invited presentation titled "Farming for Health" at the No-Till on the Plains meeting held in Salina, Kansas from Jan. 30-31, 2006.
Dr. Welch gave an invited seminar titled "Harvesting Better Nutrition: the HarvestPlus Biofortification Plant Breeding Program" on April 20, 2006 to the faculty and students in the Department of Horticulture at Michigan State University, East Lansing, MI.
Dr. Welch was invited to participate in the World Bank Leadership Dialogue on Taking Joint Action on Malnutrition – Business as a Partner held in Washington, D.C. June 22, 2006.
Dr. Welch gave an invited presentation titled "Biofortification: Enhancing Micronutrient Concentrations in Staple Food Crops Through Plant Breeding" at the Food Fortification in Developing Countries Symposium held during the Institute of Food Technologist annual meeting in Orlando, on June 25, 2006.
Dr. Welch gave the invited Opening Lecture titled "Farming for Health – Agricultural Perspectives on the Global Iron Deficiency Crisis – the Ways and Means to Sustainable Solutions" at the 13th International Symposium on Iron Nutrition and Interactions in Plants held in Montpellier, France from July 3-7, 2006.
Dr. Glahn’s research on the bioavailability of ferritin iron using the in vitro Caco-2 cell digestion model was presented at the Experimental Biology meetings in San Francisco, CA, in April of 2006. In addition, members of Dr. Glahn’s and Dr. Welch’s research team presented an additional 8 abstracts at this meeting covering research presented in this report.
Huynh, L., Stangoulis, J., Welch, R.M., Graham, R. 2004. Qtls for phytate in rice grain and their relationship with iron. International Crop Science Congress Proceedings. p. 13-27.
Wu, J., Norvell, W.A., Welch, R.M. 2006. Kriging on highly skewed data for dtpa-extractable soil zn with auxiliary information for ph and organic carbon. Geoderma. 134:187-199.
Franzen, D., Nanna, T., Norvell, W.A. 2006. A survey of soil attributes in north dakota by landscape position. Agronomy Journal. 98:1015-1022.
Aguirre-Gomez, A., Mcbride, M.B., Norvell, W.A. 2006. A voltammetric method for determining free metal activities in aqueous solutions: part i, direct current voltammetry of cd, cu, and pb. International Journal of Environment and Pollution. 26:41-67.
Aguirre-Gomez, A., Mcbride, M., Norvell, W.A. 2006. A voltammetric method for determining free metal activities in aqueous solutions: part ii, anodic stripping voltammetry of cd, cu, pb and zn in synthetic and soil solutions. International Journal of Environment and Pollution. 26:68-89.
Engle-Stone, R., Yeung, A., Welch, R.M., Glahn, R.P. 2005. Meat and ascorbic acid can promote fe availability from fe-phytate but not from fe-tannic acid complexes. Journal of Agricultural and Food Chemistry. 53:10276-10284.
Beisiegel, J.M., Glahn, R.P., Welch, R.M., Menkir, A., Maziya-Dixon, B.B., Hunt, J.R. 2006. A caco-2 cell model predicts relative iron absorption from tropical maize by women [abstract]. FASEB J. 20(4):A624.
Kim, T., Mullaney, E.J., Porres, J.M., Roneker, K.R., Crowe, S., Rice, S., Ko, T., Ullah, A.H., Daly, C.B., Welch, R.M., Lei, X.G. 2006. Shifting the ph profile of Aspergillus niger PhyA phytase to match the stomach ph enhances its effectiveness as an animal feed additive. Applied and Environmental Microbiology.
Fairweather-Tait, S., Lynch, Sean, Hunt, J.R., et al. 2005. The usefulness of in vitro models to predict the bioavailability of iron and zinc: a consensus statement from the HarvestPlus expert consultation. International Journal for Vitamin and Nutrition Research.75(6):371-374.