amino acid chelation in human and animal nutrition

AMINO ACID
CHEL ATION
IN
HUMAN AND
ANIMAL
NUTRITION


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AMINO ACID
CHEL ATION
IN
HUMAN AND
ANIMAL
NUTRITION
H. DeWAYNE ASHMEAD

Boca Raton London New York

CRC Press is an imprint of the
Taylor & Francis Group, an informa business


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CRC Press
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© 2012 by 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

v


vi

Contents

Chapter 14 Toxicity of Amino Acid Chelates...................................................... 223

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


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Foreword
Mineral bioavailability has historically been “the black box” of micronutrient metabolism. 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 ultimately 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 mineral. 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 supplement. Mineral nutritionists have long sought chemical forms of minerals that evoke
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 influence 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.
Enter the concept of supplying the mineral in an ionic or covalently bound protective 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
“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 manner “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 intestinal 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 absorption and metabolism, specifically focusing on the formation and absorption of amino
acid metal chelates.
The progress and development of amino acid mineral chelates has not been without controversy. Although the improved bioavailability of some amino acid mineral
chelates is generally accepted, it has not been clearly understood exactly why these
vii


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viii

Foreword

chelates provide improved absorption. Early studies of the nutritional aspects of the
bioavailability of mineral chelates occurred during the 1960s and 1970s when analytical 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 bioavailability 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 accessible 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 mineral 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 mineral 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 mineral chelates in considerable detail. The book contains a well-developed introduction 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 interpretation 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 optimal health and productivity.
Wayne Askew, PhD
Professor, Division of Nutrition
University of Utah


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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 carboxylate 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 reactions that produce precipitation of the metals are reactions with phosphates, phytic
acid, and other substances commonly found in the gut. This protection of the metals 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

ix


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x

Introduction

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 survival 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 bioavailability 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.
Boyd R. Beck, PhD
Retired Professor of Chemistry
Snow College, Ephraim, Utah


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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
father, the late Dr. Harvey Ashmead, directed

have been published in seven books authored
by Dr. Ashmead. He has also published over
25 peer-reviewed journal articles and over
60 magazine articles on the same subject. In
addition, he has authored chapters on chelation 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 clinical 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 organi­zations.

xi


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1

The Fundamentals
of Mineral Nutrition

During the Italian Renaissance, Leonardo da Vinci (1452–1519) wrote, “If you do
not supply nourishment equal to the nourishment departed, life will fail in vigor; and
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 proteins, carbohydrates, lipids, vitamins, minerals, and water. Depending on the authority 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.
A closer examination of these functions reveals that energy comes from the catabolism 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 provides a simplified illustration of those relationships.2
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
1

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2

Amino Acid Chelation in Human and Animal Nutrition
Vitamins

Minerals

BIOTIN
Lipid Metabolism

CALCIUM
Pancreatic Lipase

NIACIN
Lipid Metabolism

PHOSPHORUS
ATP

Energy

RIBOFLAVIN
Glycogenesis
PANTOTHENIC ACID
Activates Coenzyme A
THIAMIN

Glucose Metabolism
FOLACIN
Amino Acid Metabolism
VITAMIN A, D & E
Oxidative Stress
VITAMIN B6
Transamination
VITAMIN B12
Conversion of
Monosaccharides to Energy
VITAMIN C
Carnitine Synthesis
VITAMIN E
Transamination

Carbohydrate
Protein

MAGNESIUM
Energy Expenditure
SULFUR
Fatty Acids Catabolism

Fat

IODINE
Thyroxin
POTASSIUM
Glucogenesis
SODIUM

Glucose Absorption

Enzymes
Water

MANGANESE
Fatty Acid Synthesis
COPPER
Cytochrome Oxidase
IRON
Oxidation
ZINC
Protein Synthesis
CHROMIUM
Glucose Tolerance
VANADIUM
Glucose & Lipid Metabolism

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.)

A small number of enzymes, such as pepsin or trypepsin, are composed exclusively 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.
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 soluble and require the presence of water to enter into the enzyme system.


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The Fundamentals of Mineral Nutrition

3

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 biochemical 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 adulthood, 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 consumption, digestion, and reconstruction of protein for body tissues require enzymatic processes. 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 regulation 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 reproduction. Energy is required; creation of new tissue is required; and hormonal changes
must take place for reproduction to occur. Thus, protein, carbohydrates, lipids, vitamins, 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
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 deficiency 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.


4

Amino Acid Chelation in Human and Animal Nutrition
Optimal

0%

Marginal

Marginal

Percent of Body Performance

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100%

Death

Death

Deficiency

Toxicity

Concentration Nutrient Intake

FIGURE 1.2  A typical dose response to nutrient intake. The shape of the curve can change
depending on the nutrient need as well as the nutrient involved.

The previous discussion, of course, assumes that each nutrient operates in a vacuum. 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 preferentially absorbed in the presence of other amino acids.7 Certain minerals can also be
antagonistic to other minerals during metabolism. To illustrate, calcium and magnesium are mutually antagonistic. Calcium is also antagonistic to manganese, but
manganese has no effect on calcium.8 In another example, Wilson’s disease, a metabolic error resulting in copper toxicity, is treated by high doses of zinc, which tend to
reduce or prevent copper absorption.
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 balance 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.”9 Figure 1.3
clearly 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


5

The Fundamentals of Mineral Nutrition
Threonine

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Histidine

Glutamic Acid

Lysine

Cystine

Glycine

Alanine

Phenylalanine

Arginine

Leucine

Serine

Valine

Methionine
Proline

Isoleucine

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.)

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.11
Figure 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


6

Amino Acid Chelation in Human and Animal Nutrition

E
(Tocopherol)

A (β-carotene)
B1 (Thiamin)
Riboflavin

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Pantothenic Acid

C
(Ascorbic Acid)

B6
(Pyridoxine)

Niacin

B12
(Cobalamine)

Folic Acid

D
(Calciferol)
K

(Phylloquinone)

Biotin

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.)

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 implications.”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


7

The Fundamentals of Mineral Nutrition

Na

Ca

Ag

Cd
Be

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Se

Al

Fe

Cu

N
Mn

Co
K
P

Mo
S

I
F

As

Mg
Zn

FIGURE 1.5  Mineral relationships in the body. The absorption or metabolism of an individual 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,
eds., Animal Growth and Nutrition (Philadelphia: Lea & Febiger) 313, 1969.)






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 dissimilar 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 reaction 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.
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- → MoS42-

The tetrathiomolybdate (MoS42-) ion is then able to bind with dietary copper ions and
render them insoluble and unavailable.


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Amino Acid Chelation in Human and Animal Nutrition

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 suggested 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 similar 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
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 copper.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
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 certain 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 manganese or magnesium is displaced by iron, lead, mercury, or copper, the enzymatic
activity of aminopeptidase is blocked.23


9

The Fundamentals of Mineral Nutrition

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TABLE 1.1
A Partial List of the Electromotive
Series of Minerals and Oxides
Metal
V+3
Fe+3
In+3
Th+4
Hg+2
Ti+3
Ga+3
Cu+2
VO+2

Ni+2
Pd+2

Y+3
Pb+2
TiO+2
Zn+2
Cd+2
Co+2
Al+3

Fe+2
Mn+2
V+2
Ca+2
Sc+3
Mg+2
Sr +2
Ba+2
Rare Earths

Source: Data from Ashmead, H, “Tissue transportation of organic trace minerals,”
J Appl Nutr 22:42–51, Spring 1970.

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 peptide 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.27
While this example is somewhat synergistic, the following is exactly the opposite.
As was pointed out above the trace element, molybdenum, can interfere with copper 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
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 summarizes 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.19
The 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


10

Amino Acid Chelation in Human and Animal Nutrition

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TABLE 1.2
Effect of Molybdenum on Copper Concentrations
in Liver and Blood Serum (%)
Liver Cu
Liver Mo
Serum Cu
Serum Mo

Initial

60 Days

90 Days

100
100
100
100

  39.37
140.00
  83.3
118.50

  26.90
147.00
  78.13
418.33

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.

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 metabolism, but these interactions can also influence the metabolic response to other nutrients. To illustrate, in the biochemical utilization of valine, coenzyme A (CoA) is
required. It is produced in adequate quantities provided that a sufficient amount
of available magnesium is present as a cofactor to catalyze the enzyme activity.28
Pantothenic 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 magnesium may be reduced or prevented. If that were to occur, then again, there is interference 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, phosphorous, or magnesium and deficiencies of riboflavin, folic acid, vitamin B12, pantothenic 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, physiological, and agricultural chemistry.29 As a result of his studies, he advanced the law
of the minimum, which states that the nutrient that is the relative minimum determines the rate of growth.30 This law coupled with Voisin’s law of the maximum (the
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


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The Fundamentals of Mineral Nutrition

11

The following theoretical example illustrates the possible consequence of ingesting 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
in unwanted consequences. Monsen reported that ascorbic acid will enhance nonheme iron absorption three- to sixfold when consumed concomitantly with the

iron.35 Furthermore, the ascorbic acid will mobilize iron from the ferritin molecule by reducing it from Fe+3 to Fe+2. This becomes significant at concentrations of
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 negatively 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 deposition 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.


Amino Acid Chelation in Human and Animal Nutrition

In the case of minerals, it is extremely difficult to predict with any certainty the percentage 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.56
Much 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 justification for that position, even under controlled conditions intestinal absorption of
metal ions can vary depending on the source of the mineral.57,58
Brise and Hallberg conducted a study in 80 human volunteers in which they compared 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 similarly 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
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 nutrient groups, minerals tend to be the most limiting. As seen in the previous examples,

140
Percent Absorption
Compared to FeSO4

120
100
80
60
40
20

ro
u

ss
ul
ro
fa
us
te
su
cc
Fe
i
n
rr
at
ou
e
Fe

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rr
a
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Fe
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ta
rr
ou s f u t e
m
sg
ar
ly
at
e
Fe cine
rr
s
u
ou
l
fa
sg
te
Fe
lu
ta
rr
m
ou

at
sg
e
lu
c
on
Fe
rr
at
ou
e
sc
F
Fe
er
itr
r
rr
ou ous ate
sp
t
yr artr
Fe
o
ph ate
rr
ic
os
ch
ph

ol
at
in
e
iso
ci
tr
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at
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e
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su
lfa
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te
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at
N
e
aF
eE
D
TA

0

Fe
r

Fe
r

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12

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


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The Fundamentals of Mineral Nutrition

13

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 mammals, 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.