Document reference: http://namls.info/Zinc/Hambidge.html (August 2003)
PLANT BREEDING TO IMPROVE THE CONTENT & BIOAVAILABILITY OF ZINC IN CEREALS
K.M. Hambidge*, N.F. Krebs, J.E. Westcott, L.V. Miller, S. Lei, J. Lavely, M. Mazariegos, N.W. Solomons
*Section of Nutrition, Department of Pediatrics,
Box C225, University of Colorado Health Sciences Center,
Denver, Colorado 80262, USA
This paper reviews potential plant breeding strategies for the prevention of zinc deficiency in populations dependent on a grain-based diet. The major focus of this review is on the evaluation of the effect of consuming low phytic acid maize on human zinc bioavailability. Results of pilot studies in Colorado utilizing maize with different levels of phytic acid reduction are reviewed. Also included are preliminary baseline results of more detailed, long-term studies in progress in a Guatemalan community for whom maize is the habitual major dietary stable. It is concluded that low phytic acid grains have potential as a primary or complementary strategy for the prevention of human zinc deficiency.
Human zinc deficiency is a recognized public health challenge of global dimensions  . Among the strategies for preventing zinc deficiency, supplementation and fortification are currently the most popular. An alternative that has been demonstrated to work is community education to better utilize local produce  . Increasing the intake of animal products, especially meats, is an excellent means of increasing the intake of bioavailable zinc. Improvements in soil and the use of zinc-containing fertilizer can increase the zinc content of grains by at least 50%. In our experience, this magnitude of difference is observed serendipitously in the same genotypes grown in different soils. However, neither increasing consumption of meats nor improving soil quality is economically practical for many populations.
In contrast, plant genetics and breeding  , offers exciting possibilities as an alternative or complementary strategy to food fortification programs. While this approach has significant potential to improve mineral nutriture in all populations, it offers special advantages to the many communities that depend on homegrown or local produce and do not purchase fortified flour or other basic food commodities. Moreover, this is a potentially sustainable strategy.
Cereal grains are the most widely consumed food staples. One plant breeding strategy is to increase the zinc content of grains. Breeding for zinc-dense staple food crops has been successful in increasing zinc content of grain by 50%  . This has been achieved by selecting for crop genotypes that have the greatest ability to take up zinc from zinc-deficient soils  .
Prior to food processing, grains typically have quite substantial quantities of zinc and iron, but bioavailability of these minerals is poor. If the human could effectively utilize the zinc and iron present in unprocessed cereal grains, the intake of these minerals from the grain(s) that provide the major food staple(s) of many populations could contribute, in some circumstances very substantially, to meeting requirements for zinc. This applies, for example, to maize [2, 6], which is the major food staple for numerous populations in Central America and in Sub-Saharan Africa.
Phytic acid (inositol hexaphosphate) is widely regarded as the major factor in plant-based diets that inhibits absorption of zinc, especially when cereal grains or/and legumes are a major food staple, Reduction in phytic acid is expected to improve bioavailability of multiple minerals in addition to zinc, including iron  and calcium  . This confers an additional attraction to this strategy. Though research is currently in progress to introduce endogenous phytase enzyme into grains, genetic selection for low phytic acid is currently the established method of choice.
Plant genetic research  has been successful in identifying and breeding grains that have a major reduction in phytic acid content, but no reduction in total seed phosphorus. These grains are rice, barley and maize  . Low phytic acid soybeans are also available. These low phytic acid grains have already proved very useful in agriculture  and large quantities of these low phytic acid grains are now being planted in North America. They also offer a novel approach to improving human mineral, including zinc, nutrition in many populations.
As a first essential step, these grains also greatly facilitate research designed to determine quantitatively the long-term effects of phytic acid reduction on zinc homeostasis. This paper provides a summary overview of our studies in progress to compare zinc bioavailability from different low phytic acid maize and from isohybrid wild type or local control maize. Full reports have been [12-14] or will be published elsewhere. This paper will give emphasis to selected aspects of methodology.
Maize: two low phytic acid maize and their isohybrid wild-types were used in these studies. Lpa1-1 has approximately 60% phytic acid reduction and was supplied, together with its isohybrid wild type by Dr Victor Raboy's nursery or by Pioneer Hi-Bred Inc., Iowa (Dupont). Nutridense Low Phytate (NDLP), with approximately 80% phytic acid reduction, and its matching wild type hybrid were supplied by the Exseed Co., Iowa (subsidiary of BASF, Germany).
Research Sites: Three pilot studies have been undertaken at the University of Colorado Health Sciences Center. One study is being undertaken in the Western Highlands of Guatemala.
Colorado Study Design & Subjects: A total of 16 healthy adult volunteers, whose habitual diets were typically North American, served as their own controls in a cross-over design. They consumed a maize-only diet ad lib for a two-day period provided in the form of polenta (study 1) or tortillas (studies 2 and 3). On each of the two days (order alternated between subjects for each study), one of the two different maize being compared for that particular study was consumed. It was assumed that the precise status of zinc homeostasis in the period preceding these study days would not change to a measurable extent over the 2-day study period. Therefore, there was no prior adjustment period on a constant daily diet. The intake of food on test days was weighed accurately. Fractional absorption from lpa1-1 was compared with that from its wild-type isohybrid in study 1 and with that from NDLP in study 2. Study 3 was a comparison of fractional absorption from the two wild type maize.
Guatemala Study Design & Subjects: This is a community-based study in the village of Buena Vista, San Pedro, Sacatapequez, Guatemala and the participants are 60 post-Mayan Native American families with the index subject being a child of either sex aged 6-10 years. The habitual diet is maize-based, with maize, primarily as tortillas, providing approximately 50% of energy. This is a randomized, controlled study in which participating families receive either a low phytate maize (lpa1-1), the isohybrid wild type for lpa1-1 or a local Guatemalan maize. Participating families receive a free supply of maize adequate to meet the needs of the entire family for a three-month period. For the final 10 days of this period, the index children participate in a zinc stable isotope / metabolic study. Tracer is administered in the home of a community leader and metabolic collections are undertaken in participants' village homes. Results available from this study currently are limited to data for 13 children consuming the wild-type isohybrid for lpa1-1 or the local village maize. These have been included to give information on zinc homeostasis in this population prior to long-term phytic acid reduction.
Human Subjects: Each of these studies was approved by the Colorado Multiple Institutional Review Board. The study in Guatemala was also approved by the CeSSIAM Ethics Committee in Guatemala City.
Isotope tracer techniques: For the Colorado studies, an extrinsic labeling technique was employed. A preparation enriched with one of three zinc stable isotopes (67Zn; 68Zn or 70Zn) was administered in precisely measured quantities in water gradually during the second part of each meal. In study 1, fractional absorption of zinc was determined by a cumulative fecal enrichment method  . In the second and third studies, absorption was determined by a double isotope tracer ratio technique , which required the intravenous administration of a third zinc stable isotope tracer . The former required quantitative collection of all feces excreted for eight days after administration of the first extrinsic label. The latter required the collection of spot, timed urine samples between days 4-9 post-isotope administration. Weighed aliquots of homogenized fecal samples (study 1) or spot urine samples were ashed and zinc was separated either by ion exchange chromatography  (study 1) or by chelation  (studies 2 and 3). Stable isotope ratios were measured by either fast atom bombardment mass spectrometry (study 1) or by inductive coupled plasma mass spectrometry (ICP-MS) (studies 2 and 3).
In Guatemala, all meals for one day were extrinsically labeled with one zinc stable isotope. A second isotope was administered intravenously in the afternoon of that day. Fractional absorption of dietary zinc is being measured using the dual isotope tracer ratio technique based on urine enrichment  . Endogenous zinc excreted via the intestine is being measured using an isotope dilution technique  . Other measurements include zinc in duplicate diets and in 24-hour urines. All isotope ratios are being measured by ICP-MS  .
Data Processing and Analyses: For the Colorado studies, the primary outcome was the difference in fractional absorption of zinc from the two maizes being compared in each pilot study. Results were compared by paired t-test. Results for fractional absorption of zinc for all three studies combined were examined in relation to the molar ratio of phytic acid (inositol penta- and hexa-phosphate) to zinc in the diet using a linear mixed regression model to account for repeated measures (Proc Mixed, SAS Statistical Package, SAS Institute, Cary, NC).
For the Guatemalan studies, end points being measured or calculated include: dietary zinc; fractional absorption of zinc; the quantity of zinc absorbed each day which was calculated from these two measurements; intestinal excretion of endogenous zinc; 24 hour urine zinc  . Integumental zinc was assumed to be 86% of urine zinc  . Retention required for new tissue was assumed to be 20µg Zn/g new tissue  . Daily weight gain was estimated from average growth rates of comparably aged children  .
Secondary processing of initial Guatemalan data has included: [a]: linear regression of endogenous fecal zinc vs. total absorbed zinc analogous to that which co-authors have previously performed  and [b] calculation of the physiologic requirement for zinc (total endogenous zinc losses plus zinc required to be retained for new tissue) following the method of the Food and Nutrition Board, Institute of Medicine  ; and [c] plotting of total absorbed zinc vs dietary zinc and, from there, deriving an estimated average dietary zinc requirement for individuals in this population as recently described in a generalized context  .
This report will be limited to a selected synopsis that will provide a basis for the discussion to follow:
1. In studies 1 and 2, each individual subject had a higher fractional absorption of zinc from the phytic acid reduced maize than from the wild-type control maize [12, 13] . The mean differences for both studies were significantly different from zero.
2. There was a highly significant negative linear regression between fractional absorption of zinc for all three Colorado studies combined and dietary phytate: zinc molar ratios for the test meals. The slope of this regression, uncorrected for any covariates was 0.007 with a y-intercept (extrapolated) at 0.43. The slope was significantly different from zero (P<0.001). Phytate:zinc molar ratios over which this linear relationship was observed ranged from 7:1 to 36:1.
3. For the Guatemalan children, key dietary data, key variables of zinc homeostasis and approximate calculations of both physiologic and average dietary requirements are included in the table (all figures are means plus standard deviations). The values for intestinal excretion of endogenous zinc, calculated physiologic requirement and average dietary requirements are all substantially higher than the levels for the corresponding age groups that have been calculated recently by the Food and Nutrition Board  .
Absorption of Zn
Endogenous Fecal Zn
Calculated Zn Requirements
7.64 (2.57) 23:1 (6) 0.26 (0.07) 1.96 (0.71) 1.79 (0.72) 2.38 8.9
The final stage in the calculation of dietary zinc requirements, following the calculation of the quantity of absorbed zinc required (physiologic requirement), is depicted in the figure.
The results of the Colorado pilot studies indicated that the substitution of low phytic acid maize for maize with typical phytic acid content is associated with the anticipated increase in fractional absorption of zinc. There is an apparent linear relationship between fractional absorption of zinc and the dietary phytate:zinc molar ratio. These observations in a free-living but closely monitored group of highly motivated volunteer adults have provided strong support for more extensive studies directed to populations that have a habitual diet that is high in phytic acid. We have completed studies of dietary phytic acid reduction in one such population with the short-term use of phytase enzyme [24, 25] and have long-term studies in progress in the Western Highlands of Guatemala that are made possible by a large supply of low phytic acid maize.
Currently, the only available information from the latter studies are partial baseline data on zinc homeostasis in a population that has a habitual maize-based diet that is very high in phytic acid. As indicated in the methods section, however, these initial data serve to illustrate both the complexity and exciting potential of studies of zinc homeostasis employing stable isotope techniques. The estimate of dietary zinc requirements is an example of the possible extent to which these zinc stable isotope data can be used which ranges far beyond relatively straight forward measurements of fractional zinc absorption. The estimate of the average dietary zinc requirement is about 40% higher than that recently calculated for North American children of the same age  . This is explicable in part by the high dietary phytic acid and also possibly by relatively high losses of endogenous zinc via the intestine. It is also pertinent to note that, in contrast to these data, the FNB  had to rely on extrapolation from adult data because of the lack of any experimental data for this age group.
The principal focus of this review is on the application of tracer techniques to advance our understanding of human zinc homeostasis and, thence, zinc requirements. Advantages of stable vs. radio-tracer studies for this research are not only their more ready acceptance by ethics committees, communities and individual participants but also the availability of three stable isotopes of zinc that are in sufficiently low natural abundance to be utilized as tracers. This unusual abundance of tracers offers the potential for both versatility and complexity in study design. Though, with a well-equipped laboratory, the techniques required appear quite straightforward, in practice the ingredients for success are both subtle and demanding. They include a willingness and ability to undertake rigorous metabolic collections and analyses, the most challenging of which is the quantitative collection and laboratory preparation of fecal samples. Moreover, if we are to reach many of the populations of greatest concern, these metabolic collections need to be undertaken under field conditions, which may be quite remote. Finally, reliable studies almost inevitably depend on an effective long-term collaboration between a center experienced in the necessary analytical techniques and details of isotope / metabolic studies and a local high quality research facility. This typically implies international collaborative research teams with complementary skills and experience.
Acknowledgements: USDA, NRICGP #9900663, The Thrasher Research Fund, The International Atomic Energy Agency, The Global Network 1 U01 HD 40657 Colorado CNRU 5P 30 DK48520.
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