amino acid chelation in human and animal nutrition

solu-CALCIUM Pancreatic Lipase NIACIN Lipid Metabolism RIBOFLAVIN Glycogenesis PANTOTHENIC ACID Activates Coenzyme A THIAMIN Glucose Metabolism FOLACIN Amino Acid Metabolism VITAMIN B6 T

AMINO ACID CHELATION

IN HUMAN AND

ANIMAL

NUTRITION

AMINO ACID CHELATION

IN HUMAN AND

ANIMAL NUTRITION

H DeWAYNE ASHMEAD

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Contents

Foreword vii

Introduction ix

About.the.Author xi

Chapter 1 The.Fundamentals.of.Mineral.Nutrition 1

Chapter 2 The.Chemistry.of.Chelation 19

Chapter 3 The.History.of.Nutritional.Chelates 35

Chapter 4 The.Requirements.for.a.Nutritionally.Functional.Chelate 49

Chapter 5 The.Development.of.Analytical.Methods.to.Prove.Amino.Acid. Chelation 61

Chapter 6 Absorption.of.Amino.Acid.Chelates.from.the.Alimentary.Canal 81

Chapter 7 The.Pathways.for.Absorption.of.an.Amino.Acid.Chelate 97

Chapter 8 The.Absorption.of.Amino.Acid.Chelates.by.Active.Transport 117

Chapter 9 The.Absorption.of.Amino.Acid.Chelates.by.Facilitated.Diffusion 135

Chapter 10 The.Fate.of.Amino.Acid.Chelates.in.the.Mucosal.Cell 153

Chapter 11 The.Uptake.of.Amino.Acid.Chelates.into.and.out.of.the.Plasma 171

Chapter 12 Tissue.Metabolism.of.Amino.Acid.Chelates 185

Chapter 13 Some.Metabolic.Responses.of.the.Body.to.Amino.Acid.Chelates 201

Chapter 14 Toxicity.of.Amino.Acid.Chelates 223 Chapter 15 The.Absorption.and.Metabolism.of.Amino.Acid.Chelates 233

Foreword

olism Dietary intake of a mineral micronutrient in sufficient quantities to meet.dietary reference intakes does not always ensure adequate metabolizable mineral.at.the.tissue.level Minerals.are.by.nature.ionic.and.form.complexes.and.chemical.compounds.quite.readily The.pathway.from.the.food.or.supplement.in.which.they.are.contained.to.their.target.cells.in.the.body.provides.multitudinous.opportunities.to.interact.with.their.immediate.chemical.environments The.foodstuffs.with.which.they.are.ingested,.the.acidic.and.chemical.milieu.of.the.digestive.tract,.the.absorptive.surface.and.interface.of.the.gastrointestinal.tract,.the.ions.in.the.plasma,.and.ulti-mately.the.cellular.matrix.to.which.they.are.delivered.can.interact.to.influence.the.ultimate.efficacy.of.the.structural,.metabolic,.or.catalytic.roles.of.the.dietary.min-eral The.seemingly.large.doses.of.mineral.supplements.needed.to.correct.a.dietary.mineral deficiency can be explained in terms of the “inefficiency of absorption”.or,.in.broader.terms,.the.lack.of.“bioavailability”.of.the.particular.mineral.supple-ment Mineral.nutritionists.have.long.sought.chemical.forms.of.minerals.that.evoke

Mineral.bioavailability.has.historically.been.“the.black.box”.of.micronutrient.metab-a greater or more positive response at the target tissue Two important historical.examples.of.mineral.nutrition.research.that.continue.to.be.pursued.today.are.calcium.supplementation.to.influence.bone.mineralization.and.iron.supplementation.to.influ-ence.blood.hemoglobin.levels Not.all.covalently.bound.minerals.ionize.sufficiently.to.release.their.mineral.counterpart.optimally.at.the.sites.of.absorption.in.the.gut Mineral.absorption.from.the.gut.is.a.complex.topic,.considering.the.various.routes.that.are.available.(e.g.,.passive.absorption,.facilitated.absorption,.active.transport).to.account.for.the.disappearance.of.the.mineral.from.the.gut.and.its.appearance.in.the.plasma

tective.amino.acid.matrix.(chelate).with.a.stability.factor.that.helps.to.circumvent.ionization.issues.and.delivers.the.mineral.to.sites.of.absorption.in.the.intestinal.brush.border Certain.amino.acids.form.soluble.complex.molecules.with.metal.ions,.thus

Enter.the.concept.of.supplying.the.mineral.in.an.ionic.or.covalently.bound.pro-“protecting”.the.ions.so.that.they.cannot.react.with.other.elements.or.ions.prior.to.arriving.at.the.absorptive.site.in.the.gut The.chelated.mineral.ligand.can.then.be.either.passively.absorbed,.subsequently.released.to.its.transporter,.or.in.some.man-ner.“escorted”.through.the.absorptive.surface.of.the.gut.to.permit.a.more.rapid.and.quantitative.transfer.of.the.mineral.from.the.intestinal.contents,.across.the.intesti-nal.villi.and.into.the.blood The.principle.of.chelation.extends.well.beyond.amino.acid.chelates.and.is.well.documented.in.organic.and.inorganic.chemistry This.book.explores.the.chelation.principles.as.applied.to.the.biochemistry.of.mineral.absorp-tion.and.metabolism,.specifically.focusing.on.the.formation.and.absorption.of.amino.acid.metal.chelates

out.controversy Although.the.improved.bioavailability.of.some.amino.acid.mineral.chelates.is.generally.accepted,.it.has.not.been.clearly.understood.exactly.why.these

chelates.provide.improved.absorption Early.studies.of.the.nutritional.aspects.of.the.bioavailability.of.mineral.chelates.occurred.during.the.1960s.and.1970s.when.ana-lytical techniques suggested, but did not permit, direct implication of chelates in.improved absorption and transfer of mineral.across the.gut Over.the.intervening.years,.considerable.indirect.evidence.and.some.direct.evidence.of.enhanced.bioavail-ability.was.gained.through.numerous.animal.and.a.few.human.feeding.trials Much.

of this early experimental information was initially studied with an agricultural.emphasis.and.published.in.related.animal.nutrition.venues.and.proprietary.in-house.publications sponsored by early innovators of chelated mineral products such as.Albion.Laboratories Some.of.these.publications.were.not.widely.read.by.or.acces-sible.to.mineral.researchers.due.to.the.early.emphasis.in.livestock.applications.and.publication.venues.that.were.not.readily.available.or.read.by.those.in.the.human.min-eral.nutrition.field By.publishing.this.book,.Ashmead.makes.this.information.more.readily.available.to.a.wide.audience

In.this.book,.DeWayne.Ashmead.provides.a.historical.account.of.the.theory.and.application.of.chelates.to.mineral.nutrition Much.of.the.pioneering.early.work.was.accomplished.by.DeWayne’s.father,.the.late.Harvey.Ashmead Albion.Laboratories.is.a.family-owned.and.operated.business,.and.at.first.glance,.one.might.imagine.that.the.content.of.this.book.would.be.a.treatise.on.the.nutritional.superiorities.of.min-eral.amino.acid.chelates That.preconceived.notion.would.be.a.mistake This.book.is.a.scholarly.compendium.that.not.only.provides.the.historical.context.of.chelates.but.also.explains.the.chemistry.of.chelation.and.the.formation.of.amino.acid.min-eral.chelates.in.considerable.detail The.book.contains.a.well-developed.introduc-tion and discussion to the complexities of mineral bioavailability Ashmead then.progresses.to.review.the.analytical.methodology.necessary.to.establish.that.one.is.indeed.working.with.a.true.chelate.prior.to.engaging.in.direct.feeding.comparisons.of.amino.acid.mineral.chelates.versus.inorganic.forms.of.the.mineral.in.question Tabular.and.graphical.data.from.feeding.trials.previously.published.in.the.literature.as.well.as.some.extracted.from.some.difficult-to-access.publications.and.previously.unpublished.work.are.presented.in.the.chapters.on.amino.acid.mineral.chelates The.concept.and.criteria.for.the.development.of.a.“nutritionally.functional”.metal.chelate.are.presented.and.discussed

Although.the.main.focus.of.this.book.is.on.the.ingestion.of.amino.acid.metal.chelates as a way to optimize mineral absorption, the book also provides a good.fundamental.discussion.of.chelation.chemistry Ashmead.provides.not.only.his.inter-pretation.of.the.results.of.numerous.studies.of.animal.and.human.amino.acid.mineral.chelate.digestion.and.absorption.but.also.alternative.interpretations One.cannot.help.but.admire.the.clarity.of.writing.and.the.logical.and.stepwise.development.of.the.material.in.this.book This.reference.should.be.invaluable.to.bioinorganic.mineral.researchers.and.others.seeking.to.enhance.mineral.bioavailability.to.support.opti-mal.health.and.productivity

Introduction

In.the.early.1960s,.a.study.was.conducted.in.which.gestating.rats.were.given.diets.containing.the.same.mineral.content.of.mineral.salts.or.amino.acid.chelates The.young.from.the.group.that.was.given.amino.acid.chelates.had.a.much.higher.survival.rate.and.grew.faster This.type.of.study.was.then.extended.to.dairy.cows Here,.it.was.found.that.both.milk.and.butterfat.productions.were.higher.in.the.group.receiving.amino.acid.chelates This.type.of.study.was.then.extended.to.laying.hens;.greater.production.and.fewer.broken.eggs.were.observed.from.the.group.receiving.amino.acid chelated minerals Other researchers conducted a study with gestating sows This study showed that the group receiving amino acid chelated iron had higher.birth.weights,.lower.mortality,.and.greater.weight.gains.than.those.given.the.normal.iron dextran treatment These studies initiated many others on the absorption of.amino.acid.chelated.metals The.studies.consistently.demonstrated.that.amino.acid.chelates.were.absorbed.better.and.improved.some.aspect.of.health.in.humans.and.other.treated.animals

Although.chelation.was.first.observed.over.100.years.ago,.it.has.only.been.in.the.last.50.years.that.scientists.discovered.the.nutritional.benefits.of.amino.acid.chelates This.book.examines.the.reasons.for.those.benefits,.the.chemistry.of.chelation,.the.analytical.methods.that.have.been.used.to.prove.or.verify.chelation,.and.a.detailed.discussion.of.the.absorption.and.metabolism.of.various.metal.amino.acid.chelates.compared.to.mineral.salts The.requirements.for.nutritionally.functional.chelates.and.their.absorption.are.discussed.in.this.text For.a.chelate.to.be.formed,.a.metal.must.be.a.member.of.a.heterocyclic.ring When.an.amino.acid.forms.a.chelate,.the.car-boxylate.anion.forms.a.bond.with.a.positively.charged.metal This.places.the.amine.group.in.perfect.position.to.share.its.pair.of.electrons.with.the.metal.to.form.a.bond.to.the.metal.and.create.a.heterocyclic.ring.or.chelate Depending.on.the.charge.on.the.metal,.this.process.can.be.repeated.one.or.more.times The.structure.of.this.chelate.can.be.proven.by.x-ray.crystallography.and.strongly.indicated.by.Fourier.transform.infrared.(FT-IR).spectroscopy

It.is.logical.to.conclude.that.the.amino.acids,.which.surround.the.metal,.protect.the.metal.from.reactions.that.can.greatly.inhibit.its.absorption Some.of.the.reac-tions.that.produce.precipitation.of.the.metals.are.reactions.with.phosphates,.phytic.acid,.and.other.substances.commonly.found.in.the.gut This.protection.of.the.met-als is related to the stability of different amino acid chelates More stable amino.acid.chelates.provide.better.protection.against.precipitation It.is.also.logical.that.in.lower.pH.environments.the.amine.portion.of.the.amino.acid.could.accept.a.proton The.pair.of.electrons.that.provided.the.bond.to.the.metal.is.now.used.to.bond.to.the.proton When.this.happens,.the.protonated.amine.carries.a.positive.charge.and.the.chelate.ring.is.broken This.produces.a.chelate/complex.rather.than.a.chelate,.but.Dr Ashmead.explains.how.this.allows.the.metal.amino.acid.chelate/complex.to.be.attracted.to.negatively.charged.transport.molecules.and.thus.be.absorbed.through

active transport The relationship between absorption through passive diffusion,.facilitated.diffusion,.as.well.as.active.transport.is.explained.

A.study.to.determine.the.fate.of.amino.acid.chelates.used.a.radioactive.isotope

of the metal and another radioactive isotope in the amino acids There appeared.to.be.some.division.of.the.metal.and.the.amino.acids.in.the.mucosal.tissue.due.to.hydrolysis Differences.in.the.amount.of.hydrolysis.of.the.amino.acid.chelates.in.the.mucosal.tissue.are.explained.on.the.basis.of.the.stability.of.the.amino.acid.chelates Regardless.of.how.much.hydrolysis.occurs.in.the.mucosal.tissue,.some.of.the.amino.acid.chelate.or.chelate/complex.appeared.to.be.transferred.to.the.plasma.intact The.metabolism.of.these.amino.acid.chelates.has.been.shown.to.produce.responses.in.performance.or.production.of.the.animals.being.tested,.and.because.of.greater.tissue.retention,.these.amino.acid.chelates.can.provide.long-term.positive.responses.Increased.absorption.of.amino.acid.chelates.has.been.observed.many.times.in.tests.where.a.radioactive.isotope.of.the.metal.is.given.to.the.animal.as.an.amino.acid.chelate.or.as.a.mineral.salt After.dosing,.the.amount.of.mineral.that.is.absorbed.by.various.tissues.and.organs.can.be.accurately.determined These.tests.demonstrate.that.amino.acid.chelates.provide.better.mineral.absorption.than.when.these.minerals.are.given.as.salts Even.though.amino.acid.chelated.minerals.have.greater.absorption.than.mineral.salts,.to.be.effective.these.amino.acid.chelates.must.be.bioavailable A.detailed.explanation.of.why.this.occurs.is.found.in.this.book

Bioavailability.of.minerals.is.sometimes.more.difficult.to.determine,.but.this.is.usually.done.by.comparing.some.aspect.of.health.or.production.when.different.types.of.minerals.are.given Many.studies.are.reviewed.that.range.from.improving.iron.deficiency.anemia.in.human.infants,.to.milk.production.in.cows,.to.improved.sur-vival.of.baby.pigs These.studies.all.showed.that.when.amino.acid.chelated.minerals.are.in.the.diet,.the.response.is.improved.health.or.production

Although.introduction.of.amino.acid.chelates.in.mineral.nutrition.initially.met.with.considerable.skepticism.and.controversy,.greater.absorption.and.bioavailabil-ity of amino acid chelated minerals compared to nonchelated minerals has been.well.documented This.book.reviews.many.of.the.studies.that.provided.information.on.the.comparison.of.amino.acid.chelates.and.nonchelated.minerals These.studies.were.conducted.using.many.different.animals,.including.humans,.under.a.variety.of.conditions,.and.amino.acid.chelates.consistently.provided.improved.responses.that.resulted.from.better.absorption.and.bioavailability.of.the.minerals.being.tested

About the Author

Dr H DeWayne Ashmead, president of.

Albion Laboratories Incorporated, has been

involved in research related to amino acid

chelates since the 1960s The results of his

research and the research that he and his

addition, he has authored chapters on

chela-tion in several books His research has also

led.to.18.patents

Dr Ashmead received his BS degree in

business.in.1969.and.his.PhD.degree.in.clini-cal nutrition in 1981 He sits on the board

of directors of his own company, Albion

Laboratories,.as.well.as.the.boards.of.a.bank,.a.hospital,.and.two.universities He.has.been.recognized.with.an.honorary.doctorate.of.humanities.by.Weber.State.University In.2006,.he.was.honored.by.Ernst.&.Young.as.the.regional.Entrepreneur.of.the.Year.in.the.area.of.health.sciences In.2008,.he.received.the.State.of.Utah.Governor’s.Medal.for.Science.and.Technology He.is.a.member.of.several.professional.organi.zations

if you take away this nourishment, life is utterly destroyed.”1 The science of nutrition

is thus the science of nourishing the body

The body is, to a degree, the product of its nutrition Nutrition begins with the intake of foodstuffs They undergo digestion, which transforms those foodstuffs into basic nutrients The nutrients are then passed through the gastrointestinal tract wall into the blood and ultimately the cells that compose the body, where these nutrients carry out their life- and health-sustaining functions If the foodstuffs contain inadequate or unbalanced nutrients, the body responds by not performing

at peak efficiency, which is another way of saying that the metabolic processes within the body cells are compromised This interruption of function is manifest

as insufficient energy, poor growth, morbidity, and if too severe, mortality of the whole body

When considered in its most basic terms, nutrition is the optimal intake of teins, carbohydrates, lipids, vitamins, minerals, and water Depending on the author-ity consulted, these six nutrient groups carry out three or four basic functions: (1) They serve as a source of energy for the body; (2) they are essential for the growth and maintenance of body tissue; (3) they regulate body processes; and (4) they are required for sexual reproduction of the body

pro-A closer examination of these functions reveals that energy comes from the olism of carbohydrates, lipids, and protein The metabolic processes required to extract the energy requires the presence of certain vitamins and minerals in specific enzymes along with sufficient water to facilitate the resultant enzymatic reactions required to convert the carbohydrates, lipids, and protein into energy Figure 1.1 pro-vides a simplified illustration of those relationships.2

catab-Enzymes are proteinaceous molecules that catalyze biochemical reactions The presence of specific amino acids and their exact order in the enzyme molecule will govern the reaction that the enzyme molecule catalyzes Each amino acid contains

a carboxyl group, an amine group, and its radical which is attached to the α-carbon The radical, or R group, is the unique portion of the molecule that separates each kind

of amino acid from every other kind The active site in the enzyme is so arranged that it can bind to a specific substrate (the reactants, i.e., the energy nutrients) through the amino acid R groups In some enzymes, the active site will promote the bending

of the substrate in such a way that it accelerates a certain reaction In other enzymes, the R groups attach to, or chemically react with, the substrate, which enhances the rate of the enzymatic reaction.3

A small number of enzymes, such as pepsin or trypepsin, are composed sively of protein and nothing else Most enzymes, however, are composed of complex proteins (the apoenzyme) linked to a nonprotein group (prosthetic groups) When the prosthetic group can be readily removed from apoenzyme, that prosthetic group is called a coenzyme The enzyme functions only when the apoenzyme and prosthetic groups are joined together.

exclu-In other enzymes, the protein portion of the molecule may have a simple metal ion attached to it When the metal is removed or substituted, the enzyme loses or decreases its activity If it is replaced, the catalytic properties of the enzyme return.Not all trace minerals function as activators in an enzyme Some are incorporated into the apoenzyme, while others are parts of the prosthetic groups The roles of specific metals in either accelerating or inactivating enzymatic activity cannot be overemphasized Excesses or deficiencies of these essential elements can affect the rate of catalytic action of the affected enzymes.4

Like the trace elements, specific vitamins also function primarily as coenzymes Structurally, most vitamins are part of the apoenzyme and are usually responsible for the attachment of the enzyme to the substrate.5

Enzymatic reactions generally require the presence of water Most minerals must

be ionized to function within the apoenzymes Many of the vitamins are water ble and require the presence of water to enter into the enzyme system

solu-CALCIUM Pancreatic Lipase

NIACIN Lipid Metabolism

RIBOFLAVIN Glycogenesis

PANTOTHENIC ACID

Activates Coenzyme A

THIAMIN Glucose Metabolism

FOLACIN Amino Acid Metabolism

VITAMIN B6 Transamination

VITAMIN C Carnitine Synthesis

VITAMIN B12 Conversion of Monosaccharides to Energy

VITAMIN A, D & E

Oxidative Stress

Water Enzymes

Protein

Carbohydrate

Fat

BIOTIN Lipid Metabolism

Energy

PHOSPHORUS ATP

MAGNESIUM Energy Expenditure

SULFUR Fatty Acids Catabolism

IODINE Thyroxin

POTASSIUM Glucogenesis

SODIUM Glucose Absorption

MANGANESE Fatty Acid Synthesis

COPPER Cytochrome Oxidase

IRON Oxidation

ZINC Protein Synthesis

CHROMIUM Glucose Tolerance

VANADIUM Glucose & Lipid Metabolism

VITAMIN E Transamination

FIGURE 1.1 The interrelationships of vitamins, minerals, and water on the enzymes required to extract energy from carbohydrates, lipids and protein (Redrawn from Ashmead,

HD, Conversations on Chelation and Mineral Nutrition (New Canaan: Keats) 26, 1989.)

As can be seen from this synopsis, while minerals are not a direct source of energy, their involvement in extracting energy from specific nutrients is critical One must think of minerals not only in the nutrient sense but also in the biochemi-cal sense Most of the roles played by minerals, particularly the trace elements, are biochemical.

An exception to the statement is the second function of nutrients, the growth and maintenance of body tissues, which is a structural role As the infant grows to adult-hood, protein, minerals, and other nutrients play direct roles Soft tissue is composed mostly of protein and to a lesser degree lipids Hard tissue is primarily created from minerals Minerals also play indirect roles in creating body tissue The consump-tion, digestion, and reconstruction of protein for body tissues require enzymatic pro-cesses As previously described, certain enzymes have specific vitamins or minerals that are integral parts of the enzymes or serve as cofactors Water also plays a role

in creating body tissues

Once maximum growth is achieved, the body must maintain itself Tissues wear out and are replaced Bones dissolve and remineralize Soft tissues, organs, and the like are continually rebuilt as nutrients are ingested and absorbed Maintenance of body tissue is a 24-hour-a-day process

Regulation of body processes will generally involve biochemical processes requiring protein, minerals, vitamins, and water All of these nutrients have one or more direct roles in establishing acid/base balance, creating hormones, controlling osmotic pressure, moving nutrients into body cells, and so on Much of the regula-tion of body processes is accomplished by enzymatic activity There are, however, requirements for some nutrients in their ionic, uncomplexed forms (e.g., sodium and potassium) in body fluids Regulatory processes can become very complicated depending on the requirements for functionality Many of these processes have need

of more than one sequential biochemical or enzymatic chain reaction to achieve the overall desired control

The final role of nutrients is for sexual reproduction In a sense, reproduction can

be included in one or more of the other three roles since all are involved in tion Energy is required; creation of new tissue is required; and hormonal changes must take place for reproduction to occur Thus, protein, carbohydrates, lipids, vita-mins, minerals, and water are all necessary to the sexual reproductive process.The number of roles that a single nutrient plays in carrying out one or more of the four basic functions of nutrition in no way determines its relative importance

reproduc-to the body A deficiency or an excess of a nutrient required in minute amounts may precipitate more severe consequences to the body than the deficiency or excess of a nutrient needed in larger amounts.6 Optimum intake is the key to nutrient efficiency Too much or too little of a given nutrient has an equally deleterious effect on the body, as illustrated in Figure 1.2

If the nutrient deficiency or toxicity is marginal, the health and well-being of the body and its performance may be impaired The degree of impairment depends on the extent of the toxicity or deficiency Whenever the body suffers an acute defi-ciency or extreme toxicity of an essential nutrient for a prolonged period, death will result When the intake of the nutrient is neither deficient nor toxic but provided in the optimal range, the responses of the body are health and peak performance

The previous discussion, of course, assumes that each nutrient operates in a uum Such is not the case The presence, absence, or even the level of presence of a specific nutrient in the diet may affect the absorption and metabolism of numerous other nutrients For example, the amino acid methionine is reported to be preferen-tially absorbed in the presence of other amino acids.7 Certain minerals can also be antagonistic to other minerals during metabolism To illustrate, calcium and mag-nesium are mutually antagonistic Calcium is also antagonistic to manganese, but manganese has no effect on calcium.8 In another example, Wilson’s disease, a meta-bolic error resulting in copper toxicity, is treated by high doses of zinc, which tend to reduce or prevent copper absorption.

vac-Balance becomes extremely important to achieve optimal nutrition There are three aspects to the concept of balance There must be balance between food groups for optimum nutrition In human nutrition, for example, there must be a balance between food groups, such as meat, dairy, fruit, vegetables, and so on When this balance is ignored, the consequences can be dramatic

Second, there must be balance between nutrient groups A high-protein diet at the expense of carbohydrates and lipids may not be the most efficient way to obtain energy Further, other problems, such as ketosis, may result from a high-protein diet

A strict vegetarian diet is frequently deficient in iron, vitamin B12, folic acid, and other nutrients These nutrients must be supplemented for balance to occur

Besides balance between food and nutrient groups, a third requirement requires that balance must exist between individual nutrients within a nutrient group For example, the essential amino acids, the building blocks of protein, must be in bal-ance one with another if efficient use of the food is to be accomplished Many years ago, Morrison observed, “A shortage of a simple [essential] amino acid will limit the use of all others, and therefore reduce the efficiency of the entire ration.”9Figure 1.3clearly shows the necessity of an amino acid balance.10 This drawing illustrates that excesses of any amino acid can interfere with the utilization of those amino acids to which the arrows emanating from the originating amino acid point For example, an

excess of threonine can interfere with the utilization of phenylalanine High levels

of glutamic acid can also affect phenylalanine If phenylalanine is in excess, it can interfere with the utilization of glutamic acid, but it has no effect on threonine.Vitamins also have definite relationships with each other For example, if the body has a vitamin B6 deficiency, it cannot utilize vitamin B12 efficiently.11 Vitamin

A and E are synergistic.12 Some of the basic interrelationships between vitamins are summarized in Figure 1.4 and are based on several published vitamin balance studies.13,14 This figure emphasizes that when there is a deficiency of one vitamin, such as B6, it results in less utilization of several other vitamins, including riboflavin, vitamins B1, A, E, C, niacin, folic acid, biotin, and vitamin B12

To further complicate the picture, it will be recalled that amino acids are essential for growth and maintenance of body tissues To regenerate the protein for body tissues, these required amino acids must be in balance Selecting three specific amino acids, valine, leucine, and isoleucine, as an example, there must be adequate amounts of biotin and pantothenic acid present for the utilization of those particular amino acids.11Figure 1.4 emphasizes that both of these vitamins cannot be utilized efficiently unless there are appropriate amounts of available riboflavin, folic acid, and vitamin B12.Referring to Figure 1.3, it can be quickly noted that both leucine and isoleucine will depress the uptake of valine If the diet were marginally deficient in biotin and pantothenic acid, they would first be utilized to meet the requirements for isoleucine uptake followed next by leucine If any of the vitamins remained after satisfying the requirements for isoleucine and leucine, they would then be utilized for valine

Threonine

Glutamic Acid Cystine Alanine Arginine Serine Methionine Isoleucine

Valine Leucine

Glycine Lysine Histidine

Phenylalanine

Proline

FIGURE 1.3 The relationships of several amino acids to each other An excess of one

of the amino acids will affect the absorption/metabolism of those amino acids to which it points A deficiency of that amino acid will allow the accumulation of those amino acids to which it points (From Graff, D, “Radioactive isotope research with chelated minerals,” in

Ashmead D, ed., Chelated Minerals Nutrition in Plants, Animals and Man (Springfield, IL:

Thomas) 275, 1982.)

absorption and utilization Thus, the body could potentially suffer from a valine deficiency due to marginal deficiency of biotin and pantothenic acid At this point, the question of balance becomes even more complicated If riboflavin, folic acid, or vitamin B12 were marginally deficient in the diet, they may cause a depression in the biotin and pantothenic acid utilization, resulting in an inadequate utilization of all three of these amino acids.

If one were to add minerals to the nutritional balance equation, the results become even more complicated For optimum nutrition, the minerals must also be in balance

An excess of any one of them could result in a depression of certain other minerals, just as excesses or individual amino acids can result in the depression of other amino acids Figure 1.5 indicates this.8

The late Professor Eric Underwood said, “Metabolic interactions among trace elements are so potent and so diverse that no consideration of the current status of nutrition would be reasonable without some account of their nutritional implica-tions.”15 Underwood went on to state that the interactions are more common among metals that share common chemical parameters and compete for common metabolic sites within the body

Suttle summarized these interactions and grouped them into six categories16:

1 The formation of insoluble complexes between dissimilar ions

2 Competition for metabolic pathways between similar ions

A (β-carotene)

B1 (Thiamin)

B6(Pyridoxine)

B12(Cobalamine)

C (Ascorbic Acid)

E (Tocopherol)

D (Calciferol) K

(Phylloquinone)

Riboflavin

Biotin Folic Acid

Niacin Pantothenic Acid

FIGURE 1.4 Synergism among several vitamins An excess or deficiency of any one of the vitamins in this figure will affect the absorption or metabolism of the other vitamins

connected to it by the lines (Redrawn from Patrick, H, and Schaible, P, Poultry Feeds and

Nutrition (Westport, CT: AVI) 144, 1980; and Levander, O, and Cheng, L, eds., Micronutrient

Interactions: Vitamins, Minerals and Hazardous Elements (New York: New York Academy

of Sciences) 80–129, 1980.)

3 The complexing of ions by metal-binding proteins

4 Changes in the metallic component of metalloenzymes

5 Facilitation of trace mineral transport

6 Codependence of trace element reactions on each other

The first category relates to the formation of insoluble complexes between ilar ions.16 In their ionic form, while in the digestive tract, minerals are able to form insoluble complexes with anionic ligands sourced from the diet, resulting in lower mineral bioavailability For example, dietary phosphorus, generally in the form of phosphates, can reduce the availability of both iron and zinc.17,18 The chemical reac-tion occurring in the gastrointestinal tract can produce either iron or zinc phosphate, both of which exhibit very poor solubility When dealing with inorganic metal salts, generally, solubility is a major key to their availability

dissim-Digestion can also lead to the formation of insoluble compounds.16 The release of phytates from grain-based foods is an excellent example The phytic acid can bond with

a cation and reduces its solubility and thus availability Another example is illustrated

in a study in which the combination of dietary molybdenum and sulfur along with iron reduced the absorption of dietary copper.19 The molybdenum-sulfur effect begins with the substitution of the sulfur in the sulfide ion for oxygen from the MoO42- ion:

MoO42- → MoO3S2- → MoO2S22- → MoOS32- → MoS4The tetrathiomolybdate (MoS42-) ion is then able to bind with dietary copper ions and render them insoluble and unavailable

2-S P

F

I Mo K Mn Cu Al Be Cd Ag Ca Na Se Fe N Co

FIGURE 1.5 Mineral relationships in the body The absorption or metabolism of an ual mineral is affected by the levels of intake of the other minerals pointing to that individual mineral (Redrawn from Dyer, IA, “Mineral requirements,” in Hafez, ESE, and Dyer, IA,

individ-eds., Animal Growth and Nutrition (Philadelphia: Lea & Febiger) 313, 1969.)

In animal diets, if molybdenum intake exceeds 10 mg/kg of dry matter, the MoS42- formed may also interfere with copper metabolism The tetrathiomolybdate ions form in the plasma following the absorption of molybdenum (associated with albumin) and sulfide ions and subsequently complex with copper ions Suttle sug-gested this and other reactions may result in the formation of insoluble inorganic complexes in the tissues.16

The second group of trace element interactions can occur chemically between ilar ions These interactions generally manifest themselves through competition for transport molecules to carry the minerals into the mucosal tissue from the lumen The competition for binding sites on transport molecules can occur between groups of trace elements or groups of macrominerals or between trace elements and macrominerals.20

sim-To illustrate this, when competing with iron ions, copper ions are preferentially bound to transferrin, which has been identified as a protein transport molecule in the intestinal mucosa Under normal circumstances, the transport mechanism is not saturated Thus, there are adequate bonding sites for both iron and copper ions However, when both copper and iron are administered in excess, iron absorption is inhibited because the copper is bound first to the transferrin, and inadequate binding sites are left for all of the iron ions.21

The third group of interactions summarized by Suttle involves the formation of metal-binding proteins When metal loading occurs, the normal biological reaction

is to synthesize proteins in the plasma and tissues to complex the increased metal load The problem is that these proteins are not specific to the metal that stimulated the production of the protein molecules in the first place These protein molecules can bind other elements as well For example, the addition of a cadmium or zinc load

to the diet will induce the formation of a soluble cysteine-rich protein in the kidney

or liver Further, it will bind not only the cadmium or zinc but also mercury and per.16 The binding of these minerals is preferential depending on the metal and its valence As shown in Table 1.1, there is a hierarchy of the minerals The metal at the top will replace all of the metals below it in the table As one moves down the electromotive series, each element will displace those metals below it Concurrently, that element can be removed by any of the minerals above it, which complicates the potential processes.22

cop-A change in the metal component of a metalloenzyme involves the fourth group

of mineral interactions As noted, most enzymes require the presence of a mineral

to function This metal can be part of an apoenzyme, but more often it is part of the cofactor within the prosthetic group Other minerals have been noted in cer-tain enzymes that have integral functions that are not yet elucidated Further, some enzymes are activated by a specific mineral, whereas the activities of other enzymes are blocked by the presence of that same mineral.23,24

Aminopeptidase is an example of this It contains manganese or magnesium

as active parts of the prosthetic group Either element will activate the enzyme Additional manganese and zinc are also found in the enzyme, but their functions are not completely understood The manganese or magnesium in the prosthetic group can replace each other and the enzyme will continue to function, but if the man-ganese or magnesium is displaced by iron, lead, mercury, or copper, the enzymatic activity of aminopeptidase is blocked.23

A second example is the enzyme carboxypeptidase This enzyme is activated by zinc When activated, the enzyme will split the peptide bonds of certain peptides and thus liberate the amino acids Replacing the zinc with cobalt in the enzyme will retard its activity.25 In the same group of proteolytic enzymes that attack the pep-tide bonds of proteins and peptides is glycyl-glycine dipeptidase It requires cobalt

or manganese for its activation.26 On the one hand, cobalt activates one peptidase enzyme; on the other hand, its presence retards a different peptidase enzyme.The fifth group of mineral interactions listed by Suttle involves the transport and excretion of trace elements.16 These relate to specific interrelationships One example

is the role of copper in ceruloplasmin Its presence will facilitate the transport of iron for normal hemopoiesis The ceruloplasmin functions as a ferroxidase and catalyzes the conversion of ferrous iron to the ferric state This allows iron that is stored in the liver and reticuloendothelial system to be transported in the plasma as ferric iron.27While this example is somewhat synergistic, the following is exactly the opposite

As was pointed out above the trace element, molybdenum, can interfere with per metabolism through the formation of highly stable CuMoS4 molecules in the plasma.19 In a ruminant study, a group of calves was fed a supplement that contained

cop-20 mg Cu and 10 mg Mo/kg of supplement Each animal received 0.68 kg of this supplement daily for 120 days At 0, 60, and 90 days, liver biopsies and blood serum samples were obtained and assayed for copper and molybdenum Table 1.2 summa-rizes the mean results as a percentage of the initial levels

This study demonstrated that as the molybdenum concentration increased in the liver

or the serum, the concentration of copper declined The molybdenum appeared to cause

a mobilization of tissue copper with a consequential increase in copper excretion.19The final group of mineral interactions involves the codependence of different reactions on each other.16 Suttle has reported that the involvement of a trace element

trans-J Appl Nutr 22:42–51, Spring 1970.

in the formation of an insoluble complex will limit the capacity of that element to interfere with the absorption or metabolism of other trace elements Referring to Figure  1.5 and considering the previously described copper/molybdenum animal study, if the molybdenum were tied up with the copper, then it cannot depress or interfere with phosphorus metabolism.

Not only do the interactions between minerals affect their absorption and lism, but these interactions can also influence the metabolic response to other nutri-ents To illustrate, in the biochemical utilization of valine, coenzyme A (CoA) is required It is produced in adequate quantities provided that a sufficient amount

metabo-of available magnesium is present as a cmetabo-ofactor to catalyze the enzyme activity.28Pantothenic acid is also needed in that same series of reactions.28 Again referring to Figure 1.5, if calcium, phosphorus, or manganese is too high, utilization of magne-sium may be reduced or prevented If that were to occur, then again, there is interfer-ence with valine utilization by the body

Thus, in this simple example relating to the utilization of valine, the optimal use may be prevented by excessive amounts of leucine, isoleucine, calcium, phospho-rous, or magnesium and deficiencies of riboflavin, folic acid, vitamin B12, panto-thenic acid, thiamin, or magnesium For purposes of illustration, this example has been kept simple Carbohydrates, fats, and water have not been considered Neither have all of the side reactions and the nutrients involved in them that are necessary to build the molecules needed for the simple primary reaction of converting valine into usable substance Nutrient balance is essential for optimum nutrition

Justus von Liebig (1803–1873) was one of the early investigators of organic, ological, and agricultural chemistry.29 As a result of his studies, he advanced the law

physi-of the minimum, which states that the nutrient that is the relative minimum mines the rate of growth.30 This law coupled with Voisin’s law of the maximum (the

deter-nutrient present in the relative maximum determines yield)31 emphasize that both positive and negative interactions between nutrients exist and that balanced nutrition can occur at various levels of nutrition.30

TABLE 1.2

Effect of Molybdenum on Copper Concentrations

in Liver and Blood Serum (%)

Source: Data from Ashmead, HD, and Ashmead, SD, “The

effects of dietary molybdenum, sulfur and iron on

absorption of three organic copper sources,” J Appl Res Vet Med 2:1–9, 2004.

The following theoretical example illustrates the possible consequence of ing excessive amounts of a specific nutrient In 1970, Pauling reported that taking several grams of ascorbic acid on a daily basis prevented the common cold.31 While several experts disputed the claim of Dr Pauling,32–34 many laypeople continue to supplement their diets with large doses of ascorbic acid, which frequently results

ingest-in unwanted consequences Monsen reported that ascorbic acid will enhance heme iron absorption three- to sixfold when consumed concomitantly with the iron.35 Furthermore, the ascorbic acid will mobilize iron from the ferritin mole-cule by reducing it from Fe+3 to Fe+2 This becomes significant at concentrations of

non-50 mM.36 Studies conducted in the United Kingdom have demonstrated that iron is

a very potent antagonist of copper metabolism.37,38 Furthermore, ascorbic acid also depresses copper bioavailability.39–43 So, the high intake of ascorbic acid could nega-tively affect copper absorption and metabolism directly through its effect on copper availability and indirectly by promoting iron uptake and mobilization Further, even with the greater uptake of iron, iron deficiency anemia may result due to the role of copper in ceruloplasmin

Copper plays many other roles within the body, including formation of bones, pigmentation of hair, keratinization, prevention of infertility, creation of elasticity in the cardiovascular system, enhancement of immunity and lipid metabolism Another role of copper is facilitating glucose metabolism.44–47 Reduced glucose tolerance is brought about by reduced lipogenesis and glucose oxidation Both reductions result from copper deficiency.48 Under normal conditions, glucose is metabolized at a rate that maintains a relatively constant concentration of glucose in the blood Excess,

or unmetabolized, glucose is stored as glycogen and ultimately as fat.49 Thus, when carbohydrate intake remains constant but glucose metabolism is impaired, fat deposi-tion increases Once deposited in the tissue, it becomes more difficult for the body to metabolize that fat in a copper-deficient state.50,51 Further, in a copper-deficient state there is elevated serum cholesterol because the cholesterol cannot be degraded.52,53 The net result of this discussion is that, while the excessive intake of ascorbic acid is not directly related to weight gain, it could potentially be one of the root causes Besides its direct effect on copper, ascorbic acid also enhances iron absorption/ metabolism, which can negatively affect copper absorption and metabolism That in turn could potentially have an impact on glucogenesis and result in increased fat deposition in the tissues All of these relationships demonstrate Voisin’s law of the maximum

While this example is a little extreme, a deficiency of a mineral can have a direct impact on overall metabolic health If, for example, zinc is deficient in what is other-wise a reasonably balanced diet, it can affect the utilization of the other nutrients

in growth In 1963, Prasad et al published their findings relating to zinc deficiency

and its effect on growth or sexual maturation.54,55 Oral zinc treatment over a period

of months corrected the growth retardation and delayed puberty The other nutrients necessary for normal growth and sexual maturity were previously present in the diet, but the deficiency (not a complete absence) of a single essential nutrient significantly reduced the efficacies of the other nutrients This clearly demonstrates Liebig’s law

of the minimum

In the case of minerals, it is extremely difficult to predict with any certainty the centage of absorption following ingestion The normal absorption of calcium has been reported to range between 20% and 50% of the dose Magnesium has an even wider range: 25% to 75% Normal absorption of iron salts is reported to be between 2% and 10% Manganese absorption fluctuates between 3% and 20% of the dose Copper absorption may be as low as 10% or as high as 97% according to the study consulted.56Much of this controversy focuses on differing environmental conditions that may influence the absorption of a specific ion at a specific time While there is some jus-tification for that position, even under controlled conditions intestinal absorption of metal ions can vary depending on the source of the mineral.57,58

per-Brise and Hallberg conducted a study in 80 human volunteers in which they pared the absorption of nonheme iron from 12 sources to ferrous sulfate absorption They dissolved 30 mg of iron from one of the 12 salts tagged with 55Fe in 25 mL of distilled water They then added 10 mg of ascorbic acid to each solution to prevent oxidation and to enhance absorption of the iron A ferrous sulfate solution was simi-larly prepared, except the iron was labeled with 59Fe The volunteers consumed the iron salt solutions assigned to them on the first day The next day, they all took the ferrous sulfate solution The following day, they took the iron salt solutions assigned

com-to them Treatments continued for 10 days and alternated daily between the two iron solutions Each solution was administered in the morning Following the last dose

on the 10th day, blood samples were obtained from each individual and assayed for

59Fe and 55Fe Figure 1.6 summarizes the results The absorption of each iron salt was compared to ferrous sulfate, which was arbitrarily set at 100%.58 As can be seen, both the valence of the iron and the anion attached to the iron ion influenced the absorption of the iron

When considering the law of the minimum and applying it to the six basic ent groups, minerals tend to be the most limiting As seen in the previous examples,

nutri-0 20 40 60 80 100 120 140

Percent Absorption Compared to FeSO

Ferrous sulfate

Ferrou

s succinateFerrou

s lactate

Ferrous f

umarate

Ferrous glycine sulfat

e

Ferrou

s glut

amateFerrous gluconateFer

rous citrateFerrou

s tartrate

ate Ferric

sulfate Ferric

citrate

NaFeE

DTA

FIGURE 1.6 The percentage of iron absorption in human subjects from different sources compared to ferrous sulfate (Redrawn from Brise, H, and Hallberg, L, “Absorbability of dif-

ferent iron compounds,” Acta Med Scan Suppl 358–366, 23–37, 1960.)

bioavailability can change depending on the source of the metal There are numerous other factors that can also affect bioavailability The fact that a mineral is present in the diet does not guarantee it is bioavailable Intrinsic, extrinsic, and luminal factors all influence mineral bioavailability Table 1.3 summarizes these factors in mam-mals, including humans.59

TABLE 1.3

Factors Affecting Mineral Bioavailability

Intrinsic Factors

1 Animal species and its genetic makeup

2 Age and sex

3 Monogastric or ruminant (intestinal microflora)

4 Physiological function: growth, maintenance, reproduction

5 Environmental stress and general health

6 Food habits and nutrition status

7 Endogenous ligands to complex metals (chelates)

Extrinsic Factors

1 Mineral status of the soil on which the plants are grown

2 Transfer of minerals from soil to food supply

3 Bioavailability of mineral elements from food to animal

a Chemical form of the mineral (inorganic salt or chelate)

b Solubility of the mineral complex

c Absorption on silicates, calcium phosphates, dietary fiber

d Electronic configuration of the element and competitive antagonism

e Coordination number

f Route of administration, oral or injection

g Presence of complexing agents such as chelates

h Theoretical (in vitro) and effective (in vivo) metal binding capacity of the chelate for the element under consideration

i Relative amounts of other mineral elements

In the Lumen

1 Interactions with naturally occurring ligands

a Proteins, peptides, amino acids

b Carbohydrates

c Lipids

d Anionic molecules

e Other metals

2 At and across the intestinal membrane

a Competition with metal-transporting ligands

b Endogenously mediating ligands

c Release to the target cell

Source: From Kratzer, F, and Vohra, P, Chelates in Nutrition (Boca Raton, FL: CRC Press) 35, 1986.

Some of the factors are outside the ability of the organism to change, such as genetics, age, or sex Others, like endogenous ligands in the diet that can complex minerals, can be modified by changes in the diet There are millions of ligands in the body The ingested metals can be attached through covalent or ionic bonding to one, two, or many ligands within the biological system Changes in diet can change the makeup of the ligands in the gastrointestinal tract, which in turn affects the binding

of the metal ions also present in the lumen

To illustrate this concept, one needs only to consider the absorption of ferrous (Fe+2) ion resulting from the ingestion of ferrous sulfate When the diet is rich in phy-tates, the intestinal absorption of the iron is reduced The ferrous ion formed in the lumen is complexed by the phytic acid from the phytates, resulting in creation of an insoluble iron complex.60,61 On the other hand, if the diet is rich in animal proteins, such as beef, pork, lamb, chicken, or fish, there is pronounced enhancement of the intestinal absorption of the iron.62,63

This discussion focused on absorption variability resulting from ingestion of different sources of the same metal Consuming different foods can cause signifi-cant differences in the absorption of the same mineral from that food For example, Layrisse and Martinez-Torres determined the percentage of iron absorption from various foodstuffs.63 As shown in Figure 1.7, iron from vegetable origins was not as bioavailable as was iron from animal origins Even so, there were wide variations within the two food groups Availability of iron from rice was less than 1%, whereas iron from soybean was more than seven times greater Iron from ferritin and hemo-globin had lower absorptive values than did veal liver, but all of them were eclipsed

by the absorption of iron from veal muscle

These data suggest that the chemical presence of a mineral in a food is no antee of its availability What the mineral is bound to affects its absorption For

guar-* All values are expressed as means plus or minus one standard deviation

FIGURE 1.7 Absorption of iron from food (Redrawn from Layrisse, M, Martinez-Torres,

C, and Roche, M, “Effects of interaction of various foods on iron absorption,” Am J Clin Nutr

21:1175–1183, 1968.)

example, calcium that is located in the wall of a plant cell is much less bioavailable compared to calcium in the cytoplasm of the plant cell Further, as noted in Table 1.2, precipitating ligands in the food can, and often do, bind with ions that are freed from the food during digestion even if those metals were not originally attached to the precipitating ligands in the food.64

The health of the individual will also affect his or her ability to absorb and, more particularly, utilize absorbed minerals in a metabolic process Besel published a schematic representation of the sequence of nutritional responses resulting from con-tracting an infectious disease.65 The very first response following exposure is depres-sion of plasma amino acids, zinc, and iron This is followed by retention of urinary zinc Ultimately, at the height of the illness, there is a negative balance of zinc, magnesium, sodium, and potassium (Figure 1.8)

There have been numerous books and countless articles written on mineral absorption Most try to explain why there are variations and how to maximize absorption through selection of specific foodstuffs or use of certain salts This work

is another attempt It will focus on ingestion of metal amino acid chelates as a way

to optimize mineral absorption

Phagocytic activity Depression of plasma amino acids, Fe and Zn

Saluresis retention of urinary PO 4 and Zn Increased secretion of glucocorticoids and growth hormone Increased deiodination of thyroxine

Increased synthesis of hepatic enzymes Secretion of “acute phase” serum proteins Carbohydrate intolerance

Increased dependence on lipids for fuel Increased secretion of aldosterone and ADH

Negative Balances Begin – N, K, Mg, PO4 , Zn, and SO4Retention of body salt and water

Increased secretion of thyroxine Diuresis

Fever Illness Moment of Exposure

Convalescent Period

Incubation Period

Return to positive balances

FIGURE 1.8 Schematic representation of the sequence of nutritional responses that evolve during the course of a “typical” generalized infectious illness (Redrawn from Besel, WR,

“Magnitude of the host nutritional responses to infection,” Am J Clin Nutr 30:1236–1247, 1977.)

3 Brody, T, Nutritional Biochemistry (San Diego, CA: Academic Press) 35, 1994.

4 Schutte, KH, The Biology of the Trace Elements (Philadelphia: Lippincott) 15, 1964.

5 Guthrie, HA, Introductory Nutrition (St Louis, MO: Mosby) 200, 1975.

6 Ibid., 13.

7 MacInnis, A, and Graff, DJ, “Specificity of amino acid transport in the tapeworm,

Hymenolepis diminuta , and its rat host,” Rice Universities Studies 62:183, 1976.

8 Dyer, IA, “Mineral requirements,” in Hafez, ESE, and Dyer, IA, eds., Animal Growth

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9 Morrison, FB, Feeds and Feeding, Abridged (Clinton, IA: Morrison) 49, 1961.

10 Graff, D, “Radioactive isotope research with chelated minerals,” in Ashmead, D, ed.,

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275, 1982.

11 Sauberlich, H, “Interactions of thiamine, riboflavin and other B-vitamins,” in Levander,

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13 Patrick, H, and Schaible, P, Poultry Feeds and Nutrition (Westport, CT: AVI) 144, 1980.

14 Levander, O, and Cheng, L, eds., Vitamins, Minerals and Hazardous Elements (New

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50 Szepesi, B, “Carbohydrates,” in Zingler, EE, and Filer, LJ, eds., Present Knowledge in

Nutrition (Washington, DC: ILSI Press) 36–38, 1996.

51 Cunnane, SC, Horrobin, DF, and Manku, MS, “Contrasting effects of low or high

copper intake on rat tissue lipid essential fatty acid composition,” Ann Nutr Metab

29:103–110, 1985.

52 Wahle, KWJ, and Davies, NT, “Effect of dietary copper deficiency in the rat on fatty acid

composition of adipose tissue and desaturase activity of liver microsomes,” Br J Nutr

34:105–112, 1975.

53 Lei, KY, “Alterations on plasma lipid, lipoprotein and apoliprotein concentrations in

copper-deficient rats,” J Nutr 113:2178–2183, 1983.

54 Prasad, AS, Miale, A, Farid, Z, Sanstead, HH, Schulert, AR, and Darby, “Biochemical

studies on dwarfism, hypogonadism,” Arch Intern Med 111:407–428, 1963.

55 Prasad, AS, Schulert, AR, Miale, A, Farid, Z, and Sanstead, HH, “Zinc and iron ciencies in male subjects with dwarfism but without ancyclostomiasis, schistosmiasis or

defi-severe anemia,” Am J Clin Nutr 12:437–444, 1963.

56 Ashmead, HD, Graff, DJ, and Ashmead, HH, Intestinal Absorption of Metal Ions and

Chelates (Springfield, IL: Thomas) 24–25, 1985.

57 Graff, DJ, Ashmead, H, and Hartley, C, “Absorption of minerals compared with chelates

made from various protein sources into rat jejunal slices in vitro,” Proc Utah Acad Arts

Lett Sci Apr 1970.

58 Brise, H, and Hallberg, L, “Absorbability of different iron compounds,” Acta Med Scan

Suppl 358–366, 23–37, 1960.

59 Kratzer, F, and Vohra, P, Chelates in Nutrition (Boca Raton, FL: CRC Press) 35, 1986.

60 Hallberg, L, and Solvell, L, “Absorption of a single dose of iron in man,” Acta Med Scan

19:358, 1960.

61 Manis, J, and Schachter, D, “Active transport of iron by intestine: Features of the

two-step mechanism,” Am J Physiol 203:73–80, 1962.

62 Johnston, FA, Frechman, R, and Burroughs, ED, “The absorption of iron from beef by

women,” J Nutr 35:453–465, 1948.

63 Layrisse, M, Martinez-Torres, C, and Roche, M, “Effects of interaction of various foods

on iron absorption,” Am J Clin Nutr 21:1175–1183, 1968.

64 Kuhn, LC, Schulman, HM, and Ponka, P, “Iron-transferrin requirements and transferring reception expression in proliferating cells,” in Ponka, P, Schulman, HM, and Woodward,

RC, eds., Iron Transport and Storage (Boca Raton, FL: CRC Press) 149–177, 1990.

65 Besel, WR, “Magnitude of the host nutritional responses to infection,” Am J Clin Nutr

30:1236–1247, 1977.

In the ensuing years Werner refined his discovery He concluded that a metal ion had two kinds of valencies The first, which Werner termed the “principal valence,” related to the oxidation state or oxidation number of the metal He called the second valence the auxiliary valence, which referred to the number of atoms in the ligand (chelating agent) that could be associated with the central metal ion.2–7 Today, the auxiliary valence is referred to as the coordination number Thus, the platinum ion illustrated in Figure 2.1 has a principal valence (oxidation number) of 2+ and an aux-iliary valence (coordination number) of 4+ In this configuration, the platinum metal lost two electrons in the valence shell, becoming Pt2+, leaving room for four electrons

or four bonds in the valence shell Thus, the platinum could be bound to the two ethylenediamine ligands at four points According to Werner, the ion was attached

to the ligands by principal valencies in the outer sphere of the combination and the amine groups to the central atom in an inner sphere of combination.8 As a result of his pioneering research, Alfred Werner received the Noble Prize in chemistry in

1913 and has subsequently been called the father of coordination chemistry.9While Werner is generally credited with the discovery of chelation chemistry, he

did not use the word chelation to characterize these complexes In 1920, Morgan and Drew coined the word chelate to describe the way the ligand bound a metallic cation The word chelate comes from the Greek word chele meaning “claw.” As indicated in

Werner’s example in Figure 2.1, each ligand is able to attach to the metal ion at two points in a claw-like fashion Morgan and Drew reasoned that this caliper-like action

of the ligand resembled the closing of a lobster or crab claw and suggested the term

to describe metal complexes in which the metal atom is held at more than one point

of attachment by a single ligand.10Figure 2.2 illustrates a metal glycinate chelate When one looks at the ligand of this chelate, it is easy to see how it could be viewed

as a claw as it chelates the metal ion

The word chelate was originally used as an adjective, but today it is also employed

as an adverb, verb, or noun The ligands are the chelating agents, and the ligand compounds that they form are metal chelates

metal-The purpose of this chapter is to review, on an elementary level, the chemical characteristics of a chelate Any metal that is chelated must meet specific criteria

If it does not meet those criteria, then it is not a chelate but may instead be a plex in which the critical (to chelation) ring structure is not formed.11 Even if the mineral is chelated, the resulting molecule is not guaranteed to enhance mineral absorption The criteria for a nutritionally functional chelate are discussed in the next chapter

com-Prior to the defining work of Morgan and Drew, Ley, another chemist, commenced the elucidation of the presumed structure of a chelate.12 He was able to synthesize a copper bisglycinate molecule by bonding two moles of glycine to one mole of copper Ley inferred that a chemical reaction had occurred in the solution of copper ions and glycine due to observing a color change He also noted that the resulting solution had very low electrical conductivity compared to the conductivity of copper ions in solu-tion When the copper-glycine solution was dried and the precipitate analyzed, it was determined that the product consisted of a ratio of two moles of glycine to one mole

of copper Ley had created an amino acid chelate, although he did not call it that Instead he gave it the name “inner metallic complex salt” and illustrated the concept (not copper glycinate) as seen in Figure 2.3

The nature of bonds between the metal ion and ligand is crucial to ing chelation The basic principles of the bond formatting are similar for transition metals (elements with partially filled d or f shells) as they are for others It is the

comprehend-d orbital in the electron shells of the transition metals that plays a major role in

FIGURE 2.2 A metal bisglycine chelate illustration showing the claw-like structure of the ligand.

Z Anorg u Allgem Chem 3:267–330, 1893 Translated by D P Mellor.)

bonding Further, bonding in the d orbital results in different characteristics pared to elements where the bonding occurs exclusively in the s or p orbitals The way the metals are bonded is called ligand field theory.13,14

com-According to this theory, when transitional metals are oxidized, if they have trons in their s shells, they generally lose those outer s shell electrons before losing the d shell electrons (Some transitional metals have partially filled d orbitals.) It is the existence of the d subshell electrons and their movement that gives the transi-tional metals their unique characteristics, such as having more than one oxidation state, colored compounds, and magnetic properties.14,15 It also allows them to partici-pate in bonds that are generally unlikely for nontransitional elements.14

elec-While over the ensuing years the following chelation criteria have been refined, they were recognized10 as early as 1920:

1 In chelation, there are two types of ion ligand bonding: ionic or electrostatic bonding, in which both atoms participating in the bond each share electrons

to form the bond, and covalent bonding, in which the bonding between the two atoms occurs when both electrons shared in the bond originate from the same atom

2 The same anion ligand may participate in either type of bonding

3 The metal that participates in the complex has a fixed number of valencies, one of which is called its coordination number

4 Coordinate covalent bonds (also known as dative or semipolar bonds) may

be formed with either neutral or ionic entities

5 Coordinate covalent bonds have definite spatial arrangements

Concurrent to Ley, Bruni and Fornara were also working with copper glycinate and arriving at similar conclusions.16 Most chelates produced by early investigators were insoluble or poorly soluble Because the copper glycinate had different characteristics

C

C

C H

H

M N

N O

FIGURE 2.3 Inner metallic complex salt illustration as conceived by H Z Ley (Redrawn

from Ley, H, “Über Innere Metall-Koomplexsalze I” Z Elektrochem Angew Physikal Chem

10:954–956, 1904.)

than most of the chelates these investigators were studying, they usually overlooked

it in favor of focusing their research on less-soluble chelates made with synthetic ligands, which they relied on to describe chelated molecules Nevertheless, the prin-ciples resulting from this research with non-amino-acid ligands can be applied to amino acid chelates Chelation is a special branch of chemistry, and the principles governing this chemistry govern all chelates, regardless of the ligand employed That does not mean all chelates behave the same The similarity in chelates only relates to the type of chemistry required to form the chelate molecule

A chelate has several distinct characteristics that can be examined based on the metal atom in the chelate molecule, the ligand used to chelate the metal, or the types

of bonds linking the metal ion and the ligand together.17

Returning to the first criterion, the properties of a chelate are influenced, to a degree, by the metal in a chelate molecule and its oxidation state The metals may be considered as Lewis acids, whereas the ligands are regarded as Lewis bases, which can share an electron pair with the metal When bonded together, the result is a neutralization of the Lewis acid and Lewis base Depending on the characteristics

of the ligand, it may be attached to a metal through two, three, four, five, or six tions—creating a bidentate, tridentate, quadridentate, quindentate, or sexadentate chelate, respectively.18

posi-The bond between a metal atom and the ligand may be electrostatic (ionic) or covalent The ionic bonds are a result of attraction between oppositely charged ions

In a metal chelate bond, the positive charge originates from the metal ion and the negative charge from a negatively charged atom in the ligand According to one theory, after the ionic bond is created through ionic attractions, a sharing of elec-trons between the metal ion and the reactive moiety of the carboxyl group occurs, and the bond becomes covalent in nature The argument to support this view is that

if the bond were purely ionic, it would dissociate in an aqueous solution Had this occurred, Ley would have observed more electrical conductivity in the copper gly-cinate chelate than was created.12 Thus, some chemists believe that no ionic bond exists in a chelate Instead, they favor the view that all of the bonds are coordinate The initial attraction between the oxygen in the carboxyl moiety is a charged attrac-tion This bond will occur in a mostly dry environment or dry mixed with a little pressure The amine moiety, which definitely forms a covalent bond, does not bond

to the metal under similar conditions Further, in a solution the first bond to form is the carboxyl bond, followed by the amine bond This suggests that an ionic bond can potentially exhibit covalent characteristics.14

After the amine moiety in the ligand bonds to the metal, the oxygen is placed in

a spatial orientation that potentially endows it with some covalent characteristics Conrad and Nakamoto reported that, following chelation of copper with glycine, the strength and shift of the amine moiety indicated that the bond between the copper and the nitrogen in the amino moiety was covalent On the other hand, the changes in the carboxyl moiety indicated that the bond between the copper and the oxygen was more ionic in nature even though the oxygen in the chelate occupied a similar posi-tion as the nitrogen and still had a covalent nature.14,19 While there is room to argue

on both sides of the issue, the position taken in this chapter is that the bond between the metal and the atom from the carboxyl moiety is primarily ionic in nature

In a covalent bond, the metal ion and an atom in the ligand share a pair of trons In the past, if both electrons were donated by an atom in the ligand into a vacant d orbital of the metal ion, the bond between them was referred to as a coordi-nate covalent bond.20 Today, it is generally called a covalent bond.

elec-In an aqueous solution, the coordination number of a metal ion is generally fied by water molecules and could be considered coordinate positioning When the water molecules are displaced by a soluble ligand, a different metal complex can result This complex may be a chelate if certain requirements are met If the ligand

satis-in the solution has more than one donor atom and the metal ion has a coordsatis-ination number of 2 or more, the resulting complex can form a heterocyclic ring In this case, the metal will be the closing member of the ring A heterocyclic ring must be formed for the resulting compound to be a chelate.18 In fact, a heterocyclic ring is an absolute requirement for the formation of a chelate If this ring structure is not produced in the chemical reaction, the resulting product cannot be a chelate

The ligand, whether it is synthetic or natural, must possess at least two functional groups, one of which is capable of donating a pair of electrons to combine with the metal and the other capable of sharing an electron with the metal.17 If the ligand does not have these functional groups, chelation cannot occur In the case of the amino acid, glycine, for example, the chemical formula is NH2CH2COOH To chelate a metal ion, the glycine must lose its carboxyl-group proton (H+) and chelate as a gly-cine ion via two functional groups, one of which contains the nitrogen atom and the other, one of the oxygen atoms (Figure 2.4)

The bond between the metal ion and the oxygen from the COOH group is ionic (electrostatic) because the metal and the amino acid share one electron from the oxy-gen in the carboxyl group of the amino acid and one electron from the metal ion.21The second bond between the metal and the nitrogen in the NH2 group is a covalent bond As noted, the metal behaves as a Lewis acid and the glycine as a Lewis base The glycine donates both electrons from the same atom in the amine group of the ligand to the metal ion The donation of electrons will go to the lowest energy orbital (s, p, and d orbitals) of the metal that is unfilled.21

Since the metal ion in this example has a +2 charge, it is capable of bonding to more than one amino acid It shares one electron from the carboxyl group of the amino acid and accepts two electrons from the amine group When the metal ion has a +2 charge, it still has an electron deficiency after chelating to a single ligand Thus, to be satisfied, it can accept another electron from another carboxyl moiety of

a second amino acid ligand such as another glycine molecule This second ligand can

H2C

H2N 2H2N

also donate a pair of electrons from its amine group Thus, two ring structures can be formed, with the single metal ion the closing member of both rings.

The greater the number of rings attached to a metal ion, the more stable the late molecule becomes, up to a point The electron configuration of the metal will limit its attachments Spatial arrangements can be tetrahedral, planar, and so on, depending on the metal.18 This is referred to as the chelation effect.22 In addition, steric hindrance is a key consideration If the ligand is a large molecule, such as the theoretical molecule illustrated in Figure 2.5, only one ring may be possible.23Chelating the metal ion with a second or third ligand to form additional rings may

che-be impossible If the ligand is small, such as in the case of glycine, it che-becomes easier

to attach two, three, and sometimes more ligands to the ion and form more than one ring depending on the valence of the cation involved and the size of the metal atom.Chelate stability is affected not only by the choice of the metal being chelated but also by the type of ligand selected for chelating purposes Chelates can be classified

as multidentate or bidenate depending on the number of donor atoms in the ligand

N

N N

N N

H H

H

H H

H

H H H H H

H

H

H H

H

H H

H H

H

H H

H H H

H H

H H

H H

H

H H

H

H H H H

H H H

H H

H

H

H

O O C C

C

C

C C C C

C C C

C C C

C C

C C C

C

C C C C

C

C C C C C OH

C

C M

C

C SH

C S

C C C

C

C

C

C C C

Multidentate chelates, chelates in which there are more than two donor atoms in the ligand, are more stable than bidentate ligands (ligands that have two basic groups, one acidic and one basic group, or two acidic groups).24 A multidentate chelate is able

to occupy more positions in the coordination shell of the metal ion.25

The coordinating groups in a ligand that are capable of donating electrons to combine with the metal are18

a Coordinating groups:

–NH2 –NOH –PR2–NH –OH (alcoholic)–N= –S– (thioether)

b After the loss of a proton:

Besides having functional groups that are capable of donating a pair of electrons, the functional groups in the ligand must be located in such a way that they allow for the formation of a ring structure with the metal as the closing member of that ring.17 The potential ring formation is greatly influenced by steric hinderances For example, the attachment of one functional group may result in too much bulk and thus prevent the attachment of a second functional group to the metal ion.18 In the case of the amino acid ligands, the side chains that are attached to the a-carbon may provide additional hindrance

The Association of American Feed Control Officials (AAFCO) is an tion composed of all 50 U.S state chemists, the U.S Food and Drug Administration, the Canadian Food Inspection Agency, the Costa Rica Ministry of Agriculture and Livestock, the U.S Department of Agriculture, and the U.S Environmental Protection Agency (EPA) plus nonvoting industry members The AAFCO publishes

organiza-a list of definitions covering organiza-all organiza-approved feed ingredients, including two chelorganiza-ated mineral sources: metal proteinates and metal amine acid chelates Metal proteinates are “the product resulting from the chelation of a soluble salt with amino acids and/or hydrolyzed protein.”11 Presumably since that definition requires chelation with amino

acids and/or hydrolyzed protein, more than one amino acid must be employed in the

chelation of a single metal ion The use of the words and/or presumably requires

that partially hydrolyzed protein be part of the chelating ligands Under this tion, the partially hydrolyzed protein could be the source of the amino acids, but individual amino acids could not be the sources Such a partially hydrolyzed protein

ligand would result in steric hindrances that could potentially interfere with the lation of the metal with additional ligands, such as illustrated in Figure 2.5 Such a chelate probably could not be absorbed through the intestinal mucosal membrane intact due to its molecular size.23 It would require further gastrointestinal digestion into a smaller molecule before any absorption could occur.

che-The AAFCO has defined the metal amino acid chelate as

the product resulting from the reaction of a metal ion from a soluble metal salt with amino acids with a mole ratio of one mole of metal to one to three (preferably two) moles of amino acids to form coordinate covalent bonds The average weight of the hydrolyzed amino acids must be approximately 150 and the resulting molecular weight

of the chelate must not exceed 800 11

In expanding this definition, the AAFCO has added that “one ligand (electron pair donor) forms two or more bonds to the central metal ion through different atoms

of the ligand A distinctive feature of a metal is a member of the ring.”11 It is this complete definition that will be used to discuss amino acid chelates throughout the remainder of this book except as otherwise noted

Besides the ligands used to chelate a metal, the choice of the metal atom in the chelate will also influence the stability of the resulting chelate The electronic struc-ture of the metal ion will determine, first, if a chelate can be formed and, second, the stability of the chelate if it is formed Monovalent metals generally cannot form chelates under the definition of binding the ion at two points A monovalent metal ion, such as potassium, does not have the electronic orbital structure to bind two sites

of the ligand If the metal is not bound at two sites, no ring structure can be formed since, by definition, the metal is the closing member of the ring structure in a chelate, and at least two bonding sites on the metal ion must be available to close the ring.The oxidation number of the metal ion can also affect the stability of the chelate The higher the valency, the more stable the chelate, assuming there is a sufficient number of ligands to satisfy all of the charges on the ion and there is adequate space for all of the ligands to bond to the metal ion Therefore, the size of the metal ion

is important Metal ions with smaller radii cannot bond to as many ligands as can larger cations Thus, chelates formed from transition metals as well as lanthanides and actinides are more stable than are alkali metal chelates.26 In the case of transition elements, the stability increases to a maximum of copper Ni2+ < Co2+ < Fe2+ < Mn2+

< Zn2+ < Cu2+.27 As a general rule, as the atomic number of the metal increases, the stability of the resulting chelate also increases It should be remembered, however, that the smaller the metal ion, the smaller must be the practical coordination number due to steric hindrance regardless of other considerations

The total number of ligand atoms that can be bound to the metal ion represents the coordination number of that metal The coordination number of the metal will influence the stability of the chelate The higher the coordination number, the more stable the chelate because, in theory, more ligands can be attached to the metal as long as there are no steric hindrances

As the coordination number changes, so do the steric considerations of the ing chelate molecule As additional ligands are bonded to the metal ion, the chelate will change shape to accommodate the new ligands This affects the stability of the

chelate as illustrated in Table 2.1.28 This table demonstrates that the chelate ring can be either symmetrical or asymmetrical based on the coordination number The coordina-tion sites have well-defined mathematical stereochemical arrangements in space.18

To illustrate, a zinc bisglycinate chelate was formed following the dissolution of glycine and zinc in water The ratio of glycine to zinc was two moles of glycine to every mole of zinc The resulting product was allowed to crystallize by controlled evaporation of the water The pure zinc bisglycinate crystals were subsequently ana-lyzed by x-ray diffraction spectrometry As seen in Figure 2.6, each glycine mol-ecule was attached to the zinc by the carboxyl group (COO-) and the amino group (NH2) with the ionic bond originating from the oxygen and the coordinate covalent bond coming from the nitrogen This structure has two heterocyclic rings, with the zinc the closing member for each ring.29 Using x-ray crystallography, inductively

Oxidation State

Coordination Number Stereochemistry

Cu II (d 9 ) 4* Square planar Co IV (d 6 ) 6 Octahedral

4 Distorted tetrahedral Fe II (d 6 ) 4 Tetrahedral

6* Distorted octahedral Fe III (d 5 ) 4 Tetrahedral

Co II (d 7 ) 4* Tetrahedral Mn IV (d 3 ) 6 Octahedral

4 Square planar Mn V (d 2 ) 4 Tetrahedral

5 Trigonal bipyramidal Mn VI (d 1 ) 4 Tetrahedral

5 Square pyramidal Mn VII (d 6 ) 3 Planar

Source: Data from Huges, M, The Inorganic Chemistry of Biological Processes (London: Wiley)

25–26, 1972.

Note: * = the most common states.

coupled plasma spectroscopy, electrospray mass spectroscopy, and combustion mental analysis, Konar et al also confirmed this same chelate structure in copper and zinc bisglycinates.30

ele-Not only are the metal and its characteristics a consideration in forming a late, so also are the characteristics of the ligand An early proponent of nutritional chelates, John Miller, grouped ligands into two categories: natural and synthetic.31

che-He stated that synthetic ligands include molecules such as acetic acid (EDTA) or synthesized salicylic acid Natural ligands, on the other hand, include carbohydrates, lipids, proteins and their derivatives, some vitamins, and cer-tain organic acids (amino acids, lactic acid, citric acid, etc.), to name a few

ethylenediaminetetra-To illustrate, Table 2.2 lists several ligands Their stability constants can change, even when they chelate the same metal.32 When the metal changes, the stability con-stants also change.28

Each functional group within a chelating ligand (the moieties in the ligand that actually bond to the metal) must be situated within the molecule in such a way that

it permits the formation of at least one heterocyclic ring with the metal being the closing member of that ring.17 To form a ring structure, the ligand has to bend, twist,

or both Thus, while in theory a peptide or even a protein can form a chelate with a metal ion, for the reasons illustrated in Figure 2.5 the likelihood is extremely small.23The flat, rigid nature of the peptide bond does not allow much flexibility for the for-mation of chelates with terminal ends of short peptides

The size of the ring resulting from creating a chelate will also affect its stability.18Five or six member rings are the most stable chelate molecules.33 In Figure 2.5, there are 38 members in the ring The stability of this chelate, if it existed, would be very low A four-member ring results in bonding angles that are too acute to form stable compounds These sharp angles encourage breaking of the chelate.17 Larger chelate rings composed of seven or eight members have been studied, but these chelates are not generally very stable.17

O

O O

O

O

O

O O

C

C

FIGURE 2.6 Zinc bisglycinate chelate as determined by x-ray diffraction spectrometry (Redrawn from Dalley, NK, “Report on x-ray diffraction crystallography of Albion ® zinc amino acid chelate,” unpublished report, Brigham Young University, Provo, UT, 1985.)