Amino Acids in Animal Nutrition

Second Edition

Amino Acids in Animal Nutrition

Second Edition

Edited by

J.P.F. D’Mello

Formerly of the Scottish Agricultural CollegeEdinburgh, UK

CABI Publishing

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Library of Congress Cataloging-in-Publication DataAmino acids in animal nutrition / edited by J.P.F. D’Mello.-- 2nd ed.

p. cm.Includes bibliographical references and index.

ISBN 0-85199-654-X (alk. paper)1. Amino acids in animal nutrition. I. D’Mello, J.P. Felix.SF98.A4 A44 2003636.08�52--dc21

2002011559

ISBN 0 85199 654 X

Typeset in 9/11 Souvenir Light by Columns Design Ltd, ReadingPrinted and bound in the UK by Biddles Ltd, Guildford and King’s Lynn

Contents

Contributors vii

Preface ix

Abbreviations xi

PART I: GENERAL ASPECTS1 Amino Acids as Multifunctional Molecules 1

J.P.F. D’Mello2 Amino Acid Analysis of Feeds 15

J. Fontaine3 Absorption of Amino Acids and Peptides 41

C.R. Krehbiel and J.C. Matthews4 An Outline of Pathways in Amino Acid Metabolism 71

J.P.F. D’Mello5 Amino Acid Metabolism in Animals: an Overview 87

B.J. Bequette6 Essential to Non-essential Amino Acid Ratios 103

J. Heger7 Adverse Effects of Amino Acids 125

J.P.F. D’Mello

PART II: PIGS8 Methionine–Cystine Relationships in Pig Nutrition 143

A.J. Lewis9 Ideal Dietary Amino Acid Profiles for Pigs 157

S. Boisen10 Digestible Amino Acids in Diet Formulation for Pigs 169

R. Mosenthin and M. Rademacher11 Modelling Amino Acid Metabolism and the Estimation of Amino 187

Acid RequirementsP.J. Moughan and M.F. Fuller

12 Amino Acid Utilization for Reproduction in Sows 203S.W. Kim and R.A. Easter

v

PART III: POULTRY13 Ideal Amino Acid Patterns for Broiler Chicks 223

D.H. Baker14 Responses of Growing Poultry to Amino Acids 237

J.P.F. D’Mello

PART IV: RUMINANTS15 Metabolism and De Novo Synthesis of Amino Acids by Rumen Microbes 265

C. Atasoglu and R.J. Wallace16 Modelling Amino Acid Metabolism in Ruminants 291

R.L. Baldwin, C.C. Calvert, P.H. Robinson and H.A. Johnson17 Amino Acid Utilization for Wool Production 309

S.M. Liu and D.G. Masters18 Amino Acid Utilization by Growing and Finishing Ruminants 329

E.C. Titgemeyer19 Mammary Uptake and Metabolism of Amino Acids by Lactating 347

RuminantsB.J. Bequette, M.D. Hanigan and H. Lapierre

20 Effects of Amino Acids on Milk Production 367D.G. Chamberlain and J.-M. Yeo

21 Predicting Dietary Amino Acid Adequacy for Ruminants 389D.G. Fox and L.O. Tedeschi

PART V: OTHER ANIMALS22 Canine and Feline Amino Acid Requirements for Different Physiological 411

FunctionsW.H. Hendriks

23 Amino Acid Requirements of Finfish and Crustaceans 427R.P. Wilson

PART VI: APPLICATIONS AND PERSPECTIVES24 Crystalline Amino Acids and Nitrogen Emission 449

M.W.A. Verstegen and A.W. Jongbloed25 Economic Assessment of Amino Acid Responses in Growing Poultry 459

M. Pack, D. Hoehler and A. Lemme26 Conclusions 485

J.P.F. D’Mello

Index 503

vi Contents

Contributors

Atasoglu, C. Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB219SB, UK. Present address: Department of Animal Science, Faculty of Agriculture,Canakkale Onsekiz Mart University, 17100 Canakkale, Turkey

Baker, D.H. Department of Animal Sciences, University of Illinois, 132 Animal SciencesLaboratory, 1207 West Gregory Drive, Urbana, IL 61801, USA

Baldwin, R.L. Department of Animal Science, University of California, Davis, CA95616–8521, USA

Bequette, B.J. Department of Animal and Avian Sciences, University of Maryland,College Park, MD 20742, USA

Boisen, S. Department of Animal Nutrition and Physiology, Research Centre Foulum, POBox 50, DK-8830, Tjele, Denmark

Calvert, C.C. Department of Animal Science, University of California, Davis, CA95616–8521, USA

Chamberlain, D.G. Hannah Research Institute, Hannah Research Park, Ayr KA6 5HL,UK

D’Mello, J.P.F. Formerly of The Scottish Agricultural College, West Mains Road,Edinburgh, EH9 3JG, UK

Easter, R.A. College of Agricultural, Consumer and Environmental Sciences, Universityof Illinois, 122 Mumford Hall, 1301 West Gregory Drive, Urbana, IL 61801, USA

Fontaine, J. Feed Additives Division, Degussa AG, Applied Technology 913-205, PO Box1345, D-63403 Hanau, Germany

Fox, D.G. Department of Animal Science, Cornell University, Ithaca, NY 14853, USAFuller, M.F. Institute of Food, Nutrition and Human Health, Massey University,

Palmerston North, New ZealandHanigan, M.D. Dairy Research Group, Purina Mills Inc., St Louis, MO 63144, USAHeger, J. Biofaktory Praha s.r.o., Generala Piky 3, 613 00 Brno, Czech RepublicJohnson, H.A. Department of Animal Science, University of California, Davis, CA

95616–8521, USAHendriks, W.H. Institute of Food, Nutrition and Human Health, Massey University,

Palmerston North, New ZealandHoehler, D. Degussa AG, Applied Technology Feed Additives, PO Box 1345, D-63457

Hanau, Germany

vii

Jongbloed, A.W. ID-TNO Animal Nutrition, Lelystad, The NetherlandsKim, S.W. Department of Animal and Food Sciences, Texas Tech University, Box 2141,

123 Animal Science Building, Lubbock, TX 79209, USAKrehbiel, C.R. Department of Animal Science, Oklahoma State University, Stillwater, OK

74078, USALapierre, H. Dairy and Swine R & D Research Centre, Lennoxville, Quebec, Canada J1M

1Z3Lemme, A. Degussa AG, Applied Technology Feed Additives, PO Box 1345, D-63457

Hanau, GermanyLewis, A.J. Department of Animal Science, University of Nebraska, Lincoln, NE 68583,

USALiu, S.M. CSIRO Livestock Industries, Private Bag 5, PO Wembley, WA 6913, AustraliaMasters, D.G. CSIRO Livestock Industries, Private Bag 5, PO Wembley, WA 6913,

AustraliaMatthews, J.C. Department of Animal Sciences, University of Kentucky, Lexington, KY

40546, USAMosenthin, R. Institute of Animal Nutrition, Hohenheim University, D-70593 Stuttgart,

GermanyMoughan, P.J. Institute of Food, Nutrition and Human Health, Massey University,

Palmerston North, New ZealandPack, M. Degussa AG, Applied Technology Feed Additives, PO Box 1345, D-63457

Hanau, GermanyRademacher, M. Feed Additives Division, Degussa AG, Applied Technology, Rodenbacher

Chaussee 4, PO Box 1345, D-63457 Hanau, GermanyRobinson, P.H. Department of Animal Science, University of California, Davis, CA

95616–8521, USATedeschi, L.O. Department of Animal Science, Cornell University, Ithaca, NY 14853,

USATitgemeyer, E.C. Department of Animal Sciences and Industry, 132 Call Hall, Kansas

State University, Manhattan, KS 66506, USAVerstegen, M.W.A. Department of Animal Sciences, Animal Nutrition Group,

Wageningen University and Research Centre, PO Box 338, 6700 AA Wageningen, TheNetherlands

Wallace, R.J. Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB219SB, UK

Wilson, R.P. Department of Biochemistry and Molecular Biology, Mississippi StateUniversity, Box 9650, Mississippi State, MS 39762, USA

Yeo, J.-M. Hannah Research Institute, Hannah Research Park, Ayr KA6 5HL, UK

viii Contributors

Preface

The previous edition of this book (published in 1994 with the title Amino Acids in FarmAnimal Nutrition) was well received by academic and commercial users and by critics undertak-ing reviews on behalf of international journals. Citation of individual chapters in variousresearch publications has also been a source of considerable satisfaction.

Amino acid metabolism and nutrition of farm animals continues to be an active area ofresearch, with new data now widely published in proceedings of international conferences andin journals. A new edition would, therefore, be justified for this reason alone. However, in addi-tion, there is a need to take into account advances in the amino acid nutrition of a wider rangeof animals, including companion animals. There is also a need to address changes in focus andto recognize the efforts of new scientists in the field and the role of our commercial colleagues. Iam now able to offer an enlarged version with the not too dissimilar title of Amino Acids inAnimal Nutrition.

In this new edition of Amino Acids in Animal Nutrition, I have attempted to retain chap-ters imparting strength to the first version, while introducing authors with new ideas and vision.I have also addressed comments I received from external reviewers, chiefly to do with overlap.The book is thematically structured. Part I includes chapters of an introductory and generalnature with applications to a wide range of animal species. The next four parts are species-related sections, including pigs (Part II), poultry (Part III), ruminants (Part IV) and other animals(Part V). The chapters in the final section (Part VI) cover applications and perspectives. A unify-ing theme emerging from these sections is the improved outlook for pure amino acids, againsta backdrop of restrictions in the use of protein feeds from animal sources. Another noticeablefeature of recent work is a significant shift from empirical supplementation studies to fundamen-tals such as signalling and molecular aspects. As ever, methodological innovations are the keyto improved understanding of the amino acid nutrition of animals; examples of recent advanceswill be found throughout all sections of this book. Comparative issues are given greater promi-nence in the new edition compared to the earlier version. The aim continues to be to improveexchange and integration of information across the species barrier. Many excellent reviews ondifferent aspects of protein and amino acid nutrition have appeared since the publication of thefirst edition. For example, two reviews on the measurement and significance of protein turnoverand inter-organ amino acid flux recently appeared in the book entitled Farm AnimalMetabolism and Nutrition published in 2000 by CABI Publishing.

ix

It is hoped that Amino Acids in Animal Nutrition will appeal to final year undergraduatesand students on Masters courses as a coherent synthesis of existing literature. Authors wereonce again asked to provide comprehensive reviews with a critical appraisal of current informa-tion and concepts and suggestions for further research. Since virtually all my authors areactively publishing refereed papers in their respective fields of research, readers can look for-ward to chapters with up-to-date information and thought-provoking ideas.

I am indebted to my team of authors who have made production of this book possible,despite their busy schedules. It is clear to me that they have invested considerable time in gath-ering and integrating data into lucid and comprehensive reviews. Their help with proof-readingand preparation of the index is also much appreciated.

The information set out within the individual chapters of Amino Acids in AnimalNutrition is presented in good faith. Although every effort has been made to verify the factsand figures, neither the editor nor CABI Publishing can assume responsibility for data pre-sented in individual chapters or for any consequences of their use. This book necessarily con-tains references to commercial products. No endorsement of these products is implied orshould be attributed to the editor or to CABI Publishing.

J.P.F. D’MelloEditor

x Preface

Abbreviations

xi

AA(s) amino acid(s)ADG average daily gainAFRC Agricultural and Food Research

CouncilAID apparent ileal digestibilityAOAC Association of Official Analytical

ChemistsASCT alanine, serine, cysteine (and

other neutral �-amino acids)transport

Asn asparagineAsp aspartateATP adenosine 5�-triphosphateBBMV brush border membrane vesiclesBBSRC Biotechnology and Biological

Sciences Research CouncilBCAA branched-chain amino acidsBCKA branched-chain keto acidsBLMV basolateral membrane vesiclesBV biological valueCAT cationic amino acid transportCit citrullineCNCPS Cornell Net Carbohydrate and

Protein SystemcNOS constitutive nitric oxide

synthaseCCK cholecystokinincDNA complementary DNACoA coenzyme ACP crude proteinCys cysteined day(s)DC digestibility coefficient

DCAM decarboxylated 5-adenosyl-methionine

DE digestible energyDM dry matterDMI dry matter intakeDNA deoxyribonucleic acidDopa 3,4-dihydroxyphenylalanineEAA essential amino acidsEAAT excitatory amino acid transportECP endogenous crude proteinEU European UnionEUN endogenous urinary nitrogenEUUN endogenous urinary urea nitro-

genFRS fractional synthetic rateGABA �-aminobutyrateGDH glutamate dehydrogenaseGH growth hormoneGIT gastrointestinal tractGln glutamineGlu glutamateGly glycineGly-Sar glycylsarcosineGS-GOGAT glutamine synthetase-glutamate

synthaseh hourHCl hydrochloric acidHI heat incrementHis histidineHMB 2-hydroxy-4-(methylthiol)butyric

acidHPLC high performance liquid chro-

matography

IAAP ideal amino acid patternIEC ion-exchange chromatographyIGF-1 insulin-like growth factor-1Ile isoleucineIMP inosine monophosphate (inosi-

nate)iNOS inducible nitric oxide synthaseLeu leucineLys lysineMCP microbial crude proteinMDV mesenteric-drained visceraME metabolizable energyMet methioninemin minutesMP metabolizable proteinN nitrogenNAD+ nicotinamide adenine dinu-

cleotide (oxidized)NADP+ nicotinamide adenine dinu-

cleotide phosphate (oxidized)NADPH nicotinamide adenine dinu-

cleotide phosphate (reduced)NDF neutral detergent fibreNE net energyNEAA non-essential amino acidsNIRS near-infrared spectroscopyNO nitric oxideNOS nitric oxide synthaseNP net proteinNPN non-protein nitrogen

NRC National Research CouncilNSC non-structural carbohydratesODC ornithine decarboxylasePDV portal-drained visceraPepT1 peptide transporter 1Phe phenylalaninePro prolinePRPP 5-phosphoribosyl-1-pyrophos-

phateRDP rumen degraded proteinRNA ribonucleic acidRPAA ruminally protected amino

acidsRUP rumen undegraded proteinSAA sulphur amino acidsSD standard deviationSer serineSMCO S-methylcysteine sulphoxideSPI soy protein isolateTDN total digestible nutrientstRNA transfer RNAThr threonineTID true ileal digestibilityTrp tryptophanTyr tyrosineUDP undegraded dietary proteinUMP uridine monophosphate (uridy-

late)Val valineVFA volatile fatty acids

xii Abbreviations

1 Amino Acids as MultifunctionalMolecules

J.P.F. D’Mello*Formerly of The Scottish Agricultural College,

Edinburgh, UK

Introduction

Reviews in animal nutrition conventionallyand necessarily focus on the involvement ofamino acids in protein synthesis and theirimpact on the production of meat, milk, wooland eggs. The nutritional classification isanother traditional feature due to dietaryrequirements for specific amino acids.However, the unique physiological and bio-chemical functions of particular amino acidsare equally critical. In this chapter, emphasis isplaced on amino acids as multifunctionalbioactive molecules. Their involvement in theprocesses of protein synthesis has beenreviewed repeatedly elsewhere and will not beconsidered here.

Classification

Amino acids may be grouped according totheir transport affinities or essential role inanimal nutrition or on the basis of catabolicfate of the carbon skeleton. Additional subsetsare also recognized, based on common struc-tural features. Thus, leucine, isoleucine andvaline are referred to as the branched-chainamino acids (BCAA), whereas phenylalanineand tyrosine are categorized as the aromaticamino acids. Assessments of requirements for

sulphur amino acids (SAA) are invariablybased on a summation of methionine andcyst(e)ine needs, but it will be recalled thathomocysteine is another member of thisgroup. Another subset, the excitatory aminoacids, includes glutamate and aspartate.

Transport groups

In the context of transport systems, threemain groups of amino acids may berecognized: anionic, cationic and neutral.Cellular protein synthesis and other metabolicprocesses demand uninterrupted delivery of allamino acids at appropriate sites. The study ofamino acid transport and associated systemsis thus of critical importance, for example, tothose interested in the processes of intestinalabsorption and brain function. Animals have awide array of cells and this diversity ismatched by a complex system of transportersof amino acids which operates on the basis ofsubstrate specificity and ion requirements(Matthews, 2000). Thus, glutamate movementin the central nervous system is mediated viadistinct Na-dependent proteins of theexcitatory amino acid transport (EAAT) class.Five such transporters (EAAT1–5) have beencloned from mammalian tissue and theirmodulation by other proteins is the subject of

© CAB International 2003. Amino Acids in Animal Nutrition,2nd edition (ed. J.P.F. D’Mello) 1

*E-mail address: f.dmello@ed.sac.ac.uk

continuing studies (Jackson et al., 2001). It isimportant to recognize, however, that aminoacids in different classes may share the sametransport system. Thus, three transportsystems recognize both lysine and leucine.Details of the biochemical and molecularcharacterization of the various transportsystems for the major classes of amino acidsare presented in Chapter 3.

Essential amino acids

Higher animals require a core of nine aminoacids for maintenance and productive pur-poses (Table 1.1). The need for these aminoacids arises from the inability of all animals tosynthesize the corresponding carbon skeletonor keto acid. These amino acids are classifiedas ‘indispensable’ or ‘essential’ and provisionof these nutrients is mandatory. Non-rumi-nants will receive the essential amino acids viathe diet, but ruminants may also acquire sub-stantial amounts of these amino acids throughthe digestion of microbial protein synthesizedin the rumen. Those amino acids which ani-mals are able to synthesize are termed ‘dis-pensable’ or ‘non-essential’.

All mammals require the core of nineessential amino acids, but may also respondto dietary arginine and possibly proline aswell, during the early phases of rapid growth.Endogenous synthesis of arginine from gluta-mate/glutamine and proline plays an impor-tant role in the provision of arginine in the pig

during the neonatal and immediate post-weaning phases (Wu et al., 1997). However,it has been estimated that 40% of the argininerequirements of the rapidly growing pig mustbe supplied by the diet. This need arises partlybecause the vast majority of arginine synthe-sized in the urea cycle is catabolized in theliver by an active arginase within this path-way. Consequently, insufficient arginine isexported for the rapid growth of extra-hepatictissues. It is relevant to recall in this contextthat sow colostrum and milk are markedlydeficient in arginine (Wu and Knabe, 1994).

The cat is unique among mammals in itsrequirement for arginine as an essential com-ponent of the diet. Indeed, Morris and Rogers(1978) observed that one meal without argi-nine was sufficient to precipitate mortality incats. Other effects included emesis, tetanicspasms and hyperammonaemia. It was con-cluded that the inability of the cat to synthesizeornithine is the basis of the dietary need forarginine. The cat also has a specific require-ment for taurine which is directly related to itsrole in the prevention of retinal degeneration.

Poultry have an absolute requirement forarginine arising from a non-functional ureacycle. This dependence translates into acutesensitivity to natural structural analogues ofarginine (Chapter 14).

Of the two aromatic amino acids requiredfor protein synthesis and other functions, onlyphenylalanine is considered to be essential.Tyrosine is regarded as dispensable as it is read-ily synthesized from phenylalanine. Although

2 J.P.F. D’Mello

Table 1.1. Nutritional classification of amino acids.

Essential

Additionalspecies-related Conditionally

Common core requirements non-essential Non-essential

Lysine Arginine (cats, poultry, fish) Cyst(e)ine GlutamateHistidine Taurine (cats) Tyrosine GlutamineLeucine Arginine GlycineIsoleucine Proline SerineValine AlanineMethionine AspartateThreonine AsparagineTryptophanPhenylalanine

this conversion is irreversible, the presence oftyrosine in the diet may reduce the requirementfor phenylalanine. However, this sparing effectof tyrosine is limited and, consequently, a mini-mum quantity of dietary phenylalanine shouldalways be ensured. With regard to growingpoultry, for example, at least 58% of the totalaromatic amino acid requirement should be sup-plied in the form of phenylalanine. An analo-gous situation exists between methionine andcyst(e)ine (see Chapter 8). The unique relation-ship between tryptophan and the B-complexvitamin, nicotinamide, represents another facetof the multifunctional roles of amino acids.

It is now widely acknowledged that high-yielding animals will not achieve their geneti-cally determined potential if the dietary N issupplied exclusively in the form of the essen-tial amino acids. Additional N is required andhighly effective sources of this non-specific Ninclude glutamate, alanine and diammoniumcitrate. However, the most effective source isa mixture of the non-essential amino acids.Consequently, although animals have specificdietary requirements for the essential aminoacids, some combination of the dispensableamino acids should also be provided in orderto maximize performance. The issue of essen-tial to non-essential amino acid ratios isreviewed in Chapter 6.

Glucogenic and ketogenic amino acids

In the degradation of amino acids, the carbonskeletons follow distinct pathways (seeChapter 4). Those amino acids that are bro-ken down to pyruvate or key intermediates ofthe tricarboxylic acid cycle have the potential

to yield glucose via phosphoenolpyruvate.These amino acids are referred to as gluco-genic. Those amino acids that yield acetylCoA or acetoacetyl CoA are classified as keto-genic since the latter two compounds are theprecursors of ketone bodies. Some aminoacids are both ketogenic and glucogenic(Table 1.2). Only leucine and lysine are exclu-sively ketogenic. In dogs, hepatic gluconeoge-nesis may be reduced by administration ofamino acids with glucose prior to and duringexercise (Hamada et al., 1998, 1999).

Structural analogues

A wide range of non-protein amino acidsoccur naturally in plants, particularly in theseeds and foliage of leguminous species.

Amino Acids as Multifunctional Molecules 3

Table 1.2. Glucogenic and ketogenic amino acids.

GlucogenicGlucogenic Ketogenic and ketogenic

Threonine Leucine IsoleucineArginine Lysine PhenylalanineMethionine TyrosineValine TryptophanHistidineCysteineGlutamateGlutamineAspartateAsparagineGlycineSerineProlineAlanine

Table 1.3. Plant non-protein amino acidsa with the potential to cause adverse effects in animals.

Aromatic amino acid Sulphur amino acidArginine analogues analogues analogues Neurolathyrogens

Canavanine Mimosine Selenomethionine �-(N-oxalylamino)alanineIndospicine 3,4-Dihydroxyphenylalanine Selenocyst(e)ine �-CyanoalanineHomoarginine S-Methylcysteine �,�-Diaminobutyric acid

sulphoxide (SMCO)Djenkolic acid

aSee Chapter 4 and D’Mello (1991) for structures and intermediary metabolism.

The non-protein group (Table 1.3) includesseveral structural analogues of the essentialamino acids. The aromatic amino acid,mimosine, occurs in the tropical legume,Leucaena leucocephala, a ubiquitousspecies yielding palatable forage for rumi-nants. A structural analogue of arginine,canavanine, is widely distributed in varioustropical legumes including Canavalia ensi-formis, Gliricidia sepium and Indigoferaspicata. In temperate regions, the factorcausing haemolytic anaemia in cattle andsheep consuming forage brassicas has beenidentified as a ruminal derivative of S-methylcysteine sulphoxide, an analogue ofmethionine, distributed throughout theplant. It has been conventional to considerselenocysteine as a member of the non-protein group; however, selenocysteine isnow recognized to be the 21st amino acidthat may be incorporated into specific pro-teins (Atkins and Gesteland, 2000). A subsetof the non-protein group contains the lathy-rogenic amino acids which are accordedwith profound neurotoxic properties.Although these neurolathyrogens have notbeen associated structurally with the essen-tial or non-essential amino acids, at leasttwo lathyrogenic amino acids exert markedeffects on the brain metabolism of gluta-mine. Consequently, some biochemicalassociation with the amino acid neurotrans-mitter system embracing glutamate and �-aminobutyrate (GABA) cannot be excluded.

Whereas some non-protein amino acidsare profoundly neurotoxic, others exert theireffects by reducing utilization of specificessential amino acids. Details of the adverseeffects and underlying mechanisms will befound in Chapter 7.

Isomers

All amino acids used in protein synthesismust be in the L-configuration. Nevertheless,animals are capable of utilizing some Denantiomorphs. Indeed the widespread com-mercial supplementation of diets withmethionine has been accomplished with theracemic mixture. Two steps are essential inthe utilization of D-amino acids: firstly, the

D-isomer must undergo oxidative deamina-tion to the corresponding �-keto acid ana-logue; secondly, this analogue must thenundergo L-specific reamination by means ofan appropriate aminotransferase reaction.There are no aminotransferases for lysineand threonine in animal tissues, hence theD-isomers of these amino acids are notnutritionally active (Table 1.4). Of all the D-amino acids that are convertible by animals,D-methionine is the most effective in replac-ing its L isomer. However, marginally betterefficacy may still be observed with L-methio-nine in comparison with D-methionine orDL-methionine when levels below therequirement are employed (see D’Mello andLewis, 1978). In addition, when racemicmixtures of other amino acids are used, D-methionine becomes less effective than theL-isomer. Species differences in D-aminoacid utilization should be recognized. Themouse utilizes D- and DL-methionine lesseffectively than other animals, whereas boththe rat and the pig use D-tryptophan moreefficiently than the chick. It is apparent thatthe mouse is an unsuitable model for validat-ing the efficacy of D-amino acids for otherspecies of animals (Table 1.4).

Deficiency

The unique sequence of amino acids in pro-teins demands that all amino acids, whetheressential or non-essential, be present at thesites of protein synthesis. In the event ofany deficit, the utilization of the remainingamino acids will be prejudiced and proteinsynthesis and other physiological processeswill be impaired. Thus, it might be assumedthat dietary deficiencies of individual essen-tial amino acids would lack distinctive fea-tures. However, the published evidenceindicates unique effects of particular aminoacids. This evidence has been derived pri-marily from studies in which the amino acidin question has been omitted altogether. Inaddition, recent investigations on determina-tion of maintenance requirements haveyielded valuable data on the effects of gradu-ated degrees of deficiency of particularessential amino acids.

4 J.P.F. D’Mello

Deletion studies

It is logical to expect that dietary deletion ofone particular essential amino acid shouldimpair growth to the same extent as the omis-sion of another essential amino acid.However, the results of many studies showdistinct differences between individual aminoacids. Thus, lysine deprivation in fish, chick-ens and rats (see D’Mello and Lewis, 1978) isaccompanied by relatively modest weightlosses, whereas isoleucine or threonine defi-ciencies inhibit growth more severely. Mediansurvival times for chicks fed diets devoid oflysine or histidine were 53 and 60 days.Isoleucine or valine deprivation reducedmedian survival times to 18 and 19 daysrespectively (Ousterhout, 1960). Chicks feddiets lacking in lysine or histidine were alsostronger and more active than those deprivedof isoleucine or valine. It has been postulatedthat certain peptides such as carnosine andproteins such as haemoglobin may serve assources of histidine and lysine in times ofrestricted supply. Appreciable reserves ofcarnosine occur in muscle (Maynard et al.,2001). Nevertheless, mortality will occur ifanimals are deprived of histidine or lysine forprolonged periods of time. Dietary omission

of a single essential amino acid might beexpected to induce effects similar to depriva-tion of all amino acids. The experimental evi-dence indicates that, for example, chicksdeprived of lysine survived for longer and lostless weight than those fed a protein-free diet.Similarly in rats fed a diet devoid of lysine,body water losses were less than in those feda protein-free diet. However, rats fed dietsdevoid of threonine, isoleucine or methionineplus cystine lost body water to the sameextent as those in the protein-free group (seeD’Mello and Lewis, 1978).

Extensive morphological changes havebeen recorded following the imposition ofsevere amino acid deficiencies. It has beenconsistently recorded that during acute aminoacid deprivation or deficiency in ad libitum-fed animals, there is a severe inhibition offood intake. Diet selection may also bealtered under certain conditions (Hrupka etal., 1997, 1999). The resulting morphologi-cal responses (Table 1.5) have been attributedto the combined effects of amino acid defi-ciency and energy restriction. Attempts have,therefore, been directed at overcoming themarked differences in food intake betweendeficient and control groups by pair-feedingor by force-feeding. However, D’Mello and

Amino Acids as Multifunctional Molecules 5

Table 1.4. Efficacy of isomers and analogues of amino acids. Values represent growth efficacyas a percentage of that of the L-isomer. (Adapted from Baker, 1994.)

Amino acid Chick Rat Mouse Dog Pig

D-lysine 0 0 0 – –D-threonine 0 0 0 – –D-tryptophan 20 100 30 35 80D-methionine 90 90 75 100 100DL-methionine 95 95 88 100 100DL-OH-methionine 80 – 70 – 100Keto-methionine 90 – – – –N-acetyl-L-methionine 100 100 90 100 –D-arginine 0 0 – – –D-histidine 10 0 10 – –D-leucine 100 50 15 – –D-valine 70 15 5 – –D-isoleucine 0 – – – –D-phenylalanine 75 70 – – –L-OH-phenylalanine 70 50 – – –Keto-phenylalanine 85 65 – – –D-tyrosine 100 100 – – –

Lewis (1978) concluded that feeding methodwas less important than duration of exposureto the deficient diet. Thus, similar morpho-logical effects were observed in long-termdeprivation of an amino acid under ad libi-tum conditions as in short-term force-feedingconditions (Table 1.5). Exposure timeappears to be a significant factor in theinduction of morphological changes duringamino acid deprivation. Although ad libitumfeeding over a period of about 8 days of adiet devoid of an essential amino acid elicitsno adverse histological changes in animals(D’Mello and Lewis, 1978), extensive aberra-tions do emerge in long-term investigationslasting 30 days or more. In many instances,these abnormalities reflect those observed inanimals force-fed incomplete amino acid dietsover a shorter duration (typically 3–8 days). Itwas noted that morphological abnormalitiesoccur even in animals deprived of lysine orhistidine (Table 1.5).

Predictably, extensive biochemicalchanges also occur during acute amino aciddeficiency, irrespective of feeding method(Table 1.6). Under these conditions, muscleprotein synthesis is rapidly reduced (Tesseraud

et al., 1996), and amino acids including theone missing from the diet are released intothe systemic circulation. These amino acids,together with those derived from intestinalabsorption, cause an increase in hepatic pro-tein synthesis. The partition of amino acids,however, depends on the dietary level andintake of carbohydrate. Low carbohydrateintake diminishes or eliminates the changes inhepatic protein synthesis. Thus, the biochemi-cal effects of acute amino acid deficienciesrepresent the consequence of a complexnutritional imbalance rather than that of asimple deficiency (D’Mello and Lewis, 1978).

Insulin-like growth factor-1 (IGF-1) exertsan important effect on whole-body proteinsynthesis. This factor is complexed with up tosix specific binding proteins, which arebelieved to modulate the biological activity ofIGF-1. The results of Takenaka et al. (2000)indicate that a single essential amino acid defi-ciency may reduce IGF-1 production in ratswithout affecting plasma IGF-1 binding pro-tein-1. The molecular action of specific aminoacids is likely to be an important area for fur-ther research.

6 J.P.F. D’Mello

Table 1.5. Morphological effects of amino acid deficienciesa. (Adapted from D’Mello and Lewis, 1978.)

Ad libitum feeding conditions Force-feeding conditionsOrgan affected (long-term studiesb) (short-term studiesc)

Liver Enhanced glycogen levels; Excess glycogen levels; lipidhepatocytes in periportal region accumulation in hepatic cells indistended by lipid droplets periportal areas; nucleolar

enlargementPancreas Reduced zymogen granules; lipid Decreased cytoplasm and zymogen

droplets in acinar cells; mitochondria granules in acinar cells; nucleolarswollen and deformed enlargement; oedema

Thymus Thymic involution; formation of giant Decrease of lymphocytes in cortex;cells; loss of normal architecture; loss of distinction between cortexdepletion of thymocytes and medulla

Muscle Degenerative changes; lack of cross- No changesstriations in fibres; damaged fibresswollen, hyalinized and fragmented

Testes Atrophy of seminiferous tubules and No datatesticular interstitial cells; inhibition ofspermatogenesis

aAs observed in rats deprived of isoleucine, threonine, lysine or histidine.bDuration: 30 days or more.cFeeding period: 3–8 days.

Maintenance studies

Induction of deficiency states is an integralcomponent of the process to determinemaintenance requirements of individualamino acids (Table 1.7). The approachinvolves the formulation of diets with varyingdegrees of deficiency. For example in theirstudies on threonine requirements for mainte-nance in the chick, Edwards et al. (1997)used diets varying in threonine concentra-tions from 5 to 95% of assumed require-ments for maximal growth. Chicks fed dietscontaining threonine at 5, 10 and 15% of

ideal requirements lost weight commensuratewith the degree of deficiency. Threonineretention was also negative for these groups,in proportion to the extent of threonine defi-ciency. Over the entire range tested, whole-body accretion of threonine occurred with anefficiency of 82%, considerably higher thanthat reported for pigs (60%; Adeola, 1995).Chicks fed lysine-deficient diets retainedlysine in carcass with an efficiency of 79%(Edwards et al., 1999). In contrast, chicks fedgraded levels of total SAA (methionine + cys-tine) from 5 to 95% of ideal requirementsgained weight, and retained SAA in carcass

Amino Acids as Multifunctional Molecules 7

Table 1.6. Effects of acute amino acid deficiency on protein metabolism. (Adapted from D’Mello andLewis, 1978.)

Aspect of protein metabolism Force-feeding studies Ad libitum-feeding studies

Protein content of:Liver No change DecreaseMuscle Decrease DecreasePancreas Decrease Decrease

Free amino acid levels in:Liver Decrease –Muscle Increase –Blood plasma Increase Increase

Protein synthesis in:Liver Increase IncreaseHeart Increase –Blood Increase IncreaseMuscle Decrease DecreaseKidney No change Marginal decrease

Table 1.7. Whole-body accretion of amino acids in chicks.

Dietary amino Whole-body accretion (mg day−1)acid level(% of ideal)a Thr Val Lys SAAb

5 −11.9 −8.7 −20.9 2.810 −6.4 −2.4 – 6.315 −2.3 – – –40 40.0 53.1 15.1 50.455 83.2 100.0 50.5 67.970 116.9 139.6 100.9 93.495 158.4 190.1 167.2 109.8Reference Edwards et al. Baker et al. Edwards et al. Edwards and

(1997) (1996) (1999) Baker (1999)

aDiets deficient in the single amino acids indicated in the next four columns.bSAA, sulphur amino acids.

in a positive and dose-related manner overthe entire range of dietary concentrations(Edwards and Baker, 1999). Furthermore,efficiency of whole-body SAA accretion wasonly 52%, presumably reflecting the diversefunctions of methionine in addition to its roleas a component of tissue proteins. Althoughchicks fed the lysine-deficient diet maintaineda stable body-weight (Edwards et al., 1999)those fed the valine-deficient diet lost weight(Baker et al., 1996) when each amino acidwas set at 5% of ideal. However, carcassretention of lysine was −20.9 mg day−1 andof valine was −8.7 mg day−1 (Table 1.7).Thus equivalent degrees of deficiency induceddifferent responses, with lysine again emerg-ing as an amino acid with unique effects (Veluet al., 1972).

Molecular Action

The biochemical and molecular actions ofamino acids are areas of increasing researchinterest. It is obvious that dietary aminoacids will stimulate muscle protein synthesisin the postabsorptive period. This anaboliceffect is partly due to increased substratesupply at the sites of protein synthesis.However, it has been proposed that individ-ual amino acids may act as signalling mole-cules that serve to regulate mRNAtranslation. The binding of initiatormethionyl-tRNA to the 40S ribosomal sub-unit is an important step subject to regula-tion in vivo (Table 1.8). However, Anthonyet al. (2000a) suggest that leucine may alsoact in a signalling role in the stimulation of

8 J.P.F. D’Mello

Table 1.8. Diverse functions of amino acids.

Amino acids Products Significance/functions

21 amino acidsa Polypeptides and proteins Hormones, enzymes and other bioactiveproteins

Methionine Formylmethionine (fMet) Initiator of protein synthesisS-Adenosylmethionine Donor of methyl groupsHomocysteine Donor of S; indicator of vitamin B12 status

Tryptophan Serotonin (5-hydroxytryptamine) NeurotransmitterNicotinamide B-complex vitamin

Tyrosine Dopamine NeurotransmitterNoradrenaline NeurotransmitterAdrenaline HormoneThyroxine Hormone

Arginine Nitric oxide Involved in vasorelaxation;neurotransmission; male reproductiveperformance; gut motility

Polyamines Regulation of RNA synthesis;maintenance of membrane stability

Histidine Histamine Potent vasodilatorGlutamate Glutamine Purine and pyrimidine synthesis;

excretion of N in avian speciesGlutathione Reduced form involved in maintenance

of cysteine residues of blood proteins ina reduced state

�-Aminobutyrate (GABA) NeurotransmitterEnergy Energy source in some tissues (mucosa)

Glycine Porphyrins Part of haemoglobin structurePurines Components of nucleic acids

Serine Sphingosine Membrane structureCysteine Important for activity of proteins

Aspartate Urea, purines and pyrimidines Donor of N3-Methylhistidine Component of actin and myosin Index of muscle protein breakdown

aIncludes selenocysteine.

muscle protein synthesis by enhancing avail-ability of specific eukaryotic initiation fac-tors. Further studies demonstrated thatleucine is unique among the BCAA in itsability to stimulate muscle protein synthesis(Anthony et al., 2000b). It is thought thatthese effects are independent of the actionof insulin. Clearly, additional work isrequired to elucidate the role of leucine andother amino acids as signalling molecules.

Amino Acids as Precursors ofBioactive Molecules

Irrespective of their position in the biochem-ical and nutritional classification, aminoacids are involved in diverse pathways lead-ing to the synthesis of important bioactivemolecules (Table 1.8). Indeed, glutamate hasbeen referred to as an amino acid of ‘partic-ular distinction’ (Young and Ajami, 2000)and selenocysteine is now recognized as the21st amino acid that may be incorporatedinto proteins (Atkins and Gesteland, 2000).Some amino acids are important precursorsof neurotransmitters and certain hormones,while others are involved in N transport andin the maintenance of integrity of cell mem-branes.

Neurotransmitters

Key neurotransmitters synthesized fromamino acids include GABA, serotonin,dopamine, noradrenaline and nitric oxide(Table 1.8). The pathways for the productionand metabolic disposition of the three bio-genic amines are now well established(Bradford, 1986); the synthesis of NO is dis-cussed below. It is logical to expect thatbrain concentrations of neurotransmittersmay be subject to dietary control of theamino acid precursors. One form of dietarymanipulation involves the imposition of anamino acid imbalance (Harrison andD’Mello, 1986, 1987). However, resultsfrom various sources are inconclusive andmay have been confounded by methodologi-cal differences among individual studies(Chapter 7).

Arginine

As indicated above, the primary direction ofarginine metabolism in mammals occurs viathe urea cycle, enabling the disposal ofexcess N from amino acids. However, theperipheral metabolism of arginine is also ofconsiderable biochemical and physiologicalsignificance. Thus, the action of argininedecarboxylase permits many organisms tosynthesize putrescine and other polyamines.In animals, putrescine is produced solely bythe action of ornithine decarboxylase(ODC). Although the specific functions ofpolyamines await elucidation, recent studiessuggest that these compounds are essentialfor normal growth and development in allliving organisms, and may regulate RNAsynthesis and stabilize membrane structures.Polyamine production appears to be anindispensable feature of all tissues activelyengaged in protein synthesis. Arginineuptake by the mammary gland from theblood supply substantially exceeds the quan-tities of this amino acid secreted in milk.This is generally attributed to the need tosynthesize non-essential amino acids, partic-ularly proline, within the gland itself.However, the excess uptake of arginine mayalso reflect the need for polyamine synthesisby tissues actively synthesizing proteins inthe mammary gland. Polyamine synthesis isan important focal point for the action ofantinutritional factors. Thus in lectin-inducedhyperplastic growth of the small intestine,levels of putrescine, spermidine, spermineand cadaverine are markedly enhanced(Pusztai et al., 1993). On the other hand,the growth-retarding effect in chicks fedC. ensiformis has been attributed to inhibi-tion of polyamine synthesis (Chapter 7).The non-protein amino acid, canavanine,present in this legume is metabolized tocanaline, a potent inhibitor of ODC(D’Mello, 1993).

A striking feature of arginine relates tothe synthesis of NO. The biosynthesis of NOinvolves the oxidation of arginine by NADPHand O2 via the action of NO-synthases. It isnow established that NO plays a key role invasorelaxation, neurotransmission, immuno-competence, male reproductive performance

Amino Acids as Multifunctional Molecules 9

and gut motility (Moncada et al., 1991). It issuggested in Chapter 7 that dietary canava-nine may inhibit NO synthesis through itsstructural antagonism with arginine. Ennekinget al. (1993) arrived at a similar conclusionfrom their studies on canavanine-induced feedintake inhibition in pigs.

Homocysteine

Homocysteine is a key intermediate in SAAmetabolism, positioned at the juncturebetween remethylation to methionine andtranssulphuration to cystathionine, yieldingcysteine and taurine. The importance ofhomocysteine in human health was highlightedat the first conference on this amino acid(Rosenberg, 1996). It is now well recognizedthat plasma homocysteine levels are higherthan normal in patients with coronary, cere-brovascular or peripheral arterial occlusive dis-ease (Malinow, 1996). Other investigatorssuggested a link between homocysteine andneural tube defects (Mills et al., 1996;Rosenquist and Finnell, 2001). Furthermore,circulating concentrations of this amino acidmay be of diagnostic value in assessing vitaminB12 status in humans (Stabler et al., 1996;Cikot et al., 2001). In pigs, prolonged vitaminB12 deficiency is associated with hyperhomo-cysteinaemia (Stangl et al., 2000a) whereas incattle a similar effect has been reported inlong-term moderate deficiency of Co (Stangl etal., 2000b). Co is required for ruminal synthe-sis of vitamin B12. It is clear that much moreeffort is required to elucidate the role of homo-cysteine in farm animals, particularly pregnantruminants and sows.

Immune modulators

A number of essential amino acids have beenimplicated in immune function. Cysteine mayfunction as an immunoregulatory signalbetween macrophages and lymphocytes. It hasbeen proposed that release of this amino acidby macrophages enhances intracellular concen-trations of the cysteine-containing tripeptide,glutathione (GSH) in lymphocytes. The latter isimportant for T-cell activity. Miller et al. (2000)

observed that cysteine infusion into the aboma-sum of sheep appeared to influence certainfacets of immune response, including antibodyresponses to non-parasitic antigens. However,the exact role of cysteine in ovine immunefunction remains elusive. Swain and Johri(2000) indicated that the methionine require-ment for optimum antibody production inbroiler chickens was greater than that for opti-mum growth. Other reports suggest thatdietary cysteine and the BCAA in particularmay exert specific effects in the modulation ofimmune responses in broiler chickens(Takahashi et al., 1997; Konashi et al., 2000).Clearly, there is a need to undertake furtherstudies to elucidate the exact role of SAA andBCAA as regulators of the immune system.

3-Methylhistidine

This unique amino acid occurs in the muscleproteins actin and myosin, deriving its methylgroup in a post-translational event (Rathmacher,2000). It is used as an index of muscle proteinbreakdown since it does not charge tRNA andis thus not re-utilized in protein synthesis. It isexcreted quantitatively in the urine of certainanimals and the major source is skeletalmuscle. Thus changes in 3-methylhistidine dis-position is predominantly a reflection of muscleprotein metabolism. However, species differ-ences are apparent. Cattle, for example, quan-titatively excrete 3-methylhistidine in urine,whereas in sheep and pigs excretion is notquantitative. Rathmacher (2000) presentedthree-compartment models of 3-methylhistidinekinetics (Fig. 1.1), based on the knowledge thatthere are pools of this amino acid in plasma, inother extracellular fluids, within muscle and inother tissues. In sheep and pigs there is a bale-nine pool in muscle. Balenine is a dipeptidecomposed of �-alanine and 3-methylhistidine inequimolar quantities.

Conclusions

In this chapter an attempt has been made toset the scene in terms of identifying anddefining basic principles such as classificationof amino acids, unique effects of deficiency

10 J.P.F. D’Mello

and utilization of isomers and precursors ofneurotransmitters. Since the previous edition(D’Mello, 1994), significant advances havebeen made in our understanding of the multi-functional roles of amino acids. These have

been highlighted in this chapter and pursuedin greater depth in other parts of this book.Emerging issues include the role of specificamino acids, such as leucine, in molecularsignalling and cysteine in modulation of

Amino Acids as Multifunctional Molecules 11

Fig. 1.1. Three-compartment models for 3-methylhistidine (3-MH) kinetics in selected animal species(Rathmacher, 2000). Tracer doses of labelled 3-methylhistidine are used in investigations with these models.

immune function. In addition, a number ofissues have reached a stage enabling a moremature assessment of their metabolic ornutritional significance. The use of 3-methyl-

histidine is a case in point, whereas the roleof homocysteine emerges as an issue worthyof future research, particularly with respect tobreeding animals.

12 J.P.F. D’Mello

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Edwards, H.M. III, Fernandez, S.R. and Baker, D.H. (1999) Maintenance lysine requirement and efficiencyof using lysine for accretion of whole-body lysine and protein in young chicks. Poultry Science 78,1412–1417.

Enneking, D., Giles, L.C., Tate, M.E. and Davies, R.L. (1993) Canavanine: a natural feed-intake inhibitorfor pigs. Journal of the Science of Food and Agriculture 61, 315–325.

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Mills, J.L., Scott, J.M., Kirke, P.N., McPartlin, J.M., Conley, M.R., Weir, D.G., Molloy, A.M. and Lee, Y.J.(1996) Homocysteine and neural tube defects. Journal of Nutrition 126, 756S–760S.

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Pusztai, A., Grant, G., Spencer, R.J., Duguid, T.J., Brown, D.S., Ewen, S.W.B., Peumans, W.J., VanDamme, E.J.M. and Bardocz, S. (1993) Kidney bean lectin-induced Escherichia coli overgrowth inthe small intestine is blocked by GNA, a mannose-specific lectin. Journal of Applied Bacteriology75, 360–368.

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Rosenberg, I.H. (1996) Homocysteine, vitamins and arterial occlusive disease: an overview. Journal ofNutrition 126, 1235S–1237S.

Rosenquist, T.H. and Finnell, R.H. (2001) Genes, folate and homocysteine in embryonic development.Proceedings of the Nutrition Society 60, 53–61.

Stabler, S.P., Lindenbaum, J. and Allen, R.H. (1996) The use of homocysteine and other metabolites in thespecific diagnosis of vitamin B-12 deficiency. Journal of Nutrition 126, 1260S–1272S.

Stangl, G.I., Roth-Maier, D.A. and Kirchgessner, M. (2000a) Vitamin B-12 deficiency and hyperhomocys-teinemia are partly ameliorated by cobalt and nickel supplementation in pigs. Journal of Nutrition130, 3038–3044.

Stangl, G.I., Schwarz, F.J., Jahn, B. and Kirchgessner, M. (2000b) Cobalt-deficiency-induced hyperhomo-cysteinaemia and oxidative status of cattle. British Journal of Nutrition 83, 3–6.

Swain, B.K. and Johri, T.S. (2000) Effect of supplemental methionine, choline and their combination onthe performance and immune response of broilers. British Poultry Science 41, 83–88.

Takahashi, K., Ohta, N. and Akiba, Y. (1997) Influence of dietary methionine and cysteine on metabolicresponses to immunological stress by Escherichia coli lipopolysaccharide injection, and mitogenicresponse in broiler chickens. British Journal of Nutrition 78, 815–821.

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Amino Acids as Multifunctional Molecules 13

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14 J.P.F. D’Mello

2 Amino Acid Analysis of Feeds

Johannes Fontaine*Feed Additives Division, Degussa AG, Applied Technology, Hanau, Germany

Introduction

Never before has the economic success offeed manufacturers been so heavily depen-dent on optimal and accurate amino acidcomposition as today. The effects on growthand feed conversion of animals are soimpressive that the annual amount of syn-thetic methionine and lysine supplementedin feeds worldwide now exceeds 400,000tonnes each. This generates growing demandfor amino acid analysis of feed raw materialsto improve the amino acid matrix for linearfeed programming, but also as a qualityassurance tool for compound feeds and pre-mixtures.

In the first edition of this book A.P.Williams reported on the state of amino acidanalysis by reviewing the literature up to1992. In this second edition the focus shallbe mainly on the developments of the last10 years. There are numerous publicationson amino acid analysis, but only a small pro-portion are concerned with test matrices thatare relevant to animal nutrition, such asfeedingstuffs, food products, plants, silages,by-products of plant and animal origin, ani-mal blood plasma, intestinal and ruminalcontents; these are the topics which will bediscussed here.

One of the most important developmentsof recent years was undoubtedly the longoverdue international standardization ofamino acid analysis in feedingstuffs. Analyticalscientists at national European supervisoryauthorities, who in the 1980s had adopteddifferent methods of analysis for the determi-nation of total amino acids as standards intheir respective countries, eventually joinedforces in an international collaborative effortto establish a common EU methodology.Recently, after extensive groundwork, officialEU methods for the determination of totaland free amino acids in animal feedingstuffsand corresponding methods for tryptophanwere passed (Commission Directives98/64/EC and 2000/45/EC). Internationalcollaboration was also the basis for the analyt-ical method adopted by AOAC Internationalfor the determination of total amino acidswith the exception of tryptophan (AOAC,1994). The official methods of analysis of theAOAC enjoy worldwide recognition as anauthorative collection of analytical methodsfar beyond the NAFTA region. The referencemethod adopted for feed analysis was thechromatographic separation of amino acidswith a cation exchanger resin followed by nin-hydrin derivatization. Sample preparation isvirtually identical for the various methods. The

© CAB International 2003. Amino Acids in Animal Nutrition,2nd edition (ed. J.P.F. D’Mello) 15

*E-mail address: johannes.fontaine@degussa.com

EU standard procedure for the determinationof tryptophan in feedingstuffs utilizes alkalinehydrolysis with barium hydroxide and HPLCanalysis with specific fluorescence detection.

A second important innovation was thedevelopment of robust, internationally usablenear-infrared spectroscopy (NIRS) calibrationsfor amino acids for the major feed rawmaterials, based on reliable wet chemicalreference analysis. These allow the rapid,simple analysis of the amino acidcomposition of raw materials today and up-to-date feed optimization. This developmentwill also be described in detail.

A review article by Kivi (2000) whichfocuses on chromatographic and detectionmethods deserves special mention because itis an excellent addition to this review.

Wet Chemical Analysis

Sample preparation

Acid hydrolysis and oxidation

The determination of amino acids requires thehydrolytic splitting of protein into its individualbuilding blocks, which behave very differentlyduring hydrolysis due to the functionality ofthe R side group. Asparagine and glutaminelose the amide residue in the side group andform aspartate and glutamate, respectively.The resulting ammonia can be determinedchromatographically, but amino acid analysisalways determines the sum Asx or Glx ofthese amino acid pairs. Tryptophan is largelydestroyed by acid hydrolysis whereas methion-ine, and especially cystine, are converted bypartial oxidation into several derivatives.Standard hydrolysis conditions in the EU andUSA are 24 h with 6 M hydrochloric acid at110°C, the boiling point of this acid. This canbe done either under reflux or in a closed ves-sel in a thermostatized oven. These conditionsare a compromise for optimal recovery of allamino acids. Serine and threonine, whichcarry one hydroxyl group, are slowly degradedwith increasing length of hydrolysis or increas-ing acid strength by the splitting off of waterand the branched-chain amino acidsisoleucine, leucine and valine, especially if

they are adjacent in the protein, are moreslowly released by steric hindrance of thehydrolysis. Albin et al. (2000a,b) recentlystudied this topic and the effect of the acidconcentration for soybean products, Rowanet al. (1992) tested the effect of the hydrolysistime (8–72 h) for diets, ileal digesta and faecessamples. The optimal recovery relative to thestandard conditions is frequently 5–10%, insome cases 20% or even higher. The authorsrecommend the use of correction factors.

Hydrolysis in a microwave oven at about150°C can reduce the time needed for split-ting the protein to a few minutes (Carisano,1992; Joergensen and Thestrup, 1995;Marconi et al., 1996; Shang and Wang,1997; Kroll et al., 1998). If only minuteamounts of protein are available hydrolysiswith gaseous hydrochloric acid (Schrijver etal., 1991; Molnar-Perl and Khalifa, 1994) isof benefit. Fountoulakis and Lahm (1998)produced a survey of protein hydrolysis tech-niques, which also includes alternative acidssuch as methan- or p-toluene-sulphonic acidor the use of enzymes (Hauck, 1990; Chenet al., 1996). Weiss et al. (1998) compareddifferent hydrolysis techniques by means ofchromatographically purified proteins. In the method of standardization of the EU(Commission Directive 98/64/EC) the use ofcorrection factors for the hydrolysis, as previ-ously practised in the Netherlands, was delib-erately ruled out, however. The side reactionsof hydrolysis are matrix-dependent and canbe considerable, especially in microwavehydrolysis. Correction factors would thereforehave to be defined individually for all feed rawmaterials, which would pose problems, espe-cially in the case of compound feeds.Moreover, differences in the use of correctionfactors would probably increase the analyticalerror when compiling tables of raw materialcomposition. The uniform application ofstandard hydrolysis conditions worldwide onthe other hand produces highly consistentamino acid analyses.

The sulphur-containing amino acids areoxidized prior to hydrolysis in the protein withperformic acid to methionine sulphone andcysteic acid, which survive acid hydrolysiswithout losses. In the official methods of theEU and the AOAC this is done by leaving a

16 J. Fontaine

sample corresponding to 10 mg nitrogen toreact overnight with 5 ml performic acid (16 h)at 0°C. The latter was previously generated insitu at room temperature from 4.5 ml of 88%formic acid and 0.5 ml of 30% hydrogen per-oxide with the addition of a little phenol andcannot be concentrated or stored. These reac-tion conditions have been standardized fordecades and are based on the work of Schramet al. (1954). Excess performic acid can laterbe destroyed by the addition of hydrogenbromide or hydrogen chloride, formingbromine or chlorine, which have to be drawnoff with a rotary evaporator. Spindler et al.(1984), Elkin and Griffith (1985) and Gehrkeet al. (1987) studied the recovery of all aminoacids under these conditions and showed thatall acid-stable amino acids, with the exceptionof phenylalanine, histidine and tyrosine, arecompletely recovered from the hydrolysateafter previous oxidation. Mason et al.(1980a,b) and Bech-Andersen et al. (1990)tried to streamline this procedure and intro-duced the addition of sodium metabisulphitesalt to destroy performic acid. The advantageis that it eliminates the need for rotaryremoval of the halogen prior to hydrolysis andthat phenylalanine and histidine can also bedetermined from the oxidized hydrolysate.This method is capable of determining allamino acids except tryptophan and tyrosine.However, it is important to remember thatthese hydrolysates now contain sulphuric acidor hydrogen sulphate formed from sulphitewhich, when concentrated for the completeremoval of the hydrochloric acid, can formsulphate esters with serine and threoninewhich interfere with cysteic acid in the aminoacid analyser. The EU method based on thisvariant therefore suggests neutralization of the hydrolysates, which can be readily automated, or the partial removal of thehydrochloric acid by distillation. Llames andFontaine (1994) compared both variants ofoxidation and acid hydrolysis in a worldwidecollaborative trial, with 28 participants, usingbroiler starter and finisher feed, maize, fish-meal and poultry meal, and obtained equiva-lent results (Table 2.1). Toran et al. (1996)also suggest oxidation for the determina-tion of cystine and methionine in infant for-mulas.

Slump and Bos (1985) describe an inter-ference of the oxidation of methionine, ifchloride levels from salts far in excess of 1%occur in for example supplementary feeds orconcentrates. This causes the performic acidto be partially degraded, while forming chlo-rine, so that only cysteic acid but not methion-ine sulphone is formed completely. If thishappens the authors suggest oxidation withdiluted performic acid for methionine determi-nation, which is not suitable for cystine. TheGerman VDLUFA (1997) has nominated thisprocedure as the official method of analysisfor methionine determination in chloride-richdiets. We should also mention the work ofTuan and Phillips (1997) who studied cystinerecovery in diets based on casein andsorghum and in digesta samples as mixeddisulphide with the addition of 3,3�-dithiodipropionic acid during hydrolysis withhydrochloric acid in evacuated ampoules at145°C and different times. At a 100-foldmolar excess of the reagent they recoveredpure cystine at the rate of 99%. Very inconsis-tent results were however obtained in feedand digesta, which makes a final assessmentof the method impossible.

Tryptophan analysis

The analysis of tryptophan, an essentialamino acid which can become limiting, espe-cially in swine diets, has been extensively stud-ied. The focus in recent years has been onalkaline hydrolysis in the total absence of oxy-gen. Simat and Steinhart (1998) recently pub-lished detailed studies on the oxidation of freeand protein-bound tryptophan and describedfollow-up products. Nielsen and Hurrell(1984), having conducted comparative tests,suggested hydrolysis with sodium hydroxide inan evacuated vessel (110°C, 20 h) with theaddition of 5-methyltryptophan as internalstandard and hydrolysed starch. Werner(1986) and Rogers and Pesti (1990) on theother hand preferred to work with lithiumhydroxide after flushing with nitrogen, withoutusing an internal standard, because sodiumhydroxide readily attacks glass, and theyreported a 97–99% tryptophan recovery.Slump et al. (1991) compared lithium hydrox-ide and barium hydroxide for hydrolysis in a

Amino Acid Analysis of Feeds 17

18J. F

ontaine

Table 2.1. Comparison of three oxidation and hydrolysis procedures in an international collaborative study with 28 participating labs and five samples in blindduplicates. The mean amino acid contents obtained were equivalent, if the respective method is applicable.

Mean contents(g kg-1)

Broiler finisher Broiler starter Maize Fishmeal Poultry meal

Oxa Ox Ox Ox Ox Ox Ox Ox Ox Ox(SO2) (HBr) Hyd (SO2) (HBr) Hyd (SO2) (HBr) Hyd (SO2) (HBr) Hyd (SO2) (HBr) Hyd

Alanine 11.7 12.0 11.8 12.8 13.1 12.9 6.1 6.1 6.2 35.0 35.6 34.9 42.6 42.8 42.6Arginine 12.8 12.2 12.5 15.7 15.1 15.7 4.0 3.9 3.7 34.0 32.4 33.6 43.5 42.4 44.0Aspartate 16.8 17.5 16.7 22.9 23.6 23.0 5.4 5.6 5.4 52.2 52.6 53.1 49.2 50.4 51.3Cystine 3.2 3.5 – 3.5 3.6 – 1.8 1.9 – 4.8 4.9 – 8.1 8.2 –Glutamate 32.5 32.3 32.4 40.4 40.4 40.4 15.1 14.9 15.4 73.7 74.9 74.5 79.7 80.5 81.8Glycine 12.7 13.0 13.0 12.7 12.9 12.9 3.3 3.3 3.3 38.4 39.1 38.8 69.0 68.6 69.8Histidine 5.0 – 5.0 6.5 – 6.1 2.7 – 2.4 13.7 – 13.9 13.1 – 13.8Isoleucine 7.6 7.6 7.4 9.5 9.8 9.6 2.8 2.9 2.8 23.2 24.0 23.5 22.4 23.1 23.0Leucine 16.6 16.9 16.6 19.7 20.3 19.8 9.9 10.0 9.9 40.7 41.5 40.7 40.9 41.8 41.0Lysine 10.7 11.0 10.6 13.5 13.9 13.5 2.6 2.6 2.5 42.2 44.6 42.5 36.3 37.2 36.7Methionine 5.3 5.4 – 6.2 6.3 – 1.8 1.9 – 16.1 16.3 – 11.7 12.0 –Phenylalanine 8.7 – 8.7 11.2 – 11.1 3.8 – 4.0 22.9 – 22.4 23.3 – 23.3Proline 13.9 14.1 14.2 14.7 14.3 15.0 7.3 7.1 7.6 26.2 25.4 26.5 45.3 44.9 47.8Serine 9.4 9.4 9.7 11.2 11.4 11.7 3.9 3.9 4.1 22.1 22.3 22.5 27.6 27.1 28.6Threonine 7.3 7.4 7.4 11.2 9.1 9.0 2.9 3.0 3.0 22.8 23.8 23.7 23.2 23.8 23.8Tyrosine – – 6.3 – – 8.4 – – 3.0 – – 18.5 – – 17.8Valine 9.2 9.3 9.3 11.1 11.2 11.1 3.8 3.9 3.9 27.8 28.9 28.2 28.2 28.5 29.0

aOx (SO2), oxidation, where performic acid is destroyed by addition of sodium metabisulphite and followed by standard hydrolysis (6 mol l-1 HCl, 110°C, 24 h);

Ox (HBr), oxidation, where performic acid is destroyed by addition of hydrogen bromide and followed by standard hydrolysis; Hyd, only standard hydrolysis.

sterilizing autoclave (130°C, 8 h), added theinternal standard only after the hydrolysis andobtained the same recovery each time, whichthey estimated at about 92% for defined pro-teins and pure tryptophan. Bech-Andersen(1991) suggested hydrolysis in an autoclavewith sodium hydroxide solution, adding lac-tose to enhance recovery and �-methyltrypto-phan as internal standard. Ranfft and Faure(1993), who conducted an intercomparisonwith compound feed and four raw materialsusing different in-house methods of 14European laboratories, found coefficients ofvariation ranging from 4 (soy) to 10% (maize)and observed no significant differences in rela-tion to the hydroxide used, temperature, timeor the hydrolysis technique. Landry andDelhaye (1992a,b, 1994a) and Delhaye andLandry (1992, 1993) conducted intensivestudies on tryptophan determination. Threehydroxides, different times and temperaturesfor hydrolysis and several assay procedureswere varied and compared. They favoured asa result the hydrolysis with barium hydroxidein an autoclave with added 5-methyltrypto-phan as internal standard to compensate forlosses. They also suggested HPLC conditionson reversed phase (RP)-silica gel with a sepa-rating time of only 10 min and fluorescencedetection. Landry and Delhaye (1994b) evalu-ated the results of two collaborative studies bynormalizing the tryptophan values of all otherparticipants relative to their own assays. Theywere convinced that the above conditionsbring 15–20% higher tryptophan recovery.Most other authors also suggested HPLCwith UV or fluorescence detection, the latterbeing preferred because of its specificity fortryptophan.

Landry and Delhaye (1992a) also com-pared separation by gel filtration onSephadex, analysis of the hydrolysates with acation exchanger or colorimetric analysis afterstaining with p-dimethylamino-cinnamalde-hyde. Other authors also determined trypto-phan colorimetrically by the acid ninhydrinmethod (Pintér-Szakács and Molnar-Perl,1990), after conversion with p-dimethyl-amino-benzaldehyde (Lee et al., 1996) ornitrous acid (Shah et al., 1996). However, oncomparing this procedure with HPLC analy-sis, the simple operation and greater precision

of the HPLC method is emphasized. The gen-eral acceptance of HPLC has therefore led tothe virtual disappearance of the previouslywidespread spectrophotometric tryptophanmethods. Molnár-Perl (1997) has compiled areview of methods of tryptophan analysis inpeptides and proteins, including descriptionsof techniques not normally used in feed-ingstuffs, such as acid or enzymatic hydrolysis,measurement by gas chromatography orderivative spectroscopy whithout chemicalderivatization. Carisano (1993), whoemployed microwave hydrolysis with lithiumhydroxide under temperature control, wasable to hydrolyse meat and fish completely inless then 60 min, obtaining results similar tothose with barium hydroxide (110°C, 12 h).He derivatized tryptophan with o-phthal-dialdehyde (OPA) prior to HPLC, whereasAlgeria et al. (1996) converted with phenyl-isothiocyanate (PITC).

An expert group of the EU Commission,DG XII, has been working for 4 years oncomparative studies with a view to establish-ing a harmonized system of feedstuff analysisin the EU. Fontaine et al. (1998) reportedthat three collaborative trials with 12–16 par-ticipants tested lithium hydroxide versussodium hydroxide and barium hydroxide, theuse of an internal standard during or afterhydrolysis, the suitability of 5-methyl- or �-methyltryptophan as internal standard and theuse of evacuated or nitrogen-purged hydroly-sis vessels versus autoclaving. Landry andDelhaye (1994b), who also participated, pre-sented the view, as mentioned above, that 5-methyltryptophan must be present duringhydrolysis with barium hydroxide and that byusing their special autoclaving conditionsabout 15% higher tryptophan concentrationsare obtained than with other techniques. Thesecond collaborative trial showed, however,that irrespective of the hydrolysis techniquethe use of this internal standard generatedanalytical values in all laboratories that were asignificant 13–20% higher than with �-methyltryptophan. A comparison of thehydrolytic stability of the two internal stan-dards revealed that the 5-methyl derivative isless stable than tryptophan, whereas �-methyltryptophan is considerably more stable.This leads to a marked overcorrection of the

Amino Acid Analysis of Feeds 19

small hydrolysis losses in the case of protein-bound tryptophan, so that the addition of aninternal standard during hydrolysis wasrejected. The best results were obtained with4 M barium hydroxide (110°C, 20 h) in anautoclave. Moreover, the addition of the stable�-methyltryptophan after hydrolysis greatlyimproved the comparability of the resultsbetween laboratories. The determination ofsupplemented tryptophan was also optimized,the best technique being extraction with 0.1M hydrochloric acid with an internal standard.Publication of the Commission Directive(2000) made the adopted method compulsoryacross the EU. The accuracy achieved wasexcellent, with a reproducibility CVR ofbetween 2.2 and 6.3%, and 1.5–4.7% in asecond test.

Supplemented or free amino acids andtaurine

Virtually all compound feeds are now supple-mented with amino acids and determinationof this additive is an important quality assur-ance tool in the feed industry. Fahnenstichand Tanner (1973) developed a method forextracting free amino acids from a few gramsof finely ground feed using diluted cold 0.1 Mhydrochloric acid. Mason et al. (1980a)described similar extraction conditions butwith the addition of thiodiglycol as stabilizerfor methionine and protein precipitation inthe extract prior to chromatography. Virtuallyidentical extraction conditions were incorpo-rated in the official EU method for the deter-mination of supplemented amino acids as aresult of the Commission Directive (1998). Inthe EU collaborative trial, where this extrac-tion method was tested on a premix, excellentrepeatability within laboratory CVr and repro-ducibility between laboratories CVR areachieved (Table 2.2). Protein hydrolysis doesnot occur with diluted hydrochloric acid. It isnot possible, however, to distinguish supple-mented from natural, non-protein-boundamino acids out of the feed raw materials. Allthat needs to be done prior to performingcation exchange chromatography is to adjustthe pH to 2.20 and no special cleaning of theextracts is necessary according to our experi-

ence. Extractions of various compound feedswith the addition of norleucine as internalstandard gave a very good intralaboratoryrepeatability CVr of 1–1.5% in our laboratory(Fontaine, 2002). Foulon et al. (1990), whocompared extraction of methionine and lysinewith water, 85% ethanol and buffer solutionwith the acid extraction described above,achieved the highest recovery with 0.1 Mhydrochloric acid. Saurina and Hernandez-Cassou (1993) described a flow-injection spec-trophotometric determination specialized forsupplemented lysine in commercial feeds.Bech-Andersen (1997) developed special alka-line/acid extraction conditions for mineralpremixes with high contents of heavy metalsin order to obtain extracts without heavymetal ions as the latter cause considerableinterference in the cation exchanger. Fontaineand Eudaimon (2000) gave a detailed descrip-tion of an acid extraction method with subse-quent determination in an amino acidanalyser, specially designed for assays of com-mercial amino acid products and concentratedpremixes, which they tested in an interna-tional collaborative trial with 17 laboratories.They achieved a between-laboratory repro-ducibility CVR of only 1.5–2.6% and a recov-ery of the amino acids in the accuratelyproduced premixes of 97.5–102.8%. Thismethod has been adopted as the Official FirstAction by AOAC International.

The effect of amino acids in the diet onfree amino acid concentrations in bloodplasma, muscles or liver, etc., has also beenthe subject of many animal experiments.These matrices require deproteinization byprecipitation (addition of acids or solvents) orby means of physical techniques (ultrafiltrationetc.). Walker and Mills (1995) wrote in areview article that in human clinical diagnosticwork precipitation is performed predomi-nantly with 4–5% final concentration of 5-sulphosalicylic acid, as also described by DeJonge and Breuer (1994) for porcine plasmaand Hagen et al. (1993) for bovine plasma.Sedgwick et al. (1991) compared protein pre-cipitation by means of acetone or acetonitrilewith that performed using perchloric acid ortrichloroacetic acid in ovine plasma andbovine serum albumin solutions. Acid precipi-tation achieved good amino acid recoveries

20 J. Fontaine

Am

ino Acid A

nalysis of Feeds

21

Table 2.2. Precision of amino acid analysis. Published results of collaborative trials on the analysis of total amino acid contents in mixed feed and ingredientsand of supplemented amino acids in premixes and pure amino acid trade products.

Llames and Fontaine (1994) Commission Directive (1998) Bütikofer et al. (1992)

Whey proteinBroiler finisher Poultry meal Fish meal Mixed pig feed Broiler compound concentrate Feed

CVr CVR CVr CVR CVr CVR CVr CVR CVr CVR CVr CVR CVr CVR

Methionine (total) 1.1 7.6 2.1 12.0 1.9 9.7 3.4 7.0 3.1 10.9 3.1 13.4 5.9 16.0Cystine (total) 3.1 11.3 4.6 17.7 4.0 19.0 3.3 9.9 2.8 8.8 5.5 44.8 13.1 77.2Lysine (total) 3.5 9.0 3.1 9.9 2.8 7.9 2.8 3.2 2.1 5.4 2.3 5.5 5.3 13.2Threonine (total) 2.7 8.2 3.2 9.1 3.6 10.7 1.9 4.1 2.1 5.3 1.5 5.2 3.1 8.4Arginine (total) 2.3 8.6 3.3 9.7 2.3 7.2 3.0 8.8 4.2 7.8Isoleucine (total) 3.2 6.8 2.7 11.7 2.1 10.3 4.2 9.4 3.1 18.7Leucine (total) 2.7 6.3 2.5 7.6 1.9 6.8 2.5 6.4 2.3 7.8Valine (total) 3.8 12.7 3.2 12.8 2.3 11.2 2.9 10.2 6.1 16.4Phenylalanine (total) 4.4 14.6 3.5 9.2 1.6 7.7 4.2 11.5 4.7 12.3

CommissionCommission Directive (2000) Dir. (1998) Fontaine and Eudaimon (2000)

Feed concentratefor pigs Soybean meal Premixture No. 3 Premix No. 5 Premix BiolysTM DL-Methionine

CVr CVR CVr CVR CVr CVR CVr CVR CVr CVR CVr CVR CVr CVR

Tryptophan (total) 1.9 2.2 1.3 4.1

Methionine (suppl.) 2.4 6.9 1.2 1.8 0.5 2.6 0.9 1.5Lysine (suppl.) 2.1 6.7 1.3 2.5 0.7 1.9 0.8 2.3Threonine (suppl.) 2.2 4.3 0.7 1.9 0.8 1.9

CVr, coefficient of variation (%) for within laboratory standard deviation Sr, repeatability CVr; CVR, coefficient of variation (%) for between laboratory standarddeviation SR, reproducibility CVR.

over a wide concentration range of the dis-solved protein (4–16%). The solvents, how-ever, included free amino acids in theprecipitate so that the recovery fell to only50% with increasing protein content. Aristoyand Toldra (1991) on the other hand obtainedequally satisfactory results in the determina-tion of free amino acids in fresh pork muscleand dry-cured ham either by protein precipita-tion with perchloric acid, trichloroacetic acid,picric acid and acetonitrile or by ultrafiltrationwith a 10 kDa exclusion limit, whereassulphosalicylic acid or ultrafiltration with 1kDa gave a poorer recovery. Peter et al.(1999) also used trichloroacetic acid for pro-tein precipitation in cell extracts of algae andBugueno et al. (1999) used acetonitrile fordeproteinization of raw salmon muscle.Antoine et al. (1999) used 75% methanol inthe analysis of free amino acids in fish sam-ples. Urine sample preparation on the otherhand was done by Hara et al. (1999) with30% sulphosalicylic acid. Van de Merbel et al.(1995) described online amino acid monitor-ing in fermentation processes employingultrafiltration for protein separation and ionexchange chromatography. There are thusmany suitable variants for the removal ofunwanted protein and the recovery of supple-mented amino acids should always be testedin preliminary experiments.

Taurine, a metabolic derivative of cys-teine, is essential for cats but it also plays arole in infant nutrition. It occurs in the non-protein-bound form and can, in principle, beanalysed in the same way as free amino acids.Balschukat and Kunesch (1989) described itsdetermination in fish meal, pet food and ani-mal raw materials following extraction with0.1 M hydrochloric acid in an amino acidanalyser. Nicolas et al. (1990) measured tau-rine concentrations in infant formulas andhuman milk following ultrafiltration also byion-exchange chromatography. McCarthy etal. (2001) tested a taurine assay in pet foodafter acid hydrolysis (6 M HCl, 110°C, 16 h)and precolumn derivatization with dansyl chlo-ride in a collaborative trial. In six samples ofwet and dry cat and dog food interlaboratoryreproducibility CVRs of 6.1–13.3% wereachieved, and the procedure was adopted asthe official method by AOAC International.

Porter et al. (1988), Amiss et al. (1990),Sakai and Nagasawa (1992) and Messing andSturmann (1993) described the determinationof taurine in feline and other blood andplasma by HPLC separation following pre-column derivatization with different reagents.

Chromatographic separation anddetection

Several recent review articles on amino acidanalysis should first be mentioned here. Morrand Ha (1995) reported in general terms onanalysis of protein and amino acids in foodproducts, whereas Rutherfurd and Moughan(2000) focused on analysis of animal feeds.Molnar-Perl (2000) described chromato-graphic techniques for the determination ofsugar, carboxylic acids and amino acids infoodstuffs and included references to tech-niques such as gas chromatography and capil-lary electrophoresis. Fekkes (1996) andWalker and Mills (1995) reported on advancesin amino acid determination in physiologicalsamples or biological fluids, respectively.Detailed descriptions of different chromato-graphic techniques were given, with examplesof the degree of separation obtained.

Ion-exchange chromatography (IEC)

The chromatographic separation of allamino acids by means of a cation exchangecolumn (polystyrene sulphonic acid as Na+

or Li+ salt) eluted with three to five citrate orsometimes acetate buffers of different ionicstrength, pH and with different modifiers,was developed way back in 1948 and auto-mated by Spackman et al. as an amino acidanalyser in 1958. More than 50 years laterthis method had become the standard inamino acid analysis owing to the outstand-ing and stable separation, which allows theanalysis of more than 45 amino acids inblood plasma (lithium buffer), the highreproducibility of retention time and peakarea and the extended stability of the cali-bration. The resin is highly resistant tomatrix effects, which is of major importancegiven the complex composition of com-pound feeds. Any increase in pressure due

22 J. Fontaine

to soiling or a reduction in the separationperformance can in many cases be cor-rected by simply reversing the flow direc-tion; in the author’s laboratory ionexchange columns are often operated con-tinuously for more than a year; they arethen cleaned, the resin regenerated and thecolumns repacked. The running costs of anamino acid analyser are therefore far lowerthan using RP-HPLC columns, which do notallow regeneration and are susceptible toattack by fats and non-polar components infeedingstuffs and therefore need to bechanged frequently. Postcolumn derivatiza-tion is carried out either with ninhydrin in aheated coil or with OPA, the latter reagentbeing about 20 times more sensitive. Thekey advantage of postcolumn derivatizationis that each amino acid, be it from sampleor standard, is always derivatized underidentical conditions in the respective elutionbuffer, which explains the high reproducibil-ity. Moreover, amino acids in the underiva-tized state are chromatographically morestrongly differentiated than the derivatives ofconventional HPLC precolumn derivatiza-tions, where a very large non-polar mole-cule is bound to the amino group. Also, thisreaction inevitably takes place in the respec-tive matrix, which can influence the reactionyield and stability of the derivative. Bütikoferet al. (1992) conducted a collaborative trialcomparing HPLC analysis after precolumnderivatization with a mixture of OPA and 9-fluorenylmethyl chloroformate (FMOC) asan alternative to the classic ion exchanger indairy products and feedingstuffs; althoughthe means obtained with both methods weresimilar, IEC delivered greater reproducibilityand precision and was recommended forcases requiring high precision. As IECequipment technology is mature few articleswith details have been published. Grunauand Swiader (1992) and Moeller (1993)used IEC columns in HPLC machines withtwo- or three-buffer gradient elution; LeBoucher et al. (1997) subjected the HitachiL-8500A, a new amino acid analyser, tointensive tests with biological fluids for per-formance in peak reproducibility, retentiontime stability and linear concentration, withgood results.

Other separation and detection techniques ofunderivatized amino acids

The se