(Advances in agronomy 115) donald l sparks (eds ) advances in agronomy 115 academic press, elsevier (2012)

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 d

Texas A&M University

Emeritus Advisory Board Members

Prepared in cooperation with the

American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America Book and Multimedia Publishing Committee

DAVID D BALTENSPERGER, CHAIR

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ISBN: 978-0-12-394276-0

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Numbers in Parentheses indicate the pages on which the authors’ contributions begin.

Nanthi S Bolan (215)

Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, Australia; Cooperative Research Centre for Contamina- tion Assessment and Remediation of the Environment, Adelaide, Australia

School of Biology, Flinders University of South Australia, Adelaide, Australia

1 Formerly with the Nicholas Institute for Environmental Policy Solutions, Duke University, Durham, North Carolina, USA.

ix

Anitha Kunhikrishnan (215)

Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, Australia; Cooperative Research Centre for Contamina- tion Assessment and Remediation of the Environment, Adelaide, Australia; Chem- ical Safety Division, Department of Agro-Food Safety, National Academy of Agricultural Science, Suwon-si, Gyeonggi-do, Republic of Korea

Nicholas Institute for Environmental Policy Solutions, Duke University, Durham, North Carolina, USA

Volume 115 contains six excellent reviews covering important global topicsincluding human health, climate change, nutrient and trace metal mobilityand bioavailability, and food production Chapter 1 is a comprehensivereview on the role of zinc deficiency on nutritional iron deficiency inhumans Chapter 2 deals with an assessment of climate impacts on hydro-logical mobilization of diffuse substances from agriculture Chapter 3 pro-vides an overview of greenhouse gas mitigation with agricultural landmanagement in the United States Chapter 4 covers the role of abioticand coupled biotic/abiotic mineral controlled redox processes in nitratereduction Chapter 5 provides a critical review of the role of wastewaterirrigation on the transformation and bioavailability of heavy metal(loids) insoil Chapter 6 addresses the development and adoption of submergence-tolerant rice varieties.

I appreciate the fine reviews of the authors

DONALDL SPARKSNewark, Delaware, USA

xi

How Much Nutritional Iron Deficiency

in Humans Globally Is due to an

Underlying Zinc Deficiency?

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

Contents

2 Agronomy of Micronutrients in Respect to the Green Revolution

3.7 Iron and zinc transporters in enterocytes of the small intestine 18 3.8 Positive role of zinc in oxidative damage and protein synthesis 21

4.3 Hepcidin regulates DMT1 and/or FPN expression and function 25

4.5 The role of zinc in decreasing systemic intestinal inflammation

4.6 Anticipated mechanism of zinc action on iron deficiency 28

ISSN 0065-2113, DOI: 10.1016/B978-0-12-394276-0.00001-9 All rights reserved.

* School of Biology, Flinders University of South Australia, Adelaide, Australia

{ Department of Crop and Soil Sciences, Cornell University, Ithaca, New York, USA

1

5 Healthy Food Systems 30

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 in

1960 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, both

by 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

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 poor

to 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

250

Cereal production Pulse production Population 200

popula-supplementation and food fortification strategies, with a major program calledfor by the end of the 1980s decade These programs were facilitated by the ease

of 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,associated with the production of the green revolution varieties of cereals; again

no 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) edited

an 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 (Graham

et 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

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 with

it 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 fortification

of 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

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 )

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 up

to general expectations, but to experienced eyes, anomalous results hinted

at 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 plants

as 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 deficient

in zinc, 31% deficient in boron, 15% deficient in molybdenum, 14% deficient

in 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 roughly

a 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 should

be 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 deficiency

in 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 respect

is well shown in the data of the UN Food and Agriculture Organization(FAO) inFig 1where 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 expansion

to 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 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, with

syner-a projected 9 billion before the numbers stsyner-abilize syner-and hopefully begin

to 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 produced

in fertilizer factories from oil/gas, and electricity in the highly demanding Haber process (Coates, 1939) This huge production of syntheticfertilizer, unique to the late decades of the twentieth century, has placed

energy-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 each

93 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 development

is 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 can

be 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 and

so 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 is

a more viable option to enhance iron levels, if needed In contrast, zinc

in seeds is easily enhanced, as, for example, the results ofGenc 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 required

by 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 up

to 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 crops

is 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, can

be 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 to

be fully established Due to simpler genetics, it may prove more effective to

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 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, vitamin

symp-A 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 perceived

as evidence of its purity/cleanliness), and iron and zinc concentrations in

Absorption

Absorption

Utilization

Absorption RBT Transport Utilization

Vitamin A

Figure 2 Synergy of iron, zinc, and vitamin A in the human gut: an increase of any one may 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 extent

of 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 half

of 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 by proteomic techniques

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 number

of 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, that

is, its concentration does not markedly decline in the blood stream as severity

of deficiency increases, in contrast to iron, a “type I” nutrient, the tration of which does decline in the blood markedly as deficiency increases

concen-in severity When zconcen-inc supply is low, the body sacrifices bone zconcen-inc 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 deficiency

in an individual (Hess et al., 2007) Until the release of the map of deficient human diets, zinc deficiency was low on the WHO 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

zinc-2.4 Vitamin A deficiency and its significance

Vitamin A is widely deficient in humans (Abed and Combs, 2001) Vitamin A

is 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 vitamin

A 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 solved

by 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 (Graham

et al., 2007) Vitamin A must be addressed by breeding or by introductioninto the food system of an additional food crop naturally rich in provitamin

A 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 fish

or meat products); and a stable, economic food system must be capable

of 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 once

a 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 effects

of 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 effect

of 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 findings

in 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 used

in clinical trials), and/or chemically bound or complexed as in food

absorp-of zinc on hemoglobin and another positive effect on plasma ferritin.Moreover, none of the trials showed a negative effect of zinc supplements

on iron status indicators and the studies looking at whether iron 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 children

supple-in Bangladesh for its benefits supple-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 may

be taken as a valid indicator of zinc status averaged across all the individuals

in 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 (Berger

et 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 and

to 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 studies

of 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 number

of 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 stronger

in anemic than nonanemic populations A study byGibson 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 Smith

et 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 deficient, and zinc is shown to be a strong predictor of hemoglobinconcentrations Moreover, iron supplementation, by itself, is not alwayseffective in treatment of anemia.

zinc-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 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.,

supplemen-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 vitamin

A 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 prevalence

of 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 benefitsfrom multimicronutrient 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 in

a multivitamin–mineral food-based supplement (Ramakrishnan et al., 2004)

3.7 Iron and zinc transporters in enterocytes of the

small 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 Fe2þto Fe3þprior

to 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 duodenal enterocyte 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 identified

as zinc transporters, indicating separate mechanisms for iron and zinc 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)

absorp-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 ofKelleher and Lonnerdal (2006), where they 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.,

inves-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 ofSmith 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 time

of 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 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 according

enter-to intestinal iron concentrations but rather in accordance enter-to changes ofintestinal zinc concentrations The fact that DMT1 is not inversely related

to 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 Exposure

to 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 predominantly

an 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 and

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

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 metal transporter 1; IREG1, basolateral iron-transporter protein 1.

and elevating the levels of reduced glutathione Further, the presence of zinc

in 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 ofBates and McClain (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 synthesis

of 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 of

Yamaji et al (2001)describe new pathways that may also play a role

Garnica (1981)hypothesized that the rate of hemoglobin synthesis could

be 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 and myoglobin, but there are in addition many 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 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 body

main-in the bloodstream bound to transferrmain-in and most is main-integrated main-into globin by developing erythrocytes in the bone marrow Old or damaged

hemo-Hypoxia

Liver -1000mg

Inflammation

Other cells and tissues -400mg

Reticuloendothelial macrophages -600mg

Bone marrow –300mg

(Fe 3+ )2 –Tf

- 3mg 1-2mg/day

DMT1 Zn FPN

RBC -1800mg

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 amount

of 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 bloodstream

is modulated by a liver-derived peptide, hepcidin Hepcidin expression

is influenced by systemic stimuli such as iron stores, the rate of 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)

erythropoie-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 there

is 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 levels

as such do not play a primary role in this process, but rather that an additionalsignal is involved This review provides evidence that Zn concentrations

in 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 applied

to 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 din action was still missing (Collins et al., 2008)

hepci-4.3 Hepcidin regulates DMT1 and/or FPN expression

and 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 byLagnel et al.(2011): hepcidin induced a significant reduction in iron transport andDMT1 protein levels but no change in ferroportin levels

Studies byFrazer et al (2003)demonstrated that liver hepcidin sion was decreased in response to dietary iron deficiency Further, the lower

Figure 6 Effects of hepcidin on iron-transporter protein expression (adapted from

Yamaji 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.Roy

et 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; Yeh

et 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 average

of 7.5-fold within 2h after infusion, whereas IL-6 knockout mice (unlikecontrol mice) failed to induce hepcidin in response to turpentine (Nemeth

et 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 (Vasto

et 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 studies

of 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 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

hepato-The most recently described regulator of hepcidin action is the 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 sequence

mem-in their metalloprotease domamem-in and are a family of zmem-inc-dependent peptidases (Knutson, 2010; Ramsay et al., 2009), details that impute a criticalrole of zinc in iron homeostasis

endo-4.5 The role of zinc in decreasing systemic intestinal

inflammation 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 H2O2and 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 have

a 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 spectrum

of 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 activity

in 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 deficiencyThe 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 silencing

in enterocytes

Dietary iron deficiency significantly alters proportional mucosal transfer

of iron to blood, whereas hepcidin injection does not affect this ter Therefore, some additional factor, other than hepcidin, may also beinvolved in the regulation of mucosal transfer of iron under conditions ofinsufficient dietary iron

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

consen-
Recently discovered regulator of hepcidin action, matriptase-2, is a dependent endopeptidase

Decreased inflamma tion Zn

Liver

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

Zinc deficiency can lead to systemic inflammation and iron-deficiencyanemia via the effects of inflammation on elevating hepcidin production

in 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 concentrations

in the body, in addition to iron, are important for treating iron-deficiencyanemia These findings are encapsulated inFig 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 Summit

on 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 as

a 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 found

in plants and a judicious combination of plant foods can satisfy mostrequirements One in particular, cobalt-containing vitamin B12, is derived

in 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 populations

in resource-poor countries are usually based on the major cereals withsupplementary pulses, vegetables, and the occasional small quantity of fish

or 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 land

in 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 cost

of 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 and C Where there is no obvious source of vitamin A, this crop can

be 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 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 crops

defi-as 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,

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), must

be 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

of 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 extent

of 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 deficiency may

be 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 by

2050 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 program

on 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

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