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Contributions of heme and nonheme iron to humannutritionCharles E. Carpenter Ph.D. a & Arthur W. Mahoney Ph.D., F.A.C.N. aa Department of Nutrition and Food Sciences, Utah State University, Logan, UT, 84322–8700Published online: 29 Sep 2009.

To cite this article: Charles E. Carpenter Ph.D. & Arthur W. Mahoney Ph.D., F.A.C.N. (1992): Contributions of heme andnonheme iron to human nutrition, Critical Reviews in Food Science and Nutrition, 31:4, 333-367

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Critical Reviews in Food Science and Nutrition, 31(4):333-367 (1992)

Contributions of Heme and Nonheme Iron toHuman Nutrition

Charles E. Carpenter, Ph.D. and Arthur W. Mahoney, Ph.D., F.A.C.N.Department of Nutrition and Food Sciences, Utah State University, Logan, UT 84322-8700

ABSTRACT: Dietary iron is present in food both in inorganic forms as ferrous and ferric compounds, and inorganic forms, the most important of these being heme iron. The purpose of this review is to evaluate thecontributions of both heme and nonheme iron in establishing and maintaining a healthful iron status. The humanrequirement for iron, bioavailability of heme and nonheme iron, and amounts of heme and nonheme iron in thediet are individually estimated after reviewing the relevant literature in Sections II, III, and IV, respectively.In Section V, the contribution of heme and nonheme iron to human nutrition, as compared to the humanrequirement for iron (Section II), is estimated after attenuating the amounts of heme and nonheme iron foundin the diet (Section IV) by their bioavailabilities (Section III).

KEY WORDS: human nutrition, nonheme iron, transferrin, iron-binding proteins, intestinal absorption.

I. INTRODUCTION

Dietary iron is present in food both in inor-ganic forms as ferrous and ferric compounds, andin organic forms, the most important of thesebeing heme iron. The purpose of this review isto evaluate the contributions of both heme andnonheme iron in establishing and maintaining ahealthful iron status. The human requirements foriron, bioavailability of heme and nonheme iron,and amounts of heme and nonheme iron in thediet are individually estimated after reviewing therelevant literature in Sections II, III, and IV,respectively. In Section V, the contributions ofheme and nonheme iron to human nutrition areestimated by attenuating the amounts of eachfound in the diet (Section IV) by their bioavail-abilities (Section III) and subsequently comparedto the human requirement for iron (Section II).

II. HUMAN DEMAND FOR IRON

A. Physiologic Roles of Iron

Iron is a necessary micronutrient that acts as

a catalytic center for a broad spectrum of meta-bolic functions.1 As present in hemoglobin, ironis required for the transport of oxygen and carbondioxide critical for cell respiration. Iron is alsoa component of various tissue enzymes such asthe cytochromes that are critical for energy pro-duction and enzymes necessary for immune sys-tem functioning. The importance of iron as anelement necessary for life derives from its redoxreactivity as it exists in two stable, interchange-able forms, ferrous (Fe2+) and ferric (Fe3+).2

Although other transition metals can similarlyexist in different oxidation states, iron appearsunique in its capacity to form complexes havingredox potentials that are highly coordination-de-pendent.3 For example, the redox potential ofeach mitochondrial cytochrome is different,thereby allowing single electron transport fromNADH and CoQ to O2 as a result of sequentialredox cycling of the iron atoms of each cyto-chrome between the ferrous and ferric states.

Unfortunately, redox reactivity of iron is atwo-edged sword; the single-electron transfer re-actions of iron can lead to the production of harm-ful free radicals and their derivatives such as per-oxide, Superoxide, and hydroxyl radical.1-2

1040-8398/92/$.50© 1992 by CRC Press, Inc.

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Clearly, a potentially harmful situation will existin the presence of iron that is freely redox active.For this reason, under normal in vivo situations,the availability of iron for participation in redoxcycling is rigorously limited during transport andstorage by an elaborate system of iron-bindingproteins including transferrin, ferritin, and he-mosiderin.1-4""6 Transferrin is an iron transportprotein that mediates exchange of iron amongbody tissues, while ferritin and hemosiderin serveto store iron within the body. These iron-bindingproteins perform an important, although perhapssecondary, function by withholding iron neces-sary for bacterial growth and thereby preventinginfectious growth of mircoorganisms.1"3

B. Iron Distribution and Balance

Since there is general consensus among sev-eral recent reviews regarding the quantitative as-pects of iron distribution and balance, we merelysummarize the aspects relevant to this reviewwithout further reference.l-3-7-11 Daily human iron(mg) and energy (kcal) requirements for severalpopulation segments have been compiled in Ta-ble 1. From these values, the dietary densities ofbioavailable iron (mg/1000 kcal) necessary toprovide the required iron, when energy balanceis being maintained, has been calculated and is

also given in Table 1. Nutrient density has re-cently evolved as a most useful expression of theadequacy of a diet with respect to specific nu-trients.1213 In the case of iron, where its avail-ability is altered by many factors including ironform, dietary composition, and physiological ironstatus, the most useful estimator of nutritionaladequacy is not total iron density, but rather den-sity of bioavailable iron in the diet.1415

The bodily iron content of the 70-kg refer-ence man is approximately 4 to 5 g. Of this,approximately two thirds is utilized as functionaliron such as hemoglobin (60%), myoglobin (5%),and various heme and nonheme enzymes (5%).The remaining iron is found in body storage asferritin (20%) and hemosiderin (10%). Only veryminor quantities of iron (<0.1 %) are found as atransit chelate with transferrin. Up to 40 mg ofiron is needed each day for internal utilizationby the body, largely to replace functional formsof iron, especially hemoglobin, that has been de-stroyed. A preponderance of the iron utilized eachday is furnished by recycling the existing ironsupplies. Physiologic recycling of iron is so ef-ficient that only 1 to 1.5 mg of the internallyutilized 40 mg is lost from the body and need tobe provided by intestinal absorption.

An adult man has obligatory iron losses ofabout 1 mg of iron daily, largely from the gas-trointestinal tract (exfoliation of epithelial cells

TABLE 1Human Iron Requirements

Populationsegment

(sex and age)

Males1-10 years

11-18 years19 + years

Females1-10 years

11-18 years19-50 years50 + yearsPregnant

Iron*(mg)

1.01.21.0

1.01.5

1.5-2.41.04-6

Dally requirementEnergy"(kcal)

180027502800

18002200220019002500

Bioavailable iron(mg/1000 kcal)

0.560.440.36

0.560.68

0.68-1.090.53

1.6-2.4

As reviewed in this manuscript; see Section II.From Recommended Daily Allowances, 10th ed.217

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and secretions), skin, and urinary tract (0.6, 0.3,and <0.1 mg, respectively). Thus, to remain re-plete with regard to iron, an average adult manneeds to absorb only 1 mg of iron from the dieton a daily basis. Similar obligatory iron lossesfor women amount to 0.8 mg daily. However,adult women experience additional iron loss dueto menstruation. When expressed in terms of me-dian daily loss, ceiling iron loss due to menstrua-tion for 90% of women is 0.6 mg. This ceilingmust be increased to 1.6 mg to encompass theremainder of the menstruating female population.Thus, the median daily iron requirements for ab-sorbed iron is 1.4 mg for 90% of menstruatingwomen, although 10% will require daily absorp-tion of at least 2.4 mg iron. The often-quotedrequirement for women is 1.5 mg/day. Pregnancycreates an additional demand for iron, especiallyduring the second and third trimesters, leadingto daily requirements of 4 to 6 mg. Growingchildren and adolescents require 0.5 mg iron/dayin excess of body losses to support growth. Dailyiron requirements for children, male adolescents,and female adolescents are 1.0, 1.2, and 1.5 mg,respectively.

C. Abnormalities in Iron Status

Abnormalities in iron status occur as bothiron deficiency and iron overload. Since iron bal-ance is primarily regulated by controlling ironabsorption, absorption of quantities of iron thatare less or more than required will lead to nutri-tional deficiency or overload, respectively. Gen-erally, iron deficiency and the associated anemiais of much greater concern than overload.8-1*-16

Symptoms frequently associated with iron defi-ciency anemia include palor, weakness, fatigue,dyspnea, palpitations, sensitivity to cold, oralcavity and gastrointestinal tract abnormalities, andreduced capacity for work.7-11 Accumulating evi-dence suggests that even mild iron deficiency,not associated with hematological liabilities, mayhave significant health consequences that can beattributed to decreases in essential body iron andlimitations in tissue oxidative capacity.2-8-16-19 Li-abilities may include decrease in work and ex-ercise performance due to enzymatic changes,learning and behavioral abnormalities due to neu-

rologic dysfunctions, increased risk of infectiondue to immune system abnormalities, and in-crease in perinatal morbidity and mortality dueto premature birth.

An estimated 30% of the population of theworld is anemic; the highest prevalence is in de-veloping countries where the most frequent causeis poor iron bioavailability from the predomi-nantly cereal diets and/or blood loss due to par-asitic infestation with hookworms. 1>81° In con-trast, in developed countries such as the U.S. andSweden, iron deficiency anemia among adults is"surprisingly low"20 (0.2% in adult men) andnearly always pathological rather than nutritionalin origin.10-21 However, even in the industrializedcountries, iron deficiency anemia has significantprevalence among populations having large ironrequirements due to rapid growth and/or iron loss,e.g. infants, adolescent girls, and pregnantwomen.8 Once adulthood is reached and growthdemand for iron subsides, enough iron can usu-ally be absorbed to prevent anemia, although inwomen iron reserves remain low until meno-pause.1-10 Iron deficiency anemia is also prevalentamong the geriatric population, likely due to acombination of factors including normal phys-iological changes that occur with aging accom-panied by relatively high frequencies of malnu-trition and chronic disease.22 Finally, there hasrecently been much concern regarding iron de-ficiency caused by exercise, especially in thosepopulations otherwise at risk such as adolescentgirls, women, and the elderly.23"25

Clinical consequences of iron overload areassociated with increases in non-protein boundiron resulting from the physiologic iron-bindingcapacity being overwhelmed.1-2 Liabilities in-clude increased risks for bacterial infection, neo-plasia, arthropathy, cardiomyopathy, and endo-crine disfunctions. Acute cases of iron overloadmost often occur due to inborn errors in metab-olism leading to hyperabsorption of iron or in-adequate synethesis of the iron-binding proteins.Iron overload resulting from hereditary hemo-chromatosis is preeminent as a cause of morbidityand mortality among the genetic diseases.1 Sur-veys in several industrialized countries have in-dicated that 0.5 to 0.88% of Caucasians arehomozygous for this disease, while the hetero-zygous frequency is 10 to 16%. Thus, for certain

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segments of these populations, e.g., adult men,iron deficiency anemia is less common than thehomozygous state for hereditary hemochromo-tosis.1-20 Overload can also result solely from ex-cessive absorption of dietary iron due to variedcauses including chronic ingestion of greater thanadequate amounts of dietary iron, especially hemeiron.1 These observations concerning the preva-lence of iron overload have sparked controversyregarding the beneficial vs. detrimental effectsresulting from the widespread fortification of in-organic iron into food.1-2627

D. Assessment of Iron Status

Iron status is most often assessed in order toidentify deficiency. Iron deficiency is usually as-sociated with one or more clinical measures suchas decreases in serum ferritin, serum transferrinsaturation, and hemoglobin concentration and/orincreases in erythrocyte protoporphyrin and serumtotal iron binding capacity.7-8-16-19-28"30 Each canbe classified either as an indicator of iron in thestorage compartment (ferritin and total iron bind-ing capacity) or as an indicator of iron in thefunctional compartment (erythrocyte protopor-phyrin, transferrin saturation, and hemoglobinconcentration). These clinical measures are bestused in combination for the accurate diagnosisof iron deficiency. Additional clinical measure-ments such as red cell distribution width, eryth-rocyte sedimentation rate, zeta-sedimentation rate,and C-reactive protein allow differentiation ofanemia of chronic disease from iron deficiencyanemia.31

Although difficult to select a single point ex-actly where iron deficiency can be said to initiallyoccur, most agree that deficiency certainly existswhen overt anemia appears.28 The onset of irondeficiency anemia progresses through at least threedistinct stages that can be identified by the mea-sures previously discussed.7-" During the initialdevelopment of iron deficiency anemia, de-creases in serum ferritin occur simultaneous withdepletion of storage iron. Since functional ironis maintained, there are probably no liabilitieswith regard to health. In the second stage, trans-ferrin saturation rapidly decreases and erythro-cyte protoporphyrin rapidly increases, indicating

iron stores are depleted to the point that levelsof functional iron become compromised. Hemo-globin levels may or may not begin to decline,but are still maintained within what is considerednormal. Since other functional forms of iron maybe utilized to maintain hemoglobin levels, healthliabilities associated with mild iron deficiencycan be experienced. During the last stage, overtanemia appears once hemoglobin levels have de-clined to below normal values. At this time, healthliabilities associated with anemia will be present.

III. BIOAVAILABILITY OF HEME ANDNONHEME IRON

Bioavailability is a commonly used term withregard to nutritional studies. However, the use-fulness of the term is often limited by the lackof a common definition. For purposes of thisreview, bioavailability is defined as the propor-tion of a nutrient present in food that the bodyis able to both absorb and utilize by incorporationinto physiologically functional pools. As such,bioavailability can be used as a factor to estimatethe amount of physiologically utilizable nutrientin the diet based on total nutrient content of thediet. However, when using the term "bioavail-ability", we must realize that it represents thesummation of effects exerted by several physi-ological processes, each of which is in turn al-tered by various physiological and physicochem-ical factors.

The amount of literature concerning ironbioavailability is most overwhelming. The readeris referred to several recent reviews that sum-marize much of our knowledge concerning ironbioavailability.7'101'-l6>32"35 Human studies haveindicated that many dietary constituents and nu-trients, exercise, physiological status, and ma-turity alter iron bioavailability such that a per-son's iron status is increased or decreased. Therelationships between these factors, with regardto iron bioavailability, can be complex and areoften not well understood. For example, thephysiologic capacity to absorb and utilize iron isgreatest during times of depleted iron status thatoften occurs concurrent with periods of increaseddemand, as due to rapid growth. However, thisincreased physiologic capacity to absorb and uti-

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lize iron may never be realized due to the pres-ence of dietary factors such as phytates, tannins,calcium, and phosphates that interfere with non-heme iron absorption. In contrast, the presenceof meat or ascorbic acid in the diet enhances ironabsorption, thereby allowing a maximal level ofiron bioavailability to be realized.

In this section, we attempt to review and/ordevelop reasonable estimates of heme and non-heme iron bioavailability. However, before theseestimates are presented, it is beneficial to providerelevant background information necessary forunderstanding the mechanisms by which bioa-vailability of heme and nonheme is altered byvarious dietary and physiological factors. Thus,in the first part of this section, information onthe physiological processing of dietary heme andnonheme iron is presented. Second, the physio-logical and physicochemical factors known to af-fect iron processing are reviewed. Third, dietaryfactors that alter iron bioavailability are re-viewed. These discussions are not intended to beexhaustive and the reader is referred to the manyexcellent reviews of specific topics for more com-prehensive coverage. In the last part of this sec-tion, estimates for bioavailability of heme andnonheme iron are developed.

A. Physiological Processing of DietaryIron

Between the time a food is consumed andthe time the nutrient is utilized, there are severalsequential steps during which nutrient bioavail-ability can be affected (i.e., digestion, absorp-tion, utilization). The first of these steps, diges-tion, involves the degradation of food intocomponents that are available for uptake by thebody. The second step, absorption, involves thepassage of nutrients across the epithelium of thedigestive tract. The final step, utilization, in-volves the incorporation of the absorbed nutrientinto physiologically functional pools. Enhance-ment or inhibition of iron bioavailability mayoccur at each of these steps dependent both onintrinsic factors, such as the chemistry of a nu-trient or a persons nutrient status, and extrinsicfactors, such as the overall composition of thediet.

1. Digestion

a. Nonheme Iron

Digestion can be defined as the process bywhich food is broken down into components thatare absorbable by the body. Since it is generallyagreed that only soluble iron is absorbed, andfood nonheme iron is not generally present inreadily soluble forms, digestion must first releasethe dietary nonheme iron in soluble form as aprerequisite for its bioavailability. Thus, diges-tion can be said to enhance the bioavailability ofdietary nonheme iron. The importance of the gas-tric phase of digestion to nonheme iron bioa-vailability has long been recognized. For ex-ample, gastrectomized rats with inducedhemolytic anemia absorbed only 5% of extrinsicradioiron from the diet, while their sham-oper-ated counterparts absorbed 43% of the radio-iron.36 These observations appear to hold true forhumans where maximal nonheme iron absorptionis realized when iron is injected directly into thestomach as compared to the duodenum or je-junum.37 Additionally, gastrectomized patientsare observed to have a high incidence of irondeficiency anemia.38

The gastric phase of digestion and its asso-ciated acid secretion and pepsin digestion re-moves inhibition to iron bioavailability by facil-itating the solubilization of nonheme iron fromfood. The importance of gastric acidity correlateswell with iron chemistry since most nonheme ironin food is present in the oxidized ferric (Fe3+)form which is soluble only at low pH unlesschelated to a suitable ligand. The acidity of gas-tric secretions has been observed to modify non-heme iron absorption.3*"43 Close correlation (r =0.84/7 <0.001) has been observed between breadiron absorption and the pH of the gastric juice.44

When antacid was given to test subjects, ironabsorption was reduced by 52%.45 Similar re-duction in absorption of nonheme has been ob-served in man and rats given drugs that inhibitgastric acid secretion.45'46 Nonheme iron absorp-tion from a bread meal was significantly in-creased in achylia patients to a value close to thatof control subjects upon the addition of normalgastric juice to the meal,47 while addition of neu-tralized gastric juice had no significant effect.40-47

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Nonheme iron is apparently solubilized fromits original forms in food into a common poolfrom which it then equilibrates with all dietaryconstituents to which iron can bind. As reviewedby Hallberg14 and Charlton and Bothwell,10 theconcept of all nonheme iron in diet entering acommon pool has been validated by a number ofstudies and appears to hold true for nonheme ironas present in most foods or added extrinsicallyas iron salts. This concept of complete exchangeof all nonheme iron in the diet, whether fromfood or added extrinsically, forms the justifica-tion for the use of extrinsic inorganic radioirontracer to follow nonheme iron absorption.48-49

However, some exceptions have been noted forsources of nonheme iron where the iron does notfreely exchange due to (1) inhibition of iron dif-fusion from natural barriers present in the foodsuch as would be provided by the dense aleuroneof unmilled, unpolished rice; (2) poor solubili-zation of iron from its original form when presentin complexes resistant to digestion like ferritin;or (3) poor inherent solubility of iron fortificationcompounds such as ferric orthophosphate andmost forms of reduced iron.50"54 Nonheme ironthat does not enter this common pool is consid-ered unavailable for absorption.14 That equili-bration of the available "pool" iron takes placewith all the dietary constituents is exemplifiedby observation that the presence of foods in thediet that increase or decrease nonheme iron ab-sorption exert their effect to a similar degree onall the nonheme iron in the diet, regardless of itssource.53>55>56

Nonheme iron entering this common pool canform complexes with various food constituentssuch as phytates, polypeptides, and fiber, or formchelates with various ligands present in food suchas ascorbic acid, citrate, sucrose, or amino acids,or is possibly bound to components of digestivesecretions such as gastrin, gastric transferrin, ormucin.8-14'35-57"63 Most of the components whichbind iron in soluble, yet easily absorbable, formhave been often termed "enhancers" of iron ab-sorption in that their presence minimizes theoverall inhibitory effects of food. Those com-ponents that bind iron in insoluble form or insoluble, but unabsorbable, form have been termed"inhibitors" and account for much of the ob-served inhibition of iron bioavailability by foods.

The more important of these dietary components,and their possible mechanism of action in en-hancing or inhibiting nonheme iron absorption,will be discussed in a later portion of this review.

The formation of this common pool and equi-libration with all dietary components must largelytake place at the low pH in the stomach whereall nonheme iron is soluble. When ferric ironreaches the iron-absorbing, but neutral pH, re-gion of the intestine, it is expected to undergohydrolysis and precipitate irreversibly.6465 Evenexchange and equilibration of any ferrous ironwill be limited in the intestine since the increasein pH also affects the thermodynamics for oxi-dation/reduction of iron such that oxidation offerrous iron to ferric is now favored.59 Thus, thesolubilization necessary for complete exchangeof iron is not expected in the intestine and mustlargely occur in the stomach.

However, iron solubility is not completelydetermined within the stomach; intestinal diges-tion and the presence of various dietary com-ponents continue to influence iron bioavailabilitywithin the intestine, often in a manner which isin dramatic opposition to what would be pre-dicted by the physicochemical state of the non-heme iron as it leaves the stomach. For example,predictive of poor nonheme iron bioavailability,nonheme iron may leave the stomach as insolublecomplexes that are not readily bioavailable.However, such inhibition to nonheme iron bioa-vailability may be removed due to digestivebreakdown of the insoluble component into sol-uble fractions. Torre et al.61 have reviewed theliterature indicating that binding of iron as in-soluble complexes to certain types of fiber, suchas the gums and related polysaccharides, doesnot inhibit iron absorption since these types offiber are degraded during digestion. Similarly,we have hypothesized that proteolytic digestionenhances iron solubility by removing inhibitionthat would otherwise be caused by binding ofiron to undigested, insoluble proteins.66 Unfor-tunately, in other cases, physicochemical changesthat occur upon binding of iron may prevent fur-ther digestion of insoluble iron complexes. Afterreviewing the literature, Champagne has sug-gested that decreased digestibility of proteinswhen complexed as protein-mineral-phytatecauses the entire complex to remain intact during

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the digestive process, thereby lowering the bioa-vailability of both protein and mineral.58 Con-verse to the previous situation, and predictive ofgood bioavailability, iron may leave the acidicstomach as soluble chelates. However, the pres-ence of certain food constituents may still bindiron in insoluble form once the food milieu isneutralized upon entering the small intestine,thereby precluding iron absorption. In vitro stud-ies have shown that even in the presence of iron-solubilizing chelators, such as ascorbate or cit-rate, iron binding to insoluble dietary fiber in-creases rapidly with increases in pH from 2 to5.5 as would occur as the food milieu enters thesmall intestine.60

b. Heme Iron

As is reviewed later, heme iron is absorbedby the intestinal mucosa as the intact heme com-plex. However, the heme complex, which islargely found in the diet as hemoglobin or myo-globin, must first be released from the globinprior to its absorption. In vivo studies have shownthat release of heme from globin occurs in theduodenum due to the action duodenal enzymes,especially trypsin.67-68 In the neutral pH condi-tions encountered in the intestine, and in the ab-sence of substances that bind heme, heme formspolymers of large molecular size and limitedsolubility which are poorly absorbed.68 The pres-ence of substances that can bind heme as mono-meric hemochromes, such as peptides from deg-radation of globin or other dietary protein, theamino acid histidine, or the vitamin niacin willprevent aggregate formation and increase ironabsorption.68"70

More recent in vitro studies concerning thedigestion of purified hemoglobin or meat suggestthat heme may be initially released as acid he-matin by peptic digestion in the stomach.71-72

Neutralization of peptic digests of purified he-moglobin resulted in the formation of large mo-lecular weight aggregates of limited solubilitythat were relatively resistant to further digestionby intestinal enzymes. In contrast, neutralizationof the meat peptic digests resulted in the for-mation of soluble protein-heme complexes thatwere susceptible to further digestion. The ap-

parent corollary to these differences was ob-served in the final digestion products where morethan 80% of the heme in meat digests was de-stroyed and the iron was found as low molecularweight nonheme complexes as compared to lessthan 5% destruction of heme in the hemoglobindigests. Iron in the heme-derived, nonheme com-plexes formed from the meat digests was wellabsorbed from rat intestinal loops. These studiessuggest that with consumption of heme iron inthe presence of meat, as would be the typicaldietary situation, significant quantities of the di-etary heme iron are degraded into complexes ofinorganic iron and may be subsequently absorbedin that form. This may explain observations thatoral administration of desferrioxamine, a pow-erful iron chelating agent and inhibitor of non-heme iron absorption, does not inhibit in vivoabsorption of purified heme iron,67-73 but doessignificantly reduce absorption of heme iron frommeat.74-75 This example illustrates the complex,and often unexpected, interaction of the diet withiron absorption and advises the use of cautionwhen extrapolation of bioavailability estimatesfor iron in the diet from data on iron bioavaila-bility from single sources.

During the process of digestion, heme ironforms a pool in the gastrointestinal tract inde-pendent of nonheme iron. As such, heme ironcannot be labeled with an extrinsic tag of inor-ganic iron, but can be labeled with biosyntheti-cally radioiron-labeled hemoglobin.49-76 In fact,since heme iron and nonheme iron exist in sep-arate pools, they can be independently and si-multaneously labeled with extrinsic tags of ra-dioheme and radioiron, respectively.50-77

2. Absorption

a. Nonheme Iron

Absorption is defined as the movement ofiron from the intestinal lumen, across the epi-thelial cells of the digestive tract, and into thecirculation. Absorption of iron occurs largely fromthe proximal small intestine in a process that ex-hibits biphasic kinetics.78"82 The initial phase ofabsorption is very rapid, begins within secondsof iron reaching the mucosal surface, and lasts

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for approximately 1 h. Absorption during the sec-ond phase is at a much slower rate, but continuesfor 12 to 24 h.

Absorption involves at least two steps: (1)uptake of iron from the intestinal lumen into themucosa, and (2) transfer across the mucosal celland serosal membrane into the circulation. Al-though these steps are closely integrated to ac-complish the absorption of iron, each is observedto be independent and specific in its function.Independence and specificity of each of the stepsis demonstrated by genetic abnormalities in ironabsorption where only one of the steps is affectedand by specific alterations in iron absorptioncaused by chemicals. For example, two mutantstrains of mice exist where decreased iron ab-sorption can be specifically attributed either to adefect in iron transfer combined with normal up-take (sla), or to a defect in iron uptake combinedwith normal transfer (/nfc).83"86 Independent andspecific functioning of uptake and mucosal trans-port is also demonstrated by the limited effectsof various chemicals exclusively on one step orthe other. The iron chelator, desferrioxamine,can inhibit either step depending on method ofadministration; oral administration prevents ironabsorption by precluding iron uptake from theintestinal lumen,73-7487 while parenteral admin-istration of desferrioxamine prevents absorptionof iron by blocking intracellular transport inde-pendent of uptake.88 The metabolic inhibitors cy-tochalsin-B and colchicine preclude transfer, butnot uptake.78

However, not all iron taken into the mucosalcell is processed through the transfer pathway andabsorbed into the mucosa. After uptake, iron maybe diverted from the transport pool into a storagepool. Kinetic studies indicate that most iron en-tering the storage pool remains there until the cellis exfoliated.89 Thus¿ diversion of iron from theabsorption pathway into the storage pool duringtimes of low iron demand provides one mecha-nism by which iron absorption can be regulated.This pathway appears to be separate and inde-pendent from uptake and mucosal transport ofiron. Inadequate or absent mucosal ferritin, aniron storage protein, has been indicated to be aspecific congenital defect contributing to the hy-perabsorption of iron in hemochromatosis.90-91

The kinetics of nonheme iron absorption in-

dicate that two uptake processes are operatingsimultaneously. 79>89>92~97 The first process ap-pears to be diffusion mediated in that uptake isdirectly proportional to the iron concentration inthe intestinal lumen. Uptake of food iron by thispathway predominantly occurs only when ironstores are replete, although ferrous salts may beabsorbed by this pathway independent of ironstatus. Uptake by the second process followsMichaelis-Menten enzyme kinetics in that it hasa rate limited capacity, displays saturation ki-netics and competitive inhibition. This pathwaypredominates when iron stores are depleted.

Perhaps due to difficulties in presenting ferriciron in soluble form, most early reports indicatedthat uptake of inorganic iron was only as theferrous form. However, as reviewed by Bezko-rovainy,32 Conrad," and Valberg and Flanagan,97

it is now clear that both ferrous and ferric ironare equally well absorbed from the intestinal lu-men provided they are chelated by suitable li-gands and presented in soluble forms. For ex-ample, the bioavailability of iron as ferrousascorbate or ferric polymaltose has been foundto be quantitatively the same when measured ineither rats or humans.98"100 Even high molecularweight ferric hydroxide polymers are well ab-sorbed in vivo.57-101 Both ferrous and ferric ironhave been shown to follow quantitatively similaruptake in vitro.102 At present, it is not possibleto determine conclusively whether uptake of allinorganic iron is via the same mechanism, oruptake of ferric and ferrous iron is by discretemechanisms.33103 It is possible that (1) uptake isvia the same ferrous-specific mechanism withprerequisite reduction of ferric,104-105 or (2) up-take of ferrous and ferric iron is by discrete mech-anisms.92-"-106

Originally, it was hypothesized that solubleferric chelates were absorbed intact.107 More re-cent evidence has shown that uptake of iron in-volves the release of iron at the cell surface, withthe subsequent transport of free iron across themucosal membrane by the endogenous uptakesystem(s), while the intact chelate is excludedfrom cell entry.99104"108 Thus, as reviewed byClydesdale,59 the strength with which iron bindsto ligands is important in defining iron bioavail-ability. Since solubility is the first prerequisitefor iron bioavailability, iron must be bound strong

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enough to maintain it in soluble form duringdigestion. However, the bond must not be toostrong so as to prevent the release of iron foruptake by the mucosa.

A specific pathway for ferric uptake by themucosa has been proposed by Huebers et al.109

They believe transferrin, a ferric binding protein,is secreted into the intestinal lumen where it cancomplex with ferric iron. The intact complex isthen taken into the mucosal cell via receptor-mediated transport. Inside, the cell iron is re-leased from the transferrin and iron-free trans-ferrin returns to the brush border to be recycled.The stomach hormone gastrin has been suggestedto play a role in the transferrin model of ferriciron uptake since gastrin can bind both ferric ironand apotransferrin. '10> u ' It has been proposed thatgastrin initially binds dietary ferric iron in thestomach. After the gastrin-ferric complex entersthe small intestine, the gastrin binds apotrans-ferrin and ferric iron is transferred to transferrin.Since gastrin does not bind saturated transferrin,the latter is released to again deliver iron to themucosa.

Other research has produced results incon-sistent with the transferrin model for ferric up-take.112"114 For example, although receptors fortransferrin have been localized in mucosal cells,they have not been found in the brush bordermembrane where they could initiate transferrintransport from the intestinal lumen.115"118 Addi-tionally, administration of diferric transferrin toachlorohydric humans has failed to promote ironabsorption.119 Another problem with the trans-ferrin model of ferric uptake is defining the originof the secreted transferrin. Luminal transferrinhas been suggested to come from the goblet cellsof the mucosa or bile from the liver. However,these sources have been questioned. Rat mucosalcells have been reported to be devoid of trans-ferrin messenger RNA.120 Transferrin is found inbile, however, the iron content of bile exceedsthe iron-binding capacity of its transferrin.121

Thus, bile transferrin would be saturated withiron and could not bind dietary iron. It is, there-fore, unlikely that the function of transferrin re-ceptors is to take up ferric iron from the intestinallumen. Rather, if transferrin receptors functionat all in iron absorption, it may be in transportingiron out of the mucosal cell. However, as is dis-

cussed later, even this possible role for transferrinreceptors in absorption of iron is questionable.

Little is known about the mechanism by whichiron is transported across the mucosa into theplasma. However, it is known that, because freeiron is a toxic oxidant, iron in the body is notfound in free ionic form in order to protect cellsfrom damage. For the same reason, iron enteringthe mucosal cell is immediately bound to variouscompounds, including proteins.11-33 Ultrastruc-tural and morphological studies of rodent smallintestine have allowed visualization of iron trans-port through the mucosa.118-122"124 Immediatelyafter uptake from the intestinal lumen, any fer-rous iron is converted to the ferric form at themicrovillus membrane, and all iron is then boundby a nonheme ferric receptor. Subsequently, someiron appears in non-membrane-bound depositsconcentrated in the apical cytoplasm, while otheriron may later appear localized in the lysosomesas ferritin deposits. Still other iron is transported,by a presently unidentified mechanism, to theserosal membrane where it is transported acrossinto the plasma.

Although many putative iron-binding pro-teins of the mucosa have been identified,11-33-124

specific roles for each have not been convincinglydemonstrated. Two of the proteins that have beenidentified are ferritin and transferrin. Based onthe time course of iron binding to ferritin andtransferrin during iron absorption, their role iniron absorption has been suggested to be similarto their function in other tissues of the body, i.e.,iron storage and transport, respectively. Duringabsorption, the ferritin pool rapidly binds ironbut has very slow turnover thereby retaining ironrather than releasing it to the body,80125126 whiletransferrin demonstrates rapid turnover duringabsorption."-125>127"129 In the iron-absorbing re-gions of the intestine, mucosal ferritin levels havebeen observed to correlate inversely with ironabsorption, while transferrin levels correlate di-rectly with iron absorption.78-80-130"133 Such ob-servations have led to the hypothesis that controlof iron absorption is regulated by levels of mu-cosal ferritin and transferrin.

However, the exact roles of ferritin and trans-ferrin in iron transport across the cell and ab-sorption into the plasma are controversial. Nei-ther ferritin nor transferrin satisfies the criteria

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necessary for intracellular iron transport,134 andthe available iron-binding capacities of ferritinand transferrin are suggested to be insufficient toaccount for differences in mucosal iron storageand absorption, respectively, between normal andiron deficient animals.93124135 Additionally, al-though mucosal ferritin levels are increased innormal as compared with iron-deficient rats, theferritin levels are similar throughout the duo-denum and ileum despite large variations in ironabsorption.132 Such observations are in agree-ment with the suggestion that ferritin itself issimply a storage protein and does not regulateiron absorption.136"138 Similarity, an actual rolefor transferrin in iron absorption has not beenproven.103-113 To the contrary, a substantial bodyof evidence is accumulating to indicate that mu-cosal transferrin is not directly involved with ironabsorption. Akin to their role in other cells in thebody, mucosal transferrin and its receptor, arebelieved to be involved with iron acquisition forcellular growth and development.91-115116 In sum-mation, it appears that although levels of mucosalferritin and transferrin are generally correlatedwith iron absorption, they may not be the actualmediators. The observed correlations are perhapsdue to parallel, although autonomous, regulationof these proteins and the actual absorption "ma-chinery" during development and/or functioningof the mucosal cell.

If transferrin does not form the mucosaltransport pool, then this pool still needs to beidentified. Observations have been made that anintracellular transport mechanism common to allabsorbed iron forms (heme and nonheme) can beinterrupted with chelating agents such as EDTAor desferrioxamine.80-88 This alludes to the pos-sibility that low molecular weight compounds suchas inorganic and organic phosphates may con-stitute a separate pool involved in iron trans-port.93-139"141 These compounds may comprise alarge, but poorly defined and labile, pool withinthe mucosal cell.11-80-124-125-142

Transport of iron across the serosal mem-brane is the final, critical phase during iron ab-sorption. The mechanism by which this occursis perhaps the least researched, and therefore leastunderstood, iron-processing step during absorp-tion. One possible mechanism is that iron transferout of the cell is accomplished by interaction of

plasma transferrin with receptors on the basola-teral membrane. Studies with isolated rat intes-tinal epithelial cells have shown that plasmatransferrin will bind to the basolateral membraneand enhance the release of radiolabeled iron fromthe cells.143"145 Johnson et al.78 have proposed amodel for iron absorption into the plasma thatrationalizes both their observations that ironmovement within the cytosol is associated withvesicle movement and not simple diffusion, andobservations that transferrin receptors are foundin the basal cytoplasm of mucosal cells.116-146 Inthis model, plasma transferrin would bind to thebasolateral membrane via its receptors, be inter-nalized in vesicles, and then be loaded with newlyabsorbed iron that has been tightly bound to pin-ocytized villous membrane. Subsequently, theiron-loaded transferrin would be released into thebloodstream.

In contrast, other evidence indicates thattransferrin receptors are not involved in transportof iron out of the intestinal mucosa; plasma trans-ferrin appears to be neither the immediate ac-ceptor of absorbed iron, nor essential for ab-sorption of iron into the plasma. Iron absorptionis not affected in children with congenital atrans-ferremia,147 or in hypotransferric mice.148 Addi-tionally, increased concentration of transferrinreceptors is not correlated with the increased ironabsorption observed in patients with hereditaryhemochromatosis,116 or in rats following acutehemolysis.115 With these observations concern-ing mucosal transferrin receptors in mind, alongwith those previously discussed, a direct role forintestinal transferrin receptors in either the uptakeor transport steps of absorption is questionable.Thus, similar to the function of transferrin re-ceptors in other cells of the body, their functionin intestinal mucosal cells may primarily be totake up iron from blood transferrin.

b. Heme Iron

Absorption of heme iron appears to occurprimarily by a single pathway.67-68-79-149"151 Thispathway involves the uptake of heme iron directlyinto the mucosal cells as the intact iron-porphyrincomplex. The heme is then enzymatically cleavedand the iron released in inorganic form.67-152 The

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enzyme responsible for splitting the heme hasbeen suggested to be xanthine oxidase or hemeoxygenäse.153-154 Kinetic studies in dogs have in-dicated that the splitting of iron from heme inthe intestinal mucosa is the rate limiting step inheme iron absorption.151 Absorption of dietaryheme iron involves processing through a mucosaltransfer pathway(s) common to both heme andnonheme iron. This is evidenced by the blockingaction of precedent or simultaneous doses of in-organic iron on heme iron absorption.67'68-150 Aftercleavage of the heme, the released inorganic ironis apparently released into a chelatable mucosalpool common to all absorbable iron. Parenteraladministration of the iron chelator, desferriox-amine, precludes the absorption of both heme andnonheme iron by chelation within the mucosalcell.88

3. Utilization

It is generally believed that all dietary irontaken up by the intestinal mucosal cells is pro-cessed through a common pool from which it istransported into the blood and subsequently uti-lized by the body.16 However, an increasingamount of evidence from research in our labo-ratories suggests that not all forms of dietary ironenter the same mucosal pool, nor is the physio-logical utilization of absorbed iron similar for allforms of dietary iron.155*166 Results from thesestudies have recently been reevaluated and sum-marized by Kalpalathika et al.167 In this perspec-tus, the concept of effectiveness of erythropoesis(EOE, the percent of absorbed iron incorporatedinto hemoglobin), was used to evaluate iron uti-lization from various dietary iron sources. Thisanalysis has suggested that the absorption of ironby the intestine and the metabolism of absorbediron are separate components affecting iron bioa-vailability. Additionally, it was indicated that di-etary iron may be absorbed in more than oneform, and utilization of these forms, as measuredby EOE, may vary greatly. Although still a novelconcept, differential utilization of the variousforms of dietary iron may have important impli-cations for the understanding of iron bioavaila-bility.

B. Physiologic Regulation of IronBioavailability

Humans are able to regulate iron bioavaila-bility within a wide range of dietary iron intakesand requirements such that healthful iron statuscan be maintained.8'10'16-19 Adaptation of bioa-vailability in response to iron deficiency or ov-erload occurs largely through alteration of ironabsorption by the intestine, with adaptation ofiron excretion playing a minor role. During timesof clinical deficiency or overload, absorption ofiron by the intestine into the plasma increasesand decreases, respectively. Compared to ironabsorption during times of normal iron status, therelative enhancement of absorption during irondeficiency is greater than inhibition during ironoverload. Congruent adaptation of absorption oc-curs for both heme and nonheme iron, althoughthe relative adaptation is greatest for nonhemeiron.

There is good correlation between body ironstores as measured by serum ferritin concentra-tions and adaptive response as measured by ironabsorption.16168-169 Thus, physiologic enhance-ment of both heme and nonheme iron absorptioninitially occurs in response to depletion of bodyiron stores in an attempt to avoid depletion offunctional iron. This physiologic adaptation ofiron absorption is remarkably effective in main-taining healthful iron levels during times of ad-equate dietary intake of available iron. Iron re-pletion is maintained when sufficient bioavailableiron is present to satisfy iron demand. Deficiencycan be caused by inadequate intake of dietaryiron, accelerated iron requirements due to growth,pregnancy, or menstruation, and decreased ab-sorption of iron due to dietary inhibitors.19

1. Nonheme Iron

Gastric control on nonheme iron bioavaila-bility may be exerted by regulating the rate ofstomach emptying, thereby controlling the rateat which iron is exposed to the intestine.170-171

For example, iron-deficient rats have been ob-served to release stomach contents more slowlythan normal rats.93-172 However, the primary con-

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trol of nonheme iron absorption appears to residewith the mucosa.173 While each of the mucosalsteps involved in iron absorption has been shownto be independent, they appear congruent in theiradaptation to altered demand for iron. Each phaseof mucosal iron absorption (uptake, transfer, orincorporation into storage) separately responds ina manner that increases iron absorption duringtimes of iron depletion and decreases iron ab-sorption during times of iron overload. For ex-ample, during times of increased demand, ironuptake increases have been shown both in vivoand in vj/ro.79-89-130-174-177 Similarly, iron transferacross the mucosa increases during iron defi-ciency.89-103-142-178-179 Mucosal concentration ofthe putative iron-transport protein, transferrin,increases in parallel.78-80-113130-134 Finally, the rateand amount of iron processed into storage de-crease during times of increased demand foriron.81-89-130 This is accompanied by a decreasein the mucosal content of the putative iron-stor-age protein, ferritin.78-130-131-133

Regulation of nonheme iron absorption bythe intestinal mucosa results from the integratedmodulation of uptake, mucosal transfer, and mu-cosal storage. Uptake determines the amount ofiron that enters the cell. Once iron is taken up,it then enters the transfer and storage pools inproportion to their relative sizes and rates of ironincorporation. Only iron entering the transfer poolis absorbed into the plasma. Iron entering thestorage pool is exfolliated with the mucosal cells.Iron absorption in iron deplete, replete, and over-loaded rats was found to directly correlate (r =0.95, p <0.001) with the ratio of the transferpool size: storage pool size, where the size ofthe pools was estimated by the mucosal concen-trations of the putative transfer and storage pro-teins transferrin and ferritin, respectively.130

Similarily, Conrad et al.124 observed direct cor-relation between the transferrin:ferritin ratio andiron absorption.

Sophisticated kinetic studies with normal andiron-deficient dogs have indicated that iron up-take is the rate-limiting step during iron absorp-tion from a dose of radiolabeled ferric chloridein both normal and iron-deficient dogs.89 If thatis the case, then the rate of iron absorption intothe plasma should be equivalent to the rate ofuptake and should only be attenuated by iron

diversion into the storage pool. Using data fromthese authors in support of this hypothesis, wefind that the ratio of iron uptake in normal andiron-deficient dogs to be 6:1. The ratios for therate of mucosal transfer to the rate of incorpo-ration into storage can be calculated to be ap-proximately 1:1 for normal iron status and 5:1for iron deficiency. From these ratios, the frac-tion of iron entering the cell that follows thetransport pathway and is absorbed into the plasmacan be calculated as 0.50 and 0.83 in the normaland iron-deficient states, respectively. Assuminga similar time course for passage of the iron dosethrough the iron-absorbing regions of the gas-trointestinal tract, relative iron absorption can becalculated by attenuating the relative rates of up-take by the fraction that follows the transportpathway. The calculations in this case are 1 x0.5 = 0.5 and 6 x 0.83 = 4.98 for the normaliron and deficient dogs, respectively. Thus, basedon these calculations, iron absorption in the iron-deficient dogs should be 10 times that in thenormal dogs. This is exactly the ratio that wasobserved by these authors for retention of theiron dose as measured by whole body countingat 8 days after ingestion when exfolliation andexcretion of iron-loaded mucosal cells should benear completion.

Since uptake is a limiting factor in absorptionof iron, its regulation may be of primary impor-tance to the adaptation of iron absorption to al-tered iron demands.89-173 Uptake, and thereforeabsorption, appears to inversely correlate withthe overall concentration of nonheme iron withinthe mucosal cell,132-173-180-181 with the quantity ofiron within the cell being determined by both irontaken up from the intestinal lumen and internalloading from the plasma.93124-173-182 Morpholog-ical studies have shown that any iron enteringthe cell, whether from the lumen or from theplasma, becomes localized in the same areas.81-83

Moreover, the amount of unbound iron receptorswithin the cell is perhaps more important in con-trolling iron absorption than nonheme iron con-tent. This concept is related to the "physiologicalsaturation" of the small intestine originally sug-gested as an explanation for the capability of themucosa to control uptake of ingested iron.183-184

During various states of iron repletion in the rat,there exists a direct relationship between unsat-

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urated iron-binding capacity of the cell and ironabsorption.124 Furthermore, the unsaturated iron-binding capacity of the mucosal cell directly cor-responds with the unsaturated iron-binding ca-pacity of the plasma,124 which itself is a reflectionof physiological iron demand. The exact natureof the mucosal iron-binding components respon-sible for regulating iron absorption is unclear.However, since various low molecular weightcompounds may be the largest iron-binding poolin the mucosal cell,11-80-124125-142 such compoundswill certainly be important in controlling intes-tinal iron absorption in response to physiologicaliron status.

As we have reviewed, several specific alter-ations in mucosal functioning are brought aboutin response to altered demands for iron by thebody. The exact manner in which the body'sdemand for iron is conveyed to the mucosal cellis obscure. As reviewed by Conrad11 and Flana-gan,33 it is tempting to speculate that the capacityto absorb iron is programmed, via modificationof the cells transcriptional and translational ma-chinery, into the undifferentiated cells present inthe intestinal crypts. This would account for theobserved time delay before iron absorption in-creases in response to a stimulus such as bleed-ing, parenteral iron loading, or feeding an iron-deficient diet.11-80-133185 The message initiatingsuch control is unknown, but has been suggestedto be related to circulating iron in the plasma thatis in contact with the molecular machinery of themucosal cell via transferrin receptors located ontheir serosal membrane. For example, synthesisof the proteins associated with control of ironabsorption by the mucosa, ferritin, transferrin,and the transferrin receptor has been shown tobe regulated by intracellular iron levels. 1>6>186~189

Even if ferritin and transferrin are not directlyinvolved in iron absorption by the mucosal cell,regulation of their synthesis by intracellular ironlevels may be congruent, and therefore repre-sentative, of regulation of iron absorption. It canbe hypothesized that mucosal cells developingduring iron deficiency will be regulated in a man-ner such that they mature with increased iron-absorbing capabilities. They also will be ex-pected to develop with enhanced ability to ac-quire iron for growth and development from thecirculation. Similar to other cells in the body,

this probably involves increased transferrin re-ceptors and transferrin. The net result would beparallel, although autonomous, increases in bothcellular transferrin levels and iron absorption dur-ing iron deficiency. Mucosal cells developingduring periods of iron overload will not fullydevelop their iron absorption capabilities. Theyalso will develop with increased levels of ferritinin order to help regulate the body's iron load;iron removed from the circulation would be storedin ferritin until its excretion with the exfolliatedcell. Thus, in this scenario, congruent regulationof ferritin and iron absorption during iron over-load results in the observed inverse relationshipbetween them.

2. Heme Iron

Heme iron absorption responds to altered ironstatus in a manner congruent to nonheme ironabsorption; increased demand results in increasedabsorption of heme iron in both humans and ex-perimental animals.16-67-151-152-154-190191 Similar tothe case for nonheme iron, the mucosal steps ofheme iron absorption all show adaption to irondemand. Both uptake of heme67-151 and mucosaltransfer67-152 increase with increasing iron de-mand. Additionally, once iron is split from hemewithin the mucosa, its absorption is apparentlysubject to regulation by diversion into storageequivalent to nonheme iron; the amount of ra-diolabeled nonheme iron remaining within theintestinal mucosa after an oral dose of radiohemeis significantly less for iron-deficient as com-pared with normal rats.152 However, since split-ting of heme is the limiting step in absorption ofheme iron, increased splitting within the mucosahas been indicated to be the quantitatively mostimportant factor in increasing heme iron absorp-tion during deficiency.151

C. Dietary Factors

/. Nonheme Iron

Many dietary factors are known to influencethe bioavailability of nonheme iron. In devel-oping a comprehensive understanding of non-

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heme iron bioavailability, there is benefit in tak-ing the view that food is generally inhibitory tononheme iron bioavailability. This viewpoint issupported by observations that bioavailability ofnonheme iron when administered with food isalmost always decreased as compared with ironadministered alone. Even bioavailability of ironas its ferrous ascorbate complex, a highly avail-able source of inorganic iron which is often usedalone as a reference dose, is significantly reducedby its consumption simultaneously with food.192

For example, individuals with absent iron storeswill absorb 40% of the radioiron from a ferrousascorbate reference, while absorption of radio-iron rarely exceeds 20%, and is often under 10%,when the reference dose is consumed with var-ious meals.15 Thus, the use of the term "enhan-cer" with respect to the effects of food compo-nents on nonheme iron bioavailability is perhapsa misnomer; the ability of dietary food constit-uents to enhance absorption of nonheme iron canperhaps only be defined in relative terms as com-pared to the overall inhibitory action of food.Using this approach, the specific mechanisms bywhich various dietary inhibitors may act to in-fluence absorption of nonheme iron and how di-etary enhancers may preclude some of this in-hibition are reviewed. This approach gives aunique perspective which is often more physio-logically relevant than the more traditional viewof food components as either enhancer or inhib-itor to iron bioavailability.

Dietary modification of iron bioavailabilitycan be largely attributed to physicochemical re-actions of iron which may preclude uptake bythe intestinal mucosa. For appreciable uptake tooccur, iron must both be soluble and in a formsuch that ionic iron is easily released to the in-testine. Although the food environment (pH, pre-vious processing, oxidoreductase activity, diges-tibility) can play a significant role in determiningthe physicochemical forms of iron present in thediet, the presence of various dietary componentsthat are iron-binding ligands may play an evenmore significant role in determining iron bio-availability.59-193 As reviewed by Clydesdale59 andFairweather-Tait,194 dietary ligands that enhanceiron absorption are those that form soluble andstable complexes yet easily release iron for up-take by the intestine. Additionally, some ligands

which are also reducing agents, such as ascorbicacid, may enhance absorption by reducing ironto its ferrous form. Other dietary ligands inhibitiron absorption by forming insoluble complexesor by forming soluble complexes which bind ironso tightly that it cannot be taken up into the in-testinal mucosal cells.

Dietary factors which alter iron bioavaila-bility have been extensively and often reviewedwith regard to their role in determining nonhemeiron bioavailability.8'14-1519-29-30-59194-197 Addi-tionally, certain dietary components such as as-corbic acid,198 meat,62-199 and both fiber and phy-tic acid35-58-61-200 are viewed as especially importantin determining absorption of iron from the dietand have each been the subject of autonomousreviews as indicated. Interaction of these dietaryenhancers and inhibitors determines the bio-availability of nonheme iron.14-15-201 Recent find-ings suggest it is possible to predict the bio-availability of dietary nonheme iron if consid-eration is given to the relative content in the dietof both the major enhancers and inhibitors of ironabsorption.201

a. Inhibitors

Dietary fiber can be defined as that compo-nent of the diet that is resistant to digestion bythe endogenous secretions of the upper digestivetract. Generally, this refers to material derivedfrom the cell walls of plants such as cellulose,hemicellulose, pectin, lignin, and gums and re-lated polysaccharides.61 In vitro, each of thesecomponents demonstrates the capacity to bindiron, suggesting that inhibition of iron absorp-tion may occur due to the presence of these com-ponents in the diet. However, reviews of the rele-vant literature have indicated that these compo-nents of fiber have little effect on iron bioavail-ability .35>61l9S Rather, the major contributor toinhibition of iron absorption appears to be theimmutable presence of other substances foundassociated with dietary fiber such as phytate andpolyphenols (tannâtes).8-35-61

Phytic acid (myoinositol hexaphosphate) ispresent in all plants where it serves as the storageform of phosphate and cations such as iron, cop-per, calcium, zinc, and magnesium. As present

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in cereals, legumes, and oleaginous seeds, it nor-mally occurs in the form of complexes with min-erals and/or proteins that are insoluble and notbiologically available for humans. The inter-action of phytate with other dietary constituentsand its role in mineral nutrition have been thesubjects of several recent comprehensive re-views.35iS8>6Ii20° Among these reviews, the gen-eral consensus seems to be that the phytate nor-mally present in high fiber diets is the primarycause of inhibition in iron absorption. Studies inhumans by Rossander202 and Hallberg15-203 haveoften been cited as evidence of the inhibitoryeffect of phytate on iron absorption. Insolubilityand/or indigestibility of complexes formed be-tween iron, phytate, and proteins or fiber aregenerally considered to be major reasons for phy-tic acid leading to reduced iron absorption.58

However, a number of factors may influence thesolubilities of these complexes and thereby alteriron bioavailability, including (1) the relativeamounts of phytic acid and iron in the diet; (2)presence of other dietary components that cancomplex with iron and phytate such as protein,fiber, and other minerals; and (3) the pH value.58-61

Fortunately, the inhibitory effects of phytate areeffectively counteracted by the inclusion of en-hancers of iron absorption in the diet, such asmeat and ascorbic acid.15-35-201-203

Another major inhibitor of nonheme iron ab-sorption are the polyphenols, or tannins, that arepresent in certain spices, fruits, vegetables, andt e a 201.204,205 Many hundreds of phenolic com-pounds, which are secondary plant metabolitesand likely part of plant defense systems, havebeen described. They vary in structure from sim-ple compounds to very large polymerized com-plexes.8-205 Inhibition of iron absorption by poly-phenols is likely due to binding of iron withsubsequent polymerization into insoluble com-plexes.206 The presence of galloyl groups, eachof which contains three phenolic hydroxyls, hasbeen suggested to be largely responsible for thebinding of iron by polyphenols.204 Similar to thecase found with phytate, the inclusion of meator ascorbic acid has been shown to effectivelycounteract the inhibitory action of polyphenolson nonheme iron absorption.201-207

b. Enhancers

Ascorbic acid and meat, fish, and poultry(MFP) are the quantitatively most important en-hancers of nonheme iron absorption found in thediet. As such, the presence of these factors inthe diet needs to be especially considered whendeveloping models which predict iron bioavail-ability.208"211 The enhancement factor used in thesemodels is based on milligrams of ascorbic acidplus the grams of MFP consumed per meal. As-corbic acid may promote food iron absorption byseveral interdependent mechanisms.63>19S First,ascorbic acid may promote acid conditions withinthe stomach such that dietary iron is efficientlysolubilized. Second, solubilized ferric iron maybe reduced to ferrous, thereby precluding the for-mation of insoluble ferric hydroxides. Third, as-corbic acid may form chelates with solubilizedferric iron within the stomach and subsequentlymaintain solubility of ferric iron when the foodenters the alkaline environment of the small in-testine. With regard to the third point, attemptshave been made to characterize the chemical be-havior and stability of iron ascorbate complexeson the suggestion that its solubility and bondstrength be used for comparison when evaluatingother enhancers.59-212 At this time, it is appro-priate to emphasize the possible snyergism be-tween these mechanisms.62 Enhancement of ironabsorption due to chelation or reduction is onlypossible after the dietary iron has been solubilizeddue to acid conditions found within the stomach.Alternatively, increased gastric acidity will onlybe able to minimally enhance iron absorption ifthe solubilized ferric iron remains unchelated andtherefore precipitates once the more alkaline pHsof the small intestine are encountered.

After reviewing the literature concerningpossible mechanisms of meat enhancement ofnonheme iron absorption, Zhang et al.62 hypoth-esized that meat enhancement of iron absorptionis by multiple mechanisms involving both gastricacidity effects and chelation. Initially, meat mayenhance nonheme iron absorption by stimulatingproduction of gastric acid, thereby promoting ironsolubilization within the stomach. The secretionof gastric acid is dependent on both the amount

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of food protein or amino acids and on the proteinsource. The relationships between increases innonheme iron absorption affected by comparableprotein sources (beef, chicken, fish, and egg) intwo independent human studies have been foundto be highly correlated with stimulation of gastricacid secretion by the same protein sources in athird study.62 Also, the capacity for normal acidproduction has been found to be critical for en-hancement of iron absorption by beef, pork, andchicken.213 Subsequently, a meat factor(s) maychelate the solubilized iron in the acidic envi-ronment of the stomach thereby maintaining ironsolubility during intestinal digestion and absorp-tion. In vitro studies have shown that meat con-tains a factor(s) that is capable of binding ferriciron and maintaining iron in solution at neutralpH.65 This meat factor(s) is available independentof proteolytic digestion and would therefore beable to chelate iron solubilized within the stom-ach. The meat factor apparently has sufficientaffinity for iron such that inhibition due to bind-ing to dietary phytate and fiber can be miti-gated.214 However, the binding is not strongenough to preclude absorption since iron boundin complex with meat factor is available for ab-sorption from ligated loops of rat intestine.213

Third, meat may enhance nonheme iron absorp-tion by stimulating gastric secretion of additionalfactors, such as gastrin and gastric transferrin,that are capable of binding iron and maintainingsolubility at neutral pH.62

2. Heme Iron

In contrast to the many dietary componentsthat affect absorption of nonheme iron, meat isthe only significant dietary influence on absorp-tion of heme iron.14-19-197 The absorption-pro-moting effect of meat has been largely attributedto the ability of the proteolytic digestion productsof meat to prevent formation of insoluble hemeaggregates, as reviewed in the previous sectionof this manuscript concerning digestion of he-moglobin. Since heme iron is usually consumedonly as that present in meat, absorption of hemeiron can be considered to be unaltered by dietarycomposition.

D. Estimates of Heme and Nonheme IronBioavailability

The percent bioavailability of dietary ironfrom a meal, as influenced by physiologic ironstores, form of iron, and presence of dietary en-hancers, is presented in Table 2. The informationpresented therein represents our best estimates ofheme and nonheme iron bioavailability ancillaryto a range of conditions with respect to iron statusand dietary factors. Bioavailability values werecalculated based upon concepts developed byMonsen et al.209"211 for estimation of bioavailabledietary iron. This model of iron bioavailabilitytakes into consideration two factors that are knownto significantly influence iron bioavailability,physiological iron status and composition of thediet. Physiological iron stores have been used asthe measure of iron status in Table 2. The rangeof storage iron values is realistic based on ironstatus of individuals in the U.S. as estimated fromresults of the Second National Health and Nu-trition Examination Survey (NHANES H).20 Bodyiron reserves for individuals between the ages of18 to 64 averaged approximately 300 mg in men-struating women, 600 mg in postmenopausalwomen, and 775 mg in men. Stores of 1000 mgwere present in >80% of men and 10% of post-menopausal women. Stores of 500 mg were foundin >80% of men and >60% of postmenopausalwomen, but <30% of menstruating women. Nostores of iron, suggesting a potential iron defi-ciency, were found in >20% of menstruatingwomen and >5% of postmenopausal women.

Also in Table 2, the effects of dietary com-position have been taken into consideration byclassifying availability of dietary nonheme ironfor absorption as low, moderate, or high. Theseclassifications are based on the dietary density ofenhancers of nonheme iron absorption and canbe used in determining nonheme iron bioavail-ability from surveys of daily food intake. Sincemost dietary surveys do not report food intakeby meals, but rather as total daily intakes, thisis a most useful classification system. In theMonsen model, meals of low, moderate, or highavailability have been classified as those con-taining <30, 30 to 75, or >75 units of enhancingfactors per meal, respectively, where one unit of

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TABLE 2Bioavailability of Heme Iron and Nonheme Iron as Influencedby Iron Status and Dietary Composition

PhysiologicIron stores

(mg)

0

250

500

1000

Dietary availabilitynonheme iron*

LowModerateHighLowModerateHighLowModerateHighLowModerateHigh

% Bioavailability6

Heme iron

353535282828232323151515

Nonheme iron

5112047

12358234

a Availability of nonheme iron for absorption classified as low, moderate, orhigh based on dietary density of enhancing units: low, <50 units/1000kcal; moderate, 50-125 units/1000 kcal; high, >125 units/1000 kcal. Oneenhancing unit = 1 mg ascorbic acid = 1 g meat, fish, or poultry.

b Values adapted from References 7, 209-211; see Section III.

enhancing factor is equal to either 1 mg ascorbicacid or 1 g of MFP.7-209 We have recently dem-onstrated that by treating the daily dietary as onelarge meal (OLM), statistically identical valuesto the Monsen model can be estimated for avail-able iron using daily food intakes.208 Availabilityof nonheme iron for absorption in this OLM modelis classified as low, moderate, or high after com-parison to the enhancer levels used for individualmeals (Monsen model) multiplied by a factor offour: low, <120 units; moderate, 120 to 300units; high, >300 units. The multiplication by afactor of four was a logical development fromour observations that daily food intake is typicallyconsumed in three meals plus a snack. In theoriginal report, bioavailable iron was calculatedfor individuals, using each model, and then av-erages were calculated for the population. Also,identical bioavailable iron values can be deter-mined using dietary data for the population. Thus,for purposes of this review, the OLM model hasbeen carried one step further by classifying dietsas low, moderate, or high based on dietary den-sity of enhancer per 1000 kcal. Using 2400 kcalas an average daily energy consumption for adults,dietaries having enhancer densities of <50, 50

to 125, >125 units enhancer per 1000 caloriesare estimated as having low, moderate, or highavailability of nonheme iron, respectively.

Since dietary enhancers must be consumedsimultaneously with nonheme iron in order toincrease absorption, estimates of bioavailable ironusing the OLM model should, in theory, bestapproximate the Monsen model when the enhan-cer-to-nonheme iron ratio is uniform amongmeals. Such a situation may be approximatedwith large populations where any differences inenhancer-to-nonheme iron between average mealsfor the population may be insignificant as a resultof high variability in the enhancer-to-nonhemeiron content among individual meals. Thus, whenput to practical use with respect to populationstudies, the OLM model has been able to estimatebioavailable iron values that are statistically sim-ilar to those generated using the Monsen modelfor three dietaries of widely divergent iron dens-ities (G to 5.99 mg/1000 kcal; 6 to 8.99 mg/1000kcal; and >9 mg/1000 kcal).208

Although the Monsen model for predictingbioavailable iron has been widely accepted, itsappropriateness should be reevalutated in light ofour current concepts of how dietary enhancers

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act to increase iron absorption. For example, ithas been suggested that enhancement of nonhemeiron absorption is due to an increase in gastricacidity. Enhancement of nonheme iron bioavail-ability by such a mechanism will have an effecton all nonheme iron in a meal. Therefore, amountsof both enhancer and nonheme iron ingested witha meal need to be considered in order to simulatethis mechanism accurately. The Monsen model,which predicts bioavailable nonheme iron as afraction of total iron in a meal, simulates thistype of enhancement mechanism. Simulation ofthis type of mechanism by models based on av-erage dietary composition will necessarily beplagued by difficulties arising from variable in-take of iron and enhancers from meal to meal.Another theory suggests that nonheme iron ab-sorption is enhanced due to nonheme iron che-lation with dietary components. For most dietarysituations, nonheme iron is probably in excess ofthe amount that can be chelated by enhancers.Therefore, enhancement of nonheme iron bioa-vailability by this mechanism will be in directproportion to the amount of enhancer consumedwith a meal. Correspondingly, the effects of thismechanism should be additive, suggesting thatsuch a mechanism could be modeled equally wellby individual meals or dietary composition. Thistype of enhancement mechanism is not presentlysimulated by the widely used Monsen model.

The individual and combined effects of phys-iological iron status and dietary composition ondietary iron bioavailability are represented by thevarying iron values given in Table 2. These ef-fects have been discussed previously in only qual-itative terms. However, the percent bioavaila-bility values given in Table 2 now allow discussionin more quantitative terms. Regarding iron status,bioavailabilities of both heme and nonheme ironare inversely influenced, although nonheme ironbioavailability is influenced to a much greaterdegree by iron status than heme iron bioavaila-bility. Bioavailability of nonheme iron varies ten-fold, from a low of 2% in iron-replete individualsconsuming a diet of low availability to 20% iniron-deficient individuals consuming a diet of highavailability. In contrast, bioavailability of hemeiron shows only slightly more than a twofoldvariation from 15% in iron-replete individuals to35% in iron deficiency. Regarding dietary com-

position and its role in determining iron bioa-vailability, bioavailabilities of nonheme iron, butnot heme iron, are directly correlated with densityof enhancers in the diet. The presence of dietaryenhancers increases nonheme iron bioavailabilityfrom two- to fourfold depending on physiologicaliron stores, the greater enhancement occurringwhen iron stores are low.

An issue of cardinal importance to this re-view can also now begin to be examined by eval-uation of Table 2. This issue is the relative con-tribution of heme and nonheme iron toward totalbioavailable iron. Regardless of iron status ordietary composition, heme iron is significantlymore bioavailable than nonheme iron. Therefore,heme iron will always comprise a disproportion-ately large portion of total bioavailable iron ascompared to its proportion of total dietary iron.However, the relative magnitude of this dispro-portion will vary greatly depending on iron sta-tus, availability of nonheme iron for absorption,and dietary content of both heme and nonhemeiron. The influences of iron status and availabilityof nonheme iron for absorption on this dispro-portion can be evaluated from information in Ta-ble 2. The disparity in the bioavailability of hemeiron to nonheme iron is large, but independentof iron status, for diets of low availability ofnonheme iron; the ratio of heme iron bioavaila-bility-to-nonheme iron bioavailability is approx-imately 7 for individuals consuming diets of lowavailability of nonheme iron, independent ofphysiological iron status. In contrast, the dispar-ity in heme and nonheme iron bioavailabilities ismarkedly less for all diets of high or moderatenonheme availability and is significantly influ-enced by iron status; in these situations, the ratiovaries from <2 (for individuals having no ironstores consuming a diet of high nonheme bioa-vailability) to 5 (for individuals having 1000 mgstorage iron consuming a diet of moderate avail-ability of nonheme iron). Between these ex-tremes, heme iron bioavailability is typically 3to 4 times nonheme iron bioavailability for theother situations regarding iron status and dietarycomposition given in Table 2. Thus, as comparedto heme iron content of the diet, heme iron pro-vides a disproportionately large contribution tohuman nutrition, especially for diets that havelow availability of nonheme iron for absorption.

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More definitive estimation of the actual contri-bution of heme and nonheme iron to human nu-trition will depend on actual dietary composition,which is evaluated for different dietaries in thenext section.

IV. DIETARY CONTENT OF HEME IRON,NONHEME IRON, AND ENHANCERS OFNONHEME IRON ABSORPTION

The model for predicting total bioavailableiron, and contributions of heme and nonhemeiron toward this total, was presented in the pre-vious section of this review. Using this model,several points regarding the contribution of hemeand nonheme iron to human nutrition have beenassessed. Hypothetical dietaries could be used asexamples to demonstrate possible contributionsof heme and nonheme iron to human nutrition.However, the use of some actual dietaries willgive credibility to the final conclusions concern-ing the contribution of heme and nonheme ironto human nutrition. Thus, for this section and thecorresponding Table 3, the information neededto make the model workable was estimated forseveral different dietaries. The specific dietarycomponents estimated for the individual dietariesinclude total iron, heme iron, nonheme iron, andunits of nonheme iron enhancers. As required bythe model, the dietary density of each component

'is reported per 1000 kcal. The diets evaluatedrepresent a wide range of situations regardingheme and nonheme iron intake as influenced byboth choice and socioeconomic influences and/or limitations. Initially, dietaries for three pop-ulation segments of the U.S. were examined: anaverage American diet; a diet typical of residentsof a single U.S. state, Utah; and a vegetariandiet. Finally, various regional diets from devel-oping countries of Central and South Americawere examined.

The dietaries examined represent several ex-isting situations respecting two factors that largelydetermine the amount of bioavailable iron in thediet, content of dietary heme iron, and nonhemeiron bioavailability. As judged by low frequen-cies of anemia, diets typical of developed na-tions, of which the average American diet maybe considered representative, are considered ad-equate in fulfilling human iron demands. One ofthe characteristics associated with diets of de-veloped countries is the generous quantities ofMFP, and therefore heme iron, that are con-sumed. Additionally, due to enhancement of non-heme iron absorption by MFP, availability ofnonheme iron for absorption in these diets gen-erally falls in the moderate or high categories.Vegetarian diets, devoid of MFP, and thereforeheme iron, are sometimes consumed in devel-oped nations by choice. These diets may haveboth total iron contents and availability of non-

TABLE 3Dietary Content of Heme Iron, Nonheme Iron, and Enhancers of Nonheme IronAbsorption

Dietary*

U.S.

Utah

Vegetarian

RegionalLatin American

IronHeme

(% total)

1.1(16)0.60)—

0.6(7)

(mg/1000 kcal)Nonheme(% total)

5.9(84)6.4(91)7.0

(100)8.4(93)

Total

7.0

7.0

7.0

9.0

Enhancers (units/1000 kcal)MFP"

(% total)

110(69)50

(45)—

24(62)

Ascorbic acidc

(% total)

50(31)60

(55)90

(100)10

(38)

Total

160

110

90

34

Dietaries as reviewed in this manuscript; see Section IV.MFP = meat, fish, or poultry. One gram MFP = one enhancing unit.One milligram ascorbic acid = one enhancing unit.

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heme iron for absorption similar to omnivorousdiets, but do not contain heme iron. Finally, dietsof developing countries are often characterizedby both low availability of nonheme iron for ab-sorption and little heme iron content. In the lastsection of this review, these dietaries are indi-vidually analyzed and selectively compared re-garding the specific contributions of heme andnonheme iron with regard to human iron nutri-ture.

A. Typical U.S. Diet — High HemeDensity and High Availability ofNonheme Iron

The average diet consumed in the U.S. rep-resents a dietary that includes both high levels ofheme iron and high availability of nonheme ironfor absorption. For purposes of our review, es-timates of heme iron, nonheme iron, and enhan-cers of nonheme iron absorption in the averageU.S. diet were based on analysis of food con-sumption data from national surveys. Iron densityof U.S. diets averages approximately 7 mg iron/1000 kcal for women, children, and men.912-215-216

Meat, eggs, fortified cereal products, and veg-etables are the principle sources of iron in theU.S. food supply.216-218 Data from the Nation-wide Food Consumption Survey 1977-1978 in-dicate that approximately 35% of the total dietaryiron is derived from MFP.216 Data collected inNHANES II indicate a similar percentage in that>30% of total dietary iron was estimated fromM F p 215,218 T h u S ) a v a l u e o f 3 5 % o f t o t a l ¡ron as

MFP seems a reasonable estimate for an averageU.S. diet.

Dietary heme iron can be estimated from theMFP iron by assuming that 45% of the dietaryMFP iron is present as heme. Although this per-centage is slightly higher than the often-quoted40% value originally used by Monsen et al.,211

it reflects a more recently used estimate of theheme iron content of MFP in the typical U.S.diet.7-209 The higher value is also more repre-sentative of recent estimates for heme iron con-tent of various meat (Table 4)219-224 and the con-sumption frequency of these meats in the U.S.dietary.225 Thus, in concurrence with previousestimates of dietary heme iron, the percentage of

total dietary iron present as heme in the U.S.dietary can be calculated as 0.45 (fraction of MFPiron present as heme) x 35% (percent total di-etary iron contributed by MFP) = 15.75%. Thisequates to a densities of 1.1 mg heme iron/1000kcal and 5.9 mg nonheme iron/1000 kcal for thetypical U.S. diet.

Both ascorbic acid and MFP are significantenhancers of iron absorption in the typical U.S.diet. Protein content of the U.S. dietary is about40 g/1000 kcal,12215 50% of which is contributedby MFP.216226 This corresponds to a dietary in-take of approximately 110 g MFP/1000 kcal.216-226

Density of ascorbic acid in the U.S. diet is ap-proximately 50 mg/1000 kcal.12-215216-225-227-228

Fruits and vegetables, along with their juices (es-pecially citrus fruits and tomatoes), are the largestcontributors of dietary ascorbic acid.216218 Totaldensity of enhancing units in the average U.S.diet is the sum of MFP-enhancing units and as-corbic acid-enhancing units, or 160 units/1000kcal. Thus, availability of nonheme iron for ab-sorption from the average U.S. dietary is high(>125 units/1000 kcal).

B. Utah Diet — Moderate Heme Densityand Moderate Availability of NonhemeIron

We have recently reported the results of foodfrequency surveys of approximately 1500 Utahresidents including 737 school age children and762 adults.208-227-229-230 Based on these surveys,the typical diet consumed by this population ischaracterized by moderate levels of heme ironand moderate availability of nonheme iron forabsorption. Their dietary was found to be similarto the average U.S. dietary regarding density oftotal iron (7 mg/1000 kcal) and total protein (38g/1000 kcal). Dietary density of ascorbic acid,at 60 mg/1000 kcal, was slightly higher than forthe U.S. dietary. However, at about 50 g MFP/1000 kcal, the density of MFP was less than halfthat found in the typical U.S. dietary. MFP werecalculated to contribute about 21% of the totaldietary iron, which is similar to the 20% contri-bution based on data collected in one nationalsurvey, the Selected Minerals in Foods Surveysof the Food and Drug Administration.231 Based

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TABLE 4Heme Iron and Total Iron Content of Various Meats

Item

Raw productsBeef, neck"Beef, heartBeef, roundBeef, mixedLamb, mixedPork, mixedChicken, breastChicken, thigh

Cooked productsBeef, steakBeef, groundBeef, groundBeef, roastBeef, roundChicken, breast

Processed productsFranks, beef and porkFranks, beefBologna, beefHamBacon

n

4165

56525644

3230

9305

50

303333

Total iron(ftg/g)b

22.0 ± 4.351.7 ± 2.722.4 ± 3.326.1 ± 6.016.4 ± 4.226.1 ± 6.03.9 ± 1.27.2 ± 1.9

34.2 ± 1.431.5 ± 0.627.6 ± 5.133.6 ± 1.034.2 ± 4.17.4 ± 2.3

31.4 ± 1.113.4 ± 2.211.5 ± 2.79.3 ± 0.22.6 ± 1.1

Hemeng/g"

15.3 ± 2.028.7 ± 2.815.2 ± 2.416.2 ± 5.2"9.4 ± 2.6d

16.2 ± 5.2"1.3 ± 0.23.7 ± 0.9

21.0 ± 1.020.0 ± 0.419.1 ± 3.116.8 ± 0.516.7 ± 4.11.6 ± 0.5

10.4 ± 0.510.0 ± 1.58.2 ± 2.36.4 ± 0.51.6 ± 0.6

iron»% of total iron"

71 ± 1056 ± 568 ± 5

62'57-49e

34 ± 643 ± 2

62 ± 164 ± 171 ± 750 ± 149 ± 1321 ± 8

33 ± 175 ± 170 ± 469 ± 764 ± 6

Ref.

219219220221221221219219

222222219222220223

222219219219219

Heme iron determined by Hornsey22"1 procedure unless otherwise noted.Values are mean ± SD as determined on a wet weight basis.Muscles were taken from six grades of carcasses.Determined as difference between total and inorganic iron.Standard deviations are not available.

on the previous figures, the percentage of totaldietary iron present as heme in the Utah dietarycan be calculated as 0.45 (fraction of MFP ironpresent as heme) x 20% (percent total dietaryiron contributed by MFP) = 9%. This translatesinto densities of heme and nonheme iron of about0.6 mg/1000 kcal and 6.4 mg/1000 kcal, re-spectively. Total density of enhancers of non-heme iron absorption in the Utah dietary is 110units/1000 kcal, suggesting nonheme iron is onlymoderately available (50 to 125 units/1000 kcal)from the Utah diet.

C. Vegetarian Diet — No Heme Iron andModerate Nonheme Availability

Vegetarian dietaries, by specification, do not

include heme iron. Thus, vegetarianism has oftenbeen associated with possible nutritional inadequa-cies, especially with regard to iron.232"237 Amongthe vegetarian populations, members of the re-ligious denomination of Seventh Day Adventists(SDA) are considered by dietitians to have a well-considered approach to vegetarian diet.235 Assuch, dietary information on adult SDA membersin the U.S. were used as a model for a vegetariandiet consumed in developed countries bychoice.237 Vegetarian members of the SDA con-sume a lacto-ovo-vegetarian diet including anabundant intake of vegetables, fruits, and wholegrains, while abstaining from coffee, tea, andalcoholic beverages. Density of total iron in thevegetarian diet is about 7 mg/1000 kcal, the sameas for the typical U.S. diet. However, being avegetarian diet, all of the iron is present in

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nonheme forms. Density of enhancers of non-heme iron absorption is 90 units/1000 kcal, com-prised solely of ascorbic acid. Thus, this vege-tarian diet is modeled by moderate nonheme ironbioavailability (50 to 125 units/1000 kcal).

D. Regional Latin American Diet —Moderate Heme Density and LowAvailability of Nonheme Iron

Dietary content of heme iron, nonheme iron,and enhancers of nonheme iron absorption havebeen reported for eight regions in various Centraland South America countries of Argentina, Brazil,Chile, Mexico, Peru, Venezuela, and Colum-bia.238-239 Dietaries for each country were re-ported to be representative of typical diets con-sumed by individuals belonging to lower middleto lower class socioeconomic strata. Among re-gions, dietary intake of MFP varied considerablyfrom 20 g/1000 kcal to 93 g/kcal. Two regionshad low MFP intake of <30 g/1000 kcal, fiveregions had moderate MFP intake of 45 to 60 g/1000 kcal, while a single region had high MFPintake of >90 g/1000 kcal. Thus, in order toinclude a dietary in this review representing mod-erate density of heme iron in combination withlow nonheme availability, average dietary infor-mation concerning only the two regions (Braziland Chile) with low MFP intake are presented.Such a dietary is representative of diets consumedby the lower socioeconomic groups in many de-veloping countries, which have long caused con-cern with respect to iron nutriture.

For the Latin American dietary, as repre-sented by regions of Brazil and Chile, density oftotal iron was almost 9 mg/1000 kcal. This ishigher than found in diets of industrialized coun-tries due to the greater iron density found in thedietary staples. Heme iron was reported directlyand contributed approximately 0.6 mg/1000 kcalor 6.6% of total iron. Dietary density of enhan-cers of nonheme iron absorption was 34 units/1000 kcal, with MFP contributing about 24 units/1000 kcal and ascorbic acid adding only 10 units/1000 kcal. Thus, availability of nonheme iron forabsorption from this dietary falls into the lowclassification (<50 units/1000 kcal). It is inter-esting to note that although the density of heme

iron for the Latin American diet is similar to thatpreviously estimated for the Utah dietary, it isassociated with only half the MFP density. Sucha large difference in the ratio of MFP density-to-heme iron density, is somewhat unexpectedeven, considering possible differences in con-sumption frequencies for various meats, whichvary in heme iron content. The difference canperhaps largely be attributed to inaccuracies inthe manner by which heme iron was estimatedfor the different dietaries. It is possible that byusing the estimation that 45% of MFP iron ispresent as heme (as used for the typical U.S. andUtah dietaries) heme iron density in the diet isbeing underestimated.222 Conversely, the methodused to measure heme iron content of MFP forthe Latin American dietaries240 has been sug-gested to overestimate heme iron.241

V. CONTRIBUTIONS OF HEME ANDNONHEME IRON TO HUMAN NUTRITION

The contributions of heme and nonheme ironto human nutrition were estimated in both ab-solute and relative terms and are reported in Table5 for each of the actual dietaries discussed above.Absolute bioavailability is given in milligrams ofbioavailable iron per 1000 kcal, while relativebioavailability is reported as a percentage of thetotal bioavailable iron in the diet. These valueswere calculated by attenuating the amounts ofheme iron and nonheme iron for the various die-taries (reported in Table 3) using the bioavaila-bility of heme and nonheme iron (reported inTable 2), respectively. Before examining Table 5in order to identify the contributions of heme andnonheme iron, the question may be raised whetherthe models and generalizations that have neces-sarily been used to develop the table are reason-ably accurate. Some idea regarding the tenabilityof the predictions in Table 5 may be acquired bycomparing the iron status for different popula-tions as suggested from the table to actual data.Since iron status of various populations in theU.S. has already been discussed, comparison willbe made only for the U.S. dietary.

Iron status for a population can be predictedto occur at a level where bioavailable dietary ironequals the iron requirement. Iron balance will be

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TABLE 5Bioavailable Iron

Dietsphysiological

iron stores (mg)

U.S.0

250

500

1000

Utah0

250

500

1000

Vegetarian0

250

500

1000

Latin American0

250

500

1000

from Various Dietaries

Bioavailable iron (mg/1000

Heme iron*(% total)

0.39(25)0.31(30)0.25(33)0.17(41)

0.21(23)0.17(27)0.14(30)0.09(31)

—

—

—:

—

0.21(33)0.17(33)0.14(36)0.09(53)

Nonheme iron"(% total)

1.18(75)0.71(70)0.47(67)0.24(59)

0.70(77)0.45(73)0.32(70)0.20(69)

0.77(100)0.49(100)0.35(100)0.21(100)

0.42(67)0.34(67)0.25(64)0.17(47)

kcal)

Total iron

1.57

1.02

0.72

0.41

0.91

0.62

0.46

0.29

0.77

0.49

0.35

0.21

0.63

0.51

0.39

0.26

Calculated by attenuating dietary heme iron (Table 3) with hemeiron bioavailability (Table 2); see Section V.Calculated by attenuating dietary nonheme iron (Table 3) withnonheme iron bioavailability (Table 2); see Section V.

maintained at this level due to the inverse rela-tionship between iron absorption and iron status.For example, if dietary bioavailable iron (whichis directly dependent on iron status) is insufficientto meet requirements, iron status will fall untildietary iron bioavailability increases to a levelsufficient to maintain this level of iron status.

Conversely, if dietary bioavailable iron is in ex-cess of requirements, iron stores will increase(with a concurrent reduction in dietary bioavail-able iron) until dietary bioavailable iron and ironrequirements are in balance. Thus, iron statuswill be maintained at a level compatible with ironrequirements provided the diet contains sufficient

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quantities of potentially available iron to meetrequirements. The level of physiological ironstores at this equilibrium state will depend on theamount of potentially bioavailabie iron in the diet;the larger the amounts of potentially bioavailabieiron in the diet the larger the iron stores will beat equilibrium. The data provided by Table 5 arepresented in graphical form in Figure 1. The levelof iron stores that can be maintained by a specificpopulation, when consuming the individual die-taries, is predicted as the body iron stores (x-axis) corresponding to the point at which the in-dividual curves intercept a level of dietary bioa-vailabie iron (y-axis) equal to the iron require-ment . Using the human iron requirementspresented in Table 1, it can be estimated from

Figure 1 that iron stores of adult males and 90%of menstruating females consuming a typical U.S.diet could be maintained at >1000 mg and 225mg, respectively. These predictions are quitecomparable to storage iron values previously re-ported for the U . S . where > 8 0 % of adult menwere found to have iron stores >1000 mg, and300 mg was the average stores for menstruatingwomen.20

Some qualitative contributions of heme andnonheme iron to human nutrition were alluded toin relative terms during the previous discussionpertaining to Table 2. However, with develop-ment of quantitative bioavailabie iron densitiesvalues for various dietaries (Table 3), we cannow give quantitative consideration regarding the

es

©oo

co

2LJ5es>eso

5ues'SQ

USAUtahVegetarianLatin AmericanFemale RequirementMale Requirement

250 500 750 1000

Body Iron Stores, mg

FIGURE 1. Relationship between dietary bioavailabie iron and body stores. The tophorizontal line is positioned at the dietary bioavailabie iron level equal to the bioavailabieiron requirement of 90% of menstruating women (see Table 1). The lower horizontal lineis positioned at the dietary bioavailabie iron level equal to the bioavailabie iron requirementof men (see Table 1). Body iron stores can be predicted to be maintained at level corre-sponding to the intersection of the iron requirement with the curve of dietary bioavailabieiron vs. body iron stores.

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adequacy of the various dietaries in meeting hu-man requirements (Table 1). Assuming adequateenergy consumption, each of the dietaries pro-vides sufficient iron density to maintain signifi-cant iron stores in adult men (Figure 1). How-ever, only the typical U.S. dietary, which hashigh heme iron density, provides sufficient bioa-vailable iron to meet the greater bioavailable irondensity required by 90% of menstruating women.In fact, the vegetarian dietary is marginal, andthe Latin American dietary is inadequate, in pro-viding even the iron density required by the av-erage adolescent female. None of the dietariesprovide adequate bioavailable iron density to meetthe needs of pregnant women.

Comparison of bioavailable heme and non-heme iron (Table 5) with dietary content of hemeand nonheme iron (Table 3) emphasizes previ-ously recognized characteristics regarding therelative contributions of heme and nonheme ironto human nutrition. Specifically, these charac-teristics are a direct reflection of the greater bioa-vailability of heme iron combined with the greaterresponse of nonheme iron absorption to changesin physiological iron status. Primary among theseis the disproportionate contribution of heme irontoward bioavailable iron relative to its contri-bution toward total dietary iron. For example,the contribution of heme iron toward total irondensity in the typical U.S. diet is 16%. However,the contribution of heme iron toward total bioa-vailable iron for men (1000 mg iron stores) andmenstruating women (300 mg iron stores) is 41and 30%, respectively. This disproportion is evengreater for dietaries having lower availability ofnonheme iron for absorption, such as the Utahand Latin American dietaries. In these diets, hemeiron contributes up to 31 and 53% of the totalbioavailable iron (for an individual with iron storesof 1000 mg) in spite of only constituting 9 and7% of total dietary iron, respectively. Additionalsurvey of the individual dietaries in Table 5, ap-ropos the relationship between iron status andbioavailable iron as heme or nonheme, illustratesthe relatively greater importance of heme iron inmaintaining increased levels of iron stores. Foreach of the dietaries containing heme iron, thepercentage contribution of heme iron toward totalbioavailable iron is approximately 50% greater

for individuals with 1000 mg of iron stores thanfor individuals with no storage iron.

The disproportionate contribution of hemeiron toward human iron nutriture, especially inmaintaining significant iron stores, is further em-phasized by comparison of total bioavailable ironfor the Utah and vegetarian diets. These dietarieshave the same dietary iron density and the sameavailability of nonheme iron for absorption. Thus,differences in total bioavailable iron for these twodietaries can be directly attributed to heme ironcontent, which is 9% of total iron for the Utahdietary and 0% for the vegetarian dietary (Table3). This greater density of dietary heme iron inthe Utah dietary brings about increases in totalbioavailable iron of 0.14 mg/1000 kcal (18% in-crease) and 0.08 mg/1000 kcal (38% increase)for individuals having 0 to 1000 mg iron stores,respectively. Based on iron requirements, an adultman could theoretically maintain iron stores of850 mg when consuming the Utah diet, while hewould be able to maintain only 500 mg of ironstores when consuming the vegetarian diet. Mostwomen could maintain significant iron storeswhen consuming the U.S. diet, but many wouldbe unable to maintain any iron stores when con-suming the vegetarian dietary.

Since dietary density of heme iron and en-hancement of nonheme iron absorption by MFPare inseparably linked in normal diets, any es-timate of the contributions of heme and nonhemeiron to human nutrition would not be completewithout consideration of the influence on totalbioavailable iron due to enhancement of nonhemeiron absorption by MFP. The importance of thecontribution of MFP to enhancement of availa-bility of nonheme iron for absorption is empha-sized by comparison of the typical U.S. and Utahdietaries. Regarding dietary composition (Table3), these dietaries have the same total dietary irondensity. However, as compared to the typicalU.S. dietary, the Utah dietary lacks in both den-sity of heme iron and density of nonheme ironabsorption-enhancing units. This difference inboth heme iron and enhancing units is due to lessMFP in the Utah dietary and results in decreasedbioavailable heme iron and nonheme iron, re-spectively (Table 5). Together, these differencesin heme iron density and enhancing unit density

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cause decreases in total bioavailable iron of 0.66and 0.12 mg/1000 kcal in individuals having ironstores of 0 and 1000 mg, respectively. Theseabsolute decreases correspond to percentage de-creases in total bioavailable iron of 42 and 29%,respectively. Thus, considering the dietary con-tribution of MFP of both heme iron and enhanc-ing units, MFP provides a relatively larger con-tribution to total bioavailable iron in individualswith low iron stores. However, for individualswith no iron stores, only 0.18 mg/1000 kcal, or27% of the decrease in total bioavailable iron,can be directly attributed to lower heme iron den-sity in the Utah dietary. Therefore, the majorityof the decreased bioavailable iron density, 0.48mg/1000 kcal, or 73% of the entire decrease, isrelated to decreased density of enhancing unitsfrom MFP. For individuals with 1000 mg of stor-age iron, decreases in total bioavailable iron thatcan be directly attributed to decreased heme irondensity or decreased MFP enhancing units are0.08 mg/1000 kcal (66% of entire decrease) and0.04 mg/1000 kcal (33% of entire decrease), re-spectively. In summary, for individuals with zeroor low iron stores, total bioavailable iron densityis largely influenced by MFP enhancement ofavailability of nonheme iron for absorption. Con-versely, for individuals with substantial ironstores, total bioavailable iron is largely influ-enced directly by heme iron density.

The typical U.S. diet and the vegetarian dietof SDAs can be considered to be the extremesregarding heme iron consumption for industrial-ized countries. Evaluation of these dietaries sug-gests that exclusion of heme iron from the dietleads to more than a 50% decrease (0.80 mg/1000 kcal) in total bioavailable iron as comparedto what otherwise is bioavailable when heme ironis included in the diet. In order for the vegetariandiet to meet the iron requirements of 90% ofmenstruating women, iron density of the vege-tarian diet would need to be increased about 3mg/1000 kcal, or 50%. Even then, iron storescould be maintained only at very minimal levels.For iron stores of these women to be maintainedat a level corresponding to the U.S. average (300mg), iron density of the vegetarian diet wouldneed to increase by almost 4.6 mg/1000 kcal, or66%. Therefore, it is probably not unreasonableto attribute at least 50% of the total bioavailable

iron from the U.S. diet to the inclusion of sourcesof heme iron. More importantly, the increase intotal bioavailable iron realized by includingsources of heme iron in the diet is sufficient toensure the nutritional adequacy, with regard toiron, of the typical U.S. diet for most popula-tions, excepting women during pregnancy. Thecontribution of heme iron to human nutrition isprobably even more significant for many devel-oping countries, such as those consuming theLatin American diet reviewed here, due to thelower bioavailability of nonheme iron.

VI. SUMMARY

Iron is a necessary micronutrient. As presentin hemoglobin, it is required for the transport ofoxygen and carbon dioxide. Iron is also a com-ponent of various tissue enzymes, such as thecytochromes that are critical for energy produc-tion and enzymes necessary for immune systemfunctioning. The 70-kg reference man requiresapproximately 1.0 mg of iron daily to fulfill ironrequirements, which can be met by a diet havinga bioavailable iron density of 0.36 mg/1000 kcal.Due to additional iron demand brought about bymenstrual blood loss, an iron density of 1.09 mg/1000 kcal is needed to assure the requirementsfor 90% of women are being met.

Two forms of iron are present in the dietbased on different mechanisms of absorption,heme and nonheme iron. The contributions ofheme and nonheme iron to human nutrition aredelineated by a few basic concepts, which arethemselves a manifestation of differences in theinherent characteristics of heme and nonheme ironregarding bioavailabilities and/or content in thediet. The first concept is that when heme iron ispresent in the diet, it will always make a dispro-portionately large contribution to iron nutritureas compared to its contribution to total dietaryiron. This disproportionate contribution of hemeiron is due to inherent differences between theabsorption of heme and nonheme iron such thatbioavailability of heme iron is always greater thanbioavailability of nonheme iron. For example,heme iron directly contributes 41% of the totalbioavailable iron for a typical adult male in the

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U.S. (iron stores of 1000 mg), although only 16%of his total dietary iron is present as heme.

The second concept is that the magnitude ofthe disproportionality of the contribution of hemeiron to total bioavailable iron, as compared to itscontribution to total dietary iron, is significantlygreater for situations where nonheme iron bioa-vailability is low. Such situations occur in re-sponse to low availability of nonheme iron forabsorption (e.g., low enhancer content in the diet)and/or increased physiological iron status. In thesetwo circumstances, the bioavailability of non-heme iron is decreased relatively more than bioa-vailability of heme iron because of inherent dif-ferences in response to dietary composition andphysiological iron status: (1) heme iron bioa-vailability is independent of dietary composition,while nonheme iron bioavailability will vary upto fourfold; and (2) bioavailability of both hemeand nonheme iron is directly correlated with ironstatus, although nonheme iron demonstrates a rel-atively greater response. For example, with re-gard to dietary influences, the maximal dispro-portionality for the Latin American diet used inthis review (low nonheme iron bioavailability) ismore than sevenfold, while the maximal dispro-portionality for the typical U.S. diet (high non-heme iron bioavailability) is only twofold. Withregard to iron status, the disproportion in thecontribution of heme iron to iron nutriture is ap-proximately 50% greater for individuals with 1000mg of iron stores than for individuals with nostorage iron.

The final concept is that dietary heme ironexerts both direct and indirect influences on totalbioavailable iron in the diet. The direct influenceis attributable to the greater dietary density ofheme iron and is a function of the greater bioa-vailability of heme iron as compared with non-heme iron. The indirect influence of heme ironis a function of the enhancement of nonheme ironabsorption by MFP, which are the dietary sourcesof heme iron.. The direct influence of increasedheme iron density is relatively more importantfor individuals with substantial iron stores, whilethe indirect influence of MFP enhancement ofnonheme iron absorption is more important forindividuals with low iron stores. For the individ-ual with high iron stores, two thirds of the in-crease in total bioavailable iron resulting from

addition of heme iron-containing foods to the dietis attributable to increased heme iron density. Indirect opposition, for the individual with no ironstores, two thirds of the increase in total bioa-vailable iron resulting from addition of heme iron-containing foods to the diet is attributable to thecorresponding increase of MFP in the diet andtheir enhancement of nonheme iron absorption.

In conclusion, heme iron contributes signif-icantly to human iron nutriture. Dietary hemeiron, directly and indirectly (through MFP en-hancement of nonheme iron absorption), oftencontributes 50% or more of the total bioavailableiron in diets typical of industrialized and manydeveloping countries. Thus, the nutritional ade-quacy of these diets with regard to iron nutritureis critically dependent on the presence of dietaryheme iron. While diets devoid of dietary hemeiron may be nutritionally adequate in supplyingthe relatively low iron requirements of adult men,such diets often will be nutritionally inadequatefor population segments with higher iron de-mands, such as women during their reproductiveyears and children.

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