AMINO ACID CHEL ATION IN HUMAN AND ANIMAL NUTRITION Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2012 by H DeWayne Ashmead CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S Government works Version Date: 20111129 International Standard Book Number-13: 978-1-4398-9768-3 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint Except as permitted under U.S Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers For permission to photocopy or use material electronically from this work, please access www.copyright com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400 CCC is a not-for-profit organization that provides licenses and registration for a variety of users For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 Contents Foreword vii Introduction ix About the Author .xi Chapter The Fundamentals of Mineral Nutrition Chapter The Chemistry of Chelation 19 Chapter The History of Nutritional Chelates 35 Chapter The Requirements for a Nutritionally Functional Chelate 49 Chapter The Development of Analytical Methods to Prove Amino Acid Chelation 61 Chapter Absorption of Amino Acid Chelates from the Alimentary Canal 81 Chapter The Pathways for Absorption of an Amino Acid Chelate 97 Chapter The Absorption of Amino Acid Chelates by Active Transport 117 Chapter 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 Chapter 15 The Absorption and Metabolism of Amino Acid Chelates 233 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 234 Amino Acid Chelation in Human and Animal Nutrition When their findings were published, controversies arose for several reasons First, as with any new scientific advancement, no peer-reviewed reference material existed substantiating Ashmead et al.’s findings Without that secondary scientific support data, it was difficult for many to accept the research conclusions immediately This led to a second reason for controversy The published reports contradicted some of the more widely accepted theories relating to mineral nutrition, particularly when it focused on bioavailability These contradictions led to criticism and the unwarranted conclusion by some that amino acid chelates were simply an expensive fraud.11,12 While disappointing to Ashmead et al., these reactions from the scientific community should have been expected Most advances in any scientific field are usually met with skepticism, particularly if they advocate new theories or observations that contradict old ones The third major problem preventing wholesale acceptance of these early reports was that, at the time, very few people comprehended the chemical or molecular requirements necessary to create a true amino acid chelate For several years, there was no peer-reviewed analytical procedure that could prove amino acid chelates even existed Later, when analytical methods proving amino acid chelation were validated through an American Organization of Analytical Chemists (AOAC) program,13 these procedures were rarely employed as a prerequisite to a research study involving AAFCO-defined amino acid chelates Many investigators continued to work as they had in the past and assumed they were studying amino acid chelates without first analyzing the molecules to confirm their assumption Consequently, the results ascribed to an amino acid chelate were frequently derived from metallic molecules that were not proven amino acid chelates To confuse the issue even more, many investigators mixed data from studies involving AAFCO-defined amino acid complexes and metal proteinates with those of amino acid chelates.14–16 All of this produced conflicting absorption and bioavailability data that were confusing at best.17 In their monograph, Leach and Patton wrote the following: Acceptance of chelates has long been hindered by the lack of an assay A buyer simply had no way of testing his purchase, having to rely exclusively on the reputation of his supplier and subjective feedback from the field The final determination is almost solely influenced by the cost per kilogram Their frustration has been that they could not determine if the high price one paid for a copper chelate was in actuality inexpensive copper sulfate.18 While both the scientific community and industry alike recognized the need for an analytical method to substantiate that the mineral being evaluated was truly chelated, no such method existed until amino acid chelate crystals could be grown and analyzed by x-ray diffraction crystallography This analytical procedure proved that these crystals were actually amino acid chelates, and ultimately these crystals were used as reference samples to validate other analytical tests.19,20 Prior to having reference amino acid chelates, several investigators attempted to develop analytical procedures that would measure a characteristic of a known chelate in the mistaken belief that if such a characteristic existed in the product being analyzed, it would prove that product was chelated, even though certain nonchelated Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 The Absorption and Metabolism of Amino Acid Chelates 235 mineral sources exhibited the same characteristics One simple technique was to mix a suspected chelate with water, filter the slurry, and then analyze the filtrate solution for metal content The belief was that the unbound metal would ionize in solution and be recovered in the filtrate solution, whereas the insoluble chelated mineral would be trapped in the insoluble precipitate.21,22 It was ultimately concluded, however, that there was no way of determining if the bound mineral in the precipitate was chelated or attached to some other type of molecule that also resulted in insoluble mineral products.21 Further, depending on the ligand used for chelation as well as the temperature of the water mixed with the suspected chelate and the resulting pH of the solution, more or less of the amino acid chelate could potentially solubilize and thus would become part of the filtrate solution, which would then be erroneously included as part of the ionized metal content Other investigators tried to develop a more sophisticated modification of this technique They subjected both the filtrate solution and the precipitate not only to a metal analysis but also to a nitrogen analysis using the Kjeldahl method.23 The assumption was that analysis for nitrogen would indicate how much of the amino acid ligand was present in each of the components While this method could demonstrate the percentages of metal and nitrogen in both the filtrate solution and the precipitate, it did not show how these two essential components were related to each other The metal may have indeed been chelated to nitrogenous matter (the amino acid), as the investigators tried to advocate with this analytical technique, but the metal could just as easily have been complexed to a protein molecule or even exist as an inorganic metal salt that had simply been admixed with the protein This analytical method did not ascertain the type of bonds that existed between the ligand and the metal ion that was the defining feature of an amino acid chelate Still other researchers attempted to employ an ultrafiltration technique to determine chelation.24 Ultrafiltration required solubility In an attempt to solubilize the product under investigation, these investigators added ethylenediaminetetraacetic acid (EDTA) to the solution containing the insoluble mineral product they suspected of being an amino acid chelate While they were somewhat successful in putting the mineral product into solution, the EDTA, with its higher potential formation constant, had a tendency to pull the metal away from the proteinaceous ligands and form EDTA chelates instead Since an EDTA chelate is a small molecular weight chelate, it was able to pass through the filter and be recovered in the filtered solution The remaining precipitate, which would not go into solution and thus not pass through the ultrafilter, was supposed to be an amino acid chelate or metal proteinate, but the precipitate could not be proven to be a chelate since the EDTA had removed much of the original mineral from the product Paper chromatographic assays also failed to prove chelation because, as indicated, not all of the analyzed chelates were soluble.25,26 When EDTA was utilized to solubilize the product just as in the case of the ultrafiltration procedure, it removed the metals from amino acid chelates or complexes and thus destroyed the products being targeted for analysis.16 Polarography was another laboratory procedure that tried to prove chelation.26,27 While polarographic analysis could demonstrate whether a reaction had occurred Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 236 Amino Acid Chelation in Human and Animal Nutrition between the metal and the ligand in the samples being analyzed, polarography could not definitely prove what type of molecule that reaction had produced, if it occurred at all The product could have been an amino acid chelate, or it could have just as easily been an amino acid complex Or, it may have been the metal ion reacting with an inorganic anion.27 In forming any of these molecules, a chemical reaction would likely have taken place, and the polarographic analysis would have reported the occurrence of that reaction Unfortunately, it did not identify the type of reaction observed as a result of the polarographic analysis Like polarography, nuclear magnetic resonance (NMR) spectroscopy could also demonstrate that a reaction had taken place between the metal and the ligand in the samples assayed.28 It was the consensus of the investigators who developed this analytical technique that NMR could demonstrate chelation, but the analysis required that the sample submitted for analysis be soluble Unfortunately, not all amino acid chelates are equally soluble Thus, NMR at that time was not a viable analytical procedure to prove chelation Another analytical procedure, electron paramagnetic resonance (EPR) spectroscopy, could demonstrate that a sample was chelated by comparing the EPR spectra of the mineral product to the spectra of a known amino acid chelate that occurred in nature.29 Use of this procedure illustrated one essential requirement for a peerreviewed analytical procedure that proved chelation The spectra of known amino acid chelates had to be used as reference spectra At the time that EPR was studied, reference spectra for all amino acid chelate metals did not exist Further, EPR was only applicable for analysis of transitional metals, so as an analytical tool to prove chelation, EPR was not particularly viable Following several years of investigation, Hartle and Ashmead published a summary of their team’s research Titled, “Bonds Important for Amino Acid Chelates,” these authors wrote: Physical characteristics alone (solubility, percent nitrogen and approximate molecular weights) not confirm the existence of chelation … The only way to verify that an amino acid chelate exists is to look at the bonds [between the metal and the amino acid ligand].30 These investigators, along with others on their team, had been able to grow pure amino acid chelate crystals The crystals were subsequently analyzed by x-ray crystallography, which determined the angles of the bonds between the amino acid ligands and the metal, the lengths of those bonds, and finally, the steric orientations of the various atoms within the chelate molecule.31 That analysis proved that the crystalline metallic molecules that they subjected to x-ray crystallography analysis were truly in the form of amino acid chelates Further, these same crystals were later subjected to electrospray mass spectroscopy, which expanded the elucidation of their structures besides confirming chelation.20 Once it was proven that these crystals were true amino acid chelates, additional samples from the same amino acid chelate crystals were submitted for analysis by Fourier transform infrared (FT-IR) spectroscopy.30 In this way, FT-IR reference spectra developed from known amino acid chelates could be compared against other Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 The Absorption and Metabolism of Amino Acid Chelates 237 suspected amino acid chelates to prove whether the unknown sample was a chelate or some other type of metallic molecule This FT-IR procedure was submitted to the AOAC using their single-laboratory validation methology.13 Knowing whether one was employing a true amino acid chelate in a research project is extremely important, particularly so in studies describing how an amino acid chelate is absorbed or metabolized because “amino acid chelate bonding characteristics are what set it apart as a highly bioavailable mineral source.”30 If one does not absolutely know if he or she is working with a true amino acid chelate, how then can one ascribe the absorption characteristics that are observed as being due to the chelation of that metal? The same is true as it relates to the unique metabolism of an amino acid chelate To claim that the metabolism of a particular metal source is due to it being chelated with amino acids, one must ensure that he or she is working with a true amino acid chelate and not a complex or some other metal form One of the characteristics of a true amino acid chelate is that it must have a coordinate covalent bond between the metal ion and the nitrogen on the amino group This bond will form at certain pH values and dissociate when subjected to other pH values.32 Further, this forming and breaking of the bond between the amino group of the ligand and the metal ion in the chelate molecule will occur at different pH values depending on the amino acid ligand employed in the chelation process.33 If the stability constant of the amino acid chelate is lower due to the selection of an amino acid ligand with a lower potential formation constant, then that amino bond will break in a less-extreme acidic or alkaline environment compared to the amino bond in an amino acid chelate formed with the higher stability constant This assumes, of course, that the only variable is the amino acid ligand Other environmental variables can and influence the stability of the chelate.34 This effect of the pH environment on an amino acid chelate is illustrated in Figure  15.1 When a low-acid pH causes the bond between the metal ion and the nitrogen in the amino moiety to be broken, the reconfigured molecule acquires a positive charge on each of its amine termini due to a donation of a hydrogen ion from the acid environment to the nitrogen The metal ion continues to remain attached to the amino acid ligand through the carboxyl bond even though the amino bond has been broken In this state, the metal is no longer chelated, but it still remains complexed to the amino acid ligand and has been termed an amino acid chelate/complex in this treatise to distinguish it from the amino acid complex that is defined by AAFCO.3 The formation of the amino acid chelate/complex is essential for active transport of this intact molecule across the mucosal cell membrane from the lumen to occur While facilitated diffusion across the membrane of an amino acid chelate can also take place, the formation of an amino acid chelate/complex is not always a pre­ requisite for that absorption to take place Further, there may occasionally be a small portion of an ingested dose of an amino acid chelate that is hydrolyzed in the lumen and the released metal ion absorbed similarly to metal ions from ingested metal salts Most of the amino acid chelate, however, that is ingested is absorbed by either facilitated diffusion or active transport.35 Both facilitated diffusion and active transport require carrier molecules to translocate the chelate and some chelate/complexes, in the case of facilitated diffusion Neither transfer process can happen unless the amino acid chelate or chelate/complex is able to be attached to the carrier molecule 238 Amino Acid Chelation in Human and Animal Nutrition O Alkaline pH of approximately and above Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 O H2N Zn O NH2 O O Neutral pH ± approximately Zn O O O +H N O Zn NH3+ O Acidic pH of approximately or below FIGURE 15.1  The effect of pH on the amino bond of a zinc bisglycinate chelate through an electronic attraction between the two molecules The carrier molecules for facilitated diffusion or active transport are different When the amino acid chelate/complex is attached to an active transport molecule, the two are taken into the cell through an enzymatic process that requires energy No energy is required for facilitated diffusion to occur Movement into the mucosal cell is dependent on a concentration gradient The carrier molecule may be water, a protein molecule, or some other ion or molecule In either case, the amino acid chelate is carried into the mucosal cell as an intact chelate or chelate/complex molecule Active transport requires an acid pH environment of approximately or below At that pH, the amino bond between the metal ion and the ligand is broken, as described, and the amine moiety on the terminal end of the amino acid ligand picks up a hydrogen ion to form NH3+.32 It is only then that the ligand of the chelate/­complex molecule acquires a positive charge and can be bonded to the negative terminus of the active transport molecule When the negative charge on the transport molecule is balanced by the positive charge, as a result of attaching the amino acid chelate/complex to the transport molecule, it initiates the enzymatic reactions, including the utilization of cellular-produced energy, that are necessary to translocate the chelate/­complex across the mucosal cell membrane.36,37 The amino acid chelate/complex that is absorbed by active transport follows the same active transport pathway employed by amino acids or peptides because amino acids and peptides behave similarly to an amino acid chelate/complex in an acid environment 239 The Absorption and Metabolism of Amino Acid Chelates Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 Lumen Amino Acid Chelate/Complex (glutathione) γ-glu-cysH-gly Gsh Synthetase γ-glutamyl Transpeptidase Membrane ADP + Pi cysH-gly γ-glutamyl-amino acid chelate/complex γ-glutamyl Cyclotransferase glycine Peptidase γ-glu-cysH cysteine γ-glu-cysh Synthetase 5-oxoproline Cytoplasm 5-Oxoprolinase ATP ATP glutamate ADP + Pi ATP ADP + Pi Amino Acid Chelate or Amino Acid Chelate/Complex FIGURE 15.2  The steps for the absorption of an amino acid chelate/complex across the intestinal cell membrane by active transport (Redrawn from Ashmead, HD, “Summary and conclusions,” in Ashmead, HD, ed., The Roles of Amino Acid Chelates in Animal Nutrition (Park Ridge, NJ: Noyes) 464, 1993.) Meister et al elucidated this active transport mechanism.36 As Figure 15.2 suggests,37 through the positive charge on its amino moiety, the amino acid chelate/ complex is bonded to the negative charge on the glutathione molecule to form γ-­glutamyl-amino acid chelate/complex plus cysteinyl-glycine This initiates the commencement of the enzymatic reactions necessary to move the chelate/­complex by active transport across the mucosal membrane The cysteinyl-glycine that results from the transpeptidation reaction of the glutathione is cleaved by dipeptidase to cysteine and glycine, which later are utilized in the re-formation of the glutathione The γ-­glutamyl transpeptidase, which is located in the brush border of the mucosal cell membrane, then acts on that glutathione and the γ-­glutamyl amino acid chelate/­ complex The resulting γ-­glutamyl amino acid chelate/complex is imbued with energy from the degradation of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) This energy is released as a result of removing the cysteinyl-glycine from the molecule The released energy is expended moving the γ-­glutamyl amino acid chelate/complex across the cell membrane into the cytoplasm Once in the cytoplasm, the γ-­glutamyl amino acid chelate/complex is hydrolyzed into 5-oxoproline plus the amino acid chelate/complex by the action of γ-­glutamyl cyclotransferase The γ-­glutamyl amino acid chelate/complex that was previously formed by the transpeptidation is subsequently converted to 5-oxoproline (which is also known as pyroglutamate or pyrrolidone carboxylate) plus the amino acid chelate/complex by the action of γ-­glutamyl cyclotransferase At the conclusion of these enzymatic reactions, the amino acid chelate/complex will have completed its active transport Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 240 Amino Acid Chelation in Human and Animal Nutrition crossing of the mucosal cell membrane and will be released into the cytoplasm of the cell, where it is then free to enter into certain assigned metabolic activities within the mucosal cell or be transferred, intact, to the plasma, while the 5-oxoproline becomes the starting material to re-form another molecule of glutathione The remainder of the reactions in the γ-­glutamyl cycle, which are illustrated in Figure 15.2, are directed toward resynthesizing the degraded glutathione and renewing the energy source for the active transport of another amino acid, peptide, or amino acid chelate/­complex molecule.36,38 The absorption of an amino acid chelate/complex by active transport can potentially occur at almost any point in the stomach or the small intestine Just as the stomach is capable of absorbing free amino acids,38 it is also capable of absorbing amino acid chelates/complexes.39,40 When introduced into the small intestine, the absorption of the amino acid chelate/­complex can continue in the acidic environment of the duodenum As the luminal pH changes from an acid to an alkaline environment near the common bile duct, the amino acid chelate/complex generally reconfigures back into an amino acid chelate At a neutral pH of plus or minus approximately 2, the amino acid chelate does not carry a charge because the molecule is in a chelated form.32,33 If intestinal motility moves the chelate near the luminal wall where the pH remains acidic, the chelate may again reconfigure into an amino acid chelate/complex.41 If the pH environment continues to remain close to neutral and does not favor the formation of an amino acid chelate/complex, the amino acid chelate can still be absorbed into the mucosal cell by solute flow or by facilitated diffusion if the concentration of the amino acid chelate becomes high enough to initiate diffusion Diffusion, in this case, is based on the amino acid concentration on both sides of the mucosal cell membrane The metal ion within the chelate molecule is unreactive36 and does not appear to play a major role in the diffusion of an amino acid chelate As the amino acid chelate enters the alkaline environment of the small intestine (the jejunum and ileum), the higher pH environment in that area of the lumen can potentially cause the amino bond of each amino acid ligand to again be broken Because the pH is alkaline, the amino moiety cannot acquire a positive charge by picking up an extra hydrogen ion Thus, the necessary ion attraction between the amino acid chelate/complex molecule and an active transport molecule cannot occur in an alkaline environment.32 The bonding of the two molecules can only take place in an acid environment where the terminal end of the amino moiety gains a positive charge by picking up an extra H+ Since most of the lumen is alkaline, this could create a potential problem for the active transport or some types of facilitated diffusion of the amino acid chelate/complex across the absorptive cell membrane The problem is resolved in the microenvironment near the membrane on the luminal side of the mucosal cell The positive charges on integral and peripheral proteins embedded in the membrane of the mucosal cell41 can potentially attract the amino acid chelate/complex molecules via ion-dipole forces As the amino acid chelate/complex formed in the alkaline environment of the lumen approaches the mucosal cell membrane, it will enter a pH environment between the microvilli of the mucosal cell in the unstirred water layer that remains acidic in spite of the alkaline pH of the lumen as a whole.42 This Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 The Absorption and Metabolism of Amino Acid Chelates 241 microacidic environment can cause the terminal end of the amine moiety to acquire a positive charge as a result of gaining a hydrogen ion, thus allowing the chelate/ complex to bond to an active transport molecule that is embedded in the mucosal cell membrane and initiate active transport Once the amino acid chelate or amino acid chelate/complex has been taken up by the absorptive cell by either active transport or facilitated diffusion, its fate is dependent on the degree of need of the body for the metal that is attached to the ligand as well as the amino acids in the ligand itself Some intracellular hydrolysis of the amino acid chelate or chelate/complex molecule into the metal ion and amino acid ligands can, and generally does, occur in the mucosal cell If the chelate or chelate/ complex is partially or totally hydrolyzed, the released metal can be sequestered into an intracellular storage molecule, such as apoferritin, or the metal ion can be bound to a cellularly produced transporter, such as transferrin, both in the case of iron, and moved to a site of need throughout the body.35 Besides the stability constant of the amino acid chelate or chelate/complex that enters the mucosal cell and the pH environment in the cytoplasm, the rate of intracellular hydrolysis of the chelate/complex in the absorptive cell partially depends on the need of the body for that specific metal at the time that the molecule is absorbed If the need of the body is substantial, more of the absorbed metallic molecules will be rapidly transferred, as an intact amino acid chelate or chelate/complex, directly into body tissues and organs If the mineral need is not as great, less of the intact molecule will be transferred to the plasma, and more will be hydrolyzed in the mucosal cell with the resultant storage of the metal ion Movement of most of the metal into the plasma from the absorptive cell is controlled by metabolic reactions that occur within the mucosal cell When the metal is removed from the amino acid ligand, the freed metal ion is subject to the same conditions as is an inorganic metal ion that has been absorbed into the absorptive cell.43 If the metal ions are separated from their amino acid ligands following cellular uptake and hydrolysis, in times of greater mineral need more transporter molecules will be produced in the cytoplasm of the absorptive cell to facilitate increased movement of the released cations into the plasma When the need for that metal is less, an increased amount of the metal ions will remain sequestered within the mucosal cell due to a lower production of transporter molecules.44 Further, the movement of metal ions from hydrolyzed amino acid chelates and chelate/complexes out of the mucosal cell into the plasma will occur at about the same rate as would the transfer of metal ions that were absorbed from an inorganic metal salt source The rate of movement in both cases is determined by the need for that particular metal by the body (Figure 15.3).45 Figure  15.3 summarizes a previously described study in which 40 infants and young children, all suffering from severe iron deficiency anemia, were matched for sex, age, and hemoglobin values before assignment to two groups Each child received a daily dose of mg of iron as either ferrous sulfate or iron bisglycine chelate per kilogram of body weight for 28 days During the test period, both groups exhibited significant improvements in their hemoglobin levels (p < 0.001) The regression analysis shown in Figure 15.3, however, indicated that the uptakes of both sources of iron into hemoglobin were similarly regulated by the body according to 242 Amino Acid Chelation in Human and Animal Nutrition Change in Hemoglobin, g/dL Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 Ferrous Bis-Glycinate chelate R = –0.6611 Ferrous Sulfate R = –0.8627 4.5 Basal Hemoglobin, g/dL 10 11 FIGURE 15.3  Regression analysis demonstrating that iron absorption values are inversely proportional to hemoglobin levels, regardless of the iron source (Redrawn from Pineda, O, and Ashmead, HD, “Effectiveness of treatment of iron-deficiency anemia in infants and young children with ferrous bis-glycinate chelate,” Nutrition 17:381–384, 2001.) changes in hemoglobin This suggested that while absorbed in greater amounts, a substantial portion of the amino acid chelate that was taken up by the mucosal cells was probably hydrolyzed while still in the mucosal cells before the iron was utilized to produce more hemoglobin molecules This is the only way that the regulatory process illustrated in Figure 15.3 could occur.45 As the hemoglobin levels increased, the uptake of iron from either source decreased similarly It should also be remembered that, in this example, the hemoglobin values controlled the rates of transfer of both sources of iron The significant difference between the two groups is not that hemoglobin repletion was similarly regulated in both groups The infants and young children used in this study also had ferritin levels that were severely depleted The ferritin values were only repleted in those subjects receiving the amino acid-chelated source of iron Iron uptake into the hemoglobin molecules was controlled by the rate of iron saturation in the hemoglobin When hemoglobin values attained sufficiency, iron transfer to the plasma from the mucosal tissue became very low (more ionic iron storage in the mucosal cells with resulting lower transfer of that form of iron to the plasma) Concurrently, however, it appeared that additional iron, absorbed into the mucosal tissue from the amino acid chelate source, was transferred to the ferritin molecules to replete their depleted status, possibly as intact amino acid chelate/complex molecules The apparent bioavailability of the iron from the amino acid chelate was calculated to be 90.9% compared to 26.7% for the iron from the ferrous sulfate Thus, in a time of acute need, more iron from the amino acid chelate was available to address all of the iron deficiency issues, including ferritin levels, compared to ferrous sulfate because some of the amino acid chelate was not hydrolyzed in the mucosal cell but transferred intact to the area of need Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 The Absorption and Metabolism of Amino Acid Chelates 243 Guthrie has suggested that nonheme iron ions must be chelated with amino acids that are sourced from the chyme prior to their being absorbed into the mucosal cell.44 If the body has an immediate need for that absorbed iron, transferrin in the plasma will come in contact with the mucosal cell The iron will be complexed, taken up from the intestine, and carried across the mucosal cell by the same amino acids that initially complexed the iron in the lumen The iron will immediately be released on the other side of the basement membrane to combine with the plasma transferrin The iron-transferrin complex can carry two atoms of iron per molecule of transferrin to the body cells requiring the iron When the available transferrin is about 33% saturated with iron, it will not pick up additional iron from the mucosal cells Instead, the ferrous iron is released from the amino acids that initially chelated it in the lumen, oxidized to ferric iron, and stored within the apoferritin molecule within the mucosal cell.44 While there may be some disagreement with the idea that all nonheme iron ions must first be chelated to amino acids from chyme prior to mucosal cell absorption, the rest of the model proposed by Guthrie could easily apply to the description of the fate of a significant portion of the amino acid chelate/complex that is absorbed into the mucosal cell The foregoing is not meant to imply that the entire dose of the amino acid chelate or chelate/complex that is taken up by the mucosal cell must be hydrolyzed prior to the metal being transferred to the plasma Some of the absorbed amino acid chelate or chelate/complex remains intact and is transferred into the plasma as such If one ignores the discussion relating to the need for a metal by the body, the percentage of amino acid chelate or chelate/complex not hydrolyzed in the mucosal cells is, to an extent, dictated by the stability constant of the molecule The higher the stability constant of the chelate or chelate/complex is, the smaller will be the percentage of mucosal cell hydrolyzation of that molecule Thus, using iron as an example, more of an iron methionate chelate will be hydrolyzed within the mucosal cells than will be iron lysinate.35 This is because a bisamino acid chelate formed from iron and methionine has a stability constant of 6.4810 compared to 9.010 for an iron bislysinate.46 Stability constants can also be dictated by the size of a chelate ring The larger the number of members in the chelate ring, the lower the stability constant of that chelate and the greater is its potential for intracellular hydrolyzation, assuming that the ­chelate/complex is absorbed intact Thus, small amino acid ligands tend to form stronger chelates than larger amino acids The number of rings of the re-formed chelate will also influence its stability constant As the number of chelate rings increases, so does the stability of the chelate and the lower the rate of mucosal cell hydrolysis will be And finally, the basicity of the amino acid will affect its stability The more basic the ligands forming the chelate, the stronger the chelate will be.34 From the discussion, one could conclude that more of an amino acid chelate that is created with more than one highly basic smaller amino acid ligand would likely be transferred to the plasma intact compared to a chelate of the same metal created with larger and less basic amino acid ligands There are several studies suggesting that some portion of an ingested amino acid chelate will arrive in the plasma intact regardless of the amino acid ligand employed.47–49 Indeed, in an in vitro double-isotope study, in which both the metal Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 244 Amino Acid Chelation in Human and Animal Nutrition and the amino acid ligand were tagged,35 it was estimated that, in that particular study, approximately 50% of the absorbed amino acid chelate was transferred to the serosa intact.50 The delivery of intact amino acid chelate or chelates/complexes to the tissue or organ results in a different biological response compared to delivery of the same minerals ingested as inorganic metal salts While these differences can be partially attributed to the greater absorption of the amino acid-chelated form of minerals, that does not account for all of the difference For reasons not completely elucidated, tissue retention of metals from ingested amino acid chelates is greater than is tissue retention of those same metals when they are ingested as inorganic metal salts.51–53 This increased retention allows for greater metabolic activity related to the metal atom in the chelate and probably its amino acid ligand As additional amino acid chelate or chelate/complex molecules are delivered, retained, and begin to initiate metabolic activity, the overall performance of the organism increases over time In a previously discussed example, Ashmead and Samford conducted a 3-year study in dairy cows from the same herd.54 Sixty-four animals received a formula containing magnesium, zinc, manganese, and copper as amino acid chelates, and 65 other cows matched for age, initial milk production, parity, and genetics received the same mineral supplement formula except the minerals were provided as inorganic metal salts.54 The mineral supplementation program, the feeding program, and so on remained the same for all of the animals in both groups throughout the entire three lactations The only difference between the two groups of animals was the source of the minerals provided in their supplement program The supplements were isomineral and isonitrogenous As Table 15.1 summarizes,54 in each successive lactation period, the difference in milk, milk fat, and protein production between the two groups became increasingly greater The initial biological response difference seen in Table 15.1 could only be TABLE 15.1 Mean Percentage Increases in Milk and Fat and Protein Production between Dairy Cows Receiving Amino Acid Chelates versus Inorganic Metal Salts Milk production Milk fat Milk protein Lactation Lactation Lactation +0.3% –3.2% N.D.* +2.6%a +0.8%a +1.8% +11.4%a +14.2%a +10.2%a Source: Redrawn from Ashmead, HD, and Samford, RA, “Effects of metal amino acid chelates or inorganic minerals on three successive lactations in dairy cows,” Int J Appl Res Vet Med 2:181–188, 2004 a Differences from previous lactation are significant (p < 0.05) * Not determined Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 The Absorption and Metabolism of Amino Acid Chelates 245 due to the greater absorption resulting from ingestion of the chelated source of minerals and their subsequent increased tissue retention, but even that difference between the two groups did not remain consistent from lactation period to lactation period If the initial difference in mineral uptake and tissue retention of the absorbed minerals had remained the same for each successive lactation period, then one would have predicted that the percentage differences in milk, fat, and protein production in lactation periods and would have been similar to the differences observed in the first lactation period Instead, the differences between the two groups of cows became increasingly greater with each successive lactation period, indicating increasingly greater mineral retention in the animals receiving the amino acid-chelated source, which allowed for the greater mineral-related metabolic activity Because a portion of the amino acid chelate/complex can be taken up by the plasma as an intact molecule, there is another benefit attached to the ingestion of this source of minerals: the ability to prepare amino acid chelates that can deliver more of a desired metal to a target tissue or organ.55,56 Certain tissues or organs require more of a specific amino acid for their metabolic purposes than certain other tissues or organs For example, arginine and zinc are both required in significant amounts for sexual activity in the male.57,58 When the zinc is chelated to the arginine, more of that zinc can potentially be delivered to the male sex organs than when the zinc is chelated to certain other amino acids.56 Certainly, some of the absorbed chelated zinc arginate in the example will be hydrolyzed into zinc ions and arginine following uptake into the mucosal cells And just as certainly, some of that absorbed zinc arginate will survive that mucosal hydrolysis and be delivered intact into the plasma When that occurs, the surviving zinc arginate will potentially be distributed to various tissues and organs throughout the body as an intact molecule But, since there is a greater requirement for arginine by the male sex organs, and because the zinc remains attached to the arginine as the zinc arginate is introduced into the plasma, more of the zinc will be delivered to the male sex organs along with the arginine.56 Of the zinc arginate that was hydrolyzed in the mucosal cell, some of that free arginine and some of that free zinc could also be sent to the male sex organs The difference is that these two individual components will be delivered separately and not as a zinc arginate One of the main problems arising from the separate delivery of the two components is that, following mucosal cell hydrolysis of zinc arginate, the rates of exit of the metal and the amino acid from the mucosal cells into the serosa are not the same.35 Thus, the metal-ligand ratio in the plasma may not be as conducive to the re-formation of a zinc arginine complex in the sexual organs and tissues If the metal and amino acid not balance each other out for purposes of metabolic activity, the nutrient that is in excess will probably be excreted as endogenous, with resulting lower overall metabolic activity as it relates to these two nutrients In conclusion, the ingestion of a proven amino acid chelate will result in greater absorption of that metal with consequentially enhanced mineral-related metabolism due to the bonding of the metal to the amino acid ligands Chelating the metal with amino acids protects it or reduces the probability of the metal entering into absorption-limiting reactions while residing in the gastrointestinal tract Most of the ingested amino acid chelate can be absorbed intact into the mucosal cells Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 246 Amino Acid Chelation in Human and Animal Nutrition from the lumen Intracellular hydrolysis will probably destroy part of the chelate or chelate/complex that has been delivered to the cytoplasm of the mucosal cell That metal derived from that intracellular hydrolysis will be treated as if it had been absorbed similarly to the absorption of a metal ion The remainder of the amino acid chelate or chelate/complex that is not hydrolyzed in the mucosal cell will traverse the mucosal cell and enter the plasma intact Following entry into the plasma, the amino acid chelate or chelate/complex will be carried to areas of need within the body There, the tissues and organs will take up the amino acid chelate or chelate/complex as if it were a proteinaceous metallic molecule that originated from ingested food As a result of this process and the higher absorption rate of the amino acid chelate, metabolic activity associated with the mineral and its ligand is augmented for a longer period of time REFERENCES Werner, A, “Beitrag zur constitution Anaorganisiher Verbindungen,” Z Anorg U Allgem Chem 3:267, 1983 Mellor, DP, “Historical background and fundamental concepts” in Dwyer, FP, and Mellor, DP, eds., Chelating Agents and Metal Chelates (New York: Academic Press) 20, 1964 Krebs, S, ed., Official Publication Association of American Feed 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701:13–15, March 31, 1997 24 Hynes, MJ, Colleran, D, Fin, J, Headon, DR, and Lyons, TP, “Quantitative chemical assessment of some mineral proteinates used in animal feeds,” J Animal Sci 68 (Supp1.):408, 1997 25 Little, P, and Ashmead, H, “Metal ionization chromatography,” unpublished research report, Albion Laboratories, Clearfield, UT, 1976 26 James, H, “A differential pulse polarography study of zinc glycinate chelate,” unpublished research report, Weber State University, Ogden, UT, 1987 27 Holwerda, RA, Albin, RC, and Madsen, FC, “Chelation effectiveness of zinc proteinates demonstrated,” Feedstuffs 19:12–13, June 19, 1995 28 Olsen, JI, and Schweized, MP, “NMR analysis of Chelazome® products,” unpublished research report, University of Utah, Salt Lake City, UT, 1987 29 Rogers, K, and Landcaster, J, “Report on electron paramagnetic resonance spectra of Albion® metal amino acid chelates,” unpublished research report, Utah State University, Salt Lake City, UT, 1985 30 Hartle, JW, and Ashmead, HD, “Bonds important for amino acid chelates,” Feedstuff 78:16–17, September 11, 2006 31 Dalley, NK, “Report on x-ray diffraction crystallography of Albion® zinc amino acid chelate,” unpublished research report, Brigham Young University, Provo, UT, 1985 32 Ericson, C, and Ashmead, SD, “A novel approach in confirming dietary amino acid chelates by the utilization of ninhydrin,” poster presentation at American Chemical Society Annual Meeting, St Louis, MO, September 2004 33 Ericson, C, “Derivatization of ninhydrin with zinc bismethionate chelate,” unpublished research report, Albion Laboratories, Clearfield, UT, 2009 34 Kratzer, FH, and Vohra, P, Chelates in Nutrition (Boca Raton, FL: CRC Press) 25, 1986 35 Ashmead, HD, Graff, DJ, and Ashmead, HH, Intestinal Absorption of Metal Ions and Chelates (Springfield, IL: Thomas) 1985 36 Meister, A, Tate, SS, and Thompson, GA, “On the function of the γ-glutamyl cycle in the transport of amino acids and peptides,” in Elliott, K, and O’Connor, M, eds., Peptide Transport and Hydrolysis (Amsterdam: Elsevier) 123–150, 1977 37 Ashmead, HD, “Summary and conclusions,” in Ashmead, HD, ed., The Roles of Amino Acid Chelates in Animal Nutrition (Park Ridge, NJ: Noyes) 464, 1993 38 White, A, Handler, P, and Smith, EL, Principles of Biochemistry (New York: McGrawHill) 633, 1973 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:18 17 November 2015 248 Amino Acid Chelation in Human and Animal Nutrition 39 Kornegay, ET, Swinkels, JWGM, Webb, KE, and Lindermann, MD, “Absorption of zinc amino acid chelate and sulfate during repletion of zinc depleted pigs,” in Anke, M, Meissner D, and Mills, CF, eds., Trace Elements in Man and Animals—TEMA8 (Gersdof, Germany: Verlag media Touristik) 398–399, 1993 40 Kim, SW, “Zinc amino acid chelates as upgraded zinc sources for monogastric animals,” paper presented at Albion Animal Nutrition Conference, Midway, UT, January 2007 41 Giese, AC, Cell Physiology (Philadelphia: Saunders College) 192, 1973 42 de Robertis, EDP, Saez, FA, and de Robertis, EMF, Cell Biology (Philadelphia: Saunders) 158–165, 1975 43 Jeppsen, RB, “Biochemistry and physiology of Albion® metal amino acid chelates as proof of chelation,” Proceedings of Albion Laboratories, Inc International Conference on Human Nutrition, Salt Lake City, UT, January 1995 44 Guthrie, HA, Introductory Nutrition (St Louis, MO: Times Mirror/Mosby College) 293–294, 1989 45 Pineda, O, and Ashmead, HD, “Effectiveness of treatment of iron-deficiency anemia in infants and young children with ferrous bis-glycinate chelate,” Nutrition 17:381–384, 2001 46 Sillen, LG, and Martell, AE, Stability Constants of Metal-Ion Complexes (London: Chemical Society) 1964 47 Bovell-Benjamin, AC, Viteri, FE, and Allen LH, “Iron absorption from ferrous bisglycinate and ferric trisglycinate in whole maize is regulated by iron status,” Am J Clin Nutr 71:1563–1569, 2000 48 Ashmead, D, and Graff, D, “Placental transport of chelated iron,” Proceedings of the International Pig Veterinary Society, Mexico City, Mexico, July 1982 49 Martin, RB, “Complexes of g-amino acids with chelatable side chain donor atoms” in Sigel, H, ed., Metal Ions in Biological Systems (New York: Dekker) v9, 1–37, 1979 50 Pineda, O, personal communication, 2001 51 Ashmead, HD, Ashmead SD, and Samford, RA, “The effects of metal amino acid chelates on milk production, reproduction and body condition in Holstein first calf heifers,” Int J Appl Res Vet Med 2:252–260, 2004 52 Jensen, NL, “Biological assimilation of metals,” U.S Patent 4,167,564 Washington, DC, September 11, 1979 53 Ashmead, HD, “Comparative intestinal absorption and subsequent metabolism of metal amino acid chelates and inorganic salts,” in Subramanian, KS, Iyengar, GV, and Okamoto, K, eds., Biological Trace Element Research (Washington, DC: American Chemical Society) 306–319, 1991 54 Ashmead, HD, and Samford, RA, “Effects of metal amino acid chelates or inorganic minerals on three successive lactations in dairy cows,” Int J Appl Res Vet Med 2:181–188, 2004 55 Ashmead, HH, and Graff, DJ, “Zinc amino acid chelates and the male sex organs,” monograph, Albion Laboratories, Clearfield, UT, July 1987 (unpublished) 56 Ashmead, HH, Ashmead, HD, and Graff, DJ, “Amino acid chelated compositions for delivery to specific biological tissue sites,” U.S Patent 4,863,898, Washington, DC, September 5, 1989 57 Fahim MS, Wang M, Sutcu MF, and Fahim, Z, “Zinc arginine, a alpha-reductase inhibitor, reduces rat ventral prostate weight and DNA without affecting testicular function,” Andrologia 25:369–375, 1993 58 Prasad, AS, “Deficiency of zinc and its toxicity” in Prasad, AS, ed., Trace Elements in Human Health and Disease (New York: Academic Press) v1, 312, 1976 [...]... 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 Histidine Glutamic Acid Lysine Cystine Glycine Alanine Phenylalanine Arginine Leucine Serine Valine Methionine Proline Isoleucine FIGURE 1.3 ... Luong Dinh Cua] at 23:14 17 November 2015 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... 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,... 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... 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 Downloaded by [Hanoi University of Agriculture], [Thu Vien Luong Dinh Cua] at 23:14 17 November 2015 Pantothenic Acid C (Ascorbic Acid) B6 (Pyridoxine) Niacin... 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... chelated minerals,” in Ashmead, D, ed., Chelated Minerals Nutrition in Plants, Animals and Man (Springfield, IL: Thomas) 275, 1982 11 Sauberlich, H, “Interactions of thiamine, riboflavin and other B-vitamins,” in Levander, O, and Cheng, L, eds., Micronutrient Interactions: Vitamins, Minerals and Hazardous Elements (New York: New York Academy of Sciences) 80, 1980 12 Arnich, L, and Arthur, V, “Interaction... 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. .. 97:321–326, 1969 21 EI-Shobaki, F, and Rummel, W, “Binding of copper to mucosal transferrin and inhibition of intestinal iron absorption in rats,” Res Exp Med 174:187–195, 1989 22 Ashmead, H, “Tissue transportation of organic trace minerals,” J Appl Nutr 22:42–51, Spring 1970 23 Herrick, JB, “Minerals in animal health,” in Ashmead, HD, ed., The Roles of Amino Acid Chelates in Animal Nutrition (Park Ridge, NJ:... 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 ... 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 The Fundamentals of Mineral Nutrition. .. requirements for isoleucine and leucine, they would then be utilized for valine Amino Acid Chelation in Human and Animal Nutrition E (Tocopherol) A (β-carotene) B1 (Thiamin) Riboflavin Downloaded by... 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