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HowMuch Nutritional Iron Deficiency

in Humans Globally Is due to an

Underlying Zinc Deficiency?

Robin D. Graham,* Marija Knez,* and Ross M. Welch†

Contents

1. In

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troduction

Agronomy, Volume 115 # 2012

-2113, DOI: 10.1016/B978-0-12-394276-0.00001-9 All rig

f Biology, Flinders University of South Australia, Adelaide, Australiaent of Crop and Soil Sciences, Cornell University, Ithaca, New York, USA

Else

hts

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2. A
gronomy of Micronutrients in Respect to the Green Revolution
1960–1980
5
2
.1. S eed nutrient content 8
2
.2. Ir on deficiency in humans 9
2
.3. Z inc deficiency and its impact on iron nutrition 10
2
.4. V itamin A deficiency and its significance 12
2
.5. F ood systems strategies 12
3. Ir
on and Zinc Interactions in Human Nutrition 14
3
.1. S ynergy or antagonism 14
3
.2. S upplementation studies 15
3
.3. F ortification studies show no antagonism 16
3
.4. Z inc and anemia 16
3
.5. T he regulation of hemoglobin levels 17
3
.6. M icronutrient deficiencies are occurring together 18
3
.7. Ir on and zinc transporters in enterocytes of the small intestine 18
3
.8. P ositive role of zinc in oxidative damage and protein synthesis 21
4. W
hole Body Regulation of Iron and Zinc in Humans 22
4
.1. Ir on homeostasis 22
4
.2. H epcidin, an iron store regulator 24
4
.3. H epcidin regulates DMT1 and/or FPN expression and function 25
4
.4. Z inc, an important regulator of iron absorption 26
4
.5. T he role of zinc in decreasing systemic intestinal inflammation
and iron deficiency
27
4
.6. A nticipated mechanism of zinc action on iron deficiency 28
vier Inc.

reserved.

1

2 Robin D. Graham et al.

5. H
ealthy Food Systems 30
6. C
onclusion 32
Ackn
owledgment 33
Refe
rences 33
Abstract

This chapter recounts the impact of the green revolution (1960–1980) on

subsequent world food supplies and its consequences in terms of human

nutrition and health via its impact on the micronutrient status of staple foods

and of diets generally. Micronutrient deficiency disorders now occur in over half

of the total human population. This chapter then reviews the recent medical

literature on the molecular physiology of the human gut in relation to micronu-

trient absorption from food and the regulation of nutrient balance from diets

heavily based on cereals that are relatively poor in micronutrients. Weaving

these two literatures together leads to the conclusion that basing the green

revolution on low micronutrient-dense cereals to replace the lower yielding but

more nutrient-dense pulses and other dicotyledonous food crops is the proba-

ble cause of the epidemics of micronutrient deficiencies in the burgeoning

human population in the years since 1980. There are lessons in this for the

implementation of new efforts to increase food production in the face of even

further increases in population forecast to 2050, especially the new effort

starting in Africa, and for improving primary health care generally in resource-

rich as well as resource-poor countries. We conclude that while complete

nutrient balance in our diets is the only satisfactory aim of a sustainable food

strategy, we focus attention on zinc deficiency and its alleviation as the most

extensive and urgent problem among several that arose as an unforeseen side

effect of the first green revolution.

1. Introduction

The first green revolution (begun in 1960) more than doubled cerealproduction worldwide (Fig. 1), an achievement that, in the face of a rapidlyrising human population, turned aside the threat of mass starvation in1960 and of continuing food shortages during the 1960s and 1970s toreach a global surplus again by 1980. The emphasis by the internationalconsortium of agricultural scientists was naturally on increasing yield, bothby plant breeding and use of NPK fertilizers, and as it was known that acrossvarieties an inverse relation existed between yield of grain and proteinconcentration in grain, the latter and other issues of nutritional qualitywere largely set aside. No attention whatever was paid to micronutrientdensity of the green revolution cereal varieties, a quality issue that was a lowpriority among nutritionists at that time.

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Figure 1 Percent changes in cereal and pulse (grain legume) production and in popula-tion, 1965–1999 (Welch, 2002a,b).

Iron and Zinc Deficiencies in Crops and Humans 3

Figure 1 shows the percentage increases of cereal and of pulse (grainlegume) production in developing countries between 1965 and 1999. Devel-oping country population doubled during this period (represented by the“100%” line). It is the great achievement of the green revolution that cerealproduction much more than doubled due to rapid technological change.However, pulse production per capita declined markedly; owing to theurgency to produce more, the new technology was not applied to theselow-yielding secondary staples or to vegetables. These changes in productionaltered the relative prices of these commodities—lower prices of cereals andhigher noncereal food prices—so it became even more difficult for the poorto achieve mineral and vitamin adequacy in their diets. In the absence ofadequate knowledge among resource-poor populations of the importance forhealth of micronutrient and vitamin intakes, diets have shifted toward increas-ing reliance on cereal staples (Graham et al., 2007), leading to micronutrientmalnutrition, poorer health, and much misery.

During the 1980s, a steady rise was noted in the extent of iron-deficiencyanemia in humans, especially among the resource-poor populations thatbenefited most from the greater productivity of the green revolution(Graham, 2008; Graham et al., 2007); however, a putative cause-and-effectassociation between the rising extent of nutritional iron deficiency and thelow micronutrient density of the expanding green revolution cereal varieties,vis-a-vis the lower-yielding crops they displaced, was not canvassed untilmuch later. The anemia was treated by the medical community using diet


supplementation and food fortification strategies, with a major program calledfor by the end of the 1980s decade. These programs were facilitated by the easeof diagnosis of iron deficiency in a small sample of peripheral blood.During thisdecade, three other micronutrient deficiencies affecting large numbers ofpeople, those of iodine, vitamin A, and selenium, were promoted and treat-ments developed (Ren et al., 2008). Deficiencies of iodine and selenium wereregional, associated with extreme low levels of the nutrients in the soil, and asneither of them was known to affect crop production, these were treatedmedically, as with anemia, by food fortification and supplementation in thedeficient regions. Vitamin A, however, was more generally associated withpopulation density, insufficiency of the food supply, and again like anemia,associatedwith the production of the green revolution varieties of cereals; againno attribution of cause and effect was made and health authorities deployedsupplementation and food fortification strategies. The new green revolutionvarieties of wheat and rice were uniformly white-floured, containing very lowconcentrations of yellow provitamin A carotenoids; however, yellow endo-sperm varieties were known and held in the germplasm banks of both crops.

A clinical deficiency of zinc in a human was reported in a remarkablyprescient paper in the 1960s (Prasad et al., 1963) and Prasad later publishedresults of a clinical trial in the 1980s (Prasad, 1991), but both efforts werelargely ignored. Only in the 1990s was a body of evidence accumulated thatattracted some recognition (Prasad, 2003), but as there was, unlike anemia, noquick and simple diagnostic for zinc deficiency in humans, the problemcontinued to be largely ignored. Not until Hotz and Brown (2004) editedan important paper on the extent of zinc-deficient diets of the world, affectingnearly half the global population, was zinc deficiency taken as a potentiallyserious public health problem. Still little has been done about it even to thepresent day, although two developments must be acknowledged: first, theappearance of zinc deficiency as a priority in public health on the WHOwebsite in 2001, and second, zinc deficiency diagnosis in blood serum by ICPatomic emission spectrometry is now deemed a valid diagnostic at a populationlevel but not for the individual; moreover, this analysis is still far from as easyand inexpensive as is the simple test for anemia (de Benoist et al., 2007).

At the same time, soil scientists and agronomists were well aware thatzinc-deficient soils are widespread on Earth, about half of the major agricul-turally productive soil types (Sillanpaa, 1982, 1990). In contrast, crops wereiron deficient on only 3% of soils (Table 1). Moreover, zinc is low in cerealgrains, now the basis of diets for the majority of people everywhere. Morezinc can be incorporated into cereal grains both by zinc fertilization of thecrop and by breeding new cereal varieties inherently richer in zinc (Grahamet al., 1992; Yilmaz et al., 1998), so the tools to solve zinc deficiency globallyhave been available, but motivation is still lacking for an integrated “FoodSystems” approach that will provide a sustainable solution on a global scale.

This chapter reviews the medical literature on zinc deficiency, iron defi-ciency, and their interactions in the human gut, and presents a physiologically

Table 1 Percentage of nutrient-deficient soils among 190 major soils worldwide(Sillanpaa, 1982) and in parts of Bangladesh for comparison (Morris et al., 1997)

Deficiency

Macronutrients Micronutrients

N P K B Cu Fe Mn Mo Zn

World

Acute 71 55 36 10 4 0 1 3 25

Latent 14 18 19 21 10 3 9 12 24

Total 85 73 55 31 14 3 10 15 49

Bangladesh

Total 100 22 2 69 3 1 24 15 85

A latent deficiency is one masked by an even more severe deficiency of another nutrient, often N or P,such that the latent deficiency becomes limiting after the other, more acute deficiency is corrected.


based case that, potentially, a significant proportion of the iron-deficiencyanemia in humans is due to zinc deficiency. This is intended to strengthenthe case for a greater effort to eliminate zinc deficiency worldwide (and withit some of the anemia) through an integrated Food Systems-based newgreen revolution (Graham, 2008).

Because of the complex of homeostatic mechanisms in the body forpreventing excess iron accumulation that in turn prevents peroxidative cellu-lar damage (Edison et al., 2008), this chapter also questions the wisdom ofsome of the supplementation, biofortification, and process fortificationof iron, that is current practice, based on blood tests for hemoglobinand ferritin alone, without showing improvements in health and physicaland mental work capacity. We therefore raise the question whether relativelymore of the global effort to relieve iron deficiency should be spent oneliminating zinc deficiency and other overt, interacting micronutrient defi-ciencies, sustainably through an agriculturally based Food Systems strategy.

In this review, we deal first with the agronomy of the green revolutioneffort and then we present a summary of a recent, extensive medicalliterature on the molecular physiology of the human intestine and on itsimplications for human nutrition. Finally, we bring these two facetstogether to develop recommendations for radical change in the currentstrategy to eliminate anemia and to propose a new Food Systems strategy.

2. Agronomy of Micronutrients in Respect

to the Green Revolution 1960–1980

In the time man has practised agriculture, crops produced on our soilshave become widely deficient in nitrogen and phosphorus and to a lesserextent, in potassium and sulfur, nutrients that, until the turn of the twentieth


century, agriculture used to solve crop production problems on otherwisefertile old-world soils. By then, European farmers were using new mineralfertilizers such as Chilean saltpeter, superphosphate, and muriate of potash,as well as sulfur, lime, and dolomite. These minerals brought production upto general expectations, but to experienced eyes, anomalous results hintedat limitations to production yet to be discovered. In the first half or so ofthe twentieth century, a suite of new essential elements was proved essentialfor all living things in smaller amount, known to agronomists as the traceelements and later as “the micronutrients” (this term to human nutritionistsalso includes the vitamins, nutrients not needed by plants). The use ofmicronutrients contributed greatly to modern mechanized agriculture.

The essential micronutrients for growth of higher plants are iron, zinc,manganese, boron, copper, cobalt, molybdenum, nickel, and chlorine, butfor animals and man, these and the additional elements, selenium, iodine,chromium, tin, fluorine, lithium, silicon, arsenic, and vanadium, are required;some of these additional elements may eventually be found necessary for plantsas well (Nielsen, 1997).

Once the macronutrient deficiencies of soils are treated, Sillanpaa (1990)estimated that, of 190 major agricultural soils of the world, 49% are deficientin zinc, 31% deficient in boron, 15% deficient in molybdenum, 14% deficientin copper, 10% deficient in manganese, and 3% deficient in iron (Table 1).These figures may be compared with corresponding figures for the humanpopulation that depends on these same soils for most of its food production.In the same broad terms, it appears that as much as a third of the humanpopulation is deficient in iron (30% of people anemic, mostly iron-deficiencyanemia—WHO website, 2011), a third is deficient in zinc, and roughlya seventh is deficient in each of iodine, selenium, and the plant-synthesizedorganic micronutrient, b-carotene (a provitamin A dimer of vitamin A).Obviously, multiple micronutrient deficiencies are common. Selenium andiodine are not known to be required by plants (Lyons et al., 2009), and theextent of boron deficiency in soils does not lead to the same high priority inhuman nutrition as it does for crop growth. Iron deficiency in humans isexceedingly complex yet it appears the iron in most foods is far more thanthe requirement but its bioavailability from staple-plant foods is consideredpoor (Hunt, 2003). Apparently, only zinc is directly linked in the food chainsuch that deficiency is extensive in both humans and their food crops. Thecomparison of crop and human micronutrient deficiencies and the nature ofzinc deficiency in humans raises the question whether zinc deficiency shouldbe the highest priority among micronutrients for agriculture to addressbecause to increase the zinc available to crops and to the food chain isachievable with current technology, and there are flow-on benefits to ironand vitamin A status in humans. An agricultural solution to zinc deficiency inhumans is all the more compelling because mild to moderate zinc deficiencyin humans is still difficult to diagnose (Fischer-Walker et al., 2007), so the use


of zinc with all macronutrient fertilizers wherever justified by productiongains is an obvious primary agricultural strategy.

Our emphasis on zinc is based on our analysis of the agronomy of thegreen revolution 1960–1980. Its features were a focus on the cereals(mainly wheat, rice, and maize) utilizing new, high-yielding varieties,coupled with the use of NPK fertilizers in large amounts to match theyields of the new varieties. For rice and wheat, the most extensive of thecereals, the new varieties had no provitamin A or related carotenoids(whereas maize has both white and yellow types). In general, besidestheir large yield advantage, these cereals had, as cereals generally do,more tolerance to extremes of stress such as heat, cold, drought, flooding,and pests and diseases, than do the crops they replaced, especially thepulses (grain legumes). The impact of the green revolution in this respectis well shown in the data of the UN Food and Agriculture Organization(FAO) in Fig. 1 where the availability of pulses per head was decreased bypopulation growth as land was given over to the high yielding and morereliable cereals. Features of the green revolution that induced or aggra-vated a low density of zinc in the grains of the cereals used, and subse-quently in human populations dependent on them, are:

� Low soil–zinc status: 49% of global soils zinc-deficient (Sillanpaa, 1990)� Use of P fertilizers that tend to decrease zinc uptake by plants (Webb andLoneragan, 1990)

� Use of N fertilizers that tend to reduce zinc retranslocation from leaves toseeds in low-zinc soil (Chaudhry and Loneragan, 1970)

� Owing to soil degradation and population growth, agricultural expansionto higher-pH, lower-rainfall soils characteristic of cereal productionwhere zinc deficiency is common

� Loss of diet diversity toward more refined cereal-based diets lower innutrients especially zinc, provitamin A carotenoids, iron, and calcium

� Low levels in rice and wheat of provitamin A carotenoids that are syner-gistic with iron in enhancing zinc absorption from cereal diets (see later).

Some ecologists have argued that the sustainable population of Earth isabout 2 billion humans (Pimentel et al., 1999; Rees, 1996), but the effect ofthe mass production of antibiotics during World War II and improvedsanitation is said to have decreased death rates so much that the populationexploded postwar to a current population in excess of 6.7 billion, witha projected 9 billion before the numbers stabilize and hopefully beginto decline by 2050 (United Nations Population Division, 2004). It is nottoo far-fetched to claim that up to 4.7 billion (i.e., 6.7–2 billion) people areliving today on synthetic urea applied to cereal crops, urea producedin fertilizer factories from oil/gas, and electricity in the highly energy-demanding Haber process (Coates, 1939). This huge production of syntheticfertilizer, unique to the late decades of the twentieth century, has placed


equally huge demands on the world’s agricultural soils to supply matchingamounts of the other essential nutrients.

The contribution to production of food for such a large populationmade by the use of micronutrients added to NPK fertilizers is undoubtedlysignificant but as yet far from the optimal that must be reached to achievesustainability because increases in productivity on land already in cultivationare needed to relieve the global warming effect of clearing of more forestedland. Because micronutrients are needed in such small amounts, the eco-nomics of their use is generally highly favorable, as in one case of the authorswhere increases in wheat production were valued at $287/ha for each93 cents worth of copper fertilizer invested in the crop (Graham et al.,1987). While the economics of micronutrient use is compelling in mostcases, the challenge is to get both the diagnosis and the delivery rightbecause adding the wrong micronutrient can seriously decrease yields. Prin-ciples for use of micronutrient fertilizers were developed in the thirdtrimester of the past century (Graham, 2008), although further developmentis certainly warranted.

2.1. Seed nutrient content

An important strategy is to increase the micronutrient content of the seeds (orother edible product), a significant factor in production as well as in nutri-tional quality for human consumption (Welch, 1986). Indeed, high nutrientcontent is one reason for the advantage of certified seed, usually grown onthe best soils, over farmers’ seed. Plant breeders can select for higher micro-nutrient content of seeds but greater enhancement of most micronutrients canbe achieved by fertilizers, either soil-applied or sprayed on the reproductiveorgans including flowers, seedpods, or ears, one to three times during seeddevelopment. Nutrient concentrations can be increased greatly, from lessthan double for zinc in rice to 100 times in the case of selenium in wheat(Lyons et al., 2004). However, while spectacular increases are possible, wecaution against aiming for increases greater than what brings the deficientnutrient up to a relative abundance that roughly matches that of the othernutrients in the system, because replacing one imbalance (zinc too low) withanother (zinc too high) will induce a deficiency of another micronutrient andso represents no progress toward healthy food.

An increment in seed content of critical micronutrients can materiallyincrease the vigor, stress tolerance, disease resistance, and grain yield of thesubsequent crop produced from those seeds on soils deficient in the targetnutrient. In Bangladesh in comparison to farmers’ seed, yields in responsivesoils over 4 years averaged 24% higher in wheat-growing soils by using seedspreviously enhanced in micronutrients by foliar sprays on the motherplants (Johnson et al., 2005). Studies of the genetics of seed-nutrient loadingtraits indicate a number of genes involved, so the genetic approach, though


it has potential, is not easy (Lonergan et al., 2009). However, iron salts arerelatively poor fertilizers even when foliar applied so the breeding strategy isa more viable option to enhance iron levels, if needed. In contrast, zincin seeds is easily enhanced, as, for example, the results of Genc et al. (2000)where on severely deficient soil, 1.5kg/ha of zinc as zinc sulfate increasedseed zinc concentration threefold.

2.2. Iron deficiency in humans

The human population is astonishingly iron deficient despite the planet, itsrocks and soils, being especially rich in iron. TheWorld Health Organization(1995, 2005, 2011) on its website estimated in 2005 the global incidence ofiron deficiency to be between 4 and 5 billion people, and the current websiteidentifies 2 billion severely deficient, that is, anemic. Apparently, more thanhalf the total problem is dietary in origin. Iron deficiency is most severe andwidespread among growing children and premenopausal women, as adultmales until old age are reasonably resistant to anemia despite poor diets inresource-poor countries (Markle et al., 2007). Most iron-deficient womenand children are debilitated to some degree in both physical and mental workcapacity. In severe cases, this results in morbidity, complications in childbirth,and mortality for both mothers and children (www.who.int/nutrition/topics/ida/). Iron deficiency, even when mild, can increase the food requiredby 5–10% for the same amount of physical work done (Zhu and Haas, 1997);a similar increment in yield of 5–10% by modern plant breeding may take upto 10 years to achieve.

Iron deficiency is an epidemic that exists in spite of few problems in cropplants. For example, iron deficiency in humans is severe in the acidiclateritic soil areas of the Asian wet tropics where iron deficiency in cropsis rare, and if anything, it is iron toxicity that is better known, especially inrice (Phattiyakul et al., 2009).

For humans in resource-poor populations heavily dependent on cerealsfor their sustenance, at least 10 times their needs of iron are ingested dailyfrom those cereal products (other than white rice), but the bioavailability ofthat iron is reportedly low (Fairweather-Tait and Hurrell, 1996). The reasonfor the low bioavailability of cereal–iron, largely in the form of solublemonoferric phytate, is thought to be the precipitation by dietary calcium ofcomplex phytates and other insoluble forms in the small intestine, making itunavailable. Absorbed and utilized iron, measured by isotopic methods, canbe as little as 1% of ingested iron (Donangelo et al., 2003). In the HarvestPlusChallenge Program (www.harvestplus.org), that aims to increase the nutritivevalue of common staple foods to eliminate iron-deficiency anemia in theworld by biofortification, increasing iron in cereals by selecting iron-densegenotypes is the main strategy. The effectiveness of this strategy is yet tobe fully established. Due to simpler genetics, it may prove more effective to

http://www.who.int/nutrition/topics/ida/

http://www.who.int/nutrition/topics/ida/

http://www.harvestplus.org


breed for increased bioavailability-promoting substances (e.g., prebiotics) toenhance the absorbability of such nonheme iron in staples than to increase theiron itself in staple food grains (Graham et al., 2007).

2.3. Zinc deficiency and its impact on iron nutrition

Older human nutrition texts identify iron-deficiency anemia as one symp-tom of zinc deficiency (Prasad et al., 1963). While subsequent studies inhumans that gave supporting results have been deemed of poor design(Prasad, 1991), this does not disprove the proposition, and studies withanimal models including monkeys have, under more controlled conditions,supported the hypothesis of zinc deficiency as one cause of iron-deficiencyanemia (Golub, 1984). Recent studies indicate that improved dietary zincfacilitates the absorption of nonheme iron (see later sections). If this were awidespread phenomenon, it could explain some of the current extent ofanemia and nutritional iron deficiency, and the failure of the gut to absorbenough of the iron ingested to meet metabolic needs. Additionally, vitaminA deficiency, also widespread in humans, can aggravate both iron and zincdeficiencies, and conversely, correcting any one of these deficiencies canmake more of the other two nutrients available from an otherwise similardiet (Thurlow et al., 2005; Fig. 2). Carotenoid pigments have been deliber-ately bred out of wheat and other staples during the twentieth century inresponse to consumer demand for white flour (whiteness may be perceivedas evidence of its purity/cleanliness), and iron and zinc concentrations in

Absorption

AbsorptionUtilization

AbsorptionRBT Transport

Utilization

VitaminA

Iron Zinc

Figure 2 Synergy of iron, zinc, and vitamin A in the human gut: an increase of any onemay enhance absorption and/or utilization of the others when all are low in the diet(Graham et al., 2000).


green revolution cereals appear to have decreased even further over time asyields have been increased by breeding (Graham et al., 2007). Intestinalinfection by Helicobacter pylori and other gut pathogens is also linked to zincand iron deficiencies in developing countries (DuBois and Kearney, 2005).Deficiencies of iodine and selenium induce poor utilization of absorbed ironthat aggravates iron deficiency in humans (Welch, 1986). Finally, vitaminB12 deficiency can cause anemia (iron-resistant or pernicious anemia), andalthough there are no extensive maps of cobalt-deficient soils (vitamin B12contains cobalt), the extent of vitamin B12 deficiency is increasing as moreextensive testing is conducted (Stabler and Allen, 2004). The collective extentof deficiencies of zinc, iodine, selenium, vitamin A, and vitamin B12 is morethan sufficient to explain some of the nutritional anemia quantified by WorldHealth Organisation. More importantly, newly published mechanisms of theregulation of iron uptake by dietary zinc in humans (Sections 3 and 4) detailthe mechanisms by which zinc deficiency could indeed be the cause of up tohalf of the global burden of iron-deficiency anemia.

The agricultural perspective on zinc is much clearer than is the humannutritional perspective. Zinc fertilizers are remarkably effective, yet halfof the world’s soils are intrinsically deficient, as well as the lithospheregenerally where zinc abundance is barely one thousandth that of iron(Chesworth, 1991). Zinc deficiency occurs in all the world’s major crop-ping areas, climates, and soil types. Copper, iron, molybdenum, chlorine,and manganese have more than one oxidation state and so are easilymanipulated by redox transitions in biological systems in the soil to releasesoluble ions of these elements even in the presence of an unfavorable pH.On the other hand, zinc, nickel, cobalt, and boron rely on coordinationchemistry for changes in solubility, movement through soil and the bio-sphere, and so these elements tend to function biologically in stable systemssuch as structural molecules like DNA, structural proteins, and enzymes,both metabolic and regulatory. Zinc has been identified to bind with 925proteins in humans and over 500 proteins in plants (Table 2), 10 timesmore than does iron in the same organisms (the opposite of their relativeabundances in the lithosphere/soil). It is not surprising, therefore, that theoccurrence of zinc deficiency is widespread, and in both plants and humanscauses a wide range of symptoms, depending on allelic variation in genes

Table 2 Metal-containing and metal-binding proteins in two species identified byproteomic techniques

Genome Total proteins Zn Cu Mg Fe Ca Ni Co Mo

Homo sapiens 25,319 925 31 74 86 59 0 4 6

Arabidopsis thaliana (plant) 27,243 536 19 51 81 14 1 4 6

From Gladyshev et al. (2004).


controlling each of the known zinc-containing/binding proteins. As such,zinc participates in almost all processes and pathways in living organisms.It can be deemed the most important metabolic promoter among theknown essential nutrients. Because zinc interacts with such a large numberof proteins, symptoms of zinc deficiency in humans may be many, varied,and somewhat indiscriminate, and consequently many disease states are notassociated with its deficiency when they should be, and in these respects, it isnot surprising that zinc deficiency is quite difficult to diagnose in humansand animals. Zinc deficiency is the ultimate “hidden hunger.”

More importantly, in humans, zinc is described as a “type II” element, thatis, its concentration does not markedly decline in the blood stream as severityof deficiency increases, in contrast to iron, a “type I” nutrient, the concen-tration of which does decline in the blood markedly as deficiency increasesin severity. When zinc supply is low, the body sacrifices bone zinc stores andskeletal muscle mass, releasing zinc to the circulation in order to maintain vitalinternal organs, whose zinc concentrations also do not fall greatly (Golden,1995). Thus, unless an individual child has been monitored for height/weightover many months, there is neither good nor easy diagnosis of zinc deficiencyin an individual (Hess et al., 2007). Until the release of the map of zinc-deficient human diets, zinc deficiency was low on theWHO list of importantnutritional problems and this may be a reason zinc deficiency has not beenidentified as a potential cause for some of the nutritional anemia reported.

2.4. Vitamin A deficiency and its significance

Vitamin A is widely deficient in humans (Abed and Combs, 2001). Vitamin Ais not a nutrient for plants as they can biosynthesize the carotenes thatthe human body converts into vitamin A. Important here is that its deficiencycan cause anemia, and solving the problem of vitamin A deficiency isimportant to eliminating anemia (Bloem et al., 1989; Suharno and Muhilal,1996). As carotenes are not nutrients for plants, there is no fertilizer strategy,and new foods must be added to vitamin A-deficient food systems or existingstaples enriched with provitamin A carotenes by plant breeding. Thesestrategies combined with a zinc strategy thereby address not only the vitaminA deficiency problem in humans but may also address more effectively theiron deficiency in humans than any iron fertilizer is likely to do. We advocateintroducing carotene-rich secondary staples and increasing zinc in diets byfertilizer use and by plant breeding of major staples where appropriate.

2.5. Food systems strategies

Nutritional anemia (iron deficiency) is promoted, among other things, bydeficiencies of a number of other nutrients, especially zinc, iodine, selenium,vitamins A, B12, C, and folate, and is reduced by synergistic interactions


among these nutrients when their supply is increased in the range fromdeficiency to adequacy.

Among various agricultural strategies, the Consultative Group onInternational Agricultural Research (CGIAR) Global Challenge Program,HarvestPlus, utilizes plant breeding to improve diets in target countries,especially for resource-poor populations, using staple food crops as avehicle for delivering more micronutrients (principally iron, zinc, provi-tamin A carotenoids). The challenge is to minimize the number of genesinvolved to accomplish this end (Graham et al., 1999). Another agricul-tural approach to help meet the challenge is supplemental use of fertilizerswhere they have a comparative advantage, especially on soils inherentlylow in these nutrients. So far, we have seen little prospect of breedingfor high selenium or iodine content (Welch, 1986), so fertilizer strategiesare appropriate for these (Cao et al., 1994, Welch, 1986) and for zinc asalready discussed.

To combine effectively the HarvestPlus strategy with the resources ofthe fertilizer industry, we need to work within individual food systemsthat collectively support the bulk of the populations at risk of micronutrientdeficiencies. Clearly, a fertilizer strategy will not sustainably solve iron defi-ciency or vitamin A deficiency in a target population. These can be solvedby breeding more iron-dense and provitamin A-dense staples, a primaryHarvestPlus strategy, but also by use of more zinc, iodine, and seleniumfertilizers where the soils of the food system are deficient in them (Grahamet al., 2007). Vitamin A must be addressed by breeding or by introductioninto the food system of an additional food crop naturally rich in provitaminA carotenoids, such as orange-fleshed sweet potato. Often, where a foodsystem is struggling to meet basic expectations for calories to avoid starva-tion, an additional food requires that land be allocated for it and to achievethis in turn means productivity needs to be increased on existing land. Thus,emphasis on macronutrients must be considered an integral component ofany holistic approach to developing micronutrient-adequate food systems.Besides selenium and iodine already mentioned, other minerals and vita-mins are likely to be limiting for humans in particular food systems and mayrequire additional fertilizers (calcium, magnesium, copper, cobalt, boron)and vitamins (from vegetables, cassava, potatoes, sweet potatoes, a little fishor meat products); and a stable, economic food system must be capableof including the preferred crops and providing at the same time sufficientcalories, and be both economic and socially acceptable. Integrating all thisrequires successful deployment of expertise in several disciplines andincludes agronomic, fertilizer, plant breeding, sociological, and nutritionalexpertise. Delivering on this complex agenda will be challenging, but oncea successful food system is established, it will be readily extended to allcomparable communities on similar soils and to new areas once their soiland crop characteristics are defined.


3. Iron and Zinc Interactions in Human

Nutrition

3.1. Synergy or antagonism

Iron and zinc deficiencies in humans occur as a consequence of inadequatedietary intake or, where intake is adequate, of low or impaired intestinalabsorption. Factors that decrease absorption include dietary inhibitors, such asphytate or certain types of fiber, drugs or other chemicals, and interactionsbetween essential nutrients (Whittaker, 1998). The interaction between ironand zinc has drawn particular attention. Meat is the best food source ofbioavailable iron and zinc, so in developing-country vegetarian populations,iron and zinc deficiencies usually coexist. However, if additional iron andzinc are to be provided together, it is important to evaluate whether, and if so,how they interact biologically.

In the past, because of their chemically similar absorption and transportmechanisms, zinc and iron were thought to compete for the same absorp-tive pathway since both are commonly absorbed as divalent cations(Solomons, 1998). There are studies which demonstrated inhibitory effectsof zinc on iron absorption and vice versa. However, most of these studiesused high doses of soluble forms of iron and zinc that are not likely to befound in food. Further, they were commonly given in a water solution oradministered in a fasting state, which further amplifies competitive (antago-nistic) interactions. An additional limitation is the fact that most of thesestudies used only serum or plasma zinc concentrations as a measure of zincabsorption. Measurements of circulating concentrations do not necessarilyindicate true zinc uptake or status, and plasma zinc concentrations arehormonally regulated (Lopez deRomana et al., 2005).

The probability of antagonistic interactions appears to be much lowerwhen zinc and iron intake are closer to “physiological” concentrations(Lonnerdal, 2000). Further, a number of studies showed no negative effectof iron fortification of food on zinc absorption and vice versa. Recently,several studies provided evidence suggestive of positive interactions betweeniron and zinc in absorption (Chang et al., 2010; Hininger-Favier et al., 2007;Smith et al., 1999). All these findings support an hypothesis of possible ironand zinc synergism, or at least no antagonism, when small or complexedsources of these minerals are used together. This section summarizes findingsin order to shed some light on ideas about the relative significance of iron andzinc synergy (as opposed to antagonism) in normal human nutrition.An important condition for expression of synergy between nutrients, in thisinstance, is that individual subjects be moving from deficiency to adequacy, orperhaps more rarely, in the reverse direction. The review mainly includes thestudies that look at iron and zinc interactions when these nutrients are supplied


in modest amounts (closer to normal consumption levels than those often usedin clinical trials), and/or chemically bound or complexed as in food.

3.2. Supplementation studies

Solomons (1986) proposed that chemically similar ions compete for thesame absorption sites in a common absorptive pathway; by his proposal,a high concentration of zinc or iron could theoretically inhibit the absorp-tion of the other. Our review of the literature suggests that his view issupported only by studies using high doses of soluble ionic forms of iron andzinc given together in unbound forms, that is, without binding ligands orfood. The summary review of Fischer-Walker et al. (2005) provided muchsupport for noncompetitive absorption of iron and zinc added together.Findings from randomized placebo-controlled trials of supplementation ofiron and zinc separately, or in combination, in children under 5 years of ageand in women of child-bearing age, including pregnant women taking quitehigh doses (Baqui et al., 2003), were included in the review. Opposing thescenario of Solomons (1986), all trials showed no adverse effect of zinc onhemoglobin or serum ferritin. One small trial even showed a positive effectof zinc on hemoglobin and another positive effect on plasma ferritin.Moreover, none of the trials showed a negative effect of zinc supplementson iron status indicators and the studies looking at whether iron supple-mentation affects zinc absorption showed no adverse effect of iron on serumzinc status. An additional benefit of zinc-with-iron supplements for smallchildren was lower rates of diarrhea (Chang et al., 2010; Smith et al., 1999;Solomons, 1986), the last recommending joint supplementation of childrenin Bangladesh for its benefits in reduced diarrhea and hospitalization. Furtherstudies have reported synergy between iron and zinc with quite high doses(Harvey et al., 2007; Penny et al., 2004; Smith et al., 1999). Serum–zinc maybe taken as a valid indicator of zinc status averaged across all the individualsin these trials, as it is on a population basis (de Benoist et al., 2007; Hotz andBrown, 2004).

Contrary results were mostly confined to studies of short duration (Bergeret al., 2006) or studies on babies (rat pups) less than 6 months old whoseabsorptive systems have not yet matured (Kelleher and Lonnerdal, 2006).Recently, Dekker and Villamor (2010) performed a systematic review ofrandomized trials that examined the effect of food-based zinc supplementa-tion on hemoglobin concentrations in healthy children aged 0–15 years.Their quantitative analysis showed no adverse effect of zinc on hemoglobinconcentrations and no evidence for effect modification by age, zinc dosage,duration of treatment, type of control, and baseline hemoglobin status. Theauthors concluded that there could be additional benefits of zinc supplemen-tation among children with severe anemia or zinc deficiency. All these


findings clearly oppose the existence of a negative interaction between ironand zinc delivered at low doses or with food.

3.3. Fortification studies show no antagonism

Iron deficiency is a common nutritional problem in infants and children andto address it, weaning cereals are routinely fortified with iron. However,the undesirable side effect of fortifying foods with iron, observed in somestudies especially in infants, is the possibility of inadequate absorption ofzinc to sustain their rapid growth (Ziegler et al., 1989; Lofti et al., 1995).Fortification with reduced iron in a weaning food for 9-month-old infants,both normal and anemic, over a wide range of iron:zinc ratios had noadverse effects on zinc absorption unless given without food (Fairweather-Tait et al., 1995; Friel et al., 1998; Lopez deRomana et al., 2005) or usingzinc oxide in lieu of sulfate (Herman et al., 2002). These results extendthe earlier results of Davidsson et al. (1994) who used chelated iron(FeNaEDTA) to prevent adverse effects of quite high iron fortification onzinc absorption.

3.4. Zinc and anemia

Although zinc deficiency and iron-deficiency anemia were causally linked(Prasad et al., 1963) in the case of a single individual, relevant literature on apossible causal relationship between them and between the correspondingelemental concentrations in blood has accumulated only more recently,involving studies of the interaction between zinc and iron in dual ormultinutrient intervention studies and physiological and molecular studiesof the absorption sites in the human gut. Iron and zinc have a similardistribution in the food supply, and the same food components affect theabsorption of both minerals, so nutritional causes of iron deficiency and zincdeficiency are without doubt related. Additionally, over the years, a numberof data sets have clearly demonstrated a positive correlation between anemiaand signs of the risk of zinc deficiency in adult males, children, and pregnantwomen (Ece et al., 1997; Ma et al., 2004). The correlations were strongerin anemic than nonanemic populations. A study by Gibson et al. (2008) withpregnant women in Sidama, Ethiopia (75% of the subjects were iron andzinc deficient) showed plasma zinc to be the strongest predictor of hemo-globin concentrations (compared to plasma ferritin, gravida, status of vita-mins B12 and A, and folate and C-reactive protein). The study of Smithet al. (1999) also showed significant responses in serum hemoglobin to eithervitamin A or zinc treatment, or both together, and zinc concentrations haddirect effects on hemoglobin levels, more so in older children; in contrast,the nil-zinc control group declined in serum hemoglobin levels over thesame 6-month period.


A large number of studies show that anemic children are often zinc-deficient, and zinc is shown to be a strong predictor of hemoglobinconcentrations. Moreover, iron supplementation, by itself, is not alwayseffective in treatment of anemia.

3.5. The regulation of hemoglobin levels

Iron deficiency has been reported to be the most common cause oflow hemoglobin concentrations. Consequently, provision of iron supple-mentation is the main focus of programs that aim to treat anemia. Increas-ingly, however, studies are showing the incomplete improvement ofhemoglobin after iron supplementation, especially in anemic children.Allen et al. (2000) showed that, after 1 year of supervised iron supplemen-tation, the children’s hemoglobin concentrations were not significantlyhigher than those of nonsupplemented children, a result that could not beattributed to short duration, noncompliance, or lack of iron absorption.Many iron-supplemented children remained anemic (30% at 6 monthsand 31% at 12 months), as was the case in other studies (Palupi et al.,1997). In a meta-analysis of the efficacy of such iron supplementationtrials in developing countries, Beaton and McCabe (1999) concludedthat “there is a suggestion in the data that ‘something other than ironmay be operating to limit hemoglobin response and anemia control.”Could this factor be zinc?

Zinc deficiency was implicated quite early. In 1976, Jameson proposedthat some refractory anemias of pregnancy are due to zinc deficiency. Lowserum zinc concentrations were found in the majority of 33 pregnantwomen whose anemia did not respond to iron, vitamin B12, or folate.In addition, 13 of 20 pregnant women selected for very low serum zinclevels had hemoglobin levels indicative of anemia (<110g/L) (Jameson,1976). Studies by Kolsteren et al. (1999) with 216 nonpregnant anemicwomen 15–45 years old in Bangladesh and Alarcon et al. (2004) withPeruvian children, both showed a positive effect of zinc or zinc plus vitaminA delivery, with iron, on hemoglobin responses, with the added benefit ofless diarrhea. Zinc may increase vitamin A concentrations through promot-ing the production of retinol-binding protein, and in this way can redressiron deficiency (Rahman et al., 2002). Nishiyama et al. (1996a,b, 1998) instudies with 52 anemic women showed parallel zinc and iron deficiencies.Marginal zinc deficiency possibly contributes to the manifestation of ane-mia, as the combined administration of ferrous citrate and zinc was the mostsuccessful in increasing the concentration of iron, red blood cells, hemoglo-bin, and albumin levels. In cases of anemia in women endurance runners,disabled patients, pregnant women, and in premature infants, combinediron and zinc interventions helped in faster recovery from anemia(Nishiyama, 1999; Nishiyama et al., 1996a).


3.6. Micronutrient deficiencies are occurring together

Deficiencies of iron and zinc remain a global problem, especially amongwomen and children in developing countries. Current intervention programsaddress mostly iron, iodine, and vitamin A deficiencies, mostly as singlenutrient interventions, with fewer programs operating for other limitingessential trace elements (Gibson, 2003). Whether there is a common under-lying cause of these micronutrient deficiencies or whether one micronutrientdeficiency leads to another deficiency cannot be answered from such studies,but it is clear micronutrient deficiencies are occurring together in manyregions of the world. A diet rich in phytate and low in animal proteins, as iscommon in most developing countries, predisposes to insufficient intake andabsorption of both iron and zinc (Kennedy et al., 2003). Dijkhuizen et al.(2001) showed that deficiencies of vitamin A, iron, and zinc occur concur-rently in lactating mothers and their infants in rural villages in West Java,Indonesia. In addition, Anderson et al. (2008) demonstrated a high prevalenceof coexisting micronutrient deficiencies in Cambodian children, with zinc(73%) and iron (71%) as the most prevalent deficiencies.

If micronutrient deficiencies are occurring together, it is essential to treatthem together, rather than separately. The positive effect of doing so wasreported in a number of studies. Shoham and Youdim (2002) investigated theeffect of 4-week iron and/or zinc treatments on neurotransmission in thehippocampal region in rats. Iron or zinc alone was not effective whereastogether they caused a significant increase in ferritin-containing mossy fibercells (cells important for memory and learning). This is the classical response tothe addition of two limiting essential nutrients acting together on a physiolog-ical or developmental pathway. Ramakrishnan et al. (2004) undertook meta-analyses of such randomized controlled interventions to assess the effects ofsingle vitamin A, iron, and multi-micronutrient (iron, zinc, vitamin A, vita-min B, and folic acid) interventions on the growth of toddler children. In theirsummary of around 40 different studies, they clearly found greater benefitsfrommultimicronutrient interventions that they explained by the high preva-lence of concurrent micronutrient deficiencies and the positive synergisticeffects between these nutrients at the level of absorption and/or metabolism(e.g., vitamin A and iron, vitamin A and zinc, iron and zinc, all three)(Ramakrishnan et al., 2004, Fig. 2). The results also suggest that competitiveinteractions between iron and zinc are not a problem when zinc is included ina multivitamin–mineral food-based supplement (Ramakrishnan et al., 2004).

3.7. Iron and zinc transporters in enterocytes of thesmall intestine

Early studies on body iron balance revealed that humans have a limitedcapacity to excrete iron so that the iron content of the body is tightlyregulated through control of absorption by the intestine (Donovan et al.,


2006). Development of cloning technology has helped identify proteins thatare involved in iron movement into and across the human enterocytes.Because most dietary iron is in the ferric (Fe3þ) form, it must be reduced toferrous ion (Fe2þ) via the ferric reductase, Dcytb (1) (Fig. 3) in order to betransported by DMT1 (2) across the brush border membrane.

Once within the enterocyte, iron may be stored within ferritin (3) ortransported across the basolateral membrane and into circulation via ferro-portin (FPN), also known as IREG1 (5). Basolateral transport of iron alsorequires the iron oxidase, hephaestin (4) which oxidizes Fe2þ to Fe3þ priorto its entry into the blood. In the past, DMT1 has been proposed as the sitefor iron–zinc antagonism (Fleming et al., 1998; Gunshin et al., 1997), butmore recent studies show that DMT1 is an unlikely site for absorptive iron–zinc interaction (Kordas and Stoltzfus, 2004). DMT1 was implicated inintestinal iron absorption when it was identified as the gene mutated inthe microcytic anemia mouse and the phenotypically similar Belgrade rats(Fleming et al., 1998). In these two animal strains, orthologous mutations inthe DMT1 gene resulted in severely decreased absorption of dietary ironand low iron uptake by erythroid cells.

An earlier view of the main role of DMT1 was iron homeostasis. In thatview, the iron status of enterocytes strongly affects DMT1 expression and soregulates the amount of iron transported into the mucosa (Tallkvist et al.,2000). Although DMT1 is known to be an iron transporter, it was originallythought that it also transported other divalent cations, including zinc.However, in one of their experiments Lopez de Romana et al. (2003)found no relation between serum ferritin and zinc absorption. Uptake ofmetals by DMT1 is dependent on a cell membrane potential and redoxstatus, but when Kþ solution was used to depolarize the cells, changes iniron uptake only were recorded, without changes in zinc absorption. Thisclearly demonstrated that zinc does not depend on DMT1 to enter intestinal

Figure 3 A summary of the main pathway by which iron crosses the duodenalenterocyte. DMT1, divalent metal transporter 1; DCYTB, cytochrome B oxidase;FPN, ferroportin; Heph, hephaestin.


cells as thought earlier (Fleming et al., 1998; Gunshin et al., 1997) and isunlikely to compete with iron for absorption (Sacher et al., 2004). Subse-quently, a family of human intestinal transporters (ZIP) has been identifiedas zinc transporters, indicating separate mechanisms for iron and zinc absorp-tion. Some ZIP transporters (Zip14 in particular) may have iron-transportactivity (Liuzzi et al., 2006); however, Zip14-mediated iron uptake does notseem to be essential in maintaining intracellular iron status (Lichten andCousins, 2009).

In an experiment with Caco-2 cell lines, Iyengar et al. (2009) examinedthe mechanism of interaction of iron and zinc using kinetic analysis andshowed remarkable differences in Km, Vmax, and uptake of iron and zinc,which negates the possibility of direct competition for a single transporter.They also showed that zinc pretreatment modulates iron uptake, highlight-ing the importance of cellular zinc as a determinant of iron uptake. Westernblot analysis showed that zinc increases DMT1 expression which probablyexplains increased iron uptake upon zinc pretreatment.

The earlier study of Kelleher and Lonnerdal (2006), where they inves-tigated the effect of zinc supplementation on iron absorption in sucklingrats, showed that, although Zn supplementation had negative effects on ironabsorption during early infancy, this effect was completely reversed in lateinfancy. This view reconciles some of the conflicting data in the literature.They postulated that the difference is caused by DMT1 and FPN localiza-tion. During early infancy, DMT1 and FPN were located intracellularly.This may be considered “immature localization,” but the possibility existsthat this reflects homeostatic control in response to high neonatal ironstores, by internalizing DMT1 and FPN in the enterocyte (Trinder et al.,2000) to prevent further uptake of iron. However, during late infancy, bothDMT1 and FPN were appropriately localized to the apical and basolateralmembranes, respectively. These age-dependent effects are consistent withthe earlier reported results of Smith et al. (1999). Additionally, liver hepcidinexpression was lower in zinc supplemented pups. These data indicate thatdecreased iron absorption during early infancy is actually a consequence ofincreased iron retention in the small intestine, facilitated through reducedbasolateral iron efflux and enterocytic iron trapping. Interestingly, by the timeof weaning, this effect is resolved, potentially as a result of the “maturation” ofiron absorptive mechanisms (Leong et al., 2003).

The idea, that DMT1 is inversely regulated through changes in enter-ocyte iron levels, suggests that during early-mid infancy, when enterocyteiron is elevated, DMT1 expression should be decreased. However, theLeong et al. study showed that DMT1 expression did not change accordingto intestinal iron concentrations but rather in accordance to changes ofintestinal zinc concentrations. The fact that DMT1 is not inversely relatedto intestinal iron concentration, but is positively associated with intestinal zincconcentration, suggests that zinc plays a direct role in positive regulation of


DMT1 expression (Kelleher and Lonnerdal, 2006). This is consistent with theobservations in Caco-2 human colonic carcinoma cells (Yamaji et al., 2001).

One explanation for the way zinc affects DMT1 expression is that thepromoter region of DMT1 contains several metal response elements suggestingthat zinc exposure can positively regulate DMT1 mRNA level via metaltranscription factor-1 activation (de Benoist et al., 2007) or perhaps throughother zinc-dependent transcription factors, such as peroxisome proliferator-activated receptor-g, nuclear factor-kB, or activator protein-1 (Meerarani et al.,2003). Yamaji et al. (2001) measured DMT1 protein and mRNA expressionfollowing exposure to high concentrations of zinc or iron for 24h. Exposureto iron decreased DMT1 protein and mRNA expression in Caco-2 TC7 cellmembranes. Interestingly, exposure to zinc for 24h significantly increasedexpression of mRNA and DMT1 (Fig. 4). In addition, it was found thatthe expression of the basolateral iron-transporter FPN was increased in zinc-treated cells. They confirmed previous findings that DMT1 is predominantlyan iron transporter, with lower affinity for other metals.

From these studies, it is quite clear that DMT1 is not a site of iron andzinc antagonism, but rather for a synergy between them by which the irontransporters, DMT1 and FPN, are stimulated by dietary/intestinal zinc.

3.8. Positive role of zinc in oxidative damage andprotein synthesis

Long term deprivation of zinc leaves an individual more susceptible tooxidative damage and loss of functional integrity of membranes (Sreedharet al., 2004; Srigirdhar and Nair, 1998). Zinc is well known as an antioxi-dant; however, it does not interact directly with the oxidant species butindirectly stabilizes cell membrane proteins by protecting sulfhydryls fromoxidation, by contributing to the structure of superoxide dismutase thatdoes react directly with oxidative free radicals, and by maintaining the tissueconcentrations of metallothionein.

Sreedhar et al. (2004) showed that combined supplementation of iron andzinc significantly attenuates oxidative stress by inducing metallothionein

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Figure 4 Effects of iron or zinc on expression of DMT1 and IREG1:(FPN) in Caco-2 cells (modified from Yamaji et al. (2001)). C, Control; DMT1, divalent metaltransporter 1; IREG1, basolateral iron-transporter protein 1.


and elevating the levels of reduced glutathione. Further, the presence of zincin situ decreased iron-induced hydroxyl radical production in the intestinalmucosa. These results detail a protective role for zinc against iron-inducedoxidative stress, which has implications in anemia control programs. In cellsthat were treated with both zinc and iron, Formigari et al. (2007) foundhigher glutathione peroxidase and lower glutathione levels than in iron-treated cells. It is suggested that this may be a result of glutathione peroxidaseutilizing glutathione in the enzymatic renovation of lipid peroxides. Theoxidation of glutathione was prevented by zinc administration, which inhibitslipid peroxidation, increasing glutathione availability.

Zinc-deficient subjects were found to have low levels of serum albumin,pre-albumin and transferrin, which could be increased rapidly by zinc supple-mentation of 10–15 days duration in the studies of Bates andMcClain (1981).This effect is probably mediated through an effect of zinc on protein synthesis,most noticeable in the depressive effect of zinc deficiency on the synthesisof retinol-binding protein (Smith et al., 1999); these authors also proposedthat zinc, through its stimulation of retinol mobilization, can promote utiliza-tion of iron stores, decreasing iron-deficiency anemia, and the results ofYamaji et al. (2001) describe new pathways that may also play a role.

Garnica (1981) hypothesized that the rate of hemoglobin synthesis couldbe decreased during zinc deficiency because a required step was reportedlymediated by a zinc-dependent enzyme, aminolevulinic acid dehydrase,which functions in hematopoiesis. Zinc is clearly involved in several otheraspects of normal hematopoiesis by virtue of its role in various enzymesystems linked to DNA synthesis (including thymidine kinase and DNApolymerases; Prasad, 1991). The binding of zinc to proteins stabilizes thefolded conformations of domains so that interactions between proteins andRNA and DNA are facilitated; zinc is essential to the zinc-finger transcrip-tion factor, GATA-1, required for erythropoiesis (Berg and Shi, 1996;Farina et al., 1995).

4. Whole Body Regulation of Iron and Zinc

in Humans

4.1. Iron homeostasis

In higher animals and humans, iron has a central role in the formation ofhemoglobin andmyoglobin, but there are in additionmany vital iron-requiringbiochemical pathways and enzyme systems including energy metabolism, celldivision, neurotransmitter production, collagen formation, and immune systemfunction (Edison et al., 2008). At the same time, in excess, iron is potentiallytoxic to cells due to its ability to catalyze the production of reactive oxygenspecies via the Fenton reaction. Therefore, tight regulation of iron uptake and


storage at both cellular and whole body levels is equally essential. For themaintenance of body iron homeostasis, there must be effective communicationbetween the key sites of iron utilization (e.g., the erythroid marrow), storage(e.g., the liver and reticuloendothelial system), and the primary site of absorp-tion in the small intestine (Steele et al., 2005). Based on segregation of ironrequirements within the body, several “regulators” for iron homeostasis havebeen hypothesized: dietary regulator (or mucosal block regulator), stores regu-lator, erythropoietic regulator, inflammatory regulator, etc. (Edison et al., 2008).New evidence is showing that the various regulators are not necessarily differentand may perhaps represent differential responses mediated by the same mole-cules (Hentze et al., 2004). One of the molecules that is thought to be a centralregulator of iron metabolism, secreted by the liver and excreted by the kidneys,is hepcidin. Hepcidin, a small peptide, acts as a functional target for all otherregulators (Edison et al., 2008).

In order to better understand how systemic iron homeostasis is main-tained, it is necessary to look at the movement of iron among various tissuesand organs of the human body (Fig. 5). Iron is transported around the bodyin the bloodstream bound to transferrin and most is integrated into hemo-globin by developing erythrocytes in the bone marrow. Old or damaged

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Figure 5 Major pathways of iron transfer between various tissues and organs includingthe likely role of zinc in iron homeostasis. Tf, transferrin; RBC, red blood cells; DMT1,divalent metal transporter 1; FPN, ferroportin.


erythrocytes are removed from the bloodstream by the macrophages, andthe iron is recycled back to plasma transferrin. All tissues take up ironfor their metabolic needs, and as it is not actively excreted, the amountof iron in the body must be controlled at the point of absorption in thesmall intestine.

In adults, dietary iron enters the body via the small intestine in quantitiesequal to the amounts of lost iron from the body, so establishing the body’siron homeostasis. Iron flux from intestinal enterocyte to the bloodstreamis modulated by a liver-derived peptide, hepcidin. Hepcidin expressionis influenced by systemic stimuli such as iron stores, the rate of erythropoie-sis, inflammation, hypoxia, and oxidative stress. Intestinal concentrations ofzinc modulate the function of DMT1 and FPN as well as the expression ofhepcidin itself (see later).

Homeostatic mechanisms regulating the absorption, transport, storage,and mobilization of cellular iron are of critical importance in iron metabo-lism, owing to the risk of free-iron-induced peroxidative damage to cellmembranes, and a rich biology and chemistry underlie all these mechanisms(Edison et al., 2008). Cellular and systemic iron imbalance is detrimentaland so these processes require tight regulation (Edison et al., 2008). As thereis no efficient pathway for iron excretion, intestinal absorption has to bemodulated to provide enough (but not too much) iron to keep stores suppliedand erythroid demands met (Prasad, 2003). Hepcidin is the major regulatorypoint of iron homeostasis and its expression is determined by the complexinterplay of various factors, and depending on the specific situation, one ofthe several stimuli will predominate (Prasad, 1991). Stimuli can signal throughmultiple pathways to regulate hepcidin expression, and the interactionbetween positive and negative stimuli is critical in determining the nethepcidin level (Darshan and Anderson, 2009). Since hepcidin expression ismostly restricted to the liver, it is highly likely that the hepatocyte is the site ofaction of the regulatory stimulus. Current data would suggest that iron levelsas such do not play a primary role in this process, but rather that an additionalsignal is involved. This review provides evidence that Zn concentrationsin the body may have that crucial role in iron homeostasis.

4.2. Hepcidin, an iron store regulator

Hepcidin is a major communicator between liver iron stores and theintestinal iron absorption and transport mechanisms (de Benoist et al.,2007). The physiological role of hepcidin as an effector of iron absorptionwas shown when changes in iron absorption coincided inversely withchanges in the amount of hepcidin expressed in liver cells. Direct evidencewas provided when recombinant hepcidin was injected into rodents or appliedto intestinal cell lines, and iron absorption was decreased (Yamaji et al., 2004).Newborn animals in which hepcidin was overproduced suffered severe iron


deficiency and they died within a few hours of birth, whereas mice thathad been engineered to overexpress hepcidin developed a severe iron-deficiency anemia (Nicolas et al., 2002). Similarly, hepcidin-expressingtumors also resulted in iron-deficiency anemia, due to reduced iron accu-mulation and availability (Rivera et al., 2005). The direct inverse linksbetween hepcidin expression and iron absorption have been shown bynumerous studies (Nicolas et al., 2002; Rivera et al., 2005; Yamaji et al.,2004). However, consistent evidence about the exact mechanism of hepci-din action was still missing (Collins et al., 2008).

4.3. Hepcidin regulates DMT1 and/or FPN expressionand function

Yamaji et al. (2004) investigated how 24-h exposure to hepcidin affectediron transport in the human intestinal epithelial Caco-2 cell. The studyshowed that hepcidin, added to the basolateral chamber of the Transwellculture system 24h prior to experimentation, significantly decreased(P<0.04) iron uptake across the apical membrane of Caco-2 cells. Totaliron efflux was decreased in direct proportion to the reduced apical uptake.Following hepcidin treatment, there was a significant decrease (P<0.01) inthe membrane expression of DMT1 protein (A, Fig. 6), whereas proteinexpression of the efflux transporter, ferroportin (IREG1) (B), was unaf-fected by hepcidin (B, Fig. 6). Changes in transporter mRNA levelsmirrored those in protein. Similar findings were provided by Lagnel et al.(2011): hepcidin induced a significant reduction in iron transport andDMT1 protein levels but no change in ferroportin levels.

Studies by Frazer et al. (2003) demonstrated that liver hepcidin expres-sion was decreased in response to dietary iron deficiency. Further, the lower

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Figure 6 Effects of hepcidin on iron-transporter protein expression (adapted fromYamaji et al., 2004). Con, Control; Hepc, hepcidin; IRE, iron responsive element;IREG1, iron regulated transporter 1.


liver hepcidin mRNA levels correlated with increased intestinal ironabsorption and elevated expression of the intestinal DMT1 transporter.Direct injection of hepcidin into mice decreased specifically the apicaluptake step of duodenal iron absorption. The authors also showed a corre-spondence between decreased hepcidin and elevated FPN expression. Royet al. (2007) and Nemeth et al. (2004) also concluded that hepcidin affectedthe expression of FPN, the protein necessary for iron efflux from theintestine and macrophages. Hepcidin is able to bind to FPN and bringabout its internalization, thus removing it from the plasma membrane andmaking it unavailable for cellular iron export. A direct suppressive effect ofhepcidin on FPN was confirmed (Yeh et al., 2004).

Although questions remain, it is clear that hepcidin plays a key role in theregulation of iron absorption. There are some studies that have demonstratedthe effect of zinc on expression and function of both intestinal transportersDMT1 and FPN (Iyengar et al., 2009; Kelleher and Lonnerdal, 2006; Yehet al., 2004). The inconsistent evidence about the exact site of hepcidin actioncould be explained by the fact that changes at the apical side of the enterocytemost likely contribute to the changes at the basolateral side and vice versa,depending on the magnitude of iron stores at the start of each study. There-fore, it is postulated that zinc somehow influences hepcidin production and inthis way indirectly affects function of iron transporters at both apical andbasolateral sides of enterocytes.

4.4. Zinc, an important regulator of iron absorption

Laftah et al. (2004) measured the effect of hepcidin injection on ironabsorption in iron-deficient and iron-adequate mice and found that,although hepcidin had no effect on iron stores or hemoglobin levels, itdecreased iron absorption by a similar proportion in both groups. Hepcidininhibited the uptake step of duodenal iron absorption but did not affect theproportion of iron transferred to the circulation. The effect was indepen-dent of iron status of mice and did not require Hfe gene product. The datasupport a key role for hepcidin in the regulation of intestinal iron uptake.Hepcidin expression is reported to be decreased in adult Hfe KO mice,despite the latter’s elevated iron stores (Nemeth et al., 2004). This impliesthat some additional factor, other than hepcidin, may also be involved in theregulation of mucosal transfer of iron under inadequate dietary iron.

Hepcidin synthesis is markedly induced by infection and inflammation(Nemeth and Ganz, 2006). These effects are mediated by inflammatorycytokines, predominantly IL-6 (Kawabata et al., 2005). In human volunteersinfused with IL-6, urinary hepcidin excretion was increased an averageof 7.5-fold within 2h after infusion, whereas IL-6 knockout mice (unlikecontrol mice) failed to induce hepcidin in response to turpentine (Nemethet al., 2004). In vitro treatment of primary hepatocytes with IL-6 directly


increased hepcidin mRNA expression (Kawabata et al., 2005), and thisinduction was blocked by treatment with anti-IL-6 antibodies. Becauseplasma zinc concentration is lowered by inflammatory cytokines (Vastoet al., 2007) and zinc levels in inflammatory conditions are often reduced,the question becomes whether zinc deficiency, directly or through action ofIL-6 cytokines, increases hepcidin action, thereby contributing to the devel-opment of anemia of inflammation. This idea has some support from studiesof hemochromatosis where the lack of upregulation of hepcidin occursdespite increased liver iron stores. Patients afflicted with hemochromatosisabsorb and store more zinc than normal. Possibly, high cellular zinc concen-trations downregulate hepcidin expression under such conditions, ultimatelyallowing more iron absorption into the mucosa.

Recently, Balesaria et al. (2009) showed that treating cultured hepato-cyte cell lines with iron (or Cu or Cd) does not have an effect on transcrip-tion of hepcidin whereas zinc does, activating the metal transcription factor,MTF-1 that utilizes zinc to bind to DNA directly, confirming the importantrole of zinc in iron homeostasis. The authors have also postulated thathepcidin belongs to the family of metallothioneins, proteins regulated byintracellular zinc ion levels.

The most recently described regulator of hepcidin action is the mem-brane-bound serine protease matriptase-2, encoded by the Tmprss6 gene andexpressed primarily in liver. Hepcidin levels are inappropriately high whenTmprss6 is mutated (Du et al., 2008; Folgueras et al., 2008), suggesting thatmatriptase-2 acts as a repressor of hepcidin expression under normal condi-tions. Mutations in matriptase 2 in mice and humans cause iron-deficiencyanemia that responds poorly to iron therapy (Folgueras et al., 2008). Cellculture studies reveal that matriptase 2 suppresses hepcidin expression byinterfering with hepcidin-activating pathway involving hemojuvelin signal-ing (Knutson, 2010). Of interest, Du et al. (2008) in their study with miceshowed that mutant mice (mice with mutation in matriptase 2), besides iron-deficiency anemia, displayed gradual hair loss and infertility, symptoms whichmimic those of inadequate zinc levels in the body. The matrix metallopro-teinases (matriptase-2) contain the consensus zinc-binding catalytic sequencein their metalloprotease domain and are a family of zinc-dependent endo-peptidases (Knutson, 2010; Ramsay et al., 2009), details that impute a criticalrole of zinc in iron homeostasis.

4.5. The role of zinc in decreasing systemic intestinalinflammation and iron deficiency

Zinc deficiency aggravates oxidative stress in cells (Powell, 2001) whereastreatment with zinc protects sulfhydryl functional groups in proteins andinhibits the formation of hydroxyl radicals (.OH) from H2O2 and O2

� byother transition metals. Systemic intestinal inflammation associated with


zinc deficiency can lead to iron-deficiency anemia (Roy, 2010). Zinc hasrecently been shown to play a role in membrane barrier function in intesti-nal cells controlling inflammatory reactions by protecting membrane pro-tein sulfhydryl groups (Finamore et al., 2008; Scrimgeour and Condlin,2009). Most recently, mild zinc deficiency has been shown to cause colitis inrats via impairment in the immune response and not through theimpairment of epithelial barrier function, effects that are prevented byzinc treatment (Iwaya et al., 2011). These findings show a critical role ofzinc in controlling inflammatory reactions in the intestine. Increased per-meability of the intestinal membrane barrier leads to diarrhea and increasedinfections from pathogenic bacteria. Thus, zinc deficiency can lead tosystemic inflammation and iron-deficiency anemia via the effects of inflam-mation on elevating hepcidin production in the liver and suppressing ironabsorption by enterocytes.

Prebiotics can promote the absorption of zinc from the colon and so havea central role in control of systemic inflammation detailed above. Prebiotics,a group of indigestible oligosaccharides (e.g., fructans and arabinoxylans)found in some foods and not others, promote the growth of beneficialbacteria (probiotics) in the intestine (Iwaya et al., 2011; Manning andGibson, 2004). Prebiotics resist digestion in the stomach and small intestineand are metabolized by probiotic bacteria primarily in the colon. A spectrumof health benefits follows the increase of probiotics such as the Gram-positivebifidobacteria and lactobacilli in the intestine, partly from the suppression ofthe less desirable Gram-negative pathogenic bacteria by competition forsubstrate. An important side effect of prebiotic activity and probiotic activityin the large intestine is the enhancement of absorption of zinc, iron, calcium,and magnesium from the gut, coupled with production of desirable short-chain fatty acids (O’Flaherty and Klaenhammer, 2010; Soccol et al., 2010).Enhanced zinc status in the colon is linked with the suite of changes associatedwith the suppression of intestinal systemic inflammation described above.

4.6. Anticipated mechanism of zinc action on iron deficiency

The major findings from the studies included in this review lead to follow-ing inferences and conclusions:

� Iron deficiency is usually accompanied by zinc deficiency.� Zinc is a strong predictor of hemoglobin concentrations.� Iron supplementation, by itself, is not always effective in treatment ofanemia.

� Zinc treatment and zinc concentrations often increase hemoglobin levels.� DMT1 is not a site for iron–zinc antagonism.� Iron transport across apical to basolateral membranes is higher in cellsexposed to high zinc than in those exposed to high iron.


� Expression and function of DMT1 protein and mRNA do not change inaccordance to iron supply in the diet, but in accordance to zinc concen-trations in the diet.

� mRNA expression of FPN, while not altered by exposure to high iron, issignificantly increased by zinc.

� Hepcidin is a major communicator between liver iron stores and theintestinal iron absorption. Hepcidin induces a significant reduction iniron transport and DMT1 protein levels.

� Hepcidin can bind to FPN and cause its internalization and silencingin enterocytes.

� Dietary iron deficiency significantly alters proportional mucosal transferof iron to blood, whereas hepcidin injection does not affect this parame-ter. Therefore, some additional factor, other than hepcidin, may also beinvolved in the regulation of mucosal transfer of iron under conditions ofinsufficient dietary iron.

� The hepcidin mRNA lacks stem-loop structures containing the consen-sus IRE motif for binding of iron-regulatory proteins.

� Recently discovered regulator of hepcidin action, matriptase-2, is a zinc-dependent endopeptidase.

Hepcidin

Ferritin

Enterocyte

Heph FPN

Fe3+

Fe2+

Fe2+

Fe2+

Fe2+

Fe3+

DCYTB

ZIP

Zn

DMT1 Duodenum

Decrease

d

inflammatio

n

Zn

Liver

Figure 7 Proposed mechanisms of zinc action in regulating iron absorption from thehuman intestine.


� Zinc deficiency can lead to systemic inflammation and iron-deficiencyanemia via the effects of inflammation on elevating hepcidin productionin the liver and suppressing iron absorption by enterocytes.

� Zinc has a critical role in maintaining membrane barrier function andcontrolling inflammatory reactions in the intestine via immune responses.

� Zinc decreases permeability of the intestinal membrane barrier therebydecreasing risk of diarrhea and infections from pathogenic bacteria.

All these findings together suggest that zinc is the critical element incontrol of intestinal iron absorption, and that adequate zinc concentrationsin the body, in addition to iron, are important for treating iron-deficiencyanemia. These findings are encapsulated in Fig. 7.

5. Healthy Food Systems

A sense of food security for old age in people of reproductive agedrives birth rates lower, more so than economic development (UN Summiton Population, Cairo, 1994). Creating this sense of food security in populousthird world countries may not only provide the basic human right to nutri-tious food but may also be the only practical way of attacking the massivepopulation growth problem. Food security (or more precisely, nutrientsecurity) thus becomes the most urgent challenge facing the human race asa whole and it immediately raises the question of healthy food systems. Thefood systems of the post-green revolution era have provided enough caloriesand protein for most people, but nutritional health has deteriorated markedlybecause the new food systems failed to deliver all the essential nutrientsrequired for good health (Welch and Graham, 2004). This is a challenge forall countries, not just for resource-poor countries: chronic diseases such ascancer, hypertension, coronary heart disease, diabetes, osteoporosis, obesity,and other diseases of western societies are also nutrition-related, owing tocalorie-rich, nutrient-poor diets.

New food systems must be able to deliver all essential nutrients requiredfor human health (at least 42 minerals and vitamins, Welch, 2002a,b) and inroughly appropriate amounts of each. Fortunately, most of them are foundin plants and a judicious combination of plant foods can satisfy mostrequirements. One in particular, cobalt-containing vitamin B12, is derivedin part from dietary microbes but mainly from animal products, and a little,such as an occasional egg, is necessary to satisfy the requirement. A dietdominated by vegetables can satisfy all other requirements but such a diet isrelatively expensive and practical, less-expensive diets for large populationsin resource-poor countries are usually based on the major cereals withsupplementary pulses, vegetables, and the occasional small quantity of fishor meat product. A nutritious diet based on cereals is achievable for areas


of high population density but requires attention to certain details, and inalmost all soils, significant use of fertilizers not only to produce adequateyields of the basic cereals and secondary staples but also to do so on less landin order to free up some plots for diverse, more nutrient-dense minor cropsthat will together balance the diet with all of the earlier-mentioned essentialnutrients. Crop protection against fungal pathogens and insect pests will benecessary for increasingly higher yields as population grows. For each andevery soil type in the food system, it is fundamental that all limiting nutrientsare supplied by fertilizer and other external sources such as animal manures,green manures, and other recycled organic wastes. Fertilizer requirementsmust be determined by experimentation and/or by soil analysis combinedwith plant tissue testing, a considerable external input but essential as the costof fertilizer can be limiting when it is not correctly prescribed. We emphasizethat standard soil analyses are not sensitive enough to safely prescribe themicronutrient needs of a soil and plant analysis is essential. Especially in Africawith old landscapes and soils, determining the optimal fertilizer rates andcombinations will be challenging and requires high-quality professional sup-port organized at a national or even international level, especially to deter-mine micronutrient requirements.

Putting together a suitable combination of crops to compose the foodsystem is also challenging. The basic cereals, with proper fertilization (basicNPK plus calcium, magnesium, sulfur and where diagnosed, the micronu-trients iron, zinc, copper, manganese, boron, cobalt, molybdenum, nickel,selenium, iodine), should provide most of the calories and the protein required.Secondary staples of pulses with higher nutrient density are highly desirable tosupplement the cereals. Sweet potatoes are common secondary staples in mostdeveloping countries and it is important that the orange-fleshed varieties beencouraged as they can supply the vitamin A requirements for people of allages. The leaves are also edible and provide additional protein, minerals, andvitamins A andC.Where there is no obvious source of vitamin A, this crop canbe encouraged, but also yellow, orange, and red maize varieties are commonlyadequate in vitamin A, though not in vitamin C.

We know from the global picture that the common micronutrient defi-ciencies in resource-poor populations are iron, zinc, vitamin A, selenium,iodine, and vitamin B12. All of them can be addressed in a balanced foodsystem through fertilizer use (zinc, selenium, iodine, cobalt), combined withnutrient-dense secondary staples such as pulses and yellow/orange root cropsas already mentioned. New varieties of cereals biofortified by plant breeding(http://harvestplus.org) should be used where available to simplify the task ofsatisfying the needs of iron, zinc, and vitamin A. In our analysis of the greenrevolution and subsequent food systems, we have identified zinc as the mostwidely distributed and critical micronutrient deficiency worldwide, that canenhance the absorption of some of the iron already in the diet. Moreover,zinc is very effective as a fertilizer (Cakmak, 2009; Graham, 2008; Welch,

http://harvestplus.org


2002a) and is widely available alone or in strategic “blends” with N, P, NPK,or in complete (balanced) fertilizers where precise requirements have notbeen defined by agronomic experimentation. We argue that getting zinc intothe food systems along with the necessary macronutrients is the most urgentchange needed and will apply to half or more of the total target area. Havingsaid that, each soil type will have its unique spectrum of deficient minerals andthese too must be addressed if the food system is to be productive andnutritious, so satisfying all the needs of the population dependent on it.Individual deficiencies that have caused severe disease, such as the calciumdeficiency rickets in Bangladesh (Abed and Combs, 2001), the iodine defi-ciency goiters and mental retardation of Xinjiang (Cao et al., 1994), and theselenium deficiency cardiomyopathies of Keshan (Coombs et al., 1987), mustbe dealt with concurrently with the more widely distributed deficiencies ofNPK and zinc. Without complete nutritional balance, regardless of cropproductivity, the food system will have failed its people.

6. Conclusion

Our focus in human nutrition is on the recent discovery of the role ofzinc in iron absorption by the duodenum, whereas in agronomy, our focus ison the importance of zinc to cereal productivity and nutritional value.Combining reviews of parts of the recent literatures of agronomy, plantnutrition, and human nutrition together demonstrate the interdependenceof these research efforts and this collective literature can inform thoseinvolved in tackling the challenge of producing nutritious food for a projectedhuman population of more than 9 billion by 2050. This task is made morechallenging by the expectation that food security must be achieved in theface of decreasing availability of productive land (erosion, desertification, soilacidification, salinization, etc.), decreasing availability of critical fertilizerscoupled with their increasing cost, and an adverse trend in climate change.

This chapter reviews the impact of the first green revolution, 1960–1980,and documents the unintended consequence of a rise in micronutrientdeficiencies in the human population dependent on the new, low-nutrient-density cereals for much of their diets. The agronomic review suggests thatzinc deficiency is the most serious of the micronutrient imbalances that aroseout of the green revolution effort, owing to the emphasis on NPK fertilizersand their interactions with zinc. In humans, we identify a rise in the extentof zinc deficiency and iron deficiency as perhaps the most significant andextensive of these side effects, with vitamin A, iodine and selenium deficien-cies also of major concern. Zinc deficiency appears more fundamental fromthe agronomic viewpoint, and in humans, the most recent molecular


physiology suggests strongly that some, if not most, of the iron deficiencymaybe a consequence of the underlying zinc deficiency.

Currently, there is much concern about how we can increase foodproduction to feed the 2–3 billion more people expected on the planet by2050. Our review findings suggest that nutrient density fell as a result of firstgreen revolution effort based on low-nutrient-density cereal cropping sys-tems. We have reported some limited evidence that people need fewer totalcalories of nutrient-dense food than they do of low-nutrient, cereal-baseddiets and we recommend more research in this area. A research programon nutrient-rich diets, their agronomic requirements, calorie requirements,health benefits, food waste levels, and the relative costs of production perperson compared to the nutrient-poor diets widely consumed today willinform the effort to achieve food security for all in 2050.

ACKNOWLEDGMENT

Support from HarvestPlus Challenge Program is acknowledged. Robin D. Graham, noconflicts of interest; Marija Knez, no conflicts of interest; Ross M. Welch, no conflictsof interest.

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